A VH032-based intracellular self-assembly protein degrader and its preparation method and application

Abstract

The present invention discloses a kind of intracellular self-assembly protein degrader based on VH032 and its preparation method and application, by using alkyl diamines of different lengths to connect Sorafenib and bio-orthogonal group norbornene, obtain target protein ligand with bio-orthogonal group norbornene; by different types of connecting chains, tetrazine group is modified on E3 ubiquitin ligase ligand, obtain E3 ubiquitin ligase ligand with bio-orthogonal group tetrazine; make target protein ligand molecule with bio-orthogonal group norbornene and E3 ubiquitin ligase ligand with bio-orthogonal group tetrazine enter cells successively, bio-orthogonal reaction occurs in cells, self-assembles to form protein degrader. The preparation method of intracellular self-assembly protein degrader constructed by the present invention is simple, easy to implement, and has high yield, can be used in the preparation of drugs for treating cancer, especially in the preparation of anti-tumor drugs with PDGFR-β as target.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of degradation agents, and relates to an intracellular self-assembling protein degradation agent based on VH032, and a preparation method and application thereof. Background Art

[0002] Sorafenib is a new multi-target anti-tumor drug that can act on tumor cells and tumor blood vessels simultaneously, and has dual anti-tumor effects: it can directly inhibit the proliferation of tumor cells by blocking the cell signal transduction pathway mediated by RAF / MEK / ERK, and it can also block the formation of tumor neovascularization by inhibiting vascular endothelial growth factor receptor VEGFR and platelet-derived growth factor receptor PDGFR, thereby indirectly inhibiting the growth of tumor cells. However, serious adverse reactions often occur in clinical treatment, and long-term use is very likely to produce drug resistance.

[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-assembling protein degrader based on VH032 and a preparation method and application thereof. The preparation method of the degrader is simple, easy to implement, and has a high yield, and can be used in the preparation of anti-tumor drugs.

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

[0006] A VH032-based intracellular self-assembling protein degrader, the structural formula of which is as follows:

[0007]

[0008] Where X = 2 to 10, Y = 2 to 10, R 1 =CH 3 or H, R 2 =-Ph- or -CH 2 -.

[0009] A method for preparing the intracellular self-assembling protein degrader as described above comprises the following steps:

[0010] Sorafenib and norbornene were linked via alkyldiamine to obtain a target protein ligand with norbornene;

[0011] The tetrazine group is modified on the E3 ubiquitin ligase ligand through a connecting chain to obtain the E3 ubiquitin ligase ligand with tetrazine;

[0012] The target protein ligand molecule with norbornene and the E3 ubiquitin ligase ligand with tetrazine are introduced into the cell successively, and a bioorthogonal reaction occurs in the cell to self-assemble into a protein degrader.

[0013] Furthermore, the structural formula of the target protein ligand with norbornene is as follows:

[0014]

[0015] Among them, X=2~10.

[0016] Further, the target protein ligand with norbornene is prepared by the following process:

[0017] Sorafenib is hydrolyzed to obtain a compound with an active reactive group carboxyl, which is then reacted with an alkyl diamine via an amide condensation reaction to obtain a compound with an active reactive group amino, and finally the compound with an active reactive group amino is reacted with 5-norbornene-2-carboxylic acid via an amide condensation reaction to obtain a target protein ligand with norbornene.

[0018] Furthermore, the structural formula of the tetrazine-containing E3 ubiquitin ligase ligand is as follows:

[0019]

[0020] Where, Y = 2 to 10, R 1 =CH 3 or H, R 2 =-Ph- or -CH 2 -.

[0021] Furthermore, the tetrazine-bearing E3 ubiquitin ligase ligand was prepared by the following process:

[0022] Acetonitrile, a cyano compound and hydrazine hydrate, under the action of zinc trifluoromethanesulfonate or nickel trifluoromethanesulfonate, or formamidine acetate, a cyano compound and hydrazine hydrate, under the action of zinc trifluoromethanesulfonate, undergo cyclization reaction and oxidative dehydrogenation 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 to the E3 ubiquitin ligase ligand VH032 through a diacid to form an E3 ubiquitin ligase ligand with tetrazine.

[0023] Furthermore, after adding a target protein ligand solution containing norbornene to a culture dish containing cells for incubation, an E3 ubiquitin ligase ligand solution containing tetrazine is added and incubated to form a protein degrader through self-assembly in the cells.

[0024] A use of the intracellular self-assembling protein degrader as described above in the preparation of anti-tumor drugs.

[0025] Furthermore, the anti-tumor drug is a drug that selectively induces the degradation of PDGFR-β protein.

[0026] Furthermore, the anti-tumor drug is an anti-breast cancer drug or an anti-glioma drug.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] In the present invention, 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 a bioorthogonal reaction occurs in the cell to self-assemble to form a protein degradation targeting chimera, and finally the target protein is degraded by the ubiquitin-proteasome system. The present invention enables the protein degradation agent to be self-assembled in the cell by step-by-step administration. The preparation method of the protein degradation agent self-assembled in the cell constructed by the present invention is simple, easy to implement, and has a high yield.

[0029] The self-assembling protein degradation targeting chimera constructed by the present invention can not only utilize the mechanism of action of the protein degradation targeting chimera to degrade disease-related proteins, but also can 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, enhance the effect, and has the function of inducing PDGFR-β protein degradation, and can be used to prepare new anti-tumor drugs.

[0030] The small molecule protein degradation targeting chimera of the present invention can be used to prepare drugs for treating cancer, especially to prepare anti-tumor drugs with PDGFR-β as the target. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1The high performance liquid chromatogram of the bioorthogonal reaction process (2-32h) between the target recognition molecule S4N-1 constructed in the present invention and TzA;

[0032] Figure 2 The high performance liquid chromatogram of the bioorthogonal reaction process (10-240min) between the target recognition molecule S4N-1 constructed in the present invention and TzB;

[0033] Figure 3 The high performance liquid chromatogram of the bioorthogonal reaction process (2-32h) between the target recognition molecule S4N-1 constructed in the present invention and TzC;

[0034] Figure 4 The high performance liquid chromatogram of the bioorthogonal reaction process (2-32h) between the target recognition molecule S4N-1 constructed in the present invention and TzD;

[0035] Figure 5 The high performance liquid chromatogram of the bioorthogonal reaction process (2-32h) between the target recognition molecule S4N-1 constructed in the present invention and TzE;

[0036] Figure 6 The results of investigating the protein degradation effects of the targeted recognition molecules S4N-1 and TzB constructed by the present invention for intracellular self-assembly to form protein degraders on EA.hy926 cells; wherein A is a fixed S4N-1 dosing concentration, and B is a fixed TzB dosing concentration.

[0037] Figure 7 The results of investigating the protein degradation effects of the targeted recognition molecules S4N-1 and TzA constructed by the present invention for intracellular self-assembly to form protein degraders on U87 cells; wherein A is a fixed S4N-1 dosing concentration, and B is a fixed TzA dosing concentration.

[0038] Figure 8 The results of investigating the protein degradation effects of the targeted recognition molecules S4N-1 and TzB constructed by the present invention for intracellular self-assembly to form protein degraders on U87 cells; wherein A is a fixed S4N-1 dosing concentration, and B is a fixed TzB dosing concentration.

[0039] Fig. 9 The results of investigating the protein degradation effects of the targeted recognition molecules S4N-1 and TzC constructed by the present invention for intracellular self-assembly to form protein degraders on U87 cells; wherein A is a fixed S4N-1 dosing concentration, and B is a fixed TzC dosing concentration.

[0040] Fig.10The results of investigating the protein degradation effects of the targeted recognition molecules S4N-1 and TzD constructed by the present invention for intracellular self-assembly to form protein degraders on U87 cells; wherein A is a fixed S4N-1 dosing concentration, and B is a fixed TzD dosing concentration.

[0041] Fig.11 The results of investigating the protein degradation effects of the targeted recognition molecules S4N-1 and TzE constructed by the present invention for intracellular self-assembly to form protein degraders on U87 cells; wherein A is a fixed S4N-1 dosing concentration, and B is a fixed TzE dosing concentration.

[0042] Fig.12 The results of investigating the protein degradation effects of the targeted recognition molecules S8N-1 and TzB constructed by the present invention for intracellular self-assembly to form protein degraders on U87 cells; wherein A is a fixed S8N-1 dosing concentration, and B is a fixed TzB dosing concentration. DETAILED DESCRIPTION

[0043] 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.

[0044] The invention discloses a method for preparing a self-assembling protein degrader in a cell. The target protein ligand sorafenib and the bio-orthogonal group norbornene are connected by using diamines of different lengths to obtain a target protein ligand with the bio-orthogonal group norbornene; the tetrazine group is modified on the E3 ubiquitin ligase ligand by different types of connecting chains (the connecting chains are alkyl diacids of different lengths) to obtain an E3 ubiquitin ligase ligand with the bio-orthogonal group tetrazine. By a step-by-step administration method, the target protein ligand molecule with the bio-orthogonal group norbornene and the E3 ubiquitin ligase ligand with the bio-orthogonal group tetrazine can enter the cell successively, and a bio-orthogonal reaction self-assembly occurs in the cell 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 target protein can be degraded by the ubiquitin-proteasome system.

[0045] The protein degradation agent self-assembled in cells in the present invention can be used in the treatment of cancer.

[0046] The protein degradation targeting chimeras (PROTACs) involved in the present invention can selectively induce the degradation of PDGFR-β protein.

[0047] The structural formula of the target protein ligand with the bioorthogonal group norbornene is as follows:

[0048]

[0049] Among them, X=2~10.

[0050] The structural formula of the E3 ubiquitin ligase ligand with the bioorthogonal tetrazine group is as follows:

[0051]

[0052] Where, Y = 2 to 10, R 1 =CH 3 / H,R 2 =-Ph- / -CH 2 -.

[0053] The structural formula of the intracellular self-assembling protein degrader is as follows:

[0054]

[0055] Where X = 2 to 10, Y = 2 to 10, R 1 =CH 3 / H,R 2 =-Ph- / -CH 2 -.

[0056] The protein degradation agent provided by the present invention has anti-tumor activity in vitro and can be applied to the preparation of anti-tumor drugs.

[0057] The anti-tumor drug is an anti-breast cancer drug or an anti-glioma drug.

[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 with a norbornene group comprises the following synthetic steps:

[0061]

[0062] 1) Under nitrogen protection, 1.9mmol Sorafenib, 29mmol sodium hydroxide and 20mL anhydrous ethanol were refluxed at 80°C for 10h. After the reaction was completed by TLC, the anhydrous ethanol was removed under reduced pressure, a small amount of water was added, and the pH was adjusted to 3 with 2mol / L hydrochloric acid. A yellow solid was precipitated, filtered, and the filter cake was dried to obtain a yellow-brown powder, which was compound 1 (0.8g). The structural formula is as follows, and the yield is 91.45%. LC-MS (ESI, m / z): 452.00 [M+H] + , 450.00[MH] - .

[0063]

[0064] 2) 1.33mmol of compound 1 and 2.66mmol of PyBop (Benzotriazol-1-yl-oxytripyrrolidino-phosphonium Hexafluorophosphate) were dissolved in 30mL of dry dichloromethane, and 1mL of DMF (N,N-dimethylformamide) was added to assist dissolution. Under ice bath conditions, 5.31mmol of triethylamine was added dropwise. After stirring for 15min, 2.66mmol of 1,4-butanediamine or 1,8-octanediamine was added and stirred at room temperature. After the reaction was completed by TLC, the solvent was removed under reduced pressure, water was added, and the mixture was extracted with ethyl acetate (3×), washed with saturated sodium chloride, and anhydrous Na 2 SO 4 Dry. Remove the desiccant by suction filtration, and separate by column chromatography to obtain brown oily products, namely compounds 2a-2b, 2a (0.61 g), yield 88.52%, 2b (0.66 g), the structural formula is as follows, yield 84.05%. LC-MS (ESI, m / z) 2a: 522.25 [M+H] + 2b:578.20[M+H] + , 576.00[MH] - .

[0065]

[0066] Wherein, X is 2 or 6. When X=2, compound 2a is obtained, and when X=6, compound 2b is obtained.

[0067] 3) 0.77 mmol 5-norbornene-2-carboxylic acid and 1.15 mmol HATU (2-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) were dissolved in 30 mL dry dichloromethane, and 3.1 mmol DIPEA (diisopropylethylamine) was added dropwise under ice bath conditions. After reacting for 30 min, 0.77 mmol compound 2a was added. Stirring was carried out at room temperature overnight (overnight in the present invention is 12 h). After the reaction was completed by TLC detection, the organic phase was removed under reduced pressure, water was added, and the mixture was extracted with ethyl acetate (3×). The organic phases were combined and washed once with 1 mol / L hydrochloric acid, then washed twice with saturated NaCl, and anhydrous Na 2 SO 4Dry. Remove the desiccant by suction filtration and separate and purify by column chromatography to obtain the product S4N-1 (0.10 g), the structure of which is shown below, with a yield of 20.22%. LC-MS (ESI, m / z): 642.25 [M+H] + .

[0068]

[0069] 0.69mmol 5-norbornene-2-carboxylic acid and 1.04mmol HATU were dissolved in 30mL dry dichloromethane, and 2.77mmol DIPEA was added dropwise under ice bath conditions. After 30min of reaction, 0.77mmol compound 2b was added. Stirring was carried out at room temperature overnight. After the reaction was completed by TLC, the organic phase was removed under reduced pressure, water was added, and ethyl acetate was extracted (3×). The organic phases were combined and washed once with 1mol / L hydrochloric acid, then washed twice with saturated NaCl, and anhydrous Na 2 SO 4 Dry. Remove the desiccant by suction filtration, and separate and purify by column chromatography to obtain the product S8N-1 (0.09 g), the structure of which is shown below, with a yield of 18.75%. LC-MS (ESI, m / z): 698.25 [M+H] + , 696.40[MH] - .

[0070]

[0071] The target protein ligand S12N with a bioorthogonal group norbornene can be prepared by replacing the 1,4-butanediamine or 1,8-octanediamine in Example 1 with dodecanediamine, and has the following structural formula:

[0072]

[0073] Example 2

[0074] A method for preparing an E3 ubiquitin ligase ligand molecule with a bioorthogonal tetrazine group comprises the following synthetic steps:

[0075]

[0076] 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℃ for 36h. The reaction solution was cooled to room temperature, 50mmol sodium nitrite solution (3.45g sodium nitrite dissolved in 20mL 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, the organic phases were combined, and anhydrous Na2 SO 4 The desiccant was removed by suction filtration and separated by column chromatography to obtain a purple-red powder, namely compound 3a (0.11 g), the structure of which is shown below. The yield was 14.67%, LC-MS (ESI, m / z): 302.40 [M+H] + .

[0077]

[0078] 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. Ethyl acetate was extracted twice, the organic phases were combined, and anhydrous Na 2 SO 4 The desiccant was removed by suction filtration and separated by column chromatography to obtain a red powder, namely compound 3b (0.2 g), the structure of which is shown below, with a yield of 27.78%, LC-MS (ESI, m / z): 287.25 [M+H] + .

[0079]

[0080] 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 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. Filtered, the filtrate was extracted with ethyl acetate 3 times, the organic phases were combined, and anhydrous Na 2 SO 4 The desiccant was removed by suction filtration and separated by column chromatography to obtain a red powder, namely compound 3c (0.12 g), the structure of which is shown below, with a yield of 6.67%, LC-MS (ESI, m / z): 126.00 [M+H] + .

[0081]

[0082] 1) Compound 3a (1 eq) or 3b (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, and directly spin-dried for the next step of reaction, which was mixed with succinic anhydride (1 eq) in 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-red powder, i.e., compound 5a, or a red powder, i.e., compound 5b, was separated by column chromatography, compound 5a (0.12 g), yield 93.02%, compound 5b (0.20 g), yield 64.62%. LC-MS (ESI, m / z) 5a: 302.30 [M+H] + , 300.25[MH] - . 5b:286.20[MH] - The structural formula is as follows:

[0083]

[0084] Among them, R 1 CH 3 When the reaction mixture is stirred for 2 hours, compound 5a is obtained; R 1 When it is H, compound 5b is obtained.

[0085] 2) Compound 3c (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 dried by spin drying, a small amount of water was added, and the pH was adjusted to 7 with saturated sodium bicarbonate solution, extracted with dichloromethane, and directly dried by spin drying for the next step of reaction, and the compound was mixed with succinic anhydride (1 eq), pimelic acid (1 eq), and dodecanedioic acid (1 eq) in 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 5c (0.08 g), 5d (0.12 g), and 5e (0.08 g) were obtained by column chromatography, with yields of 63.49%, 37.52%, and 24.92%, respectively. LC-MS (ESI, m / z) 5c: 224.00 [MH] - . 5d:268.30[MH] - ,5e:338.35[M+H] + , 336.25[MH] - The structural formula is as follows:

[0086]

[0087] Among them, when Y=2, intermediate 5c is obtained; when Y=5, intermediate 5d is obtained; when Y=10, intermediate 5e is obtained.

[0088] 5) Compounds 5a-5e (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 and stirred at room temperature. After the reaction was completed, the organic phase was removed under reduced pressure, an appropriate amount of water was added, and the mixture was extracted with dichloromethane (3×). Anhydrous Na 2 SO 4 Dry. Remove the desiccant by suction filtration, remove the solvent under reduced pressure, and separate the red products TzA (0.08 g), TzB (0.015 g), TzC (0.06 g), TzD (0.10 g), and TzE (0.10 g) by column chromatography. The yields are 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 shown below.

[0089]

[0090] Where R 1 =CH 3 ,R 2 =-Ph-, Y = 2, and the product TzA is obtained;

[0091] R 1 =H,R 2 =-Ph-, Y = 2, and the product TzB is obtained;

[0092] R 1 =CH 3 ,R 2 =-CH 2 -, Y = 2, and the product TzC is obtained;

[0093] R 1 =CH 3 ,R2 =-CH 2 -, Y = 5, and the product TzD is obtained;

[0094] R 1 =CH 3 ,R 2 =-CH 2 -, Y = 10, and the product TzE is obtained.

[0095] Example 3

[0096] The target protein ligand molecule with a bioorthogonal group (norbornene) and the E3 ubiquitin ligase ligand molecule with a bioorthogonal group (tetrazine) self-assemble in the cell to form a protein degrader.

[0097] 1) Bioorthogonal reaction to generate protein degraders

[0098] 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 of the same concentration 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 to Figure 5 As shown in Figure 2, all the target recognition molecules can undergo bioorthogonal reactions to form protein degraders, but the reaction rates are different. Among them, the target recognition molecule TzB (R 1 For H, R 2 The reaction rate is the fastest when Ph is 1 and Y is 2, and a bioorthogonal reaction can occur quickly to form a protein degrader.

[0099] 2) Intracellular self-assembly to generate protein degraders

[0100] After treating the cells with target protein ligand molecules with bioorthogonal groups (norbornene) (final concentration of 10 μM) for 2 h, the cells were then given E3 ubiquitin ligase ligand molecules with bioorthogonal groups (tetrazine) (final concentration of 10 μM) and placed at 37°C, 5% CO 2 Incubate in a constant temperature incubator for 24 hours, then digest the cells and collect them by centrifugation, wash the cells three times with PBS, add 1 ml of methanol:acetonitrile:water (V:V:V=2:2:1) and place them in liquid nitrogen for rapid quenching for 15 minutes, re-dissolve on ice, and disrupt them with a cell disruptor (super 2 seconds, stop 1 second, total super 20 minutes), incubate at -20°C for 1 hour to precipitate the protein, centrifuge at 4°C, 13000 rpm for 15 minutes, take the supernatant and lyophilize it, and then perform electrospray ionization mass spectrometry to verify the generation of self-assembled protein degrader.

[0101] Example 4

[0102] Screening of inhibitory activity of target protein ligand molecules with bioorthogonal groups norbornene on VEGFR-2 kinase.

[0103] The ADP-Glo ​​luminescence method was used to determine the inhibitory activity of sorafenib, a targeted recognition molecule with a bioorthogonal group (norbornene), on VEGFR-2 kinase.

[0104] Use Buffer (Tris 80mM, MgCl 2 20mM, BSA 0.2mg / mL, DTT 2mM) dilute ATP (10mM) to 250μM; ATP and substrate Poly (4:1Glu, Tyr) Peptide are prepared into ATP (125μM)-Poly (4:1Glu, Tyr) Peptide (0.5μg / μL) mixed solution in a volume ratio of 1:1; kinase is diluted to 1.5ng / μL with Buffer. The compounds to be tested are prepared into 6 concentration gradient solutions, and 2μL ATP-Poly (4:1Glu, Tyr) Peptide solution, 1μL sample solution, and 2μL enzyme solution are added to the 384-well plate to start the reaction. After incubation at 30℃ for 60min, 5μL ADP-Glo ​​reagent is added to terminate the reaction. Then, 10 μL of Kinase Detection reagent was added to convert ADP into ATP, and the mixture was incubated at 25°C for 30 min. The luminescence value was measured using the chemiluminescence module of the PerkinElmer multifunctional microplate reader, and the inhibition rate was calculated.

[0105] Numerical processing: inhibition rate = (positive value - drug administration group value) / (positive value - negative value) × 100%;

[0106] The experimental results of the compounds are shown in Table 1:

[0107] Table 1 Results of the inhibitory activity of targeted recognition molecules on VEGFR-2 kinase

[0108]

[0109] It can be seen from Table 1 that the target protein ligand molecule with the bioorthogonal group norbornene prepared in the present invention has good inhibitory activity on VEGFR-2 kinase.

[0110] Example 5

[0111] Determination of cell proliferation inhibitory activity of target protein ligand molecules with bioorthogonal groups norbornene.

[0112] The activity of Sorafenib, a targeted recognition molecule with a bioorthogonal group norbornene, was detected at the cellular level using the MTT assay. EA.hy926 cells, MDA-MB-453 cells, and 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 placed in a 37°C, 5% CO 2 Culture in a constant temperature incubator, and add drugs after 24 hours when the cells adhere to the wall. Set up 4 replicate wells for each group, and 6 concentrations of the samples to be tested. Add 20 μL / well of serum-free medium to the negative control group and the blank group, and add 20 μL / well of different concentrations of drugs to the experimental group (drugs diluted with serum-free medium), and place at 37°C, 5% CO 2 Continue to culture in a constant temperature incubator. After 48 hours of drug action, add 22 μL / well of MTT solution (final concentration of 0.5 mg / mL), incubate at 37°C for 4 hours, carefully discard the supernatant, add 150 μL / well of DMSO, and shake on a shaker for 15 minutes. Measure the absorbance (OD) value of each well at a wavelength of 490 nm using an enzyme-linked immunosorbent assay.

[0113] Numerical processing: inhibition rate = (OD 阴性组 -OD 给药组 ) / (OD 阴性组 -OD 空白组 )×100%;

[0114] The experimental results of some compounds are shown in Table 2:

[0115] Table 2 Cell proliferation inhibitory activity of targeted recognition molecules

[0116]

[0117] As can be seen from Table 2, the targeting recognition molecules S4N-1 and S8N-1 prepared by the present invention have good proliferation inhibition activity on EA.hy926 cells.

[0118] Example 6

[0119] Investigation of the degradation effect of intracellular self-assembling protein degraders on target proteins.

[0120] EA.hy926 cells or 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), 2 mL per well. Place in 37°C, 5% CO 2The cells were cultured in a constant temperature incubator, and the drugs were added after the cells were attached to the wall for 24 hours. The cells were treated with a fixed concentration (final concentration of 10 μM) or different concentrations (final concentration of 0.016 μM, 0.08 μM, 0.4 μM, 2 μM, 10 μM) of the target protein ligand molecule for 2 hours, and then treated with different concentrations (final concentration of 0.016 μM, 0.08 μM, 0.4 μM, 2 μM, 10 μM) or a fixed concentration (final concentration of 10 μM) of the tetrazine-tagged E3 ubiquitin ligase ligand, and placed at 37°C, 5% CO 2 Incubate in a constant temperature incubator for 48 hours, then extract the protein, and use Western Blot to detect the level of related proteins. Figure 6 A and B and Figure 8 In Figures A and B, it can be seen that the constructed intracellular self-assembling protein degrader has a significantly better effect on U87 cells than on EA.hy926 cells. Figure 7 to Figure 11 The protein degraders formed by the assembly of A and B in the above-mentioned target protein ligand molecule S4N-1 and various types of E3 ubiquitin ligase ligands VH032 with tetrazine tags all have good degradation effects on PDGFR-β protein, and also have certain degradation effects on VEGFR-2 and EphB4 proteins, see Fig.12 From A and B in the figure, we can see that the protein degrader formed by assembling other target protein ligand molecules (S8N-1) with the fastest reaction rate tetrazine-tagged E3 ubiquitin ligase ligand TzB has a poor effect, which is not as good as the effect of S4N-1. This shows that the self-assembling protein degrader constructed with S4N-1 as the targeting recognition molecule has good application prospects and can be used in the preparation of anti-tumor drugs such as glioma.

Claims

1. A VH032-based intracellular self-assembly protein degrader, characterized in that: The structural formula of the degradation agent is as follows: Wherein X=2~10, Y=2~10, R1=CH3 or H, R2=-Ph- or -CH2-.

2. A method for preparing the intracellular self-assembly protein degradation agent according to claim 1, characterized in that: The following steps are involved: Sorafenib and norbornene were linked via alkyldiamine to obtain a target protein ligand with norbornene; 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 and the E3 ubiquitin ligase ligand with tetrazine are introduced into the cell successively, and a bioorthogonal reaction occurs in the cell to self-assemble into a protein degrader.

3. The method for preparing an intracellular self-assembling protein degradation agent according to claim 2, characterized in that: The structural formula of the target protein ligand with norbornene is as follows: Among them, X=2~10.

4. The method for preparing an intracellular self-assembling protein degradation agent according to claim 3, characterized in that: The target protein ligand with norbornene was prepared by the following process: Sorafenib is hydrolyzed to obtain a compound with an active reactive group carboxyl, which is then reacted with an alkyl diamine via an amide condensation reaction to obtain a compound with an active reactive group amino, and finally the compound with an active reactive group amino is reacted with 5-norbornene-2-carboxylic acid via an amide condensation reaction to obtain a target protein ligand with norbornene.

5. The method for preparing an intracellular self-assembling protein degradation agent according to claim 2, characterized in that: The structural formula of the tetrazine-bearing E3 ubiquitin ligase ligand is as follows: Among them, Y=2~10, R1=CH3 or H, R2=-Ph- or -CH2-.

6. The method for preparing an intracellular self-assembling protein degradation agent according to claim 5, characterized in that: Tetrazine-bearing E3 ubiquitin ligase ligands were prepared by the following process: Acetonitrile, cyano compounds and hydrazine hydrate under the action of zinc trifluoromethanesulfonate or nickel trifluoromethanesulfonate, or formamidine acetate, cyano compounds and hydrazine hydrate under the action of zinc trifluoromethanesulfonate, undergo cyclization reaction and oxidative dehydrogenation to form 1,2,4,5-tetrazine compounds, which are then reacted under the action of trifluoroacetic acid and then connected to the E3 ubiquitin ligase ligand VH032 through a diacid to form an E3 ubiquitin ligase ligand with tetrazine.

7. The method for preparing an intracellular self-assembling protein degradation agent according to claim 2, characterized in that: After adding a target protein ligand solution with norbornene to a culture dish containing cells for incubation, an E3 ubiquitin ligase ligand solution with tetrazine is added and incubated to form a protein degrader through self-assembly in the cells.

8. Use of the intracellular self-assembling protein degradation agent as claimed in claim 1 in the preparation of anti-tumor drugs; The anti-tumor drug is a drug that selectively induces the degradation of PDGFR-β protein; The anti-tumor drug is an anti-breast cancer drug or an anti-glioma drug.

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