An intracellular self-assembly degradation agent based on bioorthogonal reaction and its preparation method and application

Abstract

The present invention discloses a kind of intracellular self-assembly degradation agent based on bioorthogonal reaction and its preparation method and application, by using linker to connect target protein ligand Linifanib or S5 and bioorthogonal group norbornene, obtain target protein ligand with norbornene group; by connecting chain, tetrazine group is modified on E3 ubiquitin ligase ligand, obtain E3 ubiquitin ligase ligand with tetrazine; by step-by-step administration method, target protein ligand molecule with norbornene group and E3 ubiquitin ligase ligand with tetrazine are successively entered into cell, bioorthogonal reaction self-assembly occurs in cell, and protein degradation agent is formed. The preparation method of intracellular self-assembly protein degradation agent constructed by the present invention is simple, easy to realize, and has high yield, can be used in the preparation of medicine for treating cancer, especially in the preparation of anti-tumor drug with PDGFR-β as target.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and relates to an intracellular self-assembly degradation agent based on bioorthogonal reaction, and a preparation method and application thereof. Background Art

[0002] Linifanib is a novel receptor tyrosine kinase (RTK) inhibitor. It is a potent inhibitor of members of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor families, with IC values ​​of KDR, Flt-1, PDGFRβ, and FLT3. 50 The values ​​were 3, 4, 66, and 4 nM, respectively. In vivo experiments showed that Linifanib showed IC 50 The values ​​range from 4 nM (KDR) to 190 nM (FLT4), but the activity against unrelated RTKs such as soluble tyrosine kinases or serine / threonine kinases is poor.

[0003] Proteolysis Targeting Chimera (PROTAC) is a bifunctional molecule that can bind to the target protein and E3 ubiquitin ligase at the same time. By binding to the target protein and E3 ubiquitin ligase at the same time, the distance between the target protein and the E3 ligase is shortened, thereby inducing ubiquitination of the target protein. The ubiquitinated target protein can be recognized and degraded by the 26S proteasome, achieving the purpose of completely eliminating disease-related proteins. Compared with small molecule inhibitors, PROTAC has the advantages of low dosage and low resistance to drug resistance, so it is booming in the field of new drug research and development. However, the inherent characteristics of protein degradation targeting chimeras - large molecular weight leads to poor physical and chemical properties and cell permeability, which limits their further development, so there is an urgent need to optimize their pharmacokinetic properties. Summary of the invention

[0004] The purpose of the present invention is to provide an intracellular self-assembly degradation agent based on bioorthogonal reaction and its preparation method and application. The target protein ligand molecule with a bioorthogonal group (norbornene) and the E3 ubiquitin ligase ligand molecule with a bioorthogonal group (tetrazine) enter the cell successively, and the bioorthogonal reaction self-assembles to form a protein degradation targeting chimera in the cell, and finally the target protein is degraded by the ubiquitin-proteasome system. The target recognition molecule constructed by the present invention can self-assemble to form a protein degradation targeting chimera, and the protein degradation targeting chimera formed by self-assembly has the function of inducing PDGFR-β protein degradation, and can be used to prepare anti-tumor drugs.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] An intracellular self-assembly degrader based on bioorthogonal reaction, the structural formula of the degrader is as follows:

[0007]

[0008] Wherein X=1~6, Y=2~10, R1=CH3 or Cl, R2=F or H, R3=CH3 or H, R4=Ph or CH2.

[0009] A method for preparing the intracellular self-assembly degradation agent based on bioorthogonal reaction as described above comprises the following steps:

[0010] The target protein ligand Linifanib and norbornene are connected by using a linker, or the target protein ligand S5 and norbornene are connected by using a linker to obtain a target protein ligand with a norbornene group; wherein the target protein ligand S5 has the following structural formula:

[0011]

[0012] The tetrazine group is modified on the E3 ubiquitin ligase ligand through a connecting chain to obtain the E3 ubiquitin ligase ligand with tetrazine;

[0013] The target protein ligand molecule with norbornene group and the E3 ubiquitin ligase ligand with tetrazine are successively introduced into the cell, and bio-orthogonal reaction self-assembly occurs in the cell to form an intracellular self-assembly degrader based on bio-orthogonal reaction.

[0014] Furthermore, the structural formula of the target protein ligand with a norbornene group is as follows:

[0015]

[0016] Among them, X=1~6, R1=CH3 or Cl, R2=F or H.

[0017] Furthermore, the target protein ligand with a norbornene group is prepared by the following process:

[0018] The target protein ligand Linifanib or the target protein ligand S5 is subjected to an amide condensation reaction with a linker to obtain a compound with a Boc protective group, and then the Boc protective group is removed by the action of hydrogen chloride in ethyl acetate, and then the compound is subjected to an amide condensation reaction with 5-norbornene-2-carboxylic acid to obtain a target protein ligand with a norbornene group.

[0019] Furthermore, the structural formula of the tetrazine-containing E3 ubiquitin ligase ligand is as follows:

[0020]

[0021] Among them, Y=2~10, R3=CH3 or H, R4=Ph or CH2.

[0022] Furthermore, the tetrazine-containing E3 ubiquitin ligase ligand is prepared by the following process:

[0023] Acetonitrile, a cyano compound and hydrazine hydrate are reacted under the action of zinc trifluoromethanesulfonate or nickel trifluoromethanesulfonate, or formamidine acetate, a cyano compound and hydrazine hydrate are reacted under the action of zinc trifluoromethanesulfonate, and then oxidative dehydrogenation is performed to form a 1,2,4,5-tetrazine compound, and then the Boc protecting group is removed under the action of trifluoroacetic acid, and then the compound is connected with an E3 ubiquitin ligase ligand VH032 through a diacid to form an E3 ubiquitin ligase ligand with tetrazine.

[0024] Furthermore, the protein degradation agent is specifically prepared by the following process:

[0025] A target protein ligand solution with a norbornene group is added to a culture dish containing cells for incubation, and then an E3 ubiquitin ligase ligand with tetrazine is added and incubated to form a protein degrader through self-assembly in the cell.

[0026] An application of the above-mentioned intracellular self-assembly degradation agent based on bioorthogonal reaction in the preparation of anti-tumor drugs.

[0027] Furthermore, the anti-tumor drug is a drug that can selectively induce the degradation of PDGFR-β protein.

[0028] Furthermore, the anti-tumor drug is an anti-glioma drug.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention constructs a class of targeted recognition molecules with bioorthogonal groups, and enables them to self-assemble in cells to form protein degraders through step-by-step administration. Compared with the overall protein degradation targeting chimera, the intracellular self-assembling protein degrader constructed by the present invention can not only utilize the mechanism of action of the protein degrader to degrade disease-related proteins, but also reduce the molecular weight of the compound in a self-assembly manner, solve the problem of large molecular weight of the overall protein degrader, increase cell permeability, optimize its physicochemical properties, and enhance the effect. The preparation method of the intracellular self-assembly protein degrader constructed by the present invention is simple, easy to implement, and has a high yield.

[0031] The small molecule protein degradation targeted degradation agent of the present invention can be used in the preparation of anti-tumor drugs, especially in the preparation of anti-tumor drugs targeting PDGFR-β. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1The high performance liquid chromatogram of the bioorthogonal reaction process (10-240min) between the target recognition molecule LN and TzB constructed in the present invention;

[0033] Figure 2 The high performance liquid chromatogram of the bioorthogonal reaction process (10-240min) of the targeting recognition molecule S5N and TzB constructed in the present invention;

[0034] Figure 3 The protein degradation effect of the protein degrading agent formed by self-assembly of the targeting recognition molecule LN and TzB constructed by the present invention on U87 cells was investigated; wherein A is LN and B is TzB.

[0035] Figure 4 The protein degradation effect of the protein degradation agent formed by the self-assembly of the target recognition molecule S5N and TzB constructed by the present invention on U87 cells was investigated. Wherein, A is S5N and B is TzB. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0037] Bioorthogonal reactions refer to a type of chemical reaction that can occur in living cells or tissues and can be carried out without interfering with the biochemical reactions of the organism itself. They are simple, efficient, and highly specific. Studies have shown that tetrazines can react quickly and efficiently with cyclic alkenes or alkynes to generate stable products without the need for a catalyst, and this reaction is harmless to cells. Therefore, it is proposed to combine protein degradation targeted chimeric technology with bioorthogonal reactions to construct compound molecules with bioorthogonal groups that target target proteins and E3 ubiquitin ligases, respectively. The two parts enter the cells one after another, undergo bioorthogonal reactions and self-assemble in the cells to form protein degraders, thereby playing a role in degrading target proteins. Based on this strategy, the molecular weight of PROTAC can be reduced, its pharmacokinetic properties can be optimized, and cell permeability can be increased, in order to improve its effect.

[0038] The present invention obtains a target protein ligand molecule with a bio-orthogonal group norbornene by connecting a target protein ligand Linifanib or S5 with a bio-orthogonal group norbornene by using Linkers of different lengths; and obtains an E3 ubiquitin ligase ligand with a bio-orthogonal group tetrazine by modifying a tetrazine group on an E3 ubiquitin ligase ligand through different types of connecting chains. Through a step-by-step administration method, the two can enter the cell one after another, and a bio-orthogonal reaction occurs in the cell to self-assemble to form a protein degrader, thereby achieving the purpose of reducing the molecular weight, increasing cell permeability, and optimizing the inherent defects of traditional protein degraders. The protein degrader formed by self-assembly in the cell in the present invention can be used in the treatment of cancer. The protein degradation targeting chimera (PROTACs) of the present invention, i.e., a degrader, can selectively induce the degradation of PDGFR-β protein.

[0039] Among them, S5 is a candidate compound with anti-angiogenic activity constructed in the early stage of the laboratory. Previous studies have shown that it has VEGFR-2 inhibitory activity comparable to sorafenib. The structural formula of S5 is as follows:

[0040]

[0041] The invention provides a self-assembling protein degradation agent with anti-tumor activity. The protein degradation agent has anti-tumor activity in vitro and can be applied to the preparation of anti-tumor drugs.

[0042] The anti-tumor drug is an anti-glioma drug.

[0043] Intracellular self-assembling protein degraders, including target protein ligand molecules with bioorthogonal groups (norbornene) and E3 ubiquitin ligase ligand molecules with bioorthogonal groups (tetrazine);

[0044] The target protein ligand molecule with a bioorthogonal group (norbornene) has the following structural formula:

[0045]

[0046] Among them, X=1~6, R1=CH3 / Cl, R2=F / H.

[0047] The E3 ubiquitin ligase ligand molecule with a bioorthogonal group (tetrazine) has the following structural formula:

[0048]

[0049] Among them, Y=2~10, R3=CH3 / H, R4=Ph / CH2.

[0050] The method for preparing a compound that self-assembles in cells to form a protein degrading agent comprises the following synthesis steps:

[0051] 1) The preparation method of the target protein ligand molecule with a bioorthogonal group (norbornene) is as follows:

[0052] The anti-tumor active molecule Linifanib or S5 is reacted with linkers of different lengths (such as Boc-protected aminobutyric acid) through an amide condensation reaction, and column chromatography purification is performed to obtain a class of compounds with Boc protective groups. The Boc protective groups are then removed through the action of hydrogen chloride and ethyl acetate to expose the active reactive group amino group, and then the target protein ligand molecules with bioorthogonal groups (norbornene) are obtained through an amide condensation reaction with 5-norbornene-2-carboxylic acid.

[0053] 2) The preparation method of the E3 ubiquitin ligase ligand molecule with a bioorthogonal group (tetrazine) is as follows:

[0054] Acetonitrile or formamidine acetate, different types of cyano compounds and hydrazine hydrate are subjected to cyclization reaction and oxidative dehydrogenation under the action of catalyst zinc trifluoromethanesulfonate or nickel trifluoromethanesulfonate to form 1,2,4,5-tetrazine compounds with different substituents, and then the Boc protecting group is removed under the action of trifluoroacetic acid to expose the amino active group, which is then connected with the E3 ubiquitin ligase ligand VH032 through diacids of different lengths to form the E3 ubiquitin ligase ligand molecule VH032 with a bioorthogonal group (tetrazine).

[0055] After adding a fixed concentration or different concentrations of a target protein ligand molecule solution with a bioorthogonal group (norbornene) to a tumor cell culture dish and incubating for 2 hours, different concentrations or a fixed concentration of an E3 ubiquitin ligase ligand molecule with a bioorthogonal group (tetrazine) is added and incubated in a constant temperature incubator at 37°C and 5% CO2 for 48 hours. The protein degrader formed by self-assembly in the cell is characterized in that its general structural formula is as follows:

[0056]

[0057] Where X = 1-6, Y = 2-10, R1 = CH3 / Cl, R2 = F / H, R3 = CH3 / H, R4 = Ph / CH2

[0058] The following is a detailed description of the preparation and activity screening methods of a type of targeted recognition molecules for intracellular self-assembly to form protein degraders provided by the present invention in conjunction with the synthetic route shown in the figure and specific synthetic examples.

[0059] Example 1

[0060] A method for preparing a target protein ligand molecule LN with a norbornene group comprises the following synthesis steps:

[0061]

[0062] 1) 48.5 mmol of γ-aminobutyric acid was dissolved in 80 mL of tetrahydrofuran and placed in an ice-water bath, 80 mL of 1 mol / L sodium hydroxide solution was added, and then 53.3 mmol of di-tert-butyl dicarbonate in tetrahydrofuran solution was added dropwise, and stirred at room temperature. The reaction progress was detected by ninhydrin. After the reaction was completed, the volatile solvent was removed under reduced pressure, and the pH was adjusted to 2-3 with 1M HCl, extracted with ethyl acetate, and the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain a light yellow compound 1, the structural formula of which is as follows, LC-MS (ESI, m / z): 204.30 [M+H] + , 202.10[M+H] + .

[0063]

[0064] 2) 0.80 mmol of compound 1 and 0.60 mmol of HATU were dissolved in dry dichloromethane, and 1.60 mmol of DIPEA was added dropwise under ice bath conditions, and the mixture was stirred at room temperature for 15 min. 0.40 mmol of Linifanib was added, and the mixture was stirred at room temperature overnight (overnight in the present invention is 12 h), and water was added. The mixture was extracted with dichloromethane, and the organic phase was washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the desiccant was removed by suction. The solvent was removed by vacuum rotation, and a transparent oily substance was obtained by column chromatography, namely compound 2 (0.22 g), with the following structural formula and a yield of 98.30%, LC-MS (ESI, m / z): 561.25 [M+Na] + , 559.15[MH] - .

[0065]

[0066] 3) 0.39 mmol of compound 2 was dissolved in 2 mol / L hydrogen chloride ethyl acetate solution, stirred at room temperature overnight, and the filter cake (white solid) obtained by suction filtration was compound 3 (0.18 g), with the following structural formula, and the yield was 99.61%, LC-MS (ESI, m / z): 461.15 [M+H] + , 459.15[MH] - .

[0067]

[0068] 4) 0.47mmol 5-norbornene-2-carboxylic acid and 0.70mmol HATU were dissolved in dry dichloromethane, 1.86mmol DIPEA was added dropwise under ice bath conditions, stirred for 10min, 0.47mmol compound 3 was added, and stirred at room temperature for 8h. After the reaction was completed, the organic phase was removed by rotation, an appropriate amount of water was added, extracted with ethyl acetate (3×), washed with saturated sodium chloride, and dried over anhydrous Na2SO4. The desiccant was removed by suction filtration, the solvent was removed by rotation under reduced pressure, and a white product was obtained by column chromatography, i.e., the target protein ligand molecule LN (90mg) with a norbornene group, the structural formula of which is as follows, and the yield is 33.33%, LC-MS (ESI, m / z): 581.25 [M+H] + , 579.15[MH] - .

[0069]

[0070] Example 2

[0071] A method for preparing a target protein ligand molecule S5N with a norbornene group comprises the following synthesis steps:

[0072]

[0073] 1) 48.5 mmol of γ-aminobutyric acid was dissolved in 80 mL of tetrahydrofuran and placed in an ice-water bath, 80 mL of 1 mol / L sodium hydroxide solution was added, and then 53.3 mmol of di-tert-butyl dicarbonate in tetrahydrofuran solution was added dropwise, and stirred at room temperature. The reaction progress was detected by ninhydrin. After the reaction was completed, the volatile solvent was removed under reduced pressure, and the pH was adjusted to 2-3 with 1M HCl, extracted with ethyl acetate, and the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain a light yellow compound 1, the structural formula of which is as follows, LC-MS (ESI, m / z): 204.30 [M+H] + , 202.10[M+H] + .

[0074]

[0075] 2) 0.794 mmol of compound 1 and 0.794 mmol of HATU were dissolved in dry dichloromethane. Under ice bath conditions, 1.588 mmol of DIPEA was added dropwise, and the mixture was stirred at room temperature for 15 min. 0.397 mmol of S5 was added, and the mixture was stirred at room temperature overnight. Water was added, and the mixture was extracted with dichloromethane. The organic phase was washed with saturated sodium chloride, and dried over anhydrous Na2SO4. The desiccant was removed by suction filtration, and the solvent was removed under reduced pressure. A transparent oily substance was obtained by column chromatography, i.e., compound 4 (0.84 g). The structural formula is as follows. The yield is 98.65%. LC-MS (ESI, m / z): 585.25 [M+Na] + , 561.15[MH] - .

[0076]

[0077] 3) 0.391 mmol of compound 4 was dissolved in 2 mol / L hydrogen chloride ethyl acetate solution, stirred at room temperature overnight, and the filter cake (white solid) obtained by suction filtration was compound 5 (0.18 g), with the following structural formula, and the yield was 99.44%, LC-MS (ESI, m / z): 463.10 [M+H] + , 461.05[MH] - .

[0078]

[0079] 4) 0.389mmol 5-norbornene-2-carboxylic acid and 0.584mmol HATU were dissolved in dry dichloromethane, 1.558mmol DIPEA was added dropwise under ice bath conditions, stirred for 10min, 0.389mmol compound 5 was added, and stirred at room temperature for 8h. After the reaction was completed, the organic phase was removed by rotation, an appropriate amount of water was added, extracted with ethyl acetate (3×), washed with saturated sodium chloride, and dried over anhydrous Na2SO4. The desiccant was removed by suction filtration, the solvent was removed by rotation under reduced pressure, and a white product was obtained by column chromatography, i.e., the target protein ligand molecule S5N (94mg) with a norbornene group, the structural formula of which is as follows, and the yield is 41.46%, LC-MS (ESI, m / z): 583.15 [M+H] + , 581.10[MH] - .

[0080]

[0081] The target protein ligand L7N (X=4) with the bioorthogonal group norbornene can be prepared by replacing the γ-aminobutyric acid in Example 1 with 7-aminoheptanoic acid, which has the following structural formula:

[0082]

[0083] The target protein ligand L9N (X=6) with the bioorthogonal group norbornene can be prepared by replacing the γ-aminobutyric acid in Example 1 with 9-aminononanoic acid, which has the following structural formula:

[0084]

[0085] Example 3

[0086] A method for preparing an E3 ubiquitin ligase ligand molecule with a bioorthogonal tetrazine group comprises the following synthetic steps:

[0087]

[0088] 1) Under nitrogen protection, 25mmol acetonitrile, 2.5mmol (4-cyanobenzyl) carbamic acid tert-butyl ester, 1.25mmol zinc trifluoromethanesulfonate and 125mmol (6.067mL) of 80% hydrazine hydrate (mass fraction) were mixed and reacted at 60°C for 36h. The reaction solution was cooled to room temperature, 50mmol sodium nitrite solution (3.45g sodium nitrite dissolved in 20mL of water) was added to the reaction solution, and then 1mol / L hydrochloric acid was slowly added dropwise until no bubbles were generated and the pH was 3. Ethyl acetate was extracted twice, and the organic phases were combined and dried over anhydrous Na2SO4. The desiccant was removed by suction filtration, and a purple-red powder, i.e., compound 6a (0.11g), was obtained by column chromatography separation. The structure is shown below, and the yield is 14.67%, LC-MS (ESI, m / z): 302.40 [M+H] + .

[0089]

[0090] 2) Under nitrogen protection, 25mmol of formamidine acetate, 2.5mmol of (4-cyanobenzyl) tert-butyl carbamate, 1.25mmol of zinc trifluoromethanesulfonate and 125mmol (6.067mL) of 80% hydrazine hydrate (mass fraction) were mixed and reacted at 30°C for 24h. The reaction solution was cooled to room temperature, 50mmol of sodium nitrite solution (3.45g of sodium nitrite dissolved in 20mL of water) was added to the reaction solution, and then 1mol / L hydrochloric acid was slowly added dropwise until no bubbles were generated and the pH was 3. The organic phases were extracted twice with ethyl acetate, and the organic phases were combined and dried over anhydrous Na2SO4. The desiccant was removed by suction filtration, and a red powder, i.e., compound 6b (0.2g), was obtained by column chromatography. The structure is shown below, and the yield is 27.78%, LC-MS (ESI, m / z): 287.25 [M+H] + .

[0091]

[0092] 3) Under nitrogen protection, 40mmol acetonitrile, 4mmol N-(tert-butoxycarbonyl)-2-aminoacetonitrile, 2mmol nickel trifluoromethanesulfonate and 200mmol (9.70mL) of 80% hydrazine hydrate (mass fraction) were mixed and reacted at 60°C for 24h. The reaction solution was cooled to room temperature, 80mmol sodium nitrite solution (5.52g sodium nitrite dissolved in 30mL water) was added to the reaction solution, and then 1mol / L hydrochloric acid was slowly added dropwise until no bubbles were generated and the pH was 3. Filtered, the filtrate was extracted with ethyl acetate three times, the organic phases were combined, and dried over anhydrous Na2SO4. The desiccant was removed by suction filtration, and a red powder, i.e., compound 6c (0.12g), was obtained by column chromatography separation. The structure is shown below, and the yield is 6.67%, LC-MS (ESI, m / z): 126.00 [M+H] + .

[0093]

[0094] 4) Compound 6a (1 eq) or compound 6b (1 eq) was dissolved in 4 mL of dry dichloromethane, 500 μL of trifluoroacetic acid was added dropwise under ice bath conditions, stirred at room temperature for 2 h, and directly spin dried for the next step of reaction. The spin dried product and succinic anhydride (1 eq) were mixed in a dichloromethane solution, triethylamine (3 eq) was added dropwise under ice bath conditions, and the reaction was carried out at room temperature overnight. After the reaction was completed, dichloromethane was removed by vacuum rotary evaporation, and a purple powder, i.e., compound 8a or a red powder, i.e., compound 8b, was separated by column chromatography, 8a (0.12 g), yield 93.02%, 8b (0.20 g), yield 64.62%. LC-MS (ESI, m / z) 8a: 302.30 [M+H] + , 300.25[MH] - 8b:286.20[MH] - The structural formula is as follows:

[0095]

[0096] When R3 is CH3, compound 8a is obtained; when R3 is H, compound 8b is obtained.

[0097] 5) Compound 6c (1 eq) was dissolved in dry dichloromethane, 500 μL trifluoroacetic acid was added dropwise under ice bath conditions, stirred at room temperature for 2 h, the organic phase was spin-dried, a small amount of water was added, and the pH was adjusted to 7 with a saturated sodium bicarbonate solution, extracted with dichloromethane, and directly spin-dried for the next step of reaction. The spin-dried product was mixed with succinic anhydride (1 eq), pimelic acid (1 eq), and dodecanedioic acid (1 eq) in a dichloromethane solution, wherein 1.5 eq HATU was added to the pimelic acid and dodecanedioic acid reaction system, and DIPEA (4 eq) was added dropwise under ice bath conditions, and the reaction was allowed to react overnight at room temperature. After the reaction was completed, dichloromethane was removed by vacuum rotary evaporation, and red powders were obtained by column chromatography, namely compound 8c (0.08 g), compound 8d (0.12 g), and compound 8e (0.08 g), with yields of 63.49%, 37.52%, and 24.92%, respectively. LC-MS (ESI, m / z) 8c: 224.00 [MH] - . 8d:268.30[MH] - 8e:338.35[M+H] + , 336.25[MH] - The structural formula is as follows:

[0098]

[0099] Among them, when Y=2, compound 8c is obtained; when Y=5, compound 8d is obtained; when Y=10, compound 8e is obtained.

[0100] 6) Compounds 8a-8e (1 eq) and HATU (1.5 eq) were dissolved in dry dichloromethane. DIPEA (4 eq) was added dropwise under ice bath conditions. The mixture was stirred for 10 min. (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH032) (1 eq) was added. The mixture was stirred at room temperature. After the reaction was completed, the organic phase was removed under reduced pressure. An appropriate amount of water was added. The mixture was extracted with dichloromethane (3×) and dried over anhydrous Na2SO4. The desiccant was removed by suction filtration. The solvent was removed under reduced pressure. The red products TzA (0.08 g), TzB (0.015 g), TzC (0.06 g), TzD (0.10 g), and TzE (0.10 g) were separated by column chromatography. The yields were 48.19%, 9.38%, 40.55%, 57.47% and 57.47% respectively. LC-MS (ESI, m / z) TzA: 714.60 [M+H] + , 712.50[MH] - . TzB:698.55[MH] - . TzC:660.40[M+Na]+ , 636.35[MH] - . TzD:680.40[M+H] + , 678.50[MH] - .TzE:750.50[M+Na] + , 748.45[MH] - The structure is as follows:

[0101]

[0102] Wherein R3=CH3, R4=-Ph-, Y=2, and the product TzA is obtained;

[0103] R3=H, R4=-Ph-, Y=2, and the product TzB is obtained;

[0104] R3=CH3, R4=-CH2-, Y=2, and the product TzC is obtained;

[0105] R3=CH3, R4=-CH2-, Y=5, and the product TzD is obtained;

[0106] R3=CH3, R4=-CH2-, Y=10, and the product TzE is obtained.

[0107] Example 4

[0108] A target protein ligand molecule with a bioorthogonal group (norbornene) and an E3 ubiquitin ligase ligand molecule with a bioorthogonal group (tetrazine) are self-assembled in cells to form a protein degrader.

[0109] 1) Bioorthogonal reaction to generate protein degraders

[0110] High performance liquid chromatography was used to monitor in vitro whether the two parts of the target recognition molecules could undergo bioorthogonal reactions, reaction rates, and reaction processes. The two parts of the target recognition molecules were placed in a mixed system of PBS and acetonitrile (V:V = 1:1) and mixed in a volume ratio (1:1), placed on a 37°C constant temperature shaker for reaction, and samples were taken at different time points for detection. The results are as follows: Figure 1-2 As shown, the targeted recognition molecules can undergo bioorthogonal reactions to form protein degraders.

[0111] 2) Intracellular self-assembly to generate protein degraders

[0112] A fixed concentration or different concentrations of target protein ligand molecule solution with a bioorthogonal group (norbornene) is added to a tumor cell culture dish and incubated for 2 hours, and then different concentrations or a fixed concentration of E3 ubiquitin ligase ligand molecule solution with a bioorthogonal group (tetrazine) is added, and the dish is placed in a constant temperature incubator at 37°C and 5% CO2 for incubation for 48 hours, and then washed with PBS. After washing, the cells are digested and collected by centrifugation, and finally the cells are disrupted with a cell disruptor, filtered through a filter membrane, and then measured by electrospray mass spectrometry to obtain the product of the intracellular reaction.

[0113] Example 5

[0114] Determination of cell proliferation inhibitory activity of target protein ligand molecules with bioorthogonal groups norbornene.

[0115] The activity detection of the targeted recognition molecules LN and S5N with bioorthogonal groups norbornene at the cellular level was performed using the MTT assay. U87 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single cell suspension, which was inoculated in a 96-well plate (4000 cells / well), with 180 μL per well. The cells were cultured in a 37°C, 5% CO2 constant temperature incubator, and the drugs were added after the cells adhered to the wall after 24 hours. Four replicate wells were set up in each group, and 20 μL / well serum-free medium was added to the negative control group and the blank group, and 20 μL / well of different concentrations of drugs were added to the experimental group (drugs were diluted with serum-free medium), and the cells were placed in a 37°C, 5% CO2 constant temperature incubator for continued culture. After the drug acted for 48 hours, 22 μL / well of MTT solution (final concentration was 0.5 mg / mL) was added, and after incubation at 37°C for 4 hours, the supernatant was carefully aspirated, 150 μL / well of DMSO was added, and the cells were placed on a shaker and shaken for 15 minutes. The absorbance (OD) value of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA).

[0116] Numerical processing: inhibition rate = (OD 阴性组 -OD 给药组 ) / (OD 阴性组 -OD 空白组 )×100%;

[0117] The experimental results of some compounds are shown in Table 1:

[0118] Table 1 Cell proliferation inhibitory activity of targeted recognition molecules

[0119]

[0120] It can be seen from Table 1 that compared with the parent compound itself, the targeting recognition molecule S5N prepared by the present invention has better inhibitory activity against U87 cells.

[0121] Example 6

[0122] Investigation of the degradation effect of intracellular self-assembling protein degraders on target proteins.

[0123] U87 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single cell suspension and inoculated in 6-well plates (5×10 5 pcs / well), 2mL per well. Place in a 37°C, 5% CO2 constant temperature incubator for culture, and add drugs after 24 hours when cells adhere to the wall. Treat cells with a fixed concentration or different concentrations of target protein ligand molecules for 2 hours, then treat cells with different concentrations or fixed concentrations of tetrazine-tagged E3 ubiquitin ligase ligands, place in a 37°C, 5% CO2 constant temperature incubator for 48 hours, then extract proteins, and use Western Blot to detect related protein levels, see Figure 3 A and B and Figure 4 In Figures A and B, the results show that the protein degrader formed by the assembly of the target protein ligand molecule S5N and the E3 ubiquitin ligase ligand TzB with a tetrazine tag has a certain degradation effect on PDGFR-β protein, indicating that the self-assembling protein degrader constructed with S5N as the targeting recognition molecule has good application prospects and can be used for the preparation of anti-tumor drugs.

Claims

1. An intracellular self-assembly degradation agent based on bioorthogonal reaction, characterized in that: The structural formula of the degradation agent is as follows: Wherein X=1~6, Y=2~10, R1=CH3 or Cl, R2=F or H, R3=CH3 or H, R4=Ph or CH2.

2. A method for preparing an intracellular self-assembly degradation agent based on bioorthogonal reaction as claimed in claim 1, characterized in that: The following steps are involved: The target protein ligand Linifanib and norbornene are connected by using a linker, or the target protein ligand S5 and norbornene are connected by using a linker to obtain a target protein ligand with a norbornene group; wherein the target protein ligand S5 has the following structural formula: The tetrazine group is modified on the E3 ubiquitin ligase ligand through a connecting chain to obtain the E3 ubiquitin ligase ligand with tetrazine; The target protein ligand molecule with norbornene group and the E3 ubiquitin ligase ligand with tetrazine are successively introduced into the cell, and bio-orthogonal reaction self-assembly occurs in the cell to form an intracellular self-assembly degrader based on bio-orthogonal reaction.

3. The method for preparing the intracellular self-assembly degradation agent based on bioorthogonal reaction according to claim 2, characterized in that: The structural formula of the target protein ligand with a norbornene group is as follows: Among them, X=1~6, R1=CH3 or Cl, R2=F or H.

4. The method for preparing the intracellular self-assembly degradation agent based on bioorthogonal reaction according to claim 2 or 3, characterized in that: The target protein ligand with a norbornene group is prepared by the following process: The target protein ligand Linifanib or the target protein ligand S5 is subjected to an amide condensation reaction with a linker to obtain a compound with a Boc protective group, and then the Boc protective group is removed by the action of hydrogen chloride in ethyl acetate, and then the compound is subjected to an amide condensation reaction with 5-norbornene-2-carboxylic acid to obtain a target protein ligand with a norbornene group.

5. The method for preparing the intracellular self-assembly degradation agent based on bioorthogonal reaction according to claim 2, characterized in that: The structural formula of the tetrazine-containing E3 ubiquitin ligase ligand is as follows: Among them, Y=2~10, R3=CH3 or H, R4=Ph or CH2.

6. The method for preparing the intracellular self-assembly degradation agent based on bioorthogonal reaction according to claim 2 or 5, characterized in that: The tetrazine-bearing E3 ubiquitin ligase ligand is prepared by the following process: Acetonitrile, a cyano compound and hydrazine hydrate are reacted under the action of zinc trifluoromethanesulfonate or nickel trifluoromethanesulfonate, or formamidine acetate, a cyano compound and hydrazine hydrate are reacted under the action of zinc trifluoromethanesulfonate, and then oxidatively dehydrogenated to form a 1,2,4,5-tetrazine compound, which is then reacted under the action of trifluoroacetic acid and then connected with an E3 ubiquitin ligase ligand VH032 via a diacid to form an E3 ubiquitin ligase ligand with tetrazine.

7. The method for preparing the intracellular self-assembly degradation agent based on bioorthogonal reaction according to claim 2, characterized in that: The intracellular self-assembly degradation agent based on bioorthogonal reaction is specifically prepared by the following process: A target protein ligand solution with a norbornene group is added to a culture dish containing cells for incubation, and then an E3 ubiquitin ligase ligand with tetrazine is added and incubated to form an intracellular self-assembled degrader based on a bioorthogonal reaction through self-assembly in the cell.

8. Use of the intracellular self-assembly degradation agent based on bioorthogonal reaction as claimed in claim 1 in the preparation of anti-tumor drugs; The anti-tumor drug is a drug that can selectively induce the degradation of PDGFR-β protein; The anti-tumor drug is an anti-glioma drug.

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