Proteolysis targeting chimera as well as preparation method and application thereof

Abstract

The invention provides a proteolysis targeting chimera as well as a preparation method and application thereof. The proteolysis targeting chimera is prepared from a target protein ligand containing a phosphoramidite structure and an E3 ubiquitin ligase ligand containing a phosphoramidite structure through a DNA (Deoxyribonucleic Acid) automatic solid-phase synthesizer, the protein hydrolysis targeting chimera is prepared by a solid-phase synthesis method, and the method has the advantages of rapidness, accuracy, modularization, automation and large-scale preparation; by selecting a proper connexon, the cell permeability of the proteolysis targeting chimera is improved, and the target protein degradation capability of the proteolysis targeting chimera is improved; an enzyme activatable group is modified on a hydroxyl group of an E3 ubiquitin ligase ligand, and only when the enzyme activatable group is removed from a corresponding enzyme, a protein hydrolysis targeting chimera can be released to play a role, so that the target protein is controllably degraded. The proteolysis targeting chimera prepared by the method provided by the invention can effectively inhibit proliferation of human cervical cancer HeLa cells, has the advantages of strong specificity, good stability and high membrane permeability, and provides a new solution for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of protein hydrolysis targeting chimeras, and in particular, relates to a protein hydrolysis targeting chimera and a preparation method and application thereof. Background Art

[0002] Proteolysis Targeting Chimera (PROTAC) is a protein degradation technology that uses the ubiquitin proteasome system to degrade target proteins to treat various diseases. The target protein ligand is combined with the E3 ubiquitin ligase ligand through a linker to form a chimera that can recognize the target protein and induce the E3 ubiquitin ligase to degrade the target protein. Conventional small molecule inhibitors affect the active function of the protein by binding to the active site of the protein. In contrast, PROTAC does not need to bind to the active site of the target protein, but only needs to form a ternary complex with the target protein ligand, E3 ubiquitin ligase ligand and the linker, which greatly expands the substrate range of the target protein, provides a new solution for the targeted degradation of undruggable proteins, and brings new breakthroughs to the treatment of related diseases. In the field of tumor treatment, many key signaling pathways and oncogenic proteins have become targets of proteolysis targeting chimeras, such as (MYC, p53, TAL1, STAT3). Compared with traditional drug approaches, proteolysis-targeted chimeras have higher targeting specificity and lower adverse reactions and have great development potential.

[0003] However, there are still some challenges and problems in the development and application of proteolysis-targeted chimeras. On the one hand, the preparation of proteolysis-targeted chimeras relies on traditional chemical synthesis methods, which involve multiple chemical reactions and have many steps, low efficiency, poor coupling accuracy, and low automation. On the other hand, conventional proteolysis-targeted chimeras have no selectivity between tumor cells and normal cells, resulting in their greater toxic side effects, especially when they need to play a role in degrading specific proteins in specific cell types, their therapeutic potential is difficult to determine. These problems have brought certain difficulties to the development, optimization, and clinical application of proteolysis-targeted chimera drugs.

[0004] Therefore, there is an urgent need to develop new synthetic methods and technologies to overcome these problems and advance the development of proteolysis-targeted chimera technology. Summary of the invention

[0005] In view of the problems of the prior art, the present invention provides a proteolysis targeting chimera and its preparation method and application, wherein the proteolysis targeting chimera is synthesized by a target protein ligand containing a phosphoramidite structure and an E3 ubiquitin ligase ligand containing a phosphoramidite structure through a DNA solid phase synthesizer; the proteolysis targeting chimera is prepared by solid phase synthesis, which has the advantages of rapid, accurate, automated and large-scale preparation; by selecting a suitable linker, the cell permeability of the proteolysis targeting chimera is increased, and its ability to degrade the target protein is improved; the hydroxyl group of the E3 ubiquitin ligase ligand can be modified with an enzyme activatable group, and only when the corresponding enzyme removes the enzyme activatable group can the proteolysis targeting chimera be released to exert its effect, thereby degrading the target protein. The proteolysis targeting chimera prepared by the method provided by the present invention can effectively degrade the target protein in human cervical cancer HeLa cells and inhibit their proliferation, has the advantages of strong specificity and high membrane permeability, and provides a new solution for tumor treatment.

[0006] In one aspect, the present invention provides a method for preparing a proteolysis-targeted chimera, which is prepared by an automated DNA solid phase synthesis method, comprising the following steps:

[0007] (1) preparing and purifying E3 ubiquitin ligase ligand monomers containing phosphoramidite structures;

[0008] (2) preparing target protein ligand monomers containing phosphoramidite structures and purifying them;

[0009] (3) synthesizing an E3 ubiquitin ligase ligand monomer containing a phosphoramidite structure and a target protein ligand monomer containing a phosphoramidite structure on a solid phase carrier by means of a DNA synthesizer, and obtaining a protein hydrolysis targeting chimera after deprotection and purification.

[0010] The principle of DNA synthesis by a DNA solid phase synthesizer is: the terminal nucleotide of the DNA chain is fixed on a solid phase carrier (CPG) through a connector to form a nucleoside-derivatized solid phase carrier; starting from the 3' end, nucleotides are added in sequence, and the 3' end of each nucleotide forms a phosphite triester with the 5' end of the previous nucleotide, and then oxidized to a phosphotriester; after each round of synthesis, the protective group DMT on the 5' terminal hydroxyl group is removed to continue the next round of synthesis. The raw material nucleotides of the DNA synthesized by the solid phase synthesis method contain a phosphoramidite structure, so the present invention draws on this principle to design an E3 ubiquitin ligase ligand containing a phosphoramidite structure. The ligand molecule is first connected to a universal solid phase carrier, and after oxidation, capping, deprotection and other treatments, it reacts with a target protein ligand containing a phosphoramidite structure to form a phosphate structure. All reactions are completed on a DNA synthesizer through automated modularization. The phosphate ester includes phosphodiester, phosphorothioate, and phosphotriester, which couples the E3 ubiquitin ligase ligand and the target protein ligand together to obtain a proteolysis targeting chimera, and finally cuts the proteolysis targeting chimera from the CPG.

[0011] Therefore, the E3 ubiquitin ligase ligand and the target protein ligand prepared in advance must have a phosphoramidite structure in order to synthesize the protein hydrolysis targeting chimera by a DNA solid phase synthesizer and a solid phase synthesis method, and then purify it by high performance liquid chromatography (HPLC) after deprotection to obtain the protein hydrolysis targeting chimera. If the target protein ligand prepared in advance does not contain a phosphoramidite monomer structure, the protein hydrolysis targeting chimera cannot be prepared by this method. Compared with the method of chemically synthesizing the protein hydrolysis targeting chimera, the solid phase synthesis method for synthesizing the protein hydrolysis targeting chimera avoids the limitations of multiple steps, long time consumption, high cost and inaccuracy, and has the advantages of rapid, accurate, modular, automated and large-scale preparation.

[0012] Furthermore, the purification methods in steps (1) and (2) are both purification by chromatography column, and the eluent of the chromatography column includes triethylamine.

[0013] In some embodiments, the chromatography column is a silica gel chromatography column, and 1% triethylamine needs to be added to the eluent during purification. If 1% triethylamine is not added to the eluent, the yield of E3 ubiquitin ligase ligand and target protein ligand will be reduced by 20%-30%; or 1% triethylamine is replaced by other substances, such as 1% ammonia water, which will also lead to a decrease in the yield of the product.

[0014] Furthermore, the solid phase carrier in step (3) comprises a universal solid phase carrier or a solid phase carrier containing an enzyme activatable group, and the enzyme activatable group comprises one or more of a phosphate group and a nitro group.

[0015] In the process of synthesizing the proteolysis targeting chimera by a DNA solid phase synthesizer, the proteolysis targeting chimera obtained by adding a universal solid phase carrier does not contain an enzyme activatable group, and it can play a role when entering tumor cells; while the proteolysis targeting chimera obtained by adding a solid phase carrier containing an enzyme activatable group contains an enzyme activatable group, and the enzyme activatable group is modified on the hydroxyl group of the E3 ubiquitin ligase ligand, and the proteolysis targeting chimera needs a corresponding enzyme to remove the enzyme activatable group before entering the tumor cell to play a role. Compared with the proteolysis targeting chimera not modified with the enzyme activatable group, the proteolysis targeting chimera modified with the enzyme activatable group is not only selective for tumor cells, but can only play a role in tumor cells that can remove the enzyme activatable group, and it is also selective for degrading proteins, and can only degrade proteins bound to the targeting protein ligand.

[0016] In another aspect, a proteolysis targeting chimera is prepared by a solid phase synthesis method, wherein the proteolysis targeting chimera has the following structural formula:

[0017]

[0018] Wherein, L is a linker, the left side of L is a targeting protein ligand, the right side of L is an E3 ubiquitin ligase ligand, and R is a substituent.

[0019] In theory, the relevant proteins in the pathogenesis of the disease can be designed with corresponding targeting protein ligands according to their structures, and the enzyme-activatable protein hydrolysis targeting chimeras can be prepared by coupling the targeting protein ligands and E3 ubiquitin ligase ligands through solid-phase synthesis methods.

[0020] The linker plays a key role in the entry of the proteolysis targeting chimera into cells. The charge and structure of the linker affect the ability of the proteolysis targeting chimera to enter cells and its stability in the in vivo environment. The present invention solves the problem of poor membrane permeability of the proteolysis targeting chimera by screening linkers.

[0021] The targeting protein ligand provided by the present invention targets BRD4 protein. BRD4 protein is bromodomain protein 4, which plays a key role in gene transcription and epigenetic regulation. Its N-terminus can recognize the specific acetylated lysine residue at the tail end of the protein and selectively bind to it, thereby regulating the binding of related proteins such as chromatin remodeling factors and transcription factors to specific gene transcription sites. It plays an important role in the infiltration and metastasis of tumor cells and the malignant development of tumors. The occurrence and development of various malignant tumors such as cervical cancer, osteosarcoma, lung cancer, breast cancer, blood system tumors and liver cancer are all related to BRD4 dysfunction, so BRD4 protein is an ideal drug target.

[0022] Furthermore, the linker includes one or more of phosphodiester, phosphorothioate, and phosphotriester.

[0023] Furthermore, the linker is a phosphotriester.

[0024] Furthermore, the substituent includes one or more of a hydroxyl group, a phosphate group, and a nitro group.

[0025] In some embodiments, the substituent is preferably a phosphate group.

[0026] Furthermore, the proteolysis targeting chimera comprises any one or more of the following structural formulas A, B, C, and D:

[0027]

[0028]

[0029] The linker of the proteolysis targeting chimera A is a phosphodiester, the linker of the proteolysis targeting chimera B is a phosphorothioate, and the linkers of the proteolysis targeting chimera C and the proteolysis targeting chimera D are phosphotriester.

[0030] Furthermore, the proteolysis targeting chimera includes any one or more of the C and D structural formulas.

[0031] In another aspect, a proteolysis targeting chimera is used for preparing an anti-tumor drug, wherein the proteolysis targeting chimera comprises a targeting protein ligand, a linker and an E3 ubiquitin ligase ligand, wherein the linker comprises one or more of phosphodiester, phosphorothioate, and phosphotriester, and the E3 ubiquitin ligase ligand can modify an enzyme-activatable group.

[0032] In some methods, the ability of proteolysis targeting chimeras A, B, C and commercially available proteolysis targeting chimeras MZ1 containing different linkers to inhibit the growth of human cervical cancer HeLa cells and degrade BRD4 protein in human cervical cancer HeLa cells is experimentally tested, and it is proved that proteolysis targeting chimeras A containing phosphodiester linkers and proteolysis targeting chimeras B containing thiophosphate linkers have poor cell permeability and poor inhibitory effects on human cervical cancer HeLa cells, while proteolysis targeting chimeras C containing phosphotriester linkers and MZ1 containing polyethylene glycol linkers have relatively good inhibitory effects on human cervical cancer HeLa cells. Proteolysis targeting chimeras C containing phosphotriester linkers have stronger inhibitory effects on human cervical cancer HeLa cells and stronger ability to degrade BRD4 protein in human cervical cancer HeLa cells than MZ1, and proteolysis targeting chimeras C have inferior inhibitory effects on other tumor cells as human cervical cancer HeLa cells.

[0033] In some methods, the ability of alkaline phosphatase-activatable protein hydrolysis targeting chimera D and unmodified enzyme-activatable group protein hydrolysis targeting chimera C to inhibit the proliferation of human osteosarcoma cells Saos-2 cells and human osteosarcoma cells U-2OS cells, respectively, was detected experimentally, demonstrating that alkaline phosphatase-activatable protein hydrolysis targeting chimera D could only effectively inhibit the proliferation of Saos-2 cells with high expression of alkaline phosphatase, while unmodified enzyme-activatable group protein hydrolysis targeting chimera C had a good inhibitory effect on the proliferation of both Saos-2 cells with high expression of alkaline phosphatase and U-2OS cells with low expression of alkaline phosphatase, indicating that alkaline phosphatase-activatable protein hydrolysis targeting chimera D can specifically inhibit the proliferation of tumor cells with high expression of alkaline phosphatase.

[0034] In some methods, the ability of alkaline phosphatase-activatable protein hydrolysis targeting chimera D and unmodified enzyme-activatable group protein hydrolysis targeting chimera C to degrade BRD4 protein in human osteosarcoma Saos-2 cells and human osteosarcoma U-2OS cells, respectively, was detected experimentally, demonstrating that alkaline phosphatase-activatable protein hydrolysis targeting chimera D only has the ability to efficiently degrade BRD4 protein in Saos-2 cells with high alkaline phosphatase expression, while unmodified enzyme-activatable group protein hydrolysis targeting chimera C has the ability to efficiently degrade BRD4 protein in both Saos-2 cells with high alkaline phosphatase expression and U-2OS cells with low alkaline phosphatase expression, indicating that alkaline phosphatase-activatable protein hydrolysis targeting chimera D has specific selection for tumor cells and efficiently degrades BRD4 protein in specific cells.

[0035] The present invention has the following beneficial effects:

[0036] 1. The present invention provides a proteolysis targeting chimera, which is synthesized by a target protein ligand containing a phosphoramidite structure and an E3 ubiquitin ligase ligand containing a phosphoramidite structure through a DNA solid phase synthesizer. The proteolysis targeting chimera is prepared by solid phase synthesis, which has the advantages of rapid, accurate, automated and large-scale preparation;

[0037] 2. By selecting linkers, the cell permeability of the proteolytic targeting chimera is increased, thereby improving its ability to degrade the target protein;

[0038] 3. The hydroxyl group of the E3 ubiquitin ligase ligand can modify an enzyme activatable group. Only when the corresponding enzyme in the cell removes the enzyme activatable group can the proteolytic targeting chimera be released to exert its effect, thereby degrading the target protein;

[0039] 4. The proteolytic targeting chimera C prepared by the method provided by the present invention can effectively degrade the targeting protein in human cervical cancer HeLa cells and inhibit their proliferation. It has the advantages of strong specificity, good stability and high membrane permeability, and provides a new solution for tumor treatment;

[0040] 5. The proteolysis targeting chimera D prepared according to the method of the present invention can specifically inhibit the proliferation of tumor cells with high expression of alkaline phosphatase. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the hydrogen spectrum of compound 2 in Example 1;

[0042] Figure 2 is the carbon spectrum of compound 2 in Example 1;

[0043] Figure 3 is the hydrogen spectrum of compound 3 in Example 1;

[0044] Figure 4 is the carbon spectrum of compound 3 in Example 1;

[0045] Figure 5 is the hydrogen spectrum of compound 4 in Example 1;

[0046] Figure 6 is the carbon spectrum of compound 4 in Example 1;

[0047] Figure 7 is the phosphorus spectrum of compound 4 in Example 1;

[0048] Figure 8 is the hydrogen spectrum of compound 6 in Example 1;

[0049] Fig. 9 is the carbon spectrum of compound 6 in Example 1;

[0050] Fig.10 is the hydrogen spectrum of compound 7 in Example 1;

[0051] Fig.11 is the carbon spectrum of compound 7 in Example 1;

[0052] Fig.12 is the hydrogen spectrum of compound 8 in Example 1;

[0053] Fig.13 is the carbon spectrum of compound 8 in Example 1;

[0054] Fig.14 is the phosphorus spectrum of compound 8 in Example 1;

[0055] Fig.15 is the hydrogen spectrum of compound 10 in Example 1;

[0056] Fig.16 is the carbon spectrum of compound 10 in Example 1;

[0057] Fig.17 is the phosphorus spectrum of compound 10 in Example 1;

[0058] Fig.18 This is a schematic diagram of the experimental results of the inhibition of HeLa cell proliferation by proteolysis targeting chimeras A, B, C and MZ1 in Example 2;

[0059] Fig.19 This is a liquid phase spectrum diagram showing the hydrophobicity comparison of the proteolytic targeting chimeras MZ1 and C in Example 2;

[0060] Fig. 20 This is a WB experiment diagram of the degradation of BRD4 protein in HeLa cells by proteolysis targeting chimeras MZ1 and C in Example 3;

[0061] Fig.21 This is a curve diagram of the degradation of BRD4 protein in HeLa cells by the proteolytic targeting chimera MZ1 in Example 3;

[0062] Fig. 22 This is a curve diagram of the degradation of BRD4 protein in HeLa cells by proteolysis targeting chimera C in Example 3;

[0063] Fig.23 is the mass spectrum of the proteolysis targeting chimera C in Example 4;

[0064] Fig.24 is the mass spectrum of the proteolysis targeting chimera D in Example 4;

[0065] Fig.25 This is the mass spectrum of the proteolysis targeting chimera C obtained by dephosphorylation of the proteolysis targeting chimera D in Example 4;

[0066] Fig.26 This is a liquid phase spectrum obtained after HPLC treatment to verify whether alkaline phosphatase can dephosphorylate the proteolysis targeting chimera D to obtain the proteolysis targeting chimera C in Example 4;

[0067] Fig. 27 This is a liquid phase spectrum obtained after HPLC treatment to verify whether different concentrations of alkaline phosphatase can dephosphorylate the proteolysis targeting chimera D to obtain the proteolysis targeting chimera C in Example 4;

[0068] Fig.28 In Example 4, the alkaline phosphatase inhibitor Na 3 VO 4Whether it is possible to inhibit the dephosphorylation of D to obtain C, and the liquid phase spectrum obtained after HPLC treatment;

[0069] Fig.29 The C liquid phase spectrum of quantitative extraction by measuring the HPLC chromatographic peak area in Example 5;

[0070] Fig.30 The stability curve of C quantitatively extracted by HPLC chromatographic peak area measurement in Example 5;

[0071] Fig.31 This is the liquid phase spectrum of the BRD4 protein degrader (MZ1) quantitatively extracted by measuring the HPLC chromatographic peak area in Example 5;

[0072] Fig.32 The stability curve of MZ1 quantitatively extracted by HPLC peak area measurement in Example 5 is shown in FIG.

[0073] Fig.33 This is a graph showing the experimental results of the test of alkaline phosphatase expression in Saos-2 cells and U-2OS cells in Example 7;

[0074] Fig.34 This is a schematic diagram of the experimental results of the inhibition of Saos-2 cell proliferation by proteolysis targeting chimeras C and D in Example 7;

[0075] Fig.35 This is a schematic diagram of the experimental results of the inhibition of U-2OS cell proliferation by proteolysis targeting chimeras C and D in Example 7;

[0076] Fig.36 This is a WB experiment diagram of proteolysis targeting chimeras C and D on BRD4 protein degradation in Saos-2 cells in Example 7;

[0077] Fig.37 This is a curve diagram of the degradation of BRD4 protein in Saos-2 cells by proteolysis targeting chimera C in Example 7;

[0078] Fig.38 This is a curve diagram of the degradation of BRD4 protein in Saos-2 cells by proteolysis targeting chimera D in Example 7;

[0079] Fig.39 This is a WB experiment diagram of proteolysis targeting chimeras C and D on BRD4 protein degradation in U-2OS cells in Example 7;

[0080] Fig.40 This is a curve diagram of the degradation of BRD4 protein in U-2OS cells by proteolysis targeting chimera C in Example 7;

[0081] Fig.41 This is a curve diagram of the degradation of BRD4 protein in U-2OS cells by proteolysis targeting chimera D in Example 7. DETAILED DESCRIPTION

[0082] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0083] Example 1. Preparation of proteolysis-targeted chimeras

[0084] 1. The preparation method of the proteolysis targeting chimera is as follows:

[0085] 1) Synthesis of E3 ubiquitin ligase ligands containing phosphoramidite structures

[0086] The reaction formula of the synthesis route is as follows:

[0087]

[0088] The specific steps are as follows:

[0089] Synthesis of compound 2: Under nitrogen protection, compound 1 (1 g, 2.08 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1.19 g, 3.12 mmol) and 6-hydroxyhexanoic acid (0.303 g, 2.29 mmol) were dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF), and the reaction was stirred overnight. After the reaction was completed, it was extracted twice with dichloromethane and saturated sodium bicarbonate solution, and the organic layer was collected. Anhydrous sodium sulfate was added for drying, and after spin drying, it was purified by chromatography to obtain a white foam solid compound 2 (0.761 g, 1.36 mmol, 65.38%). 1H NMR (400MHz, DMSO-d6) δ8.98(s,1H),8.39(d,J=7.8Hz,1H),7.79(d,J=9.3Hz,1H),7.43(d,J=8.4Hz,2H),7.38(d,J=8.3Hz,2H ),5.11(d,J=3.5Hz,1H),4.96-4.86(m,1H),4.51(d,J=9.3Hz,1H),4.42(t,J=8.1Hz,1H),4.34(t,J=5.1Hz,1H),4.30-4.25(m, 1H),3.60(d,J=3.4Hz,2H),3.39-3.35(m,2H),2.45(s,3H),2.23(dd,J=14.6,6.7Hz,1H),2.12(d,J=7.9Hz,1H),2.01(ddd,J= 10.2,7.7,2.3Hz,1H),1.78(s,1H),1.56-1.44(m,2H),1.37(d,J=7.1Hz,3H),1.27(d,J=7.7Hz,2H),1.25(s,2H),0.93(s,9H). 13 C NMR (101 MHz, DMSO-d6) δ 172.06, 170.65, 169.61, 151.52, 147.77, 144.70, 131.13, 129.69, 128.84, 126.39, 68.77, 60.64, 58.55, 56.34, 47.70, 37.75, 35.21, 34.99, 32.30, 26.46, 25.25, 22.48, 16.01. The hydrogen spectrum of compound 2 is shown in Figure 1 As shown, the carbon spectrum is Figure 2 shown.

[0090] Synthesis of compound 3: Under nitrogen protection, compound 2 (0.761 g, 1.36 mmol) was dissolved in 10 mL of anhydrous pyridine, and 4,4'-dimethoxytrityl chloride (DMT-Cl) (0.553 g, 1.632 mmol) was added, and the reaction was stirred overnight. After the reaction was completed, pyridine was removed by rotary evaporator, and then purified by chromatography column to obtain white foam solid compound 3 (0.899 g, 1.04 mmol, 76.5%). 1H NMR(400MHz,Chloroform-d)δ8.67(s,1H),7.46-7.39(m,5H),7.36(d,J=8.3Hz,2H),7.31(d,J=2.1Hz,2H),7.31-7.28(m,2H),7.2 0(d,J=7.2Hz,1H),6.90-6.71(m,4H),6.16(d,J=8.7Hz,1H),5.20-5.01(m,1H),4.69(t,J=7.9Hz,1H),4.56(d,J=8.8Hz,1H),4.49 (s,1H),4.08(d,J=11.5Hz,1H),3.78(s,6H),3.59(dd,J=11.3,3.7Hz,1H),3.03(t,J=6.5Hz,2H),2.94(d,J=7.3Hz,1H),2.52(s,3 H),2.18(t,J=7.6Hz,2H),2.07-1.99(m,1H),1.59(q,J=6.8,6.4Hz,4H),1.47(d,J=6.9Hz,3H),1.30(t,J=7.3Hz,2H),1.03(s,9H). 13 C NMR (101 MHz, Chloroform-d) δ 173.82, 172.17, 169.78, 158.39, 150.45, 148.60, 145.44, 143.23, 136.74, 130.11, 129.69, 128.28, 127.82, 126.70, 126.57, 113.09, 85.80, 70.05, 63.27, 58.56, 57.55, 56.76, 55.31, 36.60, 35.49, 35.16, 29.93, 26.63, 26.17, 25.66, 22.35, 16.21. The hydrogen spectrum of compound 3 is shown in Figure 3 As shown, the carbon spectrum is Figure 4 shown.

[0091] Synthesis of compound 4: In an anhydrous and oxygen-free environment and under nitrogen protection, compound 3 (0.899 g, 1.04 mmol) was dissolved in 15 mL of anhydrous dichloromethane, and N, N-diisopropylformamide (DIPEA) (0.403 g, 3.12 mmol) was added dropwise. After cooling in an ice bath for five minutes, 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.369 g, 1.56 mmol) was added. The reaction speed was very fast, and thin layer chromatography TLC was required to monitor the reaction process in real time. After the reaction was completed, dichloromethane and saturated potassium chloride solution were used for extraction, the organic layer was collected, anhydrous sodium sulfate was added for drying, and it was quickly spin-dried, dry-loaded, purified by silica gel chromatography column, loaded on the column and eluted with an eluent, the preparation method of the eluent was: petroleum ether and ethyl acetate were mixed in a volume ratio of 1:1, and then 1% triethylamine was added. The product needs to be evaporated at a faster speed, and after the evaporation, it needs to be evacuated with a vacuum oil pump and stored with nitrogen to prevent the product from being oxidized. The product is a white foam solid compound 4 (0.828 g, 0.78 mmol, 75%). 1 H NMR(400MHz,Chloroform-d)δ8.67(s,1H),7.45-7.39(m,4H),7.38-7.28(m,7H),7.27(s,1H),7.20(d,J=7.2Hz,1H),6 .81(d,J=8.9Hz,4H),5.16-5.01(m,1H),4.77-4.55(m,3H),3.92-3.81(m,1H),3.78(s,6H),3.76-3.64(m,2H),3.54(d dd,J=14.4,10.3,7.0Hz,2H),3.03(t,J=6.5Hz,2H),2.62(t,J=6.1Hz,2H),2.53(s,3H),2.18(q,J=7.7Hz,2H),1.75(s ,2H),1.61(td,J=11.9,9.7,4.8Hz,4H),1.47(d,J=6.9Hz,3H),1.40(d,J=4.0Hz,2H),1.18-1.09(m,12H),1.01(s,9H). 13C NMR(101MHz,Chloroform-d)δ172.19,171.94,169.52,158.30,150.30,148.51,1 45.36,143.19,143.05,136.69,130.01,129.59,128.19,127.72,126.60,126.46 ,118.15,117.80,112.98,85.69,71.50,63.21,58.79,58.08,56.73,55.21,53.46,48.89,36.29,35.07,29.91,26.49,26.22,25.80,24.57,22.25,20.29,16.13. 31 P NMR (162MHz, Chloroform-d) δ148.33, 147.84. The hydrogen spectrum of compound 4 is as follows Figure 5 As shown, the carbon spectrum is Figure 6 The phosphorus spectrum is shown in Figure 7 shown.

[0092] 2) Synthesis of target protein ligands containing phosphoramidite structures

[0093] There are two synthetic pathway reaction formulas, as follows:

[0094]

[0095] The specific steps are as follows:

[0096] Synthesis of compound 6: Under nitrogen protection, compound 5 (2 g, 4.38 mmol) was dissolved in 24 mL of dichloromethane, 6 mL of trifluoroacetic acid (8.778 g, 77.19 mmol) was added dropwise, and the mixture was stirred at room temperature for 6 h. After being spin-dried, the mixture was purified by chromatography to obtain yellow foam solid compound 6 (1.646 g, 4.11 mmol, 94.1%). 1 H NMR(400MHz,Chloroform-d)δ7.43(d,J=8.5Hz,2H),7.33(d,J=8.8Hz,2H),4.60(t,J=6.9Hz,1H), 3.71(dd,J=16.8,7.1Hz,1H),3.59(dd,J=16.8,6.7Hz,1H),2.68(s,3H),2.41(s,3H),1.69(s,3H). 13C NMR (101 MHz, Chloroform-d) δ 173.22, 164.15, 155.40, 149.99, 136.95, 136.36, 131.93, 131.21, 131.01, 130.54, 129.94, 128.76, 45.72, 14.43, 13.14, 11.74. The hydrogen spectrum of compound 6 is shown in Figure 8 As shown, the carbon spectrum is Fig. 9 shown.

[0097] Synthesis of compound 7: Under nitrogen protection, compound 6 (1.646 g, 4.11 mmol) and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (2.344 g, 6.165 mmol) were dissolved in 20 mL of anhydrous N,N-dimethylformamide (DMF), 3-amino-1-propanol (0.37 g, 4.932 mmol) and N,N-diisopropylformamide (DIPEA) (1.328 g, 10.275 mmol) were added dropwise, and the reaction was stirred overnight. After the reaction was completed, the mixture was extracted with ethyl acetate and saturated sodium chloride solution, the organic layer was collected, dried with anhydrous sodium sulfate, and purified by chromatography column to obtain a white foam solid compound 7 (1.616 g, 3.53 mmol, 85.89%). 1 H NMR (400MHz, Chloroform-d) δ7.40(d,J=8.6Hz,2H),7.32(d,J=8.8Hz,2H),4.66(dd,J=7.8,6.4Hz,1H),3.63(d,J=6.8Hz,2H),3.56(d d,J=14.5,7.8Hz,1H),3.46(q,J=6.1Hz,2H),3.40(dd,J=14.5,6.4Hz,1H),2.67(s,3H),2.40(s,3H),1.75-1.69(m,2H),1.67(s,3H). 13 C NMR (101 MHz, Chloroform-d) δ 171.36, 164.08, 155.68, 149.99, 136.84, 136.48, 131.88, 131.00, 130.65, 129.85, 128.72, 58.82, 54.28, 45.57, 39.00, 32.09, 14.41, 13.12, 11.81. The hydrogen spectrum of compound 7 is shown in Fig.10 As shown, the carbon spectrum is Fig.11 shown.

[0098] Synthesis of compound 8: In an anhydrous and oxygen-free environment and under nitrogen protection, compound 7 (0.762g, 1.66mmol) was dissolved in 15mL of anhydrous dichloromethane, and N,N-diisopropylformamide (DIPEA) (0.644g, 4.98mmol) was added dropwise. After cooling in an ice bath for five minutes, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.589g, 2.49mmol) was added. The reaction speed was very fast, and thin layer chromatography TLC was required to monitor the reaction process in real time. After the reaction was completed, it was extracted with dichloromethane and saturated potassium chloride solution, the organic layer was collected, dried with anhydrous sodium sulfate, quickly spin-dried, dry loaded, and purified by silica gel chromatography. The column was loaded and eluted with an eluent, and the preparation method of the eluent was: petroleum ether and ethyl acetate were mixed in a volume ratio of 1:1, and then 1% triethylamine was added. The product needs to be evaporated at a faster speed, and after the evaporation, it needs to be evacuated with a vacuum oil pump and stored with nitrogen to prevent the product from being oxidized. The product is a white foam solid compound 8 (0.5 g, 0.759 mmol, 45%). 1 HNMR(400MHz,Chloroform-d)δ7.42-7.36(m,2H),7.32(dd,J=8.8,1.1Hz,2H),4.63(t,J =7.0Hz,1H),3.96-3.77(m,2H),3.77-3.66(m,2H),3.65-3.55(m,2H),3.54-3.41(m,2H) ,3.35(dddd,J=17.8,14.3,6.6,3.8Hz,2H),2.68(d,J=6.4Hz,1H),2.65(s,3H),2.62(d, J=6.4Hz,1H),2.39(s,3H),1.91-1.79(m,2H),1.66(s,3H),1.17(dd,J=6.7,3.8Hz,12H). 13 C NMR(101MHz,Chloroform-d)δ170.45,163.92,155.67,149.93,136.77,136.64,132.13,130.93,130.83,130 .48,129.85,128.73,117.76,61.57,58.24,54.43,43.13,37.12,30.68,24.70,20.41,14.42,13.11,11.86. 31 P NMR (162MHz, Chloroform-d) δ147.76. The hydrogen spectrum of compound 8 is as follows Fig.12 As shown, the carbon spectrum is Fig.13 As shown, the phosphorus spectrum is Fig.14 shown.

[0099] Synthesis of compound 9: Under anhydrous and oxygen-free conditions and under nitrogen protection, compound 7 (0.74 g, 1.62 mmol) was dissolved in 24 mL of anhydrous dichloromethane, N,N-diisopropylformamide (DIPEA) (0.229 g, 1.77 mmol) was added dropwise, and the mixture was cooled in an ice-salt bath for ten minutes. Bis(diisopropylamino)chlorophosphonium (0.472 g, 1.77 mmol) was dissolved in 10 mL of anhydrous dichloromethane, and the anhydrous dichloromethane in which bis(diisopropylamino)chlorophosphonium was dissolved was slowly added dropwise. The reaction speed was very fast, and thin layer chromatography (TLC) was required to monitor the reaction progress in real time. After the reaction was complete, the crude product compound 9 was obtained and directly subjected to the next step of reaction.

[0100] Synthesis of compound 10: In anhydrous and oxygen-free conditions and under nitrogen protection, anhydrous isobutanol (0.131 g, 1.77 mmol) and 5-(ethylthio)-1H-tetrazole (0.23 g, 0.81 mmol, which need to be dissolved in anhydrous acetonitrile in advance to prepare 0.25 M, and degassed at will) are added to the crude product compound 9. Thin layer chromatography TLC is required to monitor the reaction process in real time. After the reaction, dichloromethane and saturated potassium chloride solution are used for extraction, the organic layer is collected, anhydrous sodium sulfate is added for drying, and it is quickly spin-dried, dry-loaded, and purified by silica gel chromatography. The column is loaded and eluted with an eluent, and the preparation method of the eluent is: 1% triethylamine is added to ethyl acetate. A faster speed is required when the product is rotary evaporated. After rotary evaporation, a vacuum oil pump needs to be used to dry it and it needs to be stored with nitrogen to prevent the product from being oxidized. The product is a white foamy solid compound 10 (0.426 g, 0.644 mmol, 39.8%). 1 H NMR(400MHz,Chloroform-d)δ7.38(d,J=8.5Hz,2H),7.30(d,J=8.6Hz,2H),4.63(t,J =6.9Hz,1H),3.78-3.61(m,2H),3.62-3.53(m,2H),3.48(ddd,J=19.5,10.5,4.2Hz,2H ),3.44-3.36(m,2H),3.36-3.28(m,2H),2.64(s,3H),2.38(s,3H),1.87-1.82(m,2H) ,1.65(s,3H),1.24(d,J=8.4Hz,1H),1.21-1.11(m,12H),0.89(dd,J=9.1,5.9Hz,6H). 13C NMR(101MHz,Chloroform-d)δ170.43,163.78,155.70,149.84,136.67,132.15,130.92,130.73,130.47,12 9.83,128.69,70.25,61.44,54.41,42.88,39.28,37.29,30.84,29.78,24.57,19.25,14.41,13.10,11.86. 31 P NMR (162MHz, Chloroform-d) δ145.88. The hydrogen spectrum of compound 10 is as follows Fig.15 As shown, the carbon spectrum is Fig.16 The phosphorus spectrum is shown in Fig.17 shown.

[0101] It should be noted that in the above-mentioned synthesis process of compound 4, compound 8 and compound 10, when using silica gel chromatography column to purify the product, 1% triethylamine needs to be added to the eluent. If 1% triethylamine is not added to the eluent, the yield of the product will decrease by 20%-30%. Or, if 1% triethylamine is replaced by other substances, such as 1% ammonia water, the yield of the product will also decrease.

[0102] 3) Solid phase synthesis of protein hydrolysis targeting chimera A, the synthesis route reaction formula is as follows:

[0103]

[0104] The specific steps are as follows: Under nitrogen protection, compound 4 and compound 8 are dissolved in anhydrous dichloromethane to prepare a 0.1M solution and placed in the modification bottle of the German K&A H-8DNA synthesizer. The sequence is input into the instrument, and a universal solid phase carrier (universal CPG) is used to fill the column at the 3' end of the sequence. After the synthesis is completed, the terminal protection group is removed to obtain A. 1 mL of concentrated ammonia solution is added and heated at 55°C for 8 hours to cut A from CPG. After deprotection, the concentrated ammonia solution is removed by a rotary evaporator, and 50% ACN and H are added. 2 O, filtered with a 0.22 μm filter membrane, and purified by HPLC using a C18 column. After freeze-drying, it was re-dissolved with dimethyl sulfoxide (DMSO), and the concentration at 260 nm was measured using a micro-spectrophotometer Nano Drop, and the molar weight was calculated to obtain a molecular weight of 1078.68 Da for the proteolytic targeting chimera A.

[0105] 4) Solid phase synthesis of protein hydrolysis targeting chimera B, the synthesis route reaction formula is as follows:

[0106]

[0107] The specific steps are as follows: under nitrogen protection, compound 4 and compound 8 are dissolved in anhydrous dichloromethane to prepare a 0.1M solution and placed in the modification bottle of the German K&A H-8DNA synthesizer, and the thiolation reagent is prepared into a 0.2M solution and placed in the modification bottle of the thiolation reagent. Input the sequence on the instrument, use a universal solid phase carrier (universal CPG) to fill the column at the 3' end of the sequence, and after the synthesis is completed, remove the terminal protection group to obtain B. Add 1mL of concentrated ammonia solution and heat at 55°C for 8 hours to cut B from CPG. After deprotection, remove the concentrated ammonia solution with a rotary evaporator, add 50% ACN and H 2 O, filtered with a 0.22 μm filter membrane, and purified by HPLC using a C18 column. After freeze-drying, it was reconstituted with DMSO, and the concentration at 260 nm was measured using a micro-spectrophotometer Nano Drop, and the molar weight was calculated to obtain a molecular weight of 1094.68 Da for the proteolytic targeting chimera B.

[0108] 5) Solid phase synthesis of protein hydrolysis targeting chimera C, the synthesis route reaction formula is as follows:

[0109]

[0110] The specific steps are as follows: Under nitrogen protection, compound 4 and compound 10 are dissolved in anhydrous dichloromethane to prepare a 0.1M solution and placed in the modification bottle of the German K&A H-8DNA synthesizer. The sequence is input into the instrument, and a universal solid phase carrier (universal CPG) is used to fill the column at the 3' end of the sequence. After the synthesis is completed, the terminal protecting group is removed to obtain C. 1mL 50mM potassium carbonate methanol solution is added and C is cut off from CPG at room temperature for 8 hours. After deprotection, 6μL acetic acid is added to the deprotection solution, mixed, and the potassium carbonate methanol solution is removed by a rotary evaporator, and 50% ACN and H are added. 2 O, filtered with a 0.22 μm filter membrane, and purified by HPLC using a C18 column. After freeze-drying, it was reconstituted with DMSO, and the concentration at 260 nm was measured using a micro-spectrophotometer NanoDrop, and the molar amount was calculated to obtain a molecular weight of 1134 Da for the proteolytic targeting chimera D.

[0111] 6) Solid phase synthesis of protein hydrolysis targeting chimera D, the synthesis route reaction formula is as follows:

[0112]

[0113] The specific steps are as follows: under nitrogen protection, compound 4 and compound 10 are dissolved in anhydrous dichloromethane to prepare a 0.1M solution and placed in a modified bottle of a German K&AH-8DNA synthesizer. Enter the sequence on the instrument, use a 3'-phosphate CPG solid phase carrier at the 3' end of the sequence to fill the column, and after the synthesis, remove the terminal protecting group to obtain D. Add 1mL 50mM potassium carbonate methanol solution, and cut D and C from CPG at room temperature for 8 hours. After deprotection, add 6μL acetic acid to the deprotection solution, mix well, remove the potassium carbonate methanol solution with a rotary evaporator, dissolve in ultrapure water, filter with a 0.22μm filter membrane, and use a C18 chromatographic column for HPLC purification. After freeze-drying, re-dissolve with 1% DMSO, measure the concentration at 260nm with a micro-spectrophotometer Nano Drop, calculate the molar amount, and obtain a molecular weight of 1214.3Da for the proteolytic targeting chimera D.

[0114] It should be noted that in the above-mentioned solid phase synthesis of protein hydrolysis targeting chimeras A, B, C, and D by a DNA synthesizer, changing the coupling times, coupling duration, and modified monomer concentration will affect the yield of the synthesized protein hydrolysis targeting chimeras.

[0115] Example 2: Effects of proteolysis targeting chimeras containing different linkers on the inhibition of HeLa cells

[0116] The difference between the proteolysis targeting chimeras A, B, and C prepared in Example 1 is that the linkers are different, namely, phosphodiester, phosphorothioate, and phosphotriester. This example explores the effects of these three proteolysis targeting chimeras containing different linkers and MZ1 on inhibiting human cervical cancer HeLa cells. The MZ1 is a commonly used proteolysis targeting chimera targeting BRD4, whose linker is polyethylene glycol, and its structural formula is:

[0117]

[0118] The experimental steps are as follows: HeLa cells were seeded into 96-well plates, 5*10 3 / well, cultured at 37°C, 5% carbon dioxide for 12 hours, added culture medium of proteolytic targeting chimeras (A, B, C and MZ1) at concentrations of 0nM, 10nM, 50nM, 200nM, 500nM, 1000nM, 2000nM, respectively, and cultured at 37°C, 5% carbon dioxide for 48 hours. After incubation, the drug-containing culture medium was removed, and the inhibitory effect of the drug was determined after incubation for 1 hour with a culture medium containing 10% CCK-8. The analysis method of the inhibitory effect is: CCK-8 (Cell Counting kit-8) detection method, using a multifunctional microplate reader (FlexStation) to detect the absorbance at 450nm, and the cell viability (V) was calculated according to the following formula:

[0119] V=[(RA-RE) / (RB-RE)]×100%

[0120] Note: RA, RB, and RE represent the absorbance of the experimental group, blank group, and solvent background group, respectively. Fig.18 shown.

[0121] Depend on Fig.18 It can be seen that the IC of the proteolytic targeting chimera MZ1 50 The value was 385.9 nM, and the IC of proteolytic targeting chimera C 50 The value is 242.9nM. The inhibitory effect of proteolysis-targeted chimera C on human cervical cancer HeLa cells is better than that of proteolysis-targeted chimera MZ1; while proteolysis-targeted chimeras A and B have almost no inhibitory effect on human cervical cancer HeLa cells. The presumed reason is that the phosphodiester backbone of proteolysis-targeted chimera A and the thiophosphate backbone of proteolysis-targeted chimera B are both negatively charged, resulting in poor cell permeability and difficulty in entering cells to exert their effects, while the phosphotriester backbone of proteolysis-targeted chimera C and the polyethylene glycol backbone of proteolysis-targeted chimera MZ1 are both electrically neutral and have good cell permeability, so they can enter human cervical cancer HeLa cells and have a cell proliferation inhibitory effect on human cervical cancer HeLa cells.

[0122] Depend on Fig.19 It can be seen that the comparison of the HPLC chromatograms of the proteolysis-targeted chimeras MZ1 and C shows that the proteolysis-targeted chimera C is more hydrophobic than MZ1 and is easier to penetrate the cell membrane. Therefore, the IC 50 The IC values ​​of the proteolytic targeting chimera MZ1 were compared 50 The value is lower, so it has a stronger inhibitory effect on human cervical cancer HeLa cells than the proteolysis targeting chimera C.

[0123] The above experiment proves that the proteolysis targeting chimera C containing an electrically neutral phosphotriester backbone has a stronger inhibitory effect on human cervical cancer HeLa cells. The modification group of the phosphotriester in the proteolysis targeting chimera C is isobutyl. Further, the modification group of the phosphotriester in the proteolysis targeting chimera C is changed, and the modification group includes methyl, ethyl, propyl, isopropyl, etc. The effect of the proteolysis targeting chimera containing different neutral linkers on inhibiting human cervical cancer HeLa cells is detected according to the above experimental method. The experimental results prove that the proteolysis targeting chimera prepared with the above other modification groups is not as effective as the proteolysis targeting chimera C in inhibiting human cervical cancer HeLa cells.

[0124] Preferably, the linker used to prepare the proteolysis targeting chimera is isobutyl phosphotriester.

[0125] Example 3: Effect of proteolytic targeting chimera C and MZ1 on degradation of BRD4 protein in HeLa cells

[0126] The experiment in Example 2 proved that both proteolysis targeting chimera C and MZ1 had good cell proliferation inhibition effect on human cervical cancer HeLa cells, and the proteolysis targeting chimera C had better inhibition effect. The following experiment will detect the effect of proteolysis targeting chimera C and MZ1 on degrading BRD4 protein in human cervical cancer HeLa cells by Western Blotting experiment.

[0127] The specific experimental steps are as follows:

[0128] HeLa cells were seeded in 12-well plates, 1*10 5 / well, after culturing for 12 hours at 37°C and 5% carbon dioxide, cells were treated with 3.16nM, 10nM, 31.6nM, 100nM, 316nM, and 1000nM concentrations of proteolytic targeting chimera C and MZ1, respectively. After 24 hours of drug treatment, the medium was aspirated, washed twice with PBS, and 90μl RIPA lysis buffer was added. The protein was extracted on ice. The protein concentration was measured by BCA protein quantification method, and bromophenol blue was added and boiled. The sample volume per well was 20μl protein, and the electrophoresis was stopped when the bromophenol blue dye was electrophoresed to the bottom of the gel. Wet transfer was used, and the membrane was washed 3 times with TBST after transfer, each time for 10 minutes, and blocked with 5% skim milk powder for 2 hours. BRD4 (1:1000) and GAPDH (1:1000) primary antibodies were added and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST, each time for 10 min, and the corresponding secondary antibody (1:10000) was added and incubated at room temperature for 1 h, and the membrane was washed three times with TBST, each time for 10 min. The results of proteolytic targeting of chimera C and MZ1 to degrade BRD4 protein are shown in Figure 2. Fig. 20The degradation curve of BRD4 protein in human cervical cancer HeLa cells by proteolysis targeting chimera MZ1 is shown in Fig.21 The degradation curve of BRD4 protein in human cervical cancer HeLa cells by proteolysis targeting chimera C is shown in Fig. 22 shown.

[0129] Depend on Fig. 20 It can be seen that the bands of BRD4 remaining in the cells after the cells were treated with various concentrations of protein hydrolysis targeting chimeras C and MZ1 were compared with the bands of BRD4 remaining in the cells after the cells were treated with 1% DMSO in their corresponding control group. The bands of the control group in both groups were darker, indicating that the protein hydrolysis targeting chimeras C and MZ1 can effectively degrade the BRD4 protein in the cells.

[0130] Depend on Fig.21 and Fig. 22 It can be seen that proteolysis targets the DC of chimera C 50 The value was 80.16 nM, proteolytic targeting of the chimera MZ1 DC 50 The value is 104.4 nM, which is consistent with the results of Example 2, compared with the proteolysis targeting chimera MZ1.

[0131] Example 4: Detection of the dephosphorylation performance of the proteolysis-targeted chimera D

[0132] In Example 1, proteolysis targeting chimera C and proteolysis targeting chimera D were prepared. The difference between them is that proteolysis targeting chimera D will form proteolysis targeting chimera C after dephosphorylation by alkaline phosphatase. The reaction formula is

[0133]

[0134] 1. To verify whether the proteolysis targeting chimera D is dephosphorylated to obtain the proteolysis targeting chimera C, the proteolysis targeting chimera D, the proteolysis targeting chimera D treated with alkaline phosphatase and the proteolysis targeting chimera C were compared by mass spectrometry and HPLC analysis. The proteolysis targeting chimera D and the proteolysis targeting chimera C used in the following experiments were prepared according to the method described in Example 1.

[0135] The experimental method is as follows: the proteolytic targeting chimera D was treated with a buffer solution (100 mM Tris-HCl (pH = 8.0 at 37°C), 50 mM MgCl 2, 1M KCl, 0.2% Triton X-100, 10mM 2-mercaptoethanol, 1mg / mL BSA) was diluted to a final concentration of 20μM, and then 0.5U / mL ALP was added to the above solution. After mixing, the solution was incubated at 37°C for 10 minutes and then at 75°C for 5 minutes to inactivate ALP. It was filtered with a 0.22μm filter membrane and analyzed and purified using a C18 column on a LC20AR Shimadzu HPLC.

[0136] The mass spectrum of proteolysis-targeted chimera C is shown in Fig.23 The mass spectrum of the proteolysis targeting chimera D is shown in Fig.24 The mass spectrum of the proteolysis targeted chimera D after alkaline phosphatase treatment is shown in Fig.25 As shown; the liquid phase spectra obtained after HPLC treatment of the three substances are as follows Fig.26 shown.

[0137] Depend on Fig.23 , 24 , 25, the mass spectrum of the proteolysis targeted chimera D after alkaline phosphatase treatment ( Fig.25 ) and mass spectra of proteolysis targeting chimera C ( Fig.23 ), indicating that after the proteolysis targeting chimera D was incubated with alkaline phosphatase, the proteolysis targeting chimera D was dephosphorylated to form the proteolysis targeting chimera C. Fig.26 It can be seen that the peak time of proteolysis targeting chimera D is 28.5 min, the peak time of proteolysis targeting chimera C is 32.5 min, and the peak time of proteolysis targeting chimera D after alkaline phosphatase treatment is 32.5 min, indicating that after proteolysis targeting chimera D is incubated with alkaline phosphatase, proteolysis targeting chimera D is dephosphorylated to form proteolysis targeting chimera C.

[0138] 2. Detect the effect of different concentrations of alkaline phosphatase on the dephosphorylation of proteolysis targeting chimera D to form proteolysis targeting chimera C. The experimental method is as follows: proteolysis targeting chimera D is treated with a buffer solution (100 mM Tris-HCl (pH = 8.0 at 37 ° C), 50 mM MgCl 2, 1M KCl, 0.2% Triton X-100, 10mM 2-mercaptoethanol, 1mg / mL BSA) was diluted to a final concentration of 20μM, and then 2.5U / mL, 0.5U / mL, and 0.05U / mL of ALP were added to the proteolysis targeting chimera D solution, respectively, and the proteolysis targeting chimera D solution without ALP treatment was used as a control. After mixing, the solution was incubated at 37°C for 10 minutes and then incubated at 75°C for 5 minutes to inactivate ALP. Filtered with a 0.22μm filter membrane, analyzed and purified using a C18 column on a LC20AR Shimadzu HPLC. The liquid phase spectrum obtained after HPLC treatment is shown as follows Fig. 27 As shown by Fig. 27 It can be seen that with the increase of alkaline phosphatase concentration, the proportion of proteolysis targeting chimera C obtained by dephosphorylation of proteolysis targeting chimera D becomes higher. When the alkaline phosphatase concentration is 0.5U / mL, proteolysis targeting chimera D is almost completely dephosphorylated to obtain proteolysis targeting chimera C, indicating that proteolysis targeting chimera D will generate proteolysis targeting chimera C after dephosphorylation by alkaline phosphatase.

[0139] Compared with normal cells, ALP is highly expressed on the cell membrane surface of tumor and other diseased tissue cells. Therefore, the proteolysis targeting chimera D can exert its effect after dephosphorylation of ALP on the cell membrane surface of these diseased tissue cells.

[0140] 3. Verification of alkaline phosphatase inhibitor Na 3 VO 4 Whether the dephosphorylation of proteolysis-targeted chimera D can be inhibited to obtain proteolysis-targeted chimera C, the specific experimental steps are as follows: proteolysis-targeted chimera D is treated with a buffer solution (100 mM Tris-HCl (pH = 8.0 at 37 ° C), 50 mM MgCl 2 , 1M KCl, 0.2% Triton X-100, 10mM 2-mercaptoethanol, 1mg / mL BSA) to a final concentration of 20μM, and then 0.5U / mL ALP + (2mM) Na 3 VO 4 , 0.5U / mL ALP were added to the proteolysis targeting chimera D solution, and the proteolysis targeting chimera D solution without any additives was used as a control. After mixing, the solution was incubated at 37°C for 10 minutes and then at 75°C for 5 minutes to inactivate ALP. Filtered with a 0.22μm filter membrane, analyzed and purified using a C18 column on a LC20AR Shimadzu HPLC. The liquid phase spectrum obtained after HPLC treatment is shown in the figure. Fig.28 shown.

[0141] Depend on Fig.28 It can be seen that the co-addition of ALP and Na 3 VO 4 The peak time of ALP was 28.5min, which was the same as that of the control group proteolysis targeting chimera D. The peak time of ALP added to proteolysis targeting chimera D was 32.5min, indicating that ALP had converted proteolysis targeting chimera D into proteolysis targeting chimera C. The above experimental results show that the alkaline phosphatase inhibitor Na 3 VO 4 It can effectively inhibit the dephosphorylation of proteolysis targeting chimera D, and at the same time verify that the conversion of proteolysis targeting chimera D into proteolysis targeting chimera C is due to its dephosphorylation by alkaline phosphatase.

[0142] Example 5: Detection of the stability of proteolysis-targeted chimera C formed after dephosphorylation of proteolysis-targeted chimera D in plasma

[0143] In order to verify the stability of the proteolytic targeting chimera C obtained by dephosphorylation of the proteolytic targeting chimera D in plasma, the proteolytic targeting chimera C (20 μM) and MZ1 (20 μM) prepared in Example 1 were incubated at 37°C in 1640 medium supplemented with 10% fetal bovine serum, and the control group was 1640 medium supplemented with 10% fetal bovine serum, and the incubation time was 0, 6, 12, 24 and 48 hours. After the incubation was completed, it was diluted with acetonitrile, filtered with a 0.22 μm filter membrane, and HPLC analysis was performed on a C18 column on an LC20AR Shimadzu.

[0144] The HPLC peak area was measured to determine the quantitatively extracted proteolytic targeting chimera C. Fig.29 As shown in Table 2 below, the stability curve of the proteolytic targeting chimera C is as follows Fig.30 As shown, the quantitatively extracted MZ1 liquid chromatography spectrum is as follows Fig.31 As shown in Table 3 below, the stability curve of MZ1 is Fig.32 shown.

[0145] Table 2. Liquid phase analysis results of the stability of proteolytic targeting chimera C in medium containing 10% fetal bovine serum

[0146] Experimental groups Target peak area Proportion PROTAC C-0h 3456225 100％ PROTAC C-6h 3781756 109.42％ PROTAC C-12h 2550000 73.78％ PROTAC C-24h 2314369 66.96％ PROTAC C-48h 2351451 68.04％

[0147] Table 3. Liquid phase analysis results of the stability of MZ1 in a medium containing 10% fetal bovine serum

[0148] Experimental groups Target peak area Proportion MZ1-0h 5735074 100％ MZ1-6h 5536363 96.54％ MZ1-12h 6318953 110.18％ MZ1-24h 5213027 90.90％ MZ1-48h 4909312 85.60％

[0149] According to Table 2-3 and Figure 29-32Analysis showed that within the first 24 hours, the proteolysis-targeted chimera C and MZ1 showed different degrees of degradation, but the proportion of proteolysis-targeted chimera C remained stable at around 65% after 24 hours, while the proportion of MZ1 continued to decrease after 24 hours, indicating that the proteolysis-targeted chimera C has good stability in serum-containing culture medium.

[0150] Example 6: Effects of proteolysis targeting chimera D on tumor cells expressing different levels of alkaline phosphatase

[0151] This example explores the effects of the proteolysis targeting chimera D on tumor cells that highly express alkaline phosphatase and tumor cells that lowly express alkaline phosphatase, respectively. The tumor cells that highly express alkaline phosphatase are selected from human osteosarcoma Saos-2 cells, and the tumor cells that lowly express alkaline phosphatase are selected from human osteosarcoma U-2OS cells.

[0152] 1. Determination of alkaline phosphatase activity of human osteosarcoma Saos-2 cells and human osteosarcoma U-2OS cells

[0153] The assay method is as follows: Saos-2 cells and U-2OS cells were inoculated in 6-well plates, 3*10 5 Cells / well were cultured at 37°C and 5% carbon dioxide for 24 hours, and then rinsed with Tris-HCl buffer (pH = 7.4). 200 μl RIPA lysis buffer (without enzyme inhibitors) was added, and the cells were lysed on ice to extract protein. The protein concentration was determined by BCA protein quantification method. Using the Bio-Tech Alkaline Phosphatase Detection Kit, blank control wells, standard wells and sample wells were set, and 10 μg of protein was used for detection. After incubation at 37°C for 30 minutes, the reaction stop solution was added, and the absorbance at 405 nm was measured. The results were as follows: Fig.33 shown.

[0154] Depend on Fig.33 It can be seen that under 37°C conditions, the amount of p-nitrophenol produced per minute per mg of protein in Saos-2 cells and U-2OS cells was 3.217 μmol and 0.117 μmol, respectively, indicating that alkaline phosphatase is highly expressed in Saos-2 cells and lowly expressed in U-2OS cells, and the alkaline phosphatase content in Saos-2 cells is more than 25 times higher than that in U-2OS cells.

[0155] 2. Inhibitory effect of proteolysis-targeting chimera D on Saos-2 cells and U-2OS cells

[0156] The protein hydrolysis targeting chimera D prepared in Example 1 was added as an additive to Saos-2 cells and U-2OS cells to detect its cell inhibitory effect, and the protein hydrolysis targeting chimera C prepared in Example 1 without modification of enzyme activatable groups was used as a comparison.

[0157] The inhibitory effects of proteolysis targeting chimera D and proteolysis targeting chimera C on Saos-2 and U-2OS cells were detected as follows: Saos-2 cells and U-2OS cells were inoculated into 96-well plates, 5*10 3 / well, cultured at 37°C, 5% carbon dioxide for 12 hours, added 0nM, 200nM, 500nM, 1000nM, 2000nM, 4000nM, 8000nM concentrations of proteolysis targeting chimera D and proteolysis targeting chimera C culture medium to the above two cells, respectively, and cultured at 37°C, 5% carbon dioxide for 48 hours. After the incubation, the drug-containing culture medium was removed, and the inhibitory effect of the drug was determined after incubation for 1 hour with a culture medium containing 10% CCK-8. The analysis method of the inhibitory effect is: CCK-8 (CellCounting kit-8) detection method, using a multifunctional microplate reader (FlexStation) to detect the absorbance at 450nm, and the cell viability (V) was calculated according to the following formula:

[0158] V=[(RA-RE) / (RB-RE)]×100%

[0159] Note: RA, RB and RE in the formula represent the absorbance of the experimental group, blank group and solvent background group respectively.

[0160] The inhibitory effects of proteolysis targeting chimera D and proteolysis targeting chimera C on Saos-2 cells are shown in Figure 2 Fig.34 As shown, the inhibitory effect on U-2OS cells is as follows Fig.35 shown.

[0161] Depend on Figure 34-35 It can be seen that in human osteosarcoma Saos-2 cells with high expression of alkaline phosphatase, the IC 50 The value is 1364nM, and the IC of proteolytic targeting chimera C 50 The value was 931.2nM, indicating that the proteolysis-targeted chimeras D and C had a good inhibitory effect on the proliferation of human osteosarcoma Saos-2 cells; however, in human osteosarcoma U-2OS cells with low alkaline phosphatase expression, the proteolysis-targeted chimera D could not detect the IC 50 Values, IC of proteolysis targeting chimera C 50The value is 3128nM, indicating that only proteolysis targeting chimera C has an inhibitory effect on human osteosarcoma U-2OS cells, while proteolysis targeting chimera D does not. The above experimental results show that proteolysis targeting chimera D is selective for cells and can only inhibit the growth of Saos-2 cells that highly express alkaline phosphatase.

[0162] Proteolysis targeting chimera D can only degrade BRD4 protein to inhibit cell proliferation when it is dephosphorylated to form proteolysis targeting chimera C in human osteosarcoma Saos-2 cells that highly express alkaline phosphatase; in human osteosarcoma U-2OS cells, the expression of alkaline phosphatase is extremely low, and the amount of proteolysis targeting chimera D dephosphorylated to form proteolysis targeting chimera C is very small and negligible, so proteolysis targeting chimera D cannot play a role in osteosarcoma U-2OS cells. Proteolysis targeting chimera C does not have a modified enzyme activatable group and can directly degrade BRD4 protein to inhibit cell proliferation. Therefore, proteolysis targeting chimera C has no selectivity in its inhibitory effect on cells, and has an inhibitory effect on both Saos-2 cells with high expression of alkaline phosphatase and U-2OS cells with low expression of alkaline phosphatase.

[0163] 3. Effect of proteolysis targeting chimera D on degradation of BRD4 protein in Saos-2 cells and U-2OS cells

[0164] The above 2 proves that the proteolysis-targeted chimera D is selective for cells, and only dephosphorylates and then degrades BRD4 protein in Saos-2 cells that highly express alkaline phosphatase to inhibit cell proliferation. This experiment further detected the effect of proteolysis-targeted chimera D in degrading BRD4 protein in Saos-2 cells and U-2OS cells by Western Blotting experiments, using proteolysis-targeted chimera C as a comparison.

[0165] The specific experimental steps are as follows: Saos-2 cells and U-2OS cells were inoculated in 12-well plates, 1*10 5 / well, after culturing for 12 hours at 37°C and 5% carbon dioxide, the above two cells were treated with 3.16nM, 10nM, 31.6nM, 100nM, 316nM, and 1000nM concentrations of proteolysis targeting chimera D and proteolysis targeting chimera C, respectively. After 24 hours of drug treatment, the culture medium was aspirated, washed twice with PBS, and 90μl RIPA lysis buffer was added. The protein was extracted on ice. The protein concentration was measured by BCA protein quantification method, and bromophenol blue was added and boiled. The sample volume per well was 20μl protein, and the electrophoresis was stopped when the bromophenol blue dye was electrophoresed to the bottom of the gel. The wet transfer was used, and the membrane was washed 3 times with TBST after transfer, each time for 10 minutes, and blocked with 5% skim milk powder for 2 hours. BRD4 (1:1000) and GAPDH (1:1000) primary antibodies were added and incubated overnight at 4°C. The next day, the membrane was washed 3 times with TBST, each time for 10 min, and the corresponding secondary antibody (1:10000) was added for incubation at room temperature for 1 h, and the membrane was washed 3 times with TBST, each time for 10 min. The Western Blotting results of protein hydrolysis targeting chimera D and protein hydrolysis targeting chimera C degrading BRD4 protein of Saos-2 cells are shown in Figure 2. Fig.36 The degradation curve is shown in Figure 37-38 As shown; the protein hydrolysis targeting chimera D and the protein hydrolysis targeting chimera C degraded the BRD4 protein of U-2OS cells. The Western Blotting results are shown Fig.39 The degradation curve is shown in Figure 40-41 shown.

[0166] Depend on Figure 36-38 It can be seen that the results of Western Blotting showed that the remaining BRD4 protein bands in the two groups of human osteosarcoma Saos-2 cells after being treated with 3.16nM, 10nM, 31.6nM, 100nM, 316nM, and 1000nM concentrations of proteolysis targeting chimera D and proteolysis targeting chimera C had no significant difference at different concentrations. As the concentration of proteolysis targeting chimera increased, the remaining BRD4 protein content decreased, and the DC of proteolysis targeting chimera D 50 The value was 886.7 nM, proteolytic targeting of chimera C DC 50 The value was 303 nM, indicating that both proteolysis targeting chimera D and proteolysis targeting chimera C had a good degradation effect on BRD4 protein in human osteosarcoma Saos-2 cells.

[0167] Depend on Figure 39-41It can be seen that the Western Blotting results showed that the remaining BRD4 protein bands in the two groups of human osteosarcoma U-2OS cells after being treated with different concentrations of proteolysis targeting chimera D and proteolysis targeting chimera C were significantly different at different concentrations. The amount of BRD4 protein remaining in human osteosarcoma U-2OS cells treated with different concentrations of proteolysis targeting chimera D was reduced very little with the increase of proteolysis targeting chimera D concentration, which was almost negligible, and proteolysis targeting chimera D failed to detect DC 50 The values ​​indicated that proteolysis targeting chimera D had no significant degradation effect on BRD4 protein in human osteosarcoma U-2OS cells. However, the remaining BRD4 protein in human osteosarcoma U-2OS cells treated with different concentrations of proteolysis targeting chimera C decreased significantly with the increase of proteolysis targeting chimera C concentration, and the DC of proteolysis targeting chimera C 50 The value was 261.7 nM, indicating that the proteolysis targeting chimera C had a significant degradation effect on the BRD4 protein in human osteosarcoma U-2OS cells.

[0168] Through the above experiments and analysis, it is proved that the proteolysis targeting chimera D selects to be dephosphorylated in Saos-2 cells with high expression of alkaline phosphatase to achieve efficient degradation of BRD4 protein and achieve anti-tumor effect in human osteosarcoma Saos-2 cells and human osteosarcoma U-2OS cells with significant differences in the expression of two alkaline phosphatases. It shows that the proteolysis targeting chimera provided by the present invention can modify an enzyme activatable group on the hydroxyl group of the E3 ubiquitin ligase ligand, effectively preventing the ability of the E3 ubiquitin ligase ligand to recruit E3 ubiquitin ligase to degrade protein. Only when the corresponding enzyme of the enzyme activatable group removes the enzyme activatable group can the proteolysis targeting chimera be released to exert its effect, thereby achieving the proteolysis targeting chimera to select tumor cells before degrading the target protein.

[0169] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A method for preparing a proteolysis-targeted chimera, characterized in that: Prepared by automated solid phase DNA synthesis method, comprising the following steps: (1) preparing and purifying E3 ubiquitin ligase ligand monomers containing phosphoramidite structures; (2) preparing target protein ligand monomers containing phosphoramidite structures and purifying them; (3) synthesizing an E3 ubiquitin ligase ligand monomer containing a phosphoramidite structure and a target protein ligand monomer containing a phosphoramidite structure on a solid phase carrier by means of a DNA synthesizer, and obtaining a protein hydrolysis targeting chimera after deprotection and purification.

2. The preparation method according to claim 1, characterized in that The purification methods in steps (1) and (2) are both purification by chromatography column, and the eluent of the chromatography column includes triethylamine.

3. The preparation method according to claim 2, characterized in that: The solid phase carrier in step (3) includes a universal solid phase carrier or a solid phase carrier containing an enzyme activatable group, and the enzyme activatable group includes one or more of a phosphate group and a nitro group.

4. A proteolysis targeting chimera, characterized in that Prepared by solid phase synthesis, the proteolysis targeting chimera has the following structural formula: Wherein, L is a linker, the left side of L is a targeting protein ligand, the right side of L is an E3 ubiquitin ligase ligand, and R is a substituent.

5. The proteolysis targeting chimera according to claim 4, characterized in that The linker includes one or more of phosphodiester, phosphorothioate, and phosphotriester.

6. The proteolysis targeting chimera according to claim 5, characterized in that The linker is a phosphotriester.

7. The proteolysis targeting chimera according to claim 4, characterized in that The substituents include one or more of a hydroxyl group, a phosphate group, and a nitro group.

8. The proteolysis targeting chimera according to claim 5, characterized in that The proteolysis targeting chimera includes any one or more of the following structural formulas A, B, C, and D:

9. The proteolysis targeting chimera according to claim 8, characterized in that The proteolysis targeting chimera includes any one or more of the C and D structural formulas.

10. Use of a proteolysis targeting chimera for preparing an anti-tumor drug, characterized in that: The proteolysis targeting chimera comprises a targeting protein ligand, a linker and an E3 ubiquitin ligase ligand, wherein the linker comprises one or more of phosphodiester, phosphorothioate and phosphotriester, and the E3 ubiquitin ligase ligand can modify an enzyme activatable group.

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