Spatiotemporal controllable photoinitiated in situ generated protac and application thereof

Abstract

The application discloses a kind of space-time controllable PROTAC based on photo-initiated in-situ generation and application thereof.The PROTAC is divided into two components: biorthogonal reaction group and the component of target protein ligand connection, biorthogonal reaction group and the component of E3 ubiquitin ligase ligand connection, can be synthesized in situ by light control PROTAC, for targeted protein degradation.It overcomes the difficulty of PROTAC molecule from scratch synthesis, synthesis step is complicated and the like, avoids the adverse pharmacological performance and toxic side effects caused by large molecular weight in traditional PROTAC design.At the same time, only after lightening, PROTAC molecule can be generated, the target protein is degraded, tumor cells are killed, off-target toxicity is avoided, and tumor treatment effect is realized.The method for generating protein hydrolysis targeting chimera by light triggering click provided by the application can controllably and efficiently synthesize PROTAC molecules with anticancer effect, and has the prospect of precise regulation of intracellular protein expression and treatment of tumor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a spatiotemporally controllable PROTAC based on photo-induced in-situ generation and its applications. Background Technology

[0002] Proteolytic targeting chimeras (PROTACs) utilize the body's naturally occurring protein clearance system to reduce protein levels, thereby achieving therapeutic goals. After 20 years of continuous improvement and development, PROTAC technology has become one of the hottest technologies in the field of new drug development. A PROTAC molecule is a heterobifunctional chimera composed of a target protein ligand, a linker, and an E3 ubiquitin ligase ligand. The PROTAC molecule mediates the formation of a ternary complex with the target protein and the E3 ubiquitin ligase, thereby promoting the polyubiquitination of the target protein and its degradation by the proteasome. The advantages of PROTAC molecules lie in their broader scope of action, higher activity, and ability to target "undruggable" targets. Traditional small molecules and antibodies exert their therapeutic effects by inhibiting target protein function through an "occupation-driven" mechanism. This mechanism requires high concentrations of inhibitors or antibodies to occupy the active site of the target and block downstream signal transduction. In contrast, the PROTAC strategy is "event-driven," directly mediating target protein degradation rather than affecting its function. Secondly, after degrading the target protein, PROTAC molecules can be released again to continue degrading new target proteins, making them cyclical and reusable. Therefore, the catalytic amount is sufficient to exert a therapeutic effect. Moreover, the PROTAC strategy can also degrade proteins without active sites. As long as the PROTAC molecule can bind to the target protein, it can induce the degradation of the relevant protein, which can greatly expand the range of applicable targets.

[0003] However, with in-depth research into the PROTAC strategy, its shortcomings have also been exposed. There are two main drawbacks: First, while the catalytic mechanism of PROTACs brings stronger activity, it may also complicate the pharmacological process, easily leading to severe off-target toxicity. Second, PROTAC molecules need to simultaneously possess a ligand to bind the target protein, an E3 ligase recruitment element, and a linker connecting these two, resulting in high molecular weight and polar surface area. This characteristic significantly affects the cell penetration and solubility of PROTAC molecules, leading to poor bioavailability and pharmacokinetic parameters. Therefore, it is urgent to develop strategies to address these problems.

[0004] To address the potential off-target toxicity of PROTACs, researchers have developed prodrug-based PRO-PROTACs. Targeted PROTACs (folic acid, antibodies, nucleic acid aptamers) have been created by attaching target molecules to the PROTAC molecule. Photoprotective groups have also been attached to PROTAC molecules to regulate mammalian proteins through light-mediated processes; DirkTrauner et al. provided a good example of photoactivated PROTACs. A series of redox-activated, hypoxia-activated, and enzyme-activated PROTACs also exist. However, the large molecular weight of PROTACs themselves, coupled with the corresponding photoleaking or bioorthogonal protecting groups, results in even larger molecular weights and poorer physicochemical properties, significantly limiting the application of this strategy.

[0005] Bioorthogonal coupling reactions offer a solution to overcome the large molecular weight problem of PROTACs. Following the CLIPTACs strategy of clicking trans-cyclooctene and tetrazine to generate PROTACs in 2016, Zhang Jie's research group successively reported the click generation of PROTACs from norbornene and tetrazine, and the in-situ generation of PROTACs mediated by endogenous copper ion-catalyzed azidoalkynyl cycloaddition. However, this strategy lacks controllability; the click reaction occurs whenever the bioorthogonal reaction pairs meet, making it impossible to avoid off-target toxicity of PROTACs. Bengang Xi ng et al. reported an enzyme-driven click protein hydrolysis targeting chimera. They linked a bifunctional chimera through the generation of luciferin and modified the linker chain with a nitroreductase responsive group. This degradation mode relies on nitroreductase (NTR) enzymes, selectively forming bifunctional chimeras only in hypoxic sites, enabling targeted degradation of BRD4 in vitro and in vivo, achieving a combination of controllability and in-situ PROTAC generation. However, this strategy is based on an internal environment response and is not responsive to normoxic environments, thus limiting its application scope. Therefore, it is essential to develop a spatiotemporally controllable and more universal click-generating protein hydrolysis-targeting chimera strategy. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a method and application for photo-initiated in-situ generation of PROTAC for spatiotemporally controlled targeted protein degradation. The PROTAC precursor synthesized by this method possesses natural photostability. Upon activation by ultraviolet light, the PROTAC precursor synthesized by this method can rapidly undergo a click reaction to generate PROTAC molecules for spatiotemporally controlled degradation of target proteins and killing of tumor cells, thereby achieving tumor therapeutic effects.

[0007] The first objective of this invention is to provide a protein hydrolysis-targeting chimeric synthesis precursor, the structural formula of which is shown in general formula I-1:

[0008]

[0009] in:

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

[0011] L1 is a linker chain (covalently linked to phenolic hydroxyl groups to jointly construct a PROTAC synthetic precursor that links bioorthogonal reactive groups and target protein ligands);

[0012] R1 is the target protein ligand.

[0013] Furthermore, it includes at least one of the following:

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

[0015]

[0016] Where n is selected from integers between 2 and 7;

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

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

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

[0020]

[0021] Furthermore, the protein hydrolysis-targeting chimeric synthesis precursor comprises one of the following structures:

[0022] It should be noted that all molecules in this invention were obtained after screening. For example, in the first structure described above, Cl cannot be replaced with halogens such as Br, as this would cause the molecule to lose its activity.

[0023] A second objective of this invention is to provide a method for preparing the protein hydrolysis-targeted chimeric synthesis precursor, comprising the following steps:

[0024] S1. The target protein ligand and the linker chain are reacted in the presence of a condensing agent, an organic base and an organic solvent to obtain an intermediate;

[0025] S2. The intermediate described in S1 is reacted with the compound shown in Formula I-5 in the presence of an inorganic base and an organic solvent to obtain the protein hydrolysis-targeted chimeric synthesis precursor.

[0026]

[0027] Wherein, 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 an integer between 2 and 7.

[0031] Furthermore, it includes at least one of the following:

[0032] The condensing agent includes HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate);

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

[0034] The inorganic base includes sodium hydride;

[0035] The organic solvent can be any solvent capable of dissolving the reactants, such as N,N-dimethylformamide.

[0036] Furthermore, the molar ratio of the target protein ligand, linker chain, condensing agent and 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 dibenzocyclopropene derivative I-5, the inorganic base, and the intermediate is 1:(1-3):(1-2), preferably 1:2:1.5.

[0038] Further, the intermediate and inorganic base are added to an organic solvent and stirred for at least 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℃, and the reaction time is 2-30h, preferably 12h.

[0041] A third object of the present invention is to provide a protein hydrolysis-targeting chimera, the structural formula of which is shown in the following general formula:

[0042]

[0043] in:

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

[0045] L1 is the first linker chain (covalently linked to a phenolic hydroxyl group to jointly construct a PROTAC synthetic precursor 1, which is linked to a bioorthogonal reactive group and a target protein ligand);

[0046] R1 is a target protein ligand;

[0047] L2 is the second linker chain (covalently linked to an azide group to jointly construct a PROTAC precursor II linked to a bioorthogonal reactive group and an E3 ubiquitin ligase ligand);

[0048] R2 is an E3 ubiquitin ligase ligand.

[0049] Furthermore, it includes at least one of the following:

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

[0051]

[0052] Where: n is selected from integers between 2 and 7;

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

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

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

[0056]

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

[0058]

[0059] Where: n is selected from integers between 2 and 3;

[0060] (6) The E3 ubiquitin ligand is selected from at least one of Cereblon protein ligands and VHL ligands; 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 groups;

[0064] Y is selected from O or S;

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

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

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

[0068] A fourth objective of this invention is to provide a method for preparing the protein hydrolysis-targeting chimera, comprising the following steps:

[0069] The protein hydrolysis-targeting chimera synthesis precursor was mixed with the compound shown in Formula I-4 and irradiated with light to obtain the protein hydrolysis-targeting chimera;

[0070] in:

[0071]

[0072] L2 is a connection chain;

[0073] R2 is an E3 ubiquitin ligase ligand.

[0074] Furthermore, the preparation method of the compound shown in Formula I-4 includes: reacting an azide-PEG-amine or an azide-alkyl-amine with an E3 ubiquitin ligand by heating in the presence of an organic base and an organic solvent.

[0075] Furthermore, the organic solvent is any solvent capable of dissolving the reactants, such as N,N-dimethylformamide.

[0076] Furthermore, the heating reaction temperature is 90-110℃, and the reaction time is 2-12h.

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

[0078] Furthermore, the method of targeted protein degradation based on bioorthogonal reaction involves photoactivating the precursor compound of PROTAC to undergo an in-situ photodecoupling ring strain-promoted azido-alkynyl cycloaddition reaction to generate PROTAC, which can degrade target proteins and / or treat tumors.

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

[0080] (1) Using BRD4 protein small molecule ligand or PARP1 protein small molecule ligand as target protein ligand, and dibenzocycloalkanes cyclopropenone as bioorthogonal reactive groups, a series of bioorthogonal reactive groups and target protein ligands were linked to PROTAC synthesis 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 synthesis precursor linked with the bioorthogonal reactive group and the target protein ligand is co-incubated with the PROTAC synthesis precursor linked with the bioorthogonal reactive group and the E3 ubiquitin ligase ligand, and then activated in situ by light to generate PROTAC molecules, which degrade the target protein and kill tumor cells.

[0083] A fifth objective of this invention is to provide the use of the protein hydrolysis-targeting chimera precursor or the protein hydrolysis-targeting chimera in the preparation of protein degradation products or antitumor 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 for generating protein hydrolysis-targeting chimeras through phototriggered click generation provided by this invention offers a new approach to address the shortcomings of PROTAC molecules, such as high off-target toxicity and poor physicochemical properties. It also provides a new method for the 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 pharmaceutical product.

[0088] A sixth objective of this invention is to provide a protein degradation product containing the protein hydrolysis-targeting chimera synthesis precursor or the protein hydrolysis-targeting chimera.

[0089] A seventh object of the present invention is to provide an antitumor product comprising a container containing the following separately packaged formulations: a formulation containing a first synthetic precursor; and a formulation containing a second synthetic precursor.

[0090] The first synthetic precursor includes the proteolytic targeting chimeric 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 will understand that although this embodiment only provides one method of use, in actual use, the first synthetic precursor (such as compound 1 or compound 2) and the second synthetic precursor (such as compound 3) can be injected into the body separately and then combined with phototherapy to achieve the corresponding function.

[0091] The beneficial effects of this invention are:

[0092] 1. This invention provides a method for targeted protein degradation based on bioorthogonal reactions. This method divides a protein hydrolysis-targeting chimeric compound (PROTAC) into two components: a component linked to a target protein ligand by a bioorthogonal reactive group, and a component linked to an E3 ubiquitin ligase ligand by a bioorthogonal reactive group. By activating the PROTAC synthesis precursor compound under light, an in-situ photo-decoupling ring-strain-promoted azide-alkynyl cycloaddition reaction is generated, enabling spatiotemporally controllable target protein degradation. This selectively kills tumor cells, achieving precise tumor treatment. This avoids the adverse pharmacological properties and toxic side effects caused by the large molecular weight of traditional PROTACs. It also solves the defects of high off-target toxicity and poor physicochemical properties of PROTAC molecules.

[0093] 2. The method for targeted protein degradation based on bioorthogonal reaction provided by this invention can be used for precise regulation of multiple proteins in cells. By co-incubating a PROTAC synthesis precursor with bioorthogonal reactive groups and target protein ligands linked to different target protein ligands with a PROTAC synthesis precursor linked to bioorthogonal reactive groups and E3 ubiquitin ligase ligands, and then activating in situ the generation of multiple PROTAC molecules by light irradiation, multiple degradation of target proteins can be achieved in a spatiotemporally controllable manner. Attached Figure Description

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

[0095] Figure 2The figures show the HPLC chromatograms of the PROTAC synthesis precursors prepared in Examples 1-5 of this invention reacting in 16.7% DMSO / PBS, where A is the HPLC chromatogram of the in vitro photocatalytic reaction of compounds 1 and 3; B is the HPLC chromatogram of the in vitro photocatalytic reaction of compounds 2 and 3; C is the HPLC chromatogram of the stability of compound 1; D is the HPLC chromatogram of the stability of compound 2; and E is the HPLC chromatogram of the stability of compound 3.

[0096] Figure 3 These are Western blot analysis diagrams of proteins according to embodiments of the present invention, wherein A is a Western blot analysis diagram of light-dependent protein degradation of compounds 1 and 3; B is a Western blot analysis diagram of protein degradation mechanism of compounds 1 and 3; C and D are Western blot analysis diagrams of concentration-dependent protein degradation of compounds 1 and 3; and E and F are Western blot analysis diagrams of time-dependent protein degradation of compounds 1 and 3.

[0097] Figure 4 The images shown are Western blot analysis diagrams of proteins in embodiments of the present invention. A is a Western blot analysis diagram of light-dependent dual degradation of BRD4 and PARP1 proteins in HeLa cells for compounds 1, 2, and 3; B is a Western blot analysis diagram of light-dependent dual degradation of BRD4 and PARP1 proteins in MDA-MB-231 cells for compounds 1, 2, and 3.

[0098] Figure 5 The immunofluorescence image shown is an example of the immunofluorescence imaging of compounds 1, 2 and 3 in HeLa cells during light-dependent dual degradation of BRD4 and PARP1 proteins.

[0099] Figure 6 Fluorescence images showing the inhibitory effects of compounds 1, 2, and 3 on cancer cell proliferation in zebrafish before and after co-incubation with light activation. Detailed Implementation

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

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0102] The method involved in this invention: The structure of the compounds in the following examples was determined by nuclear magnetic resonance (NMR). 1 The NMR was determined by ¹H-NMR. The NMR was measured using an ACF-300BRUK NMR spectrometer, with deuterated chloroform (CDCl₃) as the solvent and TMS as the internal standard.

[0103] Example 1

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

[0105]

[0106] The specific steps are as follows:

[0107] 2-Bromoethylamine (16 mg, 0.13 mmol) and I-2 (44 mg, 0.11 mmol) were dissolved in 2 mL of DMF, and then HATU (57 mg, 0.15 mmol) and DIPEA (53 μL, 0.33 mmol) were added. The mixture was reacted at room temperature for 1 h, and the reaction was monitored by thin-layer chromatography. After the reaction was complete, saturated ammonium chloride solution was added, and the mixture was extracted with EA and washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was separated by preparative thin-layer chromatography (eluent polarity DCM:MeOH = 25:1) to obtain a white powdery solid S1 (44 mg, 80%). The product was characterized by NMR, 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 embodiment relates to the preparation of a PROTAC synthesis 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 of DMF. HATU (156 g, 0.409 mmol) and DIPEA (143 μL, 0.819 mmol) were added. The reaction was carried out at room temperature for 1 h, and the reaction was monitored by thin-layer chromatography. After the reaction was complete, saturated ammonium chloride solution was added, followed by extraction with EA. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography (eluent polarity DCM:MeOH = 25:1) to obtain a white powdery solid S2 (80 mg, 54%). The product was characterized by NMR, and the results are 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 embodiment relates to the preparation of a PROTAC synthesis precursor intermediate, and the reaction equation is as follows:

[0116]

[0117] Under nitrogen protection, a THF solution of m-bromoanisole (10 g, 53.5 mmol in 60 mL THF) was added to a three-necked flask. Butyllithium (2.5 M in hexane, 22 mL, 55 mmol) was slowly added dropwise at -78 °C over 15 min, with stirring for 1 h. Then, a THF solution of dichlorodimethylsilane (3.55 mg, 27.5 mmol in 10 mL THF) was added to the reaction system, and the reaction was allowed to proceed overnight at room temperature. The reaction was quenched with saturated ammonium chloride solution, extracted three times with EA, and the organic phases were combined. The organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography (eluting with 100% polar PE) to obtain a clear oily liquid S5 (2.19 g, 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 embodiment relates to the preparation of a PROTAC synthesis precursor intermediate, and the reaction equation is as follows:

[0122]

[0123] Tetrachlorocyclopropene (1.41 g, 7.928 mmol) was dissolved in 45 mL of DCM, and aluminum trichloride (2.2 g, 16.5 mmol) was added. The mixture was stirred at room temperature for 15 min, then cooled to -20 °C, and a DCM solution of S5 (1.8 g, 6.607 in 10 mL DCM) was added. The reaction was allowed to proceed for 0.5 h, and then the temperature was raised to room temperature for another 4 h. The reaction was monitored by thin-layer chromatography. After the starting material had completely reacted, the reaction was quenched with ice water. The organic solution was washed with saturated saline solution, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography (eluent polarity DCM:EA = 1:1) to obtain a brown powdery solid S7 (576 mg, 27%). The product was characterized by NMR, 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 embodiment relates to the preparation of a PROTAC synthesis 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 of DCM, and aluminum trichloride (154 mg, 1.16 mmol) was added. The mixture was stirred for 15 min, then heated to 55 °C and reacted for 12 h. The reaction was monitored by thin-layer chromatography until the reactants were completely reacted. The residue was filtered off 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 powdery solid I-5 (57 mg, 27%). The product was characterized by NMR, 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 embodiment relates to the preparation of a PROTAC synthesis precursor, and the reaction equation is as follows:

[0133]

[0134] The specific steps are as follows:

[0135] I-5 (100 mg, 0.32 mmol) was dissolved in 3 mL of DMF. 60% sodium hydride (26 mg, 0.64 mmol) was added under ice bath conditions, and the reaction was continued under ice bath conditions for 15 min. The reaction was then allowed to return to room temperature for 15 min. S1 (242 mg, 0.48 mmol) was then added to the reaction system, and the mixture was heated to 55 °C and reacted overnight. The reaction was monitored by thin-layer chromatography. After the reaction was complete, it was quenched with ice water, extracted with EA, washed three times with saturated ammonium chloride and saturated brine. The organic phase was dried over 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 powdery 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 embodiment relates to the preparation of a PROTAC synthesis precursor, and the reaction equation is as follows:

[0139]

[0140] The specific steps are as follows:

[0141] I-5 (100 mg, 0.32 mmol) was dissolved in 3 mL of DMF. 60% sodium hydride (26 mg, 0.64 mmol) was added under ice bath conditions, and the reaction was continued under ice bath conditions for 15 min. The reaction was then allowed to return to room temperature for 15 min. S1 (260 mg, 0.48 mmol) was then added to the reaction system, and the mixture was heated to 55 °C and reacted overnight. The reaction was monitored by thin-layer chromatography. After the reaction was complete, it was quenched with ice water, extracted with EA, washed three times with saturated ammonium chloride and saturated brine. The organic phase was dried over 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 powdery 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 embodiment relates to the preparation of a PROTAC synthesis 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 of LDMF. DIPEA (547 μL, 3.14 mmol) was then added to the reaction mixture. The mixture was heated to 100 °C and stirred 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 dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation. 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 NMR, 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 embodiment relates to the preparation of a negative control for PROTAC synthesis precursor, and the reaction equation is as follows:

[0150]

[0151] 3 (50 mg, 0.13 mmol) was dissolved in 6 mL of THF. 60% sodium hydride (8 mg, 0.16 mmol) was added under ice bath conditions, and the reaction was continued under ice bath conditions for 15 min. The reaction was then allowed to return to room temperature for 15 min. Iodomethane (10 μL, 0.16 mmol) was then added to the reaction system, and the reaction was continued at room temperature 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 washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography (eluent polarity DCM:EA = 10:1) to obtain a bright yellow powdery solid Me-3 (17 mg, 33%). The product was characterized by NMR, 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 operations are as follows:

[0155] Solutions of compound 1 (c = 300 μM) and compound 3 (c = 450 μM) in PBS:DMSO (5:1, v / v) were subjected to a 365 nm (25 mW / cm) test. 2Irradiate with light for 3 min, then incubate at 37 °C for 3 min. Monitor the reaction by HPLC (injection 5 μL) at the provided wavelength and collect the corresponding HPLC chromatogram data. Prepare solutions of compounds 1, 2, and 3 (c = 300 μM in DMSO:PBS = 1:5, v / v) and place them under natural light. Perform HPLC analysis on the solutions at 12 h, 24 h, 48 h, and 72 h, and collect the corresponding HPLC chromatogram data.

[0156] Test results are as follows Figure 2 As shown, after mixing compounds 1 and 3 and reacting with ultraviolet light for 3 minutes, compound 1 was almost completely converted into PROTAC molecules within 3 minutes, indicating that compounds 1 and 3 rapidly undergo cycloaddition reactions to generate products after photoactivation. Furthermore, no changes in the precursor molecules of compounds 1, 2, and 3 were observed within 72 hours, suggesting that the precursor molecules have good stability in solution under natural light and do not exhibit photolysis. Figure 2 A-2E).

[0157] Example 11 BRD4 protein degradation experiment

[0158] Taking compound 1 prepared in Example 6 as an example, its ability to degrade intracellular BRD4 protein before and after photoactivation, compared with compound 3 prepared in Example 8, was studied. The specific operation is as follows:

[0159] Take tumor cells in the logarithmic growth phase and administer at a concentration of 5 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates, with 2 mL of culture medium added to each well. The plates were incubated at 37°C and 5% CO2 for 12 hours. After cell attachment, different concentrations of compounds were added and incubated for a specific time (for cells requiring light, phototoxicity irradiation was performed at 365 nm and 12.5 mW / cm²). 2 (Light intensity: Irradiate for 3 min). After 24 h, discard the culture medium, collect the cells, and lyse the cells to obtain cell lysates. Perform Western blotting analysis on the cell lysates.

[0160] The results showed that compounds 1 and 3 only degraded intracellular BRD4 protein after photoactivation. Furthermore, they significantly reduced intracellular BRD4 protein levels in HeLa cells in a concentration- and time-dependent manner. In addition, the proteasome inhibitor carfilzomib (CFZ) completely blocked this process, indicating that compounds 1 and 3 specifically induce intracellular BRD4 protein degradation via the proteasome pathway after photoactivation. Figure 3 A-3F).

[0161] Example 12: Dual Degradation Experiment of BRD4 and PARP1 Proteins

[0162] The study investigated the dual degradation abilities of compounds 1, 2, and 3 on intracellular BRD4 and PARP1 proteins after photoactivation. The specific procedures were as follows:

[0163] Take tumor cells in the logarithmic growth phase and administer at a concentration of 5 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates, with 2 mL of culture medium added to each well. The plates were incubated at 37°C and 5% CO2 for 12 hours. After cell attachment, different concentrations of compounds 1, 2, and 3 were added, and the plates were incubated for 2 hours. Then, the plates were exposed to light (using a phototoxicology irradiator at 365 nm and 12.5 mW / cm² for cells requiring light exposure). 2 (Light intensity: Irradiate for 3 min). After 24 h, discard the culture medium, collect the cells, and lyse the cells to obtain cell lysates. Perform Western blotting analysis on the cell lysates.

[0164] The results showed that compounds 1, 2, and 3 significantly reduced the protein levels of BRD4 and PARP1 in HeLa cells and MDA-MB-231 cells in a concentration-dependent manner after photoactivation. This indicates that compounds 1, 2, and 3 can undergo dual degradation of intracellular proteins after photoactivation. Figure 4 A-4B).

[0165] Example 13 Immunofluorescence Experiment

[0166] The degradation abilities of compounds 1, 2, and 3 on intracellular BRD4 and PARP1 proteins before and after light activation were determined by the following procedures:

[0167] Take tumor cells in the logarithmic growth phase and administer at a dose of 1×10⁻⁶. 5 Cells were seeded per well in 24-well plates, with 1 mL of culture medium added to each well. The plates were incubated at 37°C and 5% CO2 for 12 h. After cell attachment, 0.5 μM compound 1 and 0.5 μM compound 3 were added to HeLa cells and incubated for 2 h. One group of cells was then illuminated, while the other group was not. Similarly, 0.5 μM compound 2 and 0.5 μM compound 3 were added to HeLa cells and incubated for 2 h. One group of cells was then illuminated, while the other group was not. Simultaneously, 0.5 μM compound 1, 0.5 μM compound 2, and 1 μM compound 3 were added to HeLa cells and incubated for 2 h. One group of cells was then illuminated, while the other group was not (cells requiring illumination were irradiated using a phototoxicity analyzer at 365 nm and 12.5 mW / cm²). 2Irradiation intensity: 3 min. After 24 h, discard the culture medium and add 200 μL of 4% paraformaldehyde to fix the cells. Perform immunostaining on the cells. Add 100 μL of DAPI-containing anti-fluorescence quenching mounting medium to each well of the stained cells to stain the nuclei while preventing fluorescence quenching. Finally, image using a laser confocal microscope. Analyze and process the images using NIS Elements microscopy imaging software.

[0168] The results showed that the green fluorescence intensity of BRD4 protein (green channel) in cells in the light-exposed groups incubated with compounds 1 and 3 was significantly lower than that in the DMSO group, while no significant decrease was observed in the unexposed groups. The red fluorescence intensity of PARP1 protein in the experimental groups did not change significantly, indicating that compounds 1 and 3 selectively degrade BRD4 protein. Similarly, the red fluorescence intensity of PARP1 protein (red channel) in cells incubated with compounds 2 and 3 was significantly lower than that in the DMSO group, while no significant decrease was observed in the unexposed groups. The green fluorescence intensity of BRD4 protein in the experimental groups also did not change significantly, indicating that compounds 2 and 3 selectively degrade PARP1 protein. Furthermore, the green fluorescence intensity of BRD4 protein (green channel) and the red fluorescence intensity of PARP1 protein (red channel) in cells incubated with compounds 1, 2, and 3 were significantly lower than those in the DMSO group, indicating that the levels of both BRD4 and PARP1 proteins in HeLa cells were significantly downregulated. No significant decrease was observed in the unexposed groups. This experiment further demonstrates intuitively that this method can achieve selective and light-controlled degradation of BRD4 and PARP1 proteins in cancer cells. Figure 5 ).

[0169] Example 14 Cell proliferation inhibition experiment

[0170] The study investigated the inhibitory effects of co-incubation of compounds 1, 2, and 3 on cancer cell proliferation before and after light activation. The specific procedures were as follows:

[0171] Compounds 1, 2, and 3, the small molecule inhibitor (+)-JQ1, and olaparib were each prepared as 10 mM DMSO stock solutions. These 10 mM solutions were then diluted to 20 μM in culture medium. These solutions were then further diluted three-fold to prepare nine different concentrations of culture medium. The cultured HeLa cells and MDA-MB-231 cells were decanted, and then compound solutions of 1, 2, and 3, the small molecule inhibitor (+)-JQ1, and olaparib (100 μM or 200 μM) were added to each cell, with three wells for each concentration. The small molecule inhibitors (+)-JQ1 and olaparib, as well as compounds 1, 2, and 3, were incubated in a constant temperature incubator (37℃, 5% CO2) for 2 hours. One group was not exposed to light, while the other group was exposed to light (the group requiring light exposure was irradiated using a cell phototoxicity irradiator at 365nm and 12.5mW / cm²). 2 After 3 minutes of light exposure, the 96-well plate was placed in a constant temperature incubator (37℃, 5% CO2) and incubated for 72 hours.

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

[0173] Table 1

[0174]

[0175] a: The test results were obtained using CCK8, and the mean ± standard deviation was taken (n = 3 independent experiments);

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

[0177] c: Ultraviolet light irradiation (+L, 365nm, 12.5mW / cm²) 2 3min.

[0178] This indicates that, like the prodrug strategy, the precursor molecule acts as a prodrug, and can only exert better efficacy after photo-triggered action, which can reduce the off-target toxicity inherent in the PROTAC strategy to some extent.

[0179] Example 15: Efficacy Experiment of Zebrafish Xenotransplantation HeLa-RFP Model

[0180] The ability of compounds 1, 2, and 3 to inhibit cancer cell proliferation in zebrafish before and after co-incubation under light activation was investigated. The specific procedures are as follows:

[0181] Under a microscope, RFP-labeled HeLa cells were injected into the yolk sacs of 2-day-fiber (dpf) zebrafish, with approximately 200 cells per fish. The zebrafish were wild-type AB strain. Subsequently, the treated zebrafish were cultured at 28°C until 3 dpf. This method was used to construct a zebrafish tumor transplantation model to evaluate the antitumor activity of the compounds. To ensure the accuracy of the experimental results, zebrafish with relatively consistent HeLa cell transplantation were selected as experimental animals and randomly assigned to 6-well plates, with 30 fish per well. In the experimental groups, different drugs were dissolved in water to prepare drug solutions of varying concentrations to serve as the aquatic environment for the zebrafish (those requiring light were irradiated using a cell phototoxicity analyzer at 12.5 mW / cm²). 2 Light intensity (3 min illumination). A blank control was set up. After culturing zebrafish at 28℃ for 2 days, 7 zebrafish were randomly selected from each experimental group and control group to detect their fluorescence intensity A. The morphology of the zebrafish and the fluorescent HeLa cell tumor parts were observed under a fluorescence microscope and photographed. The obtained images were processed using 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 xenografts. Inhibition rate = (1 - A(experimental group) / A(control group)) × 100%.

[0182] The results are shown below:

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

[0184]

[0185] a: Each group contains 30 zebrafish;

[0186] b: Compounds 1, 2, and 3 were incubated at the concentrations shown in the ratio 1:1:2.

[0187] Experimental results are as follows Figure 6 As shown in the fluorescence imaging results of the zebrafish xenograft model, the intensity and area of ​​red fluorescence in the zebrafish in the non-illuminated group treated with compounds 1, 2, and 3 were smaller than those in the blank control group. Conversely, the intensity and area of ​​red fluorescence in the zebrafish in the illuminated group treated with compounds 1, 2, and 3 were smaller than those in the non-illuminated group. Based on fluorescence intensity detection, the tumor cell inhibition rates in the zebrafish xenograft model treated with compounds 1, 2, and 3 in the non-illuminated group and the groups with different concentrations of light were 76%, 96%, and 94%, respectively. In this model, this strategy demonstrated excellent safety and antitumor activity.

[0188] In summary, the PROTAC precursor prepared by this invention exhibits low cellular activity and lacks protein degradation ability before photoactivation. However, it can be activated by light to generate PROTAC molecules in situ, degrading target proteins and killing tumor cells. This method overcomes the shortcomings of high off-target toxicity and poor physicochemical properties of PROTAC molecules, while also providing a novel method for the dual or even multiple controllable degradation of target proteins.

[0189] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the implementation.

[0190] While the foregoing disclosure has discussed some inventive embodiments that are 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. Rather, the claims are intended to cover all modifications and equivalent combinations that conform to the substance and scope of the embodiments of this application.

[0191] Similarly, it should be noted that, in order to simplify the description of the present application and thus aid in the understanding of one or more embodiments of the invention, the foregoing description of the embodiments of the present application sometimes combines multiple features into a single embodiment, drawing, or description thereof. However, this disclosure method does not imply that the subject matter of the application requires more features than those mentioned in the claims. In fact, the embodiments contain fewer features than all the features of the single embodiments disclosed above.

[0192] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are sometimes modified by the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates 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 be changed according to the characteristics required by individual embodiments.

[0193] Finally, it should be understood that the embodiments described in this application are merely illustrative of the principles of the embodiments of this application. Other modifications may also fall within the scope of this application. Therefore, alternative configurations of the embodiments of this application are considered as examples and not limitations, and are regarded as consistent with the teachings of this application. Accordingly, the embodiments of this application are not limited to the embodiments explicitly described and illustrated in this application.

Claims

1. A protein hydrolysis-targeted chimeric synthesis precursor, characterized in that, The structural formula of the protein hydrolysis-targeted chimeric synthesis precursor is shown in general formula I-1: ， in: X is selected from C2 alkyl or dimethylsilyl; L1 is a connecting chain, and has one of the following structures: ， Where n is selected from integers between 2 and 7; The structural formula of R1 is shown in I-2 or I-3: 。 2. The protein hydrolysis-targeted chimeric synthesis precursor according to claim 1, characterized in that, The protein hydrolysis-targeted chimeric synthesis precursor is one of the following structures: 、 。 3. The method for preparing the protein hydrolysis-targeted chimeric synthesis precursor according to claim 1, characterized in that, Includes the following steps: S1. The target protein ligand and the linker chain are reacted in the presence of a condensing agent, an organic base and an organic solvent to obtain an intermediate; S2. The intermediate described in S1 is reacted with the compound shown in Formula I-5 in the presence of an inorganic base and an organic solvent to obtain the protein hydrolysis-targeted chimeric synthesis precursor. The connecting chain is selected from one of the following structures: ， n is an integer between 2 and 7; ， Wherein, X is selected from C2 alkyl or dimethylsilyl; The structure of the target protein ligand is as follows: or .

4. A protein hydrolysis-targeting chimera, characterized in that, The structural formula of the protein hydrolysis-targeting chimera is shown in the following general formula: ， in: X is selected from C2 alkyl or dimethylsilyl; L1 is the first connecting chain, and has one of the following structures: ， Where n is selected from integers between 2 and 7; The structural formula of R1 is shown in I-2 or I-3: ； L2 is the second connecting chain, and has one of the following structures: ， Where n is selected from integers between 2 and 3; R2 is an E3 ubiquitin ligand ligand, and the E3 ubiquitin ligand ligand has the structure shown below: ,in: W is C=O; Y is O; Z is H; G is a C1-C4 alkyl group; R3 is H.

5. The method for preparing the protein hydrolysis-targeted chimera according to claim 4, characterized in that, Includes the following steps: The protein hydrolysis-targeting chimera synthesis precursor of claim 1 or 2 or the protein hydrolysis-targeting chimera synthesis precursor prepared by the preparation method of claim 3 is mixed with the compound shown in formula I-4 and irradiated with light to obtain the protein hydrolysis-targeting chimera; in: ； L2 is the second connecting chain, and has one of the following structures: ， Where n is selected from an integer between 2 and 3; R2 is an E3 ubiquitin ligand ligand, and the E3 ubiquitin ligand ligand has the structure shown below: ,in: W is C=O; Y is O; Z is H; G is a C1-C4 alkyl group; R3 is H.

6. The use of the protein hydrolysis-targeting chimera according to claim 4 or the protein hydrolysis-targeting chimera prepared by the method according to claim 5 in the preparation of protein degradation products or antitumor products, characterized in that, The protein is either BRD4 or PARP1, and the tumor cells are either HeLa cells or MDA-MB-231 cells.

7. A protein degradation product, characterized in that, The protein degradation product contains the protein hydrolysis-targeting chimera as described in claim 4 or the protein hydrolysis-targeting chimera prepared by the preparation method described in claim 5, wherein the protein is BRD4 protein or PARP1 protein.

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