Space-time controllable PROTAC based on photo-initiation in-situ generation and application thereof

Abstract

The invention discloses a time-space controllable in-situ generation PROTAC based on photo-initiation and an application of the PROTAC. The PROTAC is divided into two components, namely a component connected with a biological orthogonal reaction group and a target protein ligand and a component connected with the biological orthogonal reaction group and an E3 ubiquitin ligase ligand, and the PROTAC can be synthesized in situ through illumination control and is used for targeted protein degradation. The defects that the PROTAC molecule de novo synthesis difficulty is large, synthesis steps are tedious and the like are overcome, and poor pharmacological properties and toxic and side effects caused by large molecular weight and the like in traditional PROTAC design are avoided. Meanwhile, PROTAC molecules can be generated only after illumination, target protein is degraded, tumor cells are killed, off-target toxicity is avoided, and the tumor treatment effect is achieved. According to the method for generating the proteolysis targeting chimera through light-triggered click, PROTAC molecules with the anti-cancer effect can be controllably and efficiently synthesized, and the method has the prospects of precise regulation and control of intracellular protein expression and tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a spatiotemporally controllable photoinduced in situ generated PROTAC and its application. Background Art

[0002] Proteolysis targeting chimeras (PROTACs) can utilize the naturally existing protein cleaning system in the body to reduce protein levels and achieve the purpose of treating diseases. After 20 years of continuous improvement and development, PROTAC technology has become one of the hottest technologies in the field of new drug research and development. PROTAC molecules are heterobifunctional chimeras, which are composed of target protein ligands, linkers, and E3 ubiquitin ligase ligands. PROTAC molecules mediate the formation of a ternary complex between themselves and the target protein and E3 ubiquitin ligase, thereby promoting the polyubiquitination of the target protein and degradation by the proteasome. The advantages of PROTAC molecules are that they have a wider range of action, higher activity, and can target "undruggable" targets. Traditional small molecules and antibodies all inhibit the function of target proteins through the "occupancy-driven" mode of action to play a role in treating diseases. This mode of action requires inhibitors or antibodies to have a high concentration to occupy the active site of the target and block the transduction of downstream signaling pathways. The PROTAC strategy is "event-driven", which does not affect the function of the target protein, but directly mediates the degradation of the target protein. Secondly, after PROTAC molecules degrade target proteins, they can be released again to continue to degrade new target proteins, and can be used repeatedly, so the catalytic amount can play a therapeutic role. Moreover, the PROTAC strategy can also degrade proteins without active sites. As long as the PROTAC molecules can bind to the target protein, they can induce the degradation of related proteins, which can greatly increase the range of applicable targets.

[0003] However, with the in-depth study of the PROTAC strategy, the defects of the PROTAC strategy have also been exposed. There are two main defects: First, the catalytic mechanism of PROTAC may complicate the process of its pharmacological action while bringing stronger activity, which is prone to serious off-target toxicity. Second, PROTAC molecules need to have both ligands that bind to the target protein and E3 ligase recruitment elements and linkers that connect the two, resulting in high molecular weight and polar surface area characteristics. This characteristic will greatly affect the cell penetration and solubility of PROTAC molecules, resulting in PROTAC molecules with poor bioavailability and pharmacokinetic parameters. Therefore, it is urgent to develop some strategies to solve these problems.

[0004] In order to address the problem that PROTACs may produce large off-target toxicity, researchers have made PROTAC molecules into prodrug forms of PRO-PROTAC. Target heads (folic acid, antibodies, nucleic acid aptamers) are connected to PROTAC molecules to make targeted PROTACs. Photoprotective groups are connected to PROTAC molecules to regulate mammalian proteins through light mediation. Dirk Trauner et al. have well summarized examples of photoactivated PROTACs. There are also a series of redox-activated, hypoxia-activated and enzyme-activated PROTACs. However, the PROTAC molecule itself has a large molecular weight, and the corresponding photoremovable groups or bioorthogonal protecting groups make this type of PROTACs have a larger molecular weight and worse physical and chemical properties, which greatly limits the application of this strategy.

[0005] Bioorthogonal coupling reactions provide a way to overcome the problem of large molecular weight of PROTACs. Following the CLIPTACs strategy of generating PROTACs by clicking trans-cyclooctene and tetrazine in 2016, Zhang Jie's research group has successively reported the generation of PROTACs by clicking norbornene and tetrazine and the in situ generation of PROTACs mediated by endogenous copper ion-catalyzed azidoalkynyl cycloaddition. However, this strategy is not controllable. When bioorthogonal reaction pairs meet, click reactions will occur, and the off-target toxicity of PROTAC cannot be avoided. Bengang Xiang et al. reported enzyme-driven click protein hydrolysis targeting chimeras. They connected bifunctional chimeras through the reaction of generating fluorescein and modified nitroreductase response groups on the connecting chain. This degradation mode depends on nitroreductase (NTR) enzymes and selectively forms bifunctional chimeras only in hypoxic sites. It can target and degrade BRD4 in vitro and in vivo, combining controllability with in situ generation of PROTACs. However, this strategy is responsive to the internal environment and is not responsive to normoxic environments, so its application range is still limited. Therefore, it is very necessary to develop a more universal click-generated proteolysis-targeting chimera strategy that is spatiotemporally controllable. Summary of the invention

[0006] To solve the above problems, the present invention provides a method and application of in situ generation of PROTAC based on light initiation for spatiotemporal controllable targeted degradation of proteins. The PROTAC synthetic precursor synthesized by this method has natural light stability; the PROTAC synthetic precursor synthesized by this method can quickly undergo a click reaction after activation by ultraviolet light to generate PROTAC molecules for spatiotemporal controllable degradation of target proteins and killing of tumor cells, thereby achieving tumor treatment effects.

[0007] The first object of the present invention is to provide a proteolysis-targeted chimera synthetic precursor, the structural formula of the proteolysis-targeted chimera synthetic precursor is shown in general formula I-1:

[0008]

[0009] in:

[0010] Said X is selected from C2 alkyl or dimethylsilyl;

[0011] The L1 is a connecting chain (connected to the phenolic hydroxyl group through a covalent bond to jointly construct a PROTAC synthetic precursor for connecting the bioorthogonal reaction group and the target protein ligand);

[0012] The R1 is a target protein ligand.

[0013] Further, at least one of the following is included:

[0014] (1) L1 comprises a group covalently linked to a phenolic hydroxyl group; preferably, L1 comprises one of the following structures:

[0015]

[0016] Wherein, n is selected from an integer between 2 and 7;

[0017] (2) the target protein includes at least one of a cell membrane surface protein, a cytoplasmic protein, and a nuclear protein;

[0018] (3) The target protein includes at least one protein highly expressed in tumors;

[0019] (4) The target protein ligand comprises a bromodomain-containing protein 4 (BRD4) ligand and / or a poly ADP ribozyme (PARP1) ligand; preferably, the structural formulas of the bromodomain-containing protein 4 ligand and the poly ADP ribozyme ligand are as shown in I-2 and I-3:

[0020]

[0021] Further, the proteolysis targeting chimera synthetic precursor comprises one of the structures shown below:

[0022] It should be particularly pointed out that all the molecules of the present invention are obtained after screening. For example, in the first structure described above, Cl cannot be replaced by halogens such as Br, which will cause the molecule to lose its activity.

[0023] The second object of the present invention is to provide a method for preparing the proteolysis targeted chimera synthetic precursor, comprising the following steps:

[0024] S1, reacting the target protein ligand with the connecting chain in the presence of a condensation reagent, an organic base and an organic solvent to obtain an intermediate;

[0025] S2, reacting the intermediate described in S1 with the compound represented by formula I-5 in the presence of an inorganic base and an organic solvent to obtain the protein hydrolysis targeted chimera synthesis precursor;

[0026]

[0027] Wherein, the X is selected from C2 alkyl or dimethylsilyl.

[0028] Furthermore, the connecting chain is selected from one of the following structures:

[0029]

[0030] n is selected from an integer between 2-7.

[0031] Further, at least one of the following is included:

[0032] The condensation reagent includes HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate);

[0033] The organic base includes DIPEA (N,N-diisopropylethylamine);

[0034] The inorganic base includes sodium hydride;

[0035] The organic solvent is any solvent that can dissolve the reaction raw materials, such as N,N-dimethylformamide.

[0036] Furthermore, the molar ratio of the target protein ligand, the connecting chain, the condensation reagent and the organic base is 1:(1-1.5):(1-2):(2-4), preferably 1:1.2:1.5:3.

[0037] Furthermore, the molar ratio of the dibenzocycloalkanecyclopropenone derivative I-5, the inorganic base and the intermediate is 1:(1-3):(1-2), preferably 1:2:1.5.

[0038] Furthermore, the intermediate and the inorganic base are added to an organic solvent and stirred for not less than 15 minutes to obtain a mixed solution.

[0039] Furthermore, in step S1, the reaction temperature is room temperature, and the reaction time is 2-12 hours.

[0040] Furthermore, in step S2, the reaction temperature is 50-60°C, and the reaction time is 2-30h, preferably 12h.

[0041] The third object of the present invention is to provide a proteolysis targeting chimera, the structural formula of the proteolysis targeting chimera is shown in the following general formula:

[0042]

[0043] in:

[0044] Said X is selected from C2 alkyl or dimethylsilyl;

[0045] The L1 is the first connecting chain (connected to the phenolic hydroxyl group through a covalent bond to jointly construct a PROTAC synthetic precursor 1 for connecting the bioorthogonal reaction group and the target protein ligand);

[0046] The R1 is a target protein ligand;

[0047] The L2 is a second connecting chain (connected to the azide group through a covalent bond to jointly construct a PROTAC synthetic precursor 2 connected to the bioorthogonal reaction group and the E3 ubiquitin ligase ligand);

[0048] The R2 is an E3 ubiquitin ligase ligand.

[0049] Further, at least one of the following is included:

[0050] (1) L1 comprises a group covalently linked to a phenolic hydroxyl group; preferably, L1 comprises one of the following structures:

[0051]

[0052] Wherein: n is selected from an integer between 2 and 7;

[0053] (2) the target protein includes at least one of a cell membrane surface protein, a cytoplasmic protein, and a nuclear protein;

[0054] (3) The target protein includes at least one protein highly expressed in tumors;

[0055] (4) The target protein ligand comprises a bromodomain-containing protein 4 (BRD4) ligand and / or a poly ADP ribozyme (PARP1) ligand; preferably, the structural formulas of the bromodomain-containing protein 4 ligand and the poly ADP ribozyme ligand are as shown in I-2 and I-3:

[0056]

[0057] (5) L2 comprises a group covalently linked to the azide group; preferably, L2 comprises one of the following structures:

[0058]

[0059] Wherein: n is selected from an integer between 2 and 3;

[0060] (6) The E3 ubiquitin ligase ligand is selected from at least one of a Cereblon protein ligand and a VHL ligand; preferably, R2 comprises one of the following structures:

[0061]

[0062] in:

[0063] W is selected from CH2, C=O, NH or N-C1-C4 alkyl;

[0064] Y is selected from O or S;

[0065] Z is selected from H, C1-C4 alkyl, C3-C6 cycloalkyl or halogen;

[0066] G, G' is selected from H, C1-C4 alkyl, -OH or C1-C4 alkyl substituted 5-10 membered heterocyclic group, wherein the heterocyclic group contains 1-3 heteroatoms of N, O or S;

[0067] R3 is selected from H, halogen, nitro, amino, cyano, hydroxy, C1-C4 alkyl or halogenated C1-C4 alkyl.

[0068] The fourth object of the present invention is to provide a method for preparing the proteolysis targeting chimera, comprising the following steps:

[0069] The proteolysis-targeted chimera synthetic precursor is mixed with the compound shown in formula I-4, and irradiated with light to obtain the proteolysis-targeted chimera;

[0070] in:

[0071]

[0072] The L2 is a connecting chain;

[0073] The R2 is an E3 ubiquitin ligase ligand.

[0074] Furthermore, the preparation method of the compound represented by formula I-4 comprises: heating azide-PEG-amine or azide-alkyl-amine and E3 ubiquitin ligase ligand in the presence of an organic base and an organic solvent to obtain the obtained compound.

[0075] Furthermore, the organic solvent is any solvent that can dissolve the reaction raw materials, such as N,N-dimethylformamide.

[0076] Furthermore, the temperature of the heating reaction is 90-110° C., and the reaction time is 2-12 hours.

[0077] Furthermore, the wavelength of the light is 200-500nm, preferably 365nm.

[0078] Furthermore, the method of targeted protein degradation based on bioorthogonal reactions activates PROTAC synthetic precursor compounds through light irradiation, and then undergoes a photo-decaged ring strain-promoted azide-alkyne cycloaddition reaction in situ to generate PROTAC, which can degrade target proteins and / or treat tumors.

[0079] Furthermore, a scheme for targeted protein degradation based on bioorthogonal reactions provided in one embodiment of the present invention comprises the following steps:

[0080] (1) Using a small molecule ligand of BRD4 protein or a small molecule ligand of PARP1 protein as a target protein ligand and a dibenzocycloalkane cyclopropenone as a bioorthogonal reaction group, a series of bioorthogonal reaction groups and target protein ligands are synthesized to connect PROTAC synthetic precursors;

[0081] Using pomalidomide as the E3 ligase ligand and azide as the bioorthogonal reactive group, a PROTAC synthetic precursor linked to the bioorthogonal reactive group and the E3 ubiquitin ligase ligand was synthesized;

[0082] (2) The PROTAC synthetic precursor linked to the bioorthogonal reaction group and the target protein ligand is co-incubated with the PROTAC synthetic precursor linked to the bioorthogonal reaction group and the E3 ubiquitin ligase ligand, and then the PROTAC molecules are generated in situ by light activation to degrade the target protein and kill tumor cells.

[0083] The fifth object of the present invention is to provide the use of the proteolysis targeting chimera synthetic precursor or the proteolysis targeting chimera in the preparation of protein degradation products or anti-tumor products.

[0084] Furthermore, the protein degradation product is used for protein degradation in vivo or in vitro.

[0085] Furthermore, the protein degradation product is used to simultaneously degrade one or more target proteins. The method of light-triggered click generation of protein hydrolysis targeting chimeras provided by the present invention provides a new idea for solving the defects of PROTAC molecules such as high off-target toxicity and poor physicochemical properties, and also provides a new method for dual or even multiple controllable degradation of target proteins.

[0086] Furthermore, the anti-tumor product is used for the treatment of tumors.

[0087] Furthermore, the product is a medicine.

[0088] The sixth object of the present invention is to provide a protein degradation product, wherein the protein degradation product contains the protein hydrolysis targeting chimera synthetic precursor or the protein hydrolysis targeting chimera.

[0089] A seventh object of the present invention is to provide an anti-tumor product, comprising a container containing the following independently packaged preparations: a preparation containing a first synthetic precursor; a preparation containing a second synthetic precursor;

[0090] Wherein, the first synthetic precursor includes the proteolysis targeting chimera synthetic precursor, and the second synthetic precursor includes an azide group and an E3 ubiquitin ligase ligand (preferably a compound shown in Formula I-4). Those skilled in the art can know that although the embodiment of the present invention only provides one method of use, in actual use, the first synthetic precursor (such as compound 1, compound 2) and the second synthetic precursor (such as compound 3) can also be injected into the body separately and then combined with light therapy to achieve the corresponding function.

[0091] Beneficial effects of the present invention:

[0092] 1. The present invention provides a method for targeted protein degradation based on bioorthogonal reactions, which divides a proteolysis targeting chimera (PROTAC) into two components: a component connected to a bioorthogonal reaction group and a target protein ligand, and a component connected to a bioorthogonal reaction group and an E3 ubiquitin ligase ligand. The PROTAC generated by the photoactivated PROTAC synthetic precursor compound undergoes a ring tension-promoted azide-alkynyl cycloaddition reaction in situ to achieve spatiotemporally controllable target protein degradation, thereby selectively killing tumor cells and achieving the effect of precise tumor treatment. It avoids the adverse pharmacological properties and toxic side effects caused by large molecular weight in the traditional PROTACs design. It solves the defects of PROTAC molecules with large off-target toxicity and poor physicochemical properties.

[0093] 2. The method for targeted protein degradation based on bioorthogonal reactions provided by the present invention can be used for the precise regulation of multiple proteins in cells. By co-incubating PROTAC synthetic precursors connected to bioorthogonal reaction groups and target protein ligands with bioorthogonal reaction groups and E3 ubiquitin ligase ligands, and then activating the generation of multiple PROTAC molecules in situ through light illumination, multiple degradations of target proteins in time and space can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 This is a schematic diagram of the protein degradation principle of the present invention;

[0095] Figure 2HPLC graphs of PROTAC synthetic precursors prepared in Examples 1-5 of the present invention reacting in 16.7% DMSO / PBS, wherein A is an HPLC graph of the in vitro illumination reaction of compound 1 and compound 3; B is an HPLC graph of the in vitro illumination reaction of compound 2 and compound 3; C is an HPLC graph of the stability of compound 1; D is an HPLC graph of the stability of compound 2; and E is an HPLC graph of the stability of compound 3;

[0096] Figure 3 The protein immunoblot analysis diagrams of the embodiments of the present invention, wherein A is a protein immunoblot analysis diagram of the light-dependent protein degradation of compound 1 and compound 3; B is a protein immunoblot analysis diagram of the protein degradation mechanism of compound 1 and compound 3; C and D are protein immunoblot analysis diagrams of the concentration-dependent protein degradation of compound 1 and compound 3; E and F are protein immunoblot analysis diagrams of the time-dependent protein degradation of compound 1 and compound 3;

[0097] Figure 4 : are protein immunoblot analysis diagrams of the embodiments of the present invention, wherein A is a protein immunoblot analysis diagram of the light-dependent BRD4 and PARP1 protein dual degradation of compound 1, compound 2 and compound 3 in Hela cells; B is a protein immunoblot analysis diagram of the light-dependent BRD4 and PARP1 protein dual degradation of compound 1, compound 2 and compound 3 in MDA-MB-231 cells;

[0098] Figure 5 This is an immunofluorescence image of an embodiment of the present invention, which is an immunofluorescence imaging image of the light-dependent dual degradation of BRD4 and PARP1 proteins of Compound 1, Compound 2 and Compound 3 in Hela cells;

[0099] Figure 6 This is a fluorescence image of the ability of Compound 1, Compound 2 and Compound 3 to inhibit cancer cell proliferation in zebrafish before and after light activation after co-incubation. DETAILED DESCRIPTION

[0100] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0102] The method of the present invention is as follows: The structures of the compounds in the following examples are determined by nuclear magnetic resonance ( 1 The NMR measurement was performed using an ACF-300BRUK nuclear magnetic resonance spectrometer, with deuterated chloroform (CDCl3) as the solvent and TMS as the internal standard.

[0103] Example 1

[0104] This example relates to the preparation of a PROTAC synthetic precursor intermediate, and the reaction equation is as follows:

[0105]

[0106] The specific operations are as follows:

[0107] Dissolve 2-bromoethylamine (16 mg, 0.13 mmol) and I-2 (44 mg, 0.11 mmol) in 2 mL DMF, add HATU (57 mg, 0.15 mmol) and DIPEA (53 μL, 0.33 mmol), react at room temperature for 1 h, monitor the reaction by thin layer chromatography, and after the reaction is complete, add saturated ammonium chloride solution, extract with EA, and wash three times with saturated brine. Dry the organic phase with anhydrous sodium sulfate, remove the solvent with a rotary evaporator, and separate the residue with preparative thin layer chromatography (eluent polarity DCM: MeOH = 25:1) to obtain a white powder solid S1 (44 mg, 80%). The product was characterized by nuclear magnetic resonance, and the results are as follows:

[0108] 1 H NMR (400MHz, CDCl3) δ7.80–7.74(m,1H),7.60–7.54(m,2H),7.48–7.42(m,2H),6.22(t,J=7.0Hz,1H),3.78–3.68(m,1H),3.68–3. 61(m,1H),3.64–3.53(m,2H),3.09(dd,J=12.4,7.0Hz,1H),3.02(dd,J=12.4,7.0Hz,1H),2.65(s,2H),2.33(s,2H),2.29(s,2H).

[0109] Example 2

[0110] This example relates to the preparation of a PROTAC synthetic precursor intermediate, and the reaction equation is as follows:

[0111]

[0112] I-3 (100 mg, 0.273 mmol) and 6-bromohexanoic acid (59 mg, 0.3 mmol) were dissolved in 3 mL DMF, HATU (156 g, 0.409 mmol) and DIPEA (143 μL, 0.819 mmol) were added, and the reaction was carried out at room temperature for 1 h. The reaction was monitored by thin layer chromatography. After the reaction was complete, a saturated ammonium chloride solution was added, and then extracted with EA. The organic phases were combined, washed with saturated brine, and dried with anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the residue was separated by silica gel column chromatography (eluent polarity DCM: MeOH = 25:1) to obtain a white powder solid S2 (80 mg, 54%). The product was characterized by NMR, and the results were as follows:

[0113] 1 H NMR (400MHz, CDCl3) δ8.51–8.40(m,1H),7.82–7.63(m,3H),7.33(q,J=5.9,7.4Hz,2H),7.03(t,J=8.9Hz,1H),4.29(s,2H),3.81–3.67(m,2H),3 .59–3.52(m,2H),3.45–3.36(m,3H),3.33–3.19(m,2H),2.42–2.23(m,2 H),1.87(d,J=9.2Hz,4H),1.66(q,J=8.2,8.8Hz,2H),1.54–1.38(m,2H).

[0114] Example 3

[0115] This example relates to the preparation of a PROTAC synthetic precursor intermediate, and the reaction equation is as follows:

[0116]

[0117] Under nitrogen protection, add a THF solution of m-bromoanisole (10g, 53.5mmol in 60mL THF) to a three-necked flask, slowly add n-butyl lithium (2.5M in hexane, 22mL, 55mmol) at -78°C, add for 15min, and stir for 1h. Then add a THF solution of dichlorodimethylsilane (3.55mg, 27.5mmol in 10mL THF) to the reaction system, and then return to room temperature and react overnight. Then quench the reaction with a saturated ammonium chloride solution, extract with EA three times, combine the organic phases, wash twice with saturated brine, dry the organic phase with anhydrous sodium sulfate, and use a rotary evaporator to remove the solvent. The residue is separated by silica gel column chromatography (eluent polarity 100% PE) to obtain a transparent oily liquid S5 (2.19g, 16%). The product was characterized by NMR, and the results are as follows:

[0118] 1 H NMR (400MHz, Chloroform-d) δ7.30 (dd, J=8.2, 7.2Hz, 2H), 7.14

[0119] –7.08(m,2H),7.08–7.04(m,2H),6.91(ddd,J=8.2,2.8,1.0Hz,2H),3.79(s,6H),0.55(s,6H).

[0120] Example 4

[0121] This example relates to the preparation of a PROTAC synthetic precursor intermediate, and the reaction equation is as follows:

[0122]

[0123] Dissolve tetrachlorocyclopropene (1.41 g, 7.928 mmol) in 45 mL DCM, add aluminum chloride (2.2 g, 16.5 mmol), stir at room temperature for 15 min, then cool to -20 ° C, add S5 (1.8 g, 6.607 in 10 mL DCM) in DCM, react for 0.5 h, and then warm to room temperature for 4 h. Monitor the reaction by thin layer chromatography. After the reaction of the raw material is complete, add ice water to quench the reaction. Wash the organic solution with saturated test brine, dry the organic phase with anhydrous sodium sulfate, and remove the solvent with a rotary evaporator. The residue is separated by silica gel column chromatography (eluent polarity DCM: EA = 1:1) to obtain brown powder solid S7 (576 mg, 27%). The product was characterized by nuclear magnetic resonance, and the results are as follows:

[0124] 1 H NMR (400MHz, Chloroform-d) δ7.90(d,J=8.5Hz,2H),7.24(d,J=2.7Hz,2H),7.03(dd,J=8.5,2.6Hz,2H),3.90(s,6H),0.49(s,6H).

[0125] Example 5

[0126] This example relates to the preparation of a PROTAC synthetic precursor intermediate, and the reaction equation is as follows:

[0127]

[0128] Under ice bath conditions, S7 (250 mg, 0.77 mmol) was dissolved in 15 mL DCM, aluminum chloride (154 mg, 1.16 mmol) was added, stirred for 15 min, then heated to 55 ° C and reacted for 12 h. The reaction was monitored by thin layer chromatography until the raw material reacted completely. The residue was filtered out with diatomaceous earth, the organic phase was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography (eluent polarity DCM: EA = 1:2) to obtain a white powder solid I-5 (57 mg, 27%). The product was characterized by nuclear magnetic resonance, and the results are as follows:

[0129] 1 HNMR (400MHz, DMSO-d6) δ7.74(dd,J=40.4,8.5Hz,2H),7.35–7.15(m,3H),7.00(d,J=8.4Hz,1H),3.89(s,3H),0.46(s,6H). 13 C NMR(100MHz,DMSO-d6)δ163.13,162.35,149.10,148.48,146.93,145.25,144.94,

[0130] 134.51,134.13,122.96,122.69,122.17,121.20,118.18,116.09,56.57,0.05(2C).

[0131] Example 6

[0132] This example relates to the preparation of a PROTAC synthetic precursor, and the reaction equation is as follows:

[0133]

[0134] The specific operations are as follows:

[0135] I-5 (100 mg, 0.32 mmol) was dissolved in 3 mL DMF, 60% sodium hydride (26 mg, 0.64 mmol) was added under ice bath and the reaction continued under ice bath for 15 min, then the reaction was restored to room temperature for 15 min. S1 (242 mg, 0.48 mmol) was then added to the reaction system, heated to 55 °C and reacted overnight. The reaction was monitored by thin layer chromatography. After the reaction was complete, the reaction was quenched with ice water, extracted with EA, and then washed with saturated ammonium chloride and saturated brine three times. The organic phase was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography (eluent polarity DCM:EA=2:1) ​​to obtain a white powder solid 1 (55 mg, 37%). The product was characterized by NMR, and the results are as follows:

[0136] 1 H NMR (400MHz, CDCl3) δ0.48 (d, J=4.4Hz, 6H), 1.64 (s, 3H), 2.35–2.40 (m, 3H), 2.60 (s, 3H), 3.40 (ddd, J=1. 8,6.5,14.4Hz,1H),3.54–3.63(m,1H),3.64–3.74(m,1H),3.78–3.88(m,1H),3.90(s,3H),4.11–4.22(m,2 H),4.57–4.64(m,1H),6.97(dd,J=2.6,8.5Hz,1H),7.03(dd,J=2.6,8.5Hz,1H),7.21(d,J=8.6Hz,2H),7.2 4(dd,J=2.6,4.1Hz,2H),7.30(t,J=5.8Hz,1H),7.35(dd,J=1.9,8.5Hz,2H),7.88(dd,J=8.4,16.9Hz,2H).

[0137] Example 7

[0138] This example relates to the preparation of a PROTAC synthetic precursor, and the reaction equation is as follows:

[0139]

[0140] The specific operations are as follows:

[0141] I-5 (100 mg, 0.32 mmol) was dissolved in 3 mL DMF, 60% sodium hydride (26 mg, 0.64 mmol) was added under ice bath and the reaction continued under ice bath for 15 min, then the reaction was restored to room temperature for 15 min. S1 (260 mg, 0.48 mmol) was then added to the reaction system, heated to 55 °C and reacted overnight. The reaction was monitored by thin layer chromatography. After the reaction was complete, the reaction was quenched with ice water, extracted with EA, and then washed with saturated ammonium chloride and saturated brine three times. The organic phase was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography (eluent polarity DCM:EA=2:1) ​​to obtain a white powder solid 1 (55 mg, 37%). The product was characterized by NMR, and the results are as follows:

[0142] 1HNMR(400MHz, CDCl3)δ8.49–8.43(m,1H),7.88(t,J=8.7Hz,2H),7.80–7.66( m,3H),7.32(t,J=6.8Hz,2H),7.25–7.18(m,2H),7.09–6.95(m,3H),4.28(s,2 H),4.10–3.99(m,2H),3.90(s,3H),3.83–3.21(m,8H),2.38(dt,J=7.4,29.0H z,2H),1.92–1.79(m,2H),1.78–1.73(m,2H),1.62–1.49(m,2H),0.48(s,6H).

[0143] Example 8

[0144] This example relates to the preparation of a PROTAC synthetic precursor, and the reaction equation is as follows:

[0145]

[0146] 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (479 mg, 1.73 mmol) and azide-alkyl-amine (202 mg, 1.57 mmol) were dissolved in 6 mL DMF, and DIPEA (547 μL, 3.14 mmol) was added to the reaction system. After heating to 100 °C, stirring was continued for 12 h. The reaction was monitored by thin layer chromatography. After the reaction was complete, the reaction solution was diluted with EA, washed with saturated sodium bicarbonate solution, washed with saturated brine, and the organic phase was dried with anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the residue was separated by silica gel column chromatography (eluent polarity DCM: EA = 10:1) to obtain a yellow powder solid 3 (130 mg, 21%). The product was characterized by nuclear magnetic resonance, and the results are as follows:

[0147] 1 H NMR (400MHz, CDCl3) δ8.08(s,1H),7.55–7.45(m,1H),7.10(dd,J=7.2,0.6Hz,1H),6.88(d,J=8.5Hz,1H),6.24(t,J=5.7Hz,1H) ,4.91(dd,J=12.1,5.4Hz,1H),3.36–3.23(m,4H),2.94–2.65(m,3H),2.19–2.09(m,1H),1.79–1.61(m,4H),1.57–1.44(m,2H).

[0148] Example 9

[0149] This example relates to the preparation of a PROTAC synthetic precursor negative control, and the reaction equation is as follows:

[0150]

[0151] Dissolve 3 (50 mg, 0.13 mmol) in 6 mL THF, add 60% sodium hydride (8 mg, 0.16 mmol) in an ice bath and continue to react in an ice bath for 15 min, then return to room temperature for 15 min. Then add iodomethane (10 μL, 0.16 mmol) to the reaction system and continue to react at room temperature overnight. Monitor the reaction by thin layer chromatography. After the reaction is complete, quench the reaction with ice water, extract with EA, and then wash with saturated brine three times. Dry the organic phase with anhydrous sodium sulfate, remove the solvent with a rotary evaporator, and separate the residue with silica gel column chromatography (eluent polarity DCM: EA = 10:1) to obtain a bright yellow powder solid Me-3 (17 mg, 33%). The product was characterized by nuclear magnetic resonance, and the results are as follows:

[0152] 1 H NMR(400MHz,Chloroform-d)δ7.49(dd,J=7.1,8.5Hz,1H),7.09(d,J=7.1Hz,1H),6.87(d,J=8.5Hz,1H),6.23(t,J=5.8Hz,1H),4.95–4.8 5(m,1H),3.34–3.24(m,4H),3.21(s,3H),3.03–2.89(m,1H),2.82–2.69(m,2H),2.15–2.05(m,1H),1.73–1.63(m,4H),1.55–1.46(m,2H).

[0153] Example 10 In vitro synthesis HPLC experiment

[0154] Taking compound 1 prepared in Example 6 as an example, its reactivity with compound 3 prepared in Example 8 and its stability under natural light were studied. The specific operation was as follows:

[0155] Solutions of compound 1 (c = 300 μM) and compound 3 (c = 450 μM) in PBS:DMSO (5:1, v / v) were illuminated at 365 nm (25 mW / cm 2) light irradiation for 3 min, and then incubated at 37 ° C for 3 min. The reaction was monitored by HPLC at the wavelength provided (injection of 5 μL), and the corresponding HPLC spectral data were collected. Compound 1, compound 2 and compound 3 were prepared into solutions (c = 300 μM in DMSO: PBS = 1: 5, v / v) and placed under natural light. The solutions were analyzed by HPLC at 12h, 24h, 48h, and 72h, and the corresponding HPLC spectral data were collected.

[0156] The test results are as follows Figure 2 As shown in the figure, after compound 1 and compound 3 were mixed and reacted with ultraviolet light for 3 minutes, compound 1 was almost completely converted into a PROTAC molecule within 3 minutes, indicating that compound 1 and compound 3 will quickly undergo a cycloaddition reaction to generate products after light activation. And no changes in the precursor molecules of compound 1, compound 2 and compound 3 were observed within 72 hours, which indicates that the precursor molecules are more stable in the solution under natural light and there is no photolysis phenomenon ( Figure 2 A-2E).

[0157] Example 11 BRD4 protein degradation experiment

[0158] Taking compound 1 prepared in Example 6 as an example, the ability of compound 1 and compound 3 prepared in Example 8 to degrade intracellular BRD4 protein before and after light activation was studied. The specific operation was as follows:

[0159] Take the tumor cells to be tested in the logarithmic growth phase and calculate 5×10 5 Each well was inoculated with 2 mL of culture medium and cultured at 37°C and 5% CO2 for 12 h. After the cells adhered to the wall, different concentrations of compounds were added for a certain period of time (if light was required, a cell phototoxicity irradiator was used at 365 nm 12.5 mW / cm 2 After 24 hours, the culture medium was discarded, the cells were collected, and the cells were lysed to obtain a cell lysate. The cell lysate was subjected to protein immunoblotting analysis.

[0160] The results showed that compound 1 and compound 3 would degrade intracellular BRD4 protein only after light activation. The protein level of BRD4 in Hela cells was significantly reduced in a concentration-dependent and time-dependent manner. In addition, the proteasome inhibitor carfilzomib (CFZ) could completely block this process, indicating that compound 1 and compound 3 specifically induced intracellular BRD4 protein degradation through the proteasome pathway after light activation ( Figure 3 A-3F).

[0161] Example 12 BRD4 and PARP1 protein dual degradation experiment

[0162] The dual degradation ability of compound 1, compound 2 and compound 3 on intracellular BRD4 and PARP1 proteins after light activation was studied. The specific operation is as follows:

[0163] Take the tumor cells to be tested in the logarithmic growth phase and calculate 5×10 5 Each well was inoculated with 2 mL of culture medium and cultured at 37°C and 5% CO2 for 12 h. After the cells attached to the wall, different concentrations of compound 1, compound 2, and compound 3 were added. After incubation for 2 h, the cells were illuminated (the cells that needed to be illuminated were illuminated with a cell phototoxicity irradiator at 365 nm 12.5 mW / cm 2 After 24 hours, the culture medium was discarded, the cells were collected, and the cells were lysed to obtain a cell lysate. The cell lysate was subjected to protein immunoblotting analysis.

[0164] The results showed that compound 1, compound 2 and compound 3 could significantly reduce the protein levels of BRD4 and PARP1 in Hela cells and MDA-MB-231 in a concentration-dependent manner after light activation. This indicates that compound 1, compound 2 and compound 3 can double degrade intracellular proteins after light activation ( Figure 4 A-4B).

[0165] Example 13 Immunofluorescence experiment

[0166] The degradation ability of compound 1, compound 2 and compound 3 on intracellular BRD4 and PARP1 proteins before and after light activation is as follows:

[0167] Take the tumor cells to be tested in the logarithmic growth phase and calculate 1×10 5 Each well was inoculated in a 24-well plate, 1 mL of culture medium was added to each well, and the cells were cultured at 37°C and 5% CO2 for 12 h. After the cells adhered to the wall, 0.5 μM compound 1 and 0.5 μM compound 3 were added to the Hela cells and incubated for 2 h, and then one group of cells was illuminated, while the other group of cells was not illuminated; 0.5 μM compound 2 and 0.5 μM compound 3 were added to the Hela cells and incubated for 2 h, and then one group of cells was illuminated, while the other group of cells was not illuminated; at the same time, 0.5 μM compound 1, 0.5 μM compound 2, and 1 μM compound 3 were added to the Hela cells and incubated for 2 h, and then one group of cells was illuminated, while the other group of cells was not illuminated (cell phototoxicity irradiator was used for cells that needed to be illuminated at 365 nm 12.5 mW / cm 2After 24 hours, discard the culture medium and add 200 μL 4% paraformaldehyde to fix the cells. Perform immunostaining on the cells. Add 100 μL DAPI anti-fluorescence quenching mounting medium to each well of the stained cells to stain the nucleus and prevent fluorescence quenching. Finally, image with laser confocal microscopy. NIS Elements microscope imaging software is used to analyze and process the images.

[0168] The results showed that the green fluorescence intensity of BRD4 protein (green channel) in the illumination group cells incubated with compound 1 and compound 3 was significantly weakened compared with the DMSO group, while there was no significant weakening in the group without illumination. The fluorescence intensity of red fluorescence of PARP1 protein in the experimental group did not change significantly, indicating that compound 1 and compound 3 can only selectively degrade BRD4 protein; the red fluorescence intensity of PARP1 protein (red channel) in the illumination group cells incubated with compound 2 and compound 3 was significantly weakened compared with the DMSO group, while there was no significant weakening in the group without illumination. The fluorescence intensity of green fluorescence of BRD4 protein in the experimental group did not change significantly, indicating that compound 2 and compound 3 can only selectively degrade PARP1 protein. At the same time, the green fluorescence intensity of BRD4 protein (green channel) in the illumination group cells incubated with compound 1, compound 2 and compound 3 was significantly weakened compared with the DMSO group, and the red fluorescence intensity of PARP1 protein (red channel) was also significantly weakened compared with the DMSO group, indicating that the levels of BRD4 protein and PARP1 protein in Hela cells were significantly downregulated. There was no significant weakening in the group without illumination. This experiment further demonstrated that this method can achieve selective light-controlled degradation of BRD4 and PARP1 proteins in cancer cells. Figure 5 ).

[0169] Example 14 Cell proliferation inhibition experiment

[0170] The ability of compound 1, compound 2 and compound 3 to inhibit cancer cell proliferation before and after light activation by co-incubation was studied. The specific operation was as follows:

[0171] Compound 1, Compound 2, Compound 3, small molecule inhibitor (+)-JQ1 and Olaparib were prepared into 10mM DMSO drug stock solutions, and the 10mM concentrations of Compound 1, Compound 2, Compound 3, small molecule inhibitor (+)-JQ1 and Olaparib were diluted into 20μM culture medium solutions, and then three times diluted to prepare 9 concentrations of culture medium solutions. The cultured Hela cells and MDA-MB-231 cells were aspirated, and compound 1, Compound 2, Compound 3, small molecule inhibitor (+)-JQ1 and Olaparib (100μM or 200μM) solutions were added, with 3 wells for each concentration. The small molecule inhibitor (+)-JQ1 and olaparib, compound 1, compound 2, and compound 3 were incubated in a constant temperature incubator (37°C, 5% CO2) for 2 h. One group was not exposed to light, and the other group was exposed to light (the cells that needed light were exposed to light using a cell phototoxicity irradiator at 365 nm 12.5 mW / cm 2 After illumination for 3 min, the 96-well plate was placed in a constant temperature incubator (37°C, 5% CO2) and incubated for 72 h.

[0172] The results are shown in Table 1 below:

[0173] Table 1

[0174]

[0175] a: The results of the test using CCK8 are the mean ± standard deviation (n = 3 independent experiments);

[0176] b: 1+2+3 means: compound 1+compound 2+compound 3;

[0177] c: UV light (+L, 365nm, 12.5mW / cm 2 )3min.

[0178] This shows that this method can be like the prodrug strategy, in which the precursor molecule is equivalent to the prodrug, and can only exert better therapeutic effects after being triggered by light, which can reduce the off-target toxicity inherent in the PROTAC strategy to a certain extent.

[0179] Example 15 Drug efficacy experiment in zebrafish xenotransplantation HeLa-RFP model

[0180] The ability of Compound 1, Compound 2 and Compound 3 to inhibit cancer cell proliferation in zebrafish before and after light activation by co-incubation was studied. The specific operation was as follows:

[0181] Under a microscope, RFP-labeled HeLa cells were injected into the yolk sac of 2dpf zebrafish, with about 200 cells transplanted per tail. The zebrafish were wild-type AB strain. Subsequently, the treated zebrafish were placed in a culture tank at 28°C and cultured until 3dpf. This method was used to construct a zebrafish tumor transplantation model to evaluate the anti-tumor activity of the compound. In order to ensure the accuracy of the experimental results, zebrafish with relatively consistent HeLa cell transplantation conditions were selected as experimental animals, randomly assigned, and cultured in 6-well plates, with 30 tails per well. In the experimental group, different drugs were dissolved in water and prepared into drug solutions of different concentrations as the living water environment for zebrafish (those that require light were exposed to a cell phototoxicity irradiator at 12.5mW / cm 2 Light intensity illumination for 3 minutes). A blank control was set. After the zebrafish were cultured at 28°C for 2 days, 7 zebrafish were randomly selected from each experimental group and control group to detect their fluorescence intensity A. The zebrafish morphology and the HeLa cell tumor part that can produce fluorescence were observed under a fluorescence microscope, and photographed and recorded. The obtained pictures were processed with NIS-Elements D 3.10 image processing software to calculate the fluorescence intensity of cancer cells, thereby calculating the inhibitory activity of the compound on zebrafish HeLa cell transplanted tumors. Inhibition rate = (1-A (experimental group) / A (control group)) × 100%.

[0182] The result is as follows:

[0183] Table 2 In vivo safety evaluation of the combination of compounds 1+2+3 in the zebrafish model a

[0184]

[0185] a: Each group contained 30 zebrafish;

[0186] b: Compounds 1, 2, and 3 were incubated at the indicated concentrations of 1:1:2.

[0187] The experimental results are as follows Figure 6 As shown, from the experimental results of fluorescence imaging of zebrafish transplant tumor model, it can be seen that the intensity and area of ​​red fluorescence in zebrafish in the non-illumination experimental group after combined treatment with compounds 1, 2, and 3 are both smaller than those in the blank control group. The intensity and area of ​​red fluorescence in zebrafish in the illumination experimental group after combined treatment with compounds 1, 2, and 3 are both smaller than those in the non-illumination experimental group after combined treatment with compounds 1, 2, and 3. Through fluorescence intensity detection, the statistical data of tumor cell inhibition rate in the zebrafish xenograft model of non-illumination experimental group and different concentrations of illumination experimental groups after combined treatment with compounds 1, 2, and 3 were 76%, 96%, and 94%, respectively. In this model, this strategy showed excellent safety and anti-tumor activity.

[0188] In summary, the PROTAC synthetic precursor prepared by the present invention has low cell activity before light activation and does not have the ability to degrade proteins, but it can generate PROTAC molecules in situ through light activation, degrade target proteins, and kill tumor cells. The above solves the defects of PROTAC molecules with large off-target toxicity and poor physicochemical properties, and is a new method for dual or even multi-controllable degradation of target proteins.

[0189] Obviously, the above embodiments are merely examples for clear description and are not limitations of the implementation methods.

[0190] Although the above disclosure discusses some of the invention embodiments currently considered useful through various examples, it should be understood that such details are for illustrative purposes only, and the appended claims are not limited to the disclosed embodiments. On the contrary, the claims are intended to cover all modifications and equivalent combinations that are consistent with the essence and scope of the embodiments of the present application.

[0191] Similarly, it should be noted that in order to simplify the description of the disclosure of this application and thus help understand one or more embodiments of the invention, in the above description of the embodiments of this application, multiple features are sometimes combined into one embodiment, figure or description thereof. However, this disclosure method does not mean that the features required by the object of this application are more than the features mentioned in the claims. In fact, the features of the embodiments are less than all the features of the single embodiment disclosed above.

[0192] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may vary according to the required features of individual embodiments.

[0193] Finally, it should be understood that the embodiments described in this application are only used to illustrate the principles of the embodiments of the present application. Other variations may also fall within the scope of the present application. Therefore, as an example and not a limitation, the alternative configurations of the embodiments of the present application may be considered to be consistent with the teachings of the present application. Accordingly, the embodiments of the present application are not limited to the embodiments explicitly introduced and described in the present application.

Claims

1. A proteolytically targeted chimera synthetic precursor, characterized in that: The structural formula of the proteolysis-targeted chimera synthesis precursor is shown in general formula I-1: in: Said X is selected from C2 alkyl or dimethylsilyl; The L1 is a connecting chain; The R1 is a target protein ligand.

2. The proteolysis-targeted chimera synthetic precursor according to claim 1, characterized in that: Contains at least one of the following characteristics: (1) L1 comprises a group covalently linked to a phenolic hydroxyl group; preferably, L1 comprises one of the following structures: Wherein, n is selected from an integer between 2 and 7; (2) the target protein includes at least one of a cell membrane surface protein, a cytoplasmic protein, and a nuclear protein; (3) The target protein includes at least one protein highly expressed in tumors; (4) The target protein ligand comprises a bromodomain-containing protein 4 ligand and / or a poly (ADP) ribozyme ligand; preferably, the structural formulas of the bromodomain-containing protein 4 ligand and the poly (ADP) ribozyme ligand are as shown in I-2 and I-3:

3. The proteolysis-targeted chimera synthetic precursor according to claim 1, characterized in that: The proteolysis targeting chimera synthetic precursor comprises one of the structures shown below:

4. The method for preparing the proteolysis-targeted chimera synthetic precursor according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, reacting the target protein ligand with the connecting chain in the presence of a condensation reagent, an organic base and an organic solvent to obtain an intermediate; S2, reacting the intermediate described in S1 with the compound represented by formula I-5 in the presence of an inorganic base and an organic solvent to obtain the protein hydrolysis targeted chimera synthesis precursor; Wherein, the X is selected from C2 alkyl or dimethylsilyl.

5. A proteolysis targeting chimera, characterized in that The structural formula of the proteolysis targeting chimera is shown in the following general formula: in: Said X is selected from C2 alkyl or dimethylsilyl; The L1 is the first connecting chain; The R1 is a target protein ligand; The L2 is the second connecting chain; The R2 is an E3 ubiquitin ligase ligand.

6. The proteolysis targeting chimera according to claim 5, characterized in that Contains at least one of the following characteristics: (1) L1 comprises a group covalently linked to a phenolic hydroxyl group; preferably, L1 comprises one of the following structures: Wherein: n is selected from an integer between 2 and 7; (2) the target protein includes at least one of a cell membrane surface protein, a cytoplasmic protein, and a nuclear protein; (3) The target protein includes at least one protein highly expressed in tumors; (4) The target protein ligand comprises a bromodomain-containing protein 4 ligand and / or a poly (ADP) ribozyme ligand; preferably, the structural formulas of the bromodomain-containing protein 4 ligand and the poly (ADP) ribozyme ligand are as shown in I-2 and I-3: (5) L2 comprises a group covalently linked to the azide group; preferably, L2 comprises one of the following structures: Wherein: n is selected from an integer between 2 and 3; (6) The E3 ubiquitin ligase ligand is selected from at least one of a Cereblon protein ligand and a VHL ligand; preferably, R2 comprises one of the following structures: in: W is selected from CH2, C=O, NH or N-C1-C4 alkyl; Y is selected from O or S; Z is selected from H, C1-C4 alkyl, C3-C6 cycloalkyl or halogen; G, G' is selected from H, C1-C4 alkyl, -OH or C1-C4 alkyl substituted 5-10 membered heterocyclic group, wherein the heterocyclic group contains 1-3 heteroatoms of N, O or S; R3 is selected from H, halogen, nitro, amino, cyano, hydroxy, C1-C4 alkyl or halogenated C1-C4 alkyl.

7. The method for preparing the proteolysis targeting chimera according to claim 5 or 6, characterized in that: The following steps are involved: Mixing the proteolysis-targeted chimera synthetic precursor according to any one of claims 1 to 3 or the proteolysis-targeted chimera synthetic precursor prepared by the preparation method according to claim 4 with the compound represented by formula I-4, and irradiating with light to obtain the proteolysis-targeted chimera; in: The L2 is a connecting chain; The R2 is an E3 ubiquitin ligase ligand.

8. Use of the synthetic precursor of the proteolysis-targeted chimera according to any one of claims 1 to 3, the synthetic precursor of the proteolysis-targeted chimera prepared by the preparation method according to claim 4, the proteolysis-targeted chimera according to claim 5 or 6, or the proteolysis-targeted chimera prepared by the preparation method according to claim 7 in the preparation of protein degradation products or anti-tumor products.

9. A protein degradation product, characterized in that: The protein degradation product contains the synthetic precursor of the protein hydrolysis targeted chimera according to any one of claims 1 to 3, the synthetic precursor of the protein hydrolysis targeted chimera prepared by the preparation method according to claim 4, the protein hydrolysis targeted chimera according to claim 5 or 6, or the protein hydrolysis targeted chimera prepared by the preparation method according to claim 7.

10. An anti-tumor product, characterized in that: The anti-tumor product includes a container containing the following independently packaged preparations: a preparation containing a first synthetic precursor; a preparation containing a second synthetic precursor; The first synthetic precursor comprises the proteolysis targeting chimera synthetic precursor according to any one of claims 1 to 3 or the proteolysis targeting chimera synthetic precursor prepared by the preparation method according to claim 4, and the second synthetic precursor comprises an azide group and an E3 ubiquitin ligase ligand; Preferably, the structural formula of the second synthetic precursor is as shown in general formula I-4: The L2 is a connecting chain; The R2 is an E3 ubiquitin ligase ligand.

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