TNIK degradation agent as well as preparation method and application thereof

Abstract

The invention discloses a TNIK degradation agent as well as a preparation method and application thereof, and relates to the technical field of organic synthesis medicines. The TNIK degradation agent provided by the invention has a structure as shown in a formula I. The invention further discloses a preparation method of the TNIK degradation agent and application of the TNIK degradation agent in preparation of drugs for treating and / or preventing cancers. The TNIK degradation agent is a PROTAC small-molecule degradation agent targeting TNIK, has certain degradation activity on TNIK, can degrade TNIK protein in cells in a dose-dependent mode, can effectively inhibit proliferation of colorectal cancer cells, and is wide in application prospect.

Description

Technical Field

[0001] This invention relates to the field of organic synthetic drug technology, specifically to a TNIK degrading agent, its preparation method, and its application. Background Technology

[0002] TNIK (Traf2 and Nck interacting protein kinase) belongs to the germinal center kinase (GCK) family and is a serine / threonine kinase. Its gene is located in the 3q26 chromosome region and is expressed in various tissues. Researchers first discovered that TNIK regulates synapse formation and its biological activity in neurons. Most importantly, TNIK regulates the cytoskeleton by promoting the ubiquitin ligase Nedd4-1 and the GTP-binding protein Rap2A. Later studies found that in the Wnt / β-catenin signaling pathway, TNIK is located in the nucleus and can phosphorylate TCF4 at the Ser154 site, thereby affecting downstream signal transduction and gene expression, and is closely related to the occurrence and development of various tumors.

[0003] Wnt is a secreted glycoprotein secreted by palmitoylation of the acyltransferase PORCN. It co-transmits downstream signals with the frizzled (FZD) receptor family and lipoprotein receptor-related protein 5 / 6 (LRP5 / 6) on the cell surface. The canonical pathway, dependent on β-catenin, is significantly associated with tumor progression. The central event in this signaling cascade is the bidirectional regulation of the hydrolysis of the transcriptional coactivator β-catenin. In the absence of Wnt signaling, the axin acts as a scaffold protein, binding to the intact APC protein (which also acts as a scaffold protein) to form a complex of Axin, APC, GSK-3β (glycogen synthase kinase-3β), and β-catenin. GSK-3β promotes β-catenin phosphorylation, which in turn interacts with β-TRCP (β-transduction protein repeat sequence protein) and is degraded, thus maintaining low β-catenin expression levels in cells. In the Wnt signaling activation state, Wnt binds to the FZD receptor, leading to LRP5 / 6 phosphorylation and the formation of the Wnt-FZD-LRP5 / 6 complex. This activates the downstream Dvl protein (Disheveled) and induces the aggregation of the multi-subunit complex towards the receptor. Dvl enhances the phosphorylation and inhibition of GSK3β and negatively regulates the degradation of β-catenin by the multi-subunit complex. This promotes the accumulation and nuclear translocation of β-catenin. Finally, β-catenin enters the nucleus and forms a complex with transcription factors CBP / TCF4 and TNIK, activating the transcriptional activity of TCF4, thereby accelerating cell division and the generation of tumor cells. Given the correlation between abnormal Wnt / β-catenin signaling and the pathogenesis of various cancers such as colorectal cancer, liver cancer, and breast cancer, this pathway has become an important area of ​​research and development for oncology drugs. TNIK is an important regulatory protein in Wnt signaling, and the proliferation and differentiation of various tumors are highly dependent on the expression level of TNIK. Therefore, targeted intervention of TNIK can block Wnt signaling and inhibit the growth and proliferation of tumor cells. Several TNIK inhibitors have been publicly reported, but most of them have unsatisfactory anti-tumor effects, which greatly limits the subsequent clinical drug development. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a TNIK degrading agent, its preparation method, and its application. The TNIK degrading agent of the present invention is a PROTAC small molecule degrading agent targeting TNIK, which has certain degrading activity against TNIK. It can dose-dependently degrade intracellular TNIK protein and effectively inhibit the proliferation of colorectal cancer cells, thus solving the problem of unsatisfactory anti-tumor effects of existing TNIK inhibitors.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A TNIK degrading agent is provided, the TNIK degrading agent having the structure shown in Formula I:

[0006] In this context, R1 is an alkyl or alkoxy group, and R2 is an E3 ubiquitin ligand.

[0007] Furthermore, in the structure shown in Equation I, R2 is , , , , , , , , or .

[0008] Furthermore, R2 is or .

[0009] Furthermore, the specific structure of the aforementioned TNIK degrading agent is as follows:

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024] .

[0025] The preparation method of the above-mentioned TNIK degrading agent includes the following steps: (1) 4-(4-fluorophenyl)-1H-imidazolium and 2-bromopropane were subjected to a substitution reaction to obtain intermediate 3; (2) Intermediate 3 was subjected to an iodination reaction to obtain intermediate 4; (3) Under an inert atmosphere, intermediate 4 and (5-formyl-2-furanyl)boronic acid were coupled to obtain intermediate 6; (4) Hydrolyze intermediate 6 to obtain intermediate 7; (5) Intermediate 7 and 4-(4-tert-butyloxycarbonylpiperazin-1-yl)aniline were subjected to a first amidation reaction to obtain intermediate 9; (6) Intermediate 9 was subjected to a first acid hydrolysis reaction to obtain intermediate 10; (7) The E3 ubiquitin ligase ligand is subjected to a nucleophilic substitution reaction or a second amidation reaction with compound 12 to obtain intermediate 13. (8) The intermediate 13 was subjected to a second acid hydrolysis reaction to obtain the intermediate 14; (9) Intermediate 10 and intermediate 14 were subjected to a third amidation reaction to obtain the compound shown in Formula I; the synthetic route is as follows: .

[0026] Further, step (1) includes the following steps: dissolving 4-(4-fluorophenyl)-1H-imidazolium in an organic solvent, adding sodium hydride, stirring at -2~2℃ for 20~40 min, adding 2-bromopropane, and then carrying out a substitution reaction at 20~30℃ for 2~4 h; step (2) the iodination reaction is carried out under the action of N-iodosuccinimide and trifluoroacetic acid at a temperature of 8~12℃ for 10~14 h; step (3) the coupling reaction is carried out under the action of K3PO4 and a catalyst at a temperature of 90~95℃ for 4~6 h; in step (4), the hydrolysis reaction is carried out under the action of an inorganic base at a temperature of 25~35℃ for 1.5~2.5 h; in step (5), the first amidation reaction is carried out under the action of a condensing agent and an organic base catalyst at a temperature of 20~30℃ for 1.5~2.5 h. h; In step (6), the first acidolysis reaction is carried out in a 1,4-dioxane solution of hydrogen chloride at room temperature for 1-2 h.

[0027] Furthermore, step (1) includes the following steps: 4-(4-fluorophenyl)-1H-imidazolium is dissolved in an organic solvent, sodium hydride is added, and the mixture is stirred at 0°C for 30 min, followed by the addition of 2-bromopropane, and then a substitution reaction is carried out at 25°C for 3 h; step (2) the iodination reaction is carried out in the presence of N-iodosuccinimide and trifluoroacetic acid at 10°C for 12 h; step (3) the coupling reaction is carried out in the presence of K3PO4 and a catalyst at 95°C for 5 h; in step (4) the hydrolysis reaction is carried out in the presence of an inorganic base at 25°C for 2 h; in step (5) the first amidation reaction is carried out in the presence of a condensing agent and an organic base catalyst at 25°C for 2 h; in step (6) the first acidolysis reaction is carried out in a solution of hydrogen chloride in 1,4-dioxane at room temperature for 1.5 h.

[0028] Furthermore, in step (1), the molar ratio of 4-(4-fluorophenyl)-1H-imidazolium, 2-bromopropane, and sodium hydride is (100~130):(200~250):(130~150); in step (2), the molar ratio of intermediate 3, N-iodosuccinimide, and trifluoroacetic acid is (100~110):(300~310):(20~40); and in step (3), the molar mass to volume ratio of intermediate 4, (5-formyl-2-furanyl)boronic acid, K3PO4, and catalyst is (1~2) mmol:(2~3) mmol:(3~4). mL:(0.1~0.2)mmol; In step (4), the molar ratio of intermediate 6 to inorganic base is (0.1~0.2):(1~2); In step (5), the molar ratio of intermediate 7, 4-(4-tert-butyloxycarbonylpiperazin-1-yl)aniline, condensing agent and organic base catalyst is (70~80):(150~160):(150~160):(200~240); In step (6), the feed-to-liquid ratio of intermediate 9 to 1,4-dioxane solution of hydrogen chloride is (40~60)mg:(5~15)mL.

[0029] Furthermore, in step (1), the molar ratio of 4-(4-fluorophenyl)-1H-imidazolium, 2-bromopropane, and sodium hydride is 123:246.66:148; in step (2), the molar ratio of intermediate 3, N-iodosuccinimide, and trifluoroacetic acid is 102.82:308.46:30.85; and in step (3), the molar mass to volume ratio of intermediate 4, (5-formyl-2-furanyl)boronic acid, K3PO4, and catalyst is 1.62 mmol:2.43 mmol:3.24 mL:0.162 mL. mmol; the molar ratio of intermediate 6 and inorganic base in step (4) is 0.18274:1.83; the molar ratio of intermediate 7, 4-(4-tert-butyloxycarbonylpiperazin-1-yl)aniline, condensing agent and organic base catalyst in step (5) is 79.54:159.08:159.08:238.62; the feed-to-liquid ratio of intermediate 9 and 1,4-dioxane solution of hydrogen chloride in step (6) is 50 mg:10 mL.

[0030] Furthermore, in step (1), the organic solvent is N,N-dimethylformamide; in step (3), the catalyst is XPhosPd G3 catalyst; and in step (4), the inorganic base is sodium hydroxide.

[0031] Further, in step (7), when the E3 ubiquitin ligase ligand contains a halogen group, R3 in compound 12 is -NH2, and the E3 ubiquitin ligase ligand undergoes a nucleophilic substitution reaction with compound 12. The nucleophilic substitution reaction is carried out under the action of an organic base catalyst at a temperature of 85~95℃ for 1.5~2.5 h; the molar ratio of E3 ubiquitin ligase ligand, compound 12, and organic base catalyst is (0.1~0.2):(0.2~0.3):(0.3~0.4); when the E3 ubiquitin ligase ligand contains an amino group, R3 in compound 12 is -COOH, and the E3 ubiquitin ligase ligand undergoes a second amidation reaction with compound 12. The second amidation reaction is carried out under the action of an organic base catalyst and a condensing agent at room temperature for 10~14 h; the feed-to-liquid ratio of E3 ubiquitin ligase ligand, compound 12, organic base catalyst, and condensing agent is (50~60). mg: (120~160) mg: (300~350) μL: (200~210) mg.

[0032] Furthermore, in step (7), when the E3 ubiquitin ligase ligand contains a halogen group, R3 in compound 12 is -NH2, and the E3 ubiquitin ligase ligand undergoes a nucleophilic substitution reaction with compound 12. The nucleophilic substitution reaction is carried out under the action of an organic base catalyst at a temperature of 90°C for 2 h. The molar ratio of E3 ubiquitin ligase ligand, compound 12, and organic base catalyst is 0.18:0.22:0.36. When the E3 ubiquitin ligase ligand contains an amino group, R3 in compound 12 is -COOH, and the E3 ubiquitin ligase ligand undergoes a second amidation reaction with compound 12. The second amidation reaction is carried out under the action of an organic base catalyst and a condensing agent at a temperature of room temperature for 12 h. The feed-to-liquid ratio of E3 ubiquitin ligase ligand, compound 12, organic base catalyst, and condensing agent is 59 mg:150 mg:330 μL:205 mg.

[0033] Furthermore, the second acidolysis reaction in step (8) is carried out under the action of trifluoroacetic acid at room temperature for 0.5 to 1.5 h; the third amidation reaction in step (9) is carried out under the action of organic base catalyst and condensing agent at room temperature for 2 to 14 h.

[0034] Furthermore, the second acidolysis reaction in step (8) is carried out under the action of trifluoroacetic acid at room temperature for 1 h.

[0035] Furthermore, in step (8), the ratio of intermediate 13 to trifluoroacetic acid is (40~80) mg:(0.2~2) mL; in step (9), the ratio of intermediate 10, intermediate 14, organic base catalyst and condensing agent is (60~120) mg:(40~150) mg:(40~300) μL:(70~150) mg.

[0036] Furthermore, in step (8), the ratio of intermediate 13 to trifluoroacetic acid is (50~70) mg:(0.5~1.5) mL; in step (9), the ratio of intermediate 10, intermediate 14, organic base catalyst and condensing agent is (76~103) mg:(65~120) mg:(65~200) μL:(85~137) mg.

[0037] Furthermore, in steps (5), (7), and (9), the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and the organic base catalyst is N,N-diisopropylethylamine.

[0038] The above-mentioned TNIK degrading agents are used in the preparation of drugs for treating and / or preventing cancer.

[0039] Furthermore, the cancer is colorectal cancer.

[0040] The present invention has the following beneficial effects: The TNIK degrader of this invention is a novel PROTAC small molecule degrader targeting TNIK, belonging to the bifunctional molecule category. This structure can utilize the body's own ubiquitin-proteasome system to label and degrade target proteins, exhibiting certain degradative activity against TNIK and dose-dependently degrading intracellular TNIK proteins. Simultaneously, the TNIK degrader of this invention can also effectively inhibit the proliferation of colorectal cancer cells, laying a solid foundation for subsequent drug development and optimization. Attached Figure Description

[0041] Figure 1 This is a graph showing the degree of inhibition of HCT116 cell proliferation by the TNIK degrading agent I-18 of this invention; Figure 2 The figure shows the results of the degradation of TNIK protein by the TNIK degrading agent I-18 of the present invention; where (a) is a protein immunoblot map; and (b) is a quantitative bar chart. Detailed Implementation

[0042] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0043] Example 1 A TNIK degrading agent having the structure shown in Formula I-1: .

[0044] The TNIK degrading agent having the structure shown in Formula I-1 in this embodiment is prepared by the following steps: (1) 4-(4-fluorophenyl)-1H-imidazolium intermediate 1 (20.0 g, 123 mmol, 1.0 equivalent) was dissolved in DMF (200 mL), and NaH (9.87 g, 246.66 mmol, 60% purity, 2.0 equivalent) was added. The mixture was stirred at 0 °C for 30 min. Then, 2-bromopropane intermediate 2 (18.20 g, 148.00 mmol, 13.90 mL, 1.2 equivalent) was added to the mixture at 0 °C. The mixture was stirred at 25 °C for 3 h. The mixture was poured into water (1000 mL), extracted with ethyl acetate (200 mL × 3), the organic phase was washed with brine (300 mL), dried with Na2SO4, filtered and concentrated to obtain the residue. The residue was purified by flash silica gel chromatography with an eluent of ethyl acetate / petroleum ether gradient of 0-50% at a flow rate of 100 mL / min to obtain a yellow oily intermediate 3, which is 4-(4-fluorophenyl)-1-isopropyl-1H-imidazole (21 g, 102.82 mmol). ¹H NMR: (400 MHz, DMSO-d6)δ 7.89 – 7.81 (m, 4H), 7.27 – 7.18 (m, 2H), 4.63 (p, J = 6.8 Hz, 1H), 1.49 (d, J = 6.8 Hz, 5H). ESI-MS (m / z): 205.1 (M+H) + . (2) Intermediate 3 (21 g, 102.82 mmol, 1.0 equivalent) was dissolved in dichloromethane (210 mL), and NIS (N-iodosuccinimide, 69.40 g, 308.46 mmol, 3.0 equivalent) and trifluoroacetic acid (3.52 g, 30.85 mmol, 2.28 mL, 0.3 equivalent) were added. The mixture was stirred at 10 °C for 12 h. After stirring, the mixture was poured into a saturated Na₂SO₃ solution (500 mL), and extracted with dichloromethane (DCM, 200 mL × 3). The organic phase was washed with a saturated Na₂SO₃ solution (300 mL × 3) and brine (300 mL), dried over Na₂SO₄, filtered, and concentrated to obtain the residue. The residue was ground with PE / EA (5:1, 100 mL) and then filtered. A yellow solid intermediate 4 was prepared, which was 4-(4-fluorophenyl)-5-iodo-1-isopropyl-1H-imidazolium (30 g, 90.87 mmol, 88.38% yield); ¹H NMR: (400 MHz, DMSO-d6)δ 7.92 (d, J = 0.6 Hz, 1H), 7.78 – 7.70 (m,2H), 7.31 – 7.23 (m, 2H), 4.81 – 4.67 (m, 1H), 1.56 (d, J = 6.7 Hz, 6H). ESI-MS (m / z): 331.0 (M + H) + .; (3) Intermediate 4 (500 mg, 1.62 mmol, 1.0 equivalent) and (5-formyl-2-furanyl)boronic acid (340 mg, 2.43 mmol, 1.5 mol·L⁻¹) were added. -1 ), K3PO4(1.5 M, 3.24 mL, 3.0 mol·L -1 ) and XPhos Pd G3 catalyst (137 mg, 162 μmol, 0.1 mol·L⁻¹) -1 The mixture was placed in DMA (dimethylamine, 10 mL) and stirred at 95 °C under a nitrogen atmosphere for 5 h. After the reaction was completed, the mixture was poured into water (40 mL) and extracted with ethyl acetate (45 mL × 2). The combined organic layers were washed with brine (15 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (eluting PE / EA = 1:1) to obtain a yellow solid intermediate 6, which was methyl 5-(1-isopropyl-4-(4-fluorophenyl)-1H-imidazol-5-yl)furan-2-carboxylate (440 mg, 1.36 mmol, 83.7% yield). ¹H NMR: (400 MHz, DMSO-d6)δ 7.83 (d, J = 0.6 Hz, 1H), 7.80 – 7.72 (m,2H), 7.31 (d, J = 7.4 Hz, 1H), 7.31 – 7.22 (m, 3H), 4.75 – 4.61 (m, 1H), 3.84(s, 2H), 1.55 (d, J = 6.7 Hz, 6H). ESI-MS (m / z): 329.0 (M + H) + .; (4) Dissolve intermediate 6 (60 mg, 182.74 μmol, 1.0 mol) in a methanol / water 2:1 system, add sodium hydroxide (16.68 mg, 1.83 mmol, 10.0 mol) at 25 °C, and stir at room temperature for 2 h. After the reaction is completed, concentrate the methanol under reduced pressure and adjust the pH to 3 with dilute hydrochloric acid to precipitate a white solid to obtain intermediate 7. Filter intermediate 7 and dry it to obtain crude product, which can be directly used in the next step of the reaction. (5) Add HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 60.49 mg, 159.08 μmol, 2.0 mol) and DIEA (N,N-diisopropylethylamine, 30.84 mg, 238.62 μmol, 41.56 μL, 3.0 mol) to a DMF (1.5 mL) solution of intermediate 7 (25 mg, 79.54 μmol, 1.0 eq) and 4-(4-tert-butyloxycarbonylpiperazin-1-yl)aniline (30.43 mg, 159.08 μmol, 2.0 mol) at 25 °C, and stir at 25 °C for 2 h. Then pour in H2O (20 mL) and use DCM (20 mL) to stir. Extract the mixture by 2 mL of water, then combine the organic layers, wash with brine (20 mL), dry with Na2SO4, filter, concentrate under reduced pressure to obtain the residue, and purify the residue by column chromatography to obtain intermediate 9 (30.05 mg, 48.65 μmol, 61.17% yield). ¹H NMR: (400 MHz, DMSO-d6)δ 9.11 (s, 1H), 7.82 (d, J = 0.6 Hz, 1H), 7.79 – 7.70 (m, 2H), 7.67 – 7.60 (m, 2H), 7.33 (d, J = 7.4 Hz, 1H), 7.27 – 7.18 (m, 3H), 6.97 – 6.90 (m, 2H), 4.71 – 4.60 (m, 1H), 3.64 – 3.56 (m, 4H), 3.49 – 3.38 (m, 2H), 3.33 (dt, J = 12.3, 7.3 Hz, 2H), 1.53 (d, J = 6.8 Hz, 6H), 1.43 (s, 6H). ESI-MS (m / z): 574.1 (M + H) + .; (6) Dissolve 50 mg of intermediate 9 in 5 mL of 1,4-dioxane, add 10 mL of dioxane hydrochloride solution, stir at room temperature for 1.5 h, monitor the reaction by TLC until the residue is obtained by vacuum concentration, and prepare intermediate 10. Intermediate 10 is used directly in the next step of the reaction without purification. (7) 3-(4-fluoro-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)piperidin-2,6-dione (50 mg, 0.18 mmol) was dissolved in DMF (10 mL), and tert-butyl 3-aminopropionate (50 mg, 0.22 mmol) and DIEA (51 μL, 0.36 mmol) were added. The mixture was stirred at 90 °C for 2 h. After the reaction was completed, the mixture was concentrated and purified by silica gel column chromatography to obtain intermediate 13. ¹H NMR: (400 MHz, DMSO-d6)δ 10.77 (s, 1H), 8.46 (t, J = 6.3 Hz, 1H), 7.66 (dd, J = 7.5, 1.5 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.26 (dd, J = 7.5,1.5 Hz, 1H), 5.50 (t, J = 7.0 Hz, 1H), 3.59 (dtd, J = 12.5, 7.2, 6.3 Hz, 1H), 3.42 (dtd, J = 12.5, 7.1, 6.2 Hz, 1H), 2.68 – 2.53 (m, 4H), 2.26 (dq, J =12.3, 7.0 Hz, 1H), 2.13 (dq, J = 12.5, 7.0 Hz, 1H), 1.41 (s, 6H). ESI-MS (m / z): 402.0 (M + H) + .; (8) Dissolve 50 mg of intermediate 13 in dichloromethane (2 mL) and trifluoroacetic acid (0.5 mL), stir at room temperature for 1 h, and concentrate under reduced pressure to obtain intermediate 14, which can be used directly in the next step of the reaction without further purification; (9) Dissolve 65 mg of intermediate 14 and 76 mg of intermediate 10 in DMF (5 mL), add HATU (85 mg) and DIEA (65 µL), stir at room temperature for 2 h, concentrate the reaction solution, and purify by preparative high performance liquid chromatography to obtain the compound shown in Formula I-1. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.11 (s, 1H), 8.52 (t, J =6.4 Hz, 1H), 7.82 (d, J = 0.6 Hz, 1H), 7.78 – 7.70 (m, 3H), 7.63 – 7.57 (m,2H), 7.49 (t, J = 7.5 Hz, 1H), 7.36 – 7.27 (m, 3H), 7.21 (d, J = 7.4 Hz, 1H), 7.13 (dd, J = 7.6, 1.4 Hz, 1H), 7.08 – 7.01 (m, 2H), 5.50 (t, J = 7.0 Hz,1H), 4.65 (dqd, J = 13.7, 6.8, 0.6 Hz, 1H), 3.59 – 3.45 (m, 5H), 3.41 – 3.24(m, 5H), 2.66 (dt, J = 12.3, 7.0 Hz, 1H), 2.62 – 2.52 (m, for C 43 H 41 FN8O7[M + H] + 8801.31158, found801.31814. Its synthetic route is as follows: .

[0045] Example 2 A TNIK degrading agent having the structure shown in Formula I-17: .

[0046] In this embodiment, the TNIK degrading agent with the structure shown in Formula I-17 is prepared using the same steps (1) to (6) as in Example 1, with the remaining steps being: (7) 4-tert-butoxy-4-oxobutyric acid (150 mg, 1.0 eq), DIEA (330 μL, 3.0 eq) and HATU (205 mg, 1.2 eq) were placed in a round-bottom flask, DMF was added and stirred for 30 min, and then (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (59 mg, 1.0 eq) was added and stirred at room temperature overnight. After the reaction was complete by TLC monitoring, the reaction solution was washed with saturated ammonium chloride aqueous solution, saturated sodium bicarbonate aqueous solution and saturated sodium chloride aqueous solution in sequence. The organic phase was evaporated to dryness, mixed and purified by column chromatography to obtain white solid intermediate 13. 1 H NMR (400 MHz, DMSO-d6) δ 8.05 – 7.94 (m, 1H), 7.55 – 7.48 (m, 1H), 7.34 (dt, J = 7.5, 1.1 Hz, 1H), 4.45 – 4.31 (m, 2H), 4.13 (d, J = 7.3 Hz,0H), 3.74 – 3.58 (m, 1H), 2.68 – 2.54 (m, 1H), 2.57 – 2.46 (m, 1H), 2.44 (s,1H), 2.19 – 2.04 (m, 1H), 1.41 (s, 3H), 0.97 (s, 3H).ESI-MS (m / z): 587.1 (M +H) + . (8) Dissolve 70 mg of intermediate 13 in dichloromethane (5 mL) and trifluoroacetic acid (1.5 mL), stir at room temperature for 1 h, and then concentrate under reduced pressure to obtain intermediate 14, which can be used directly in the next step of the reaction without further purification; (9) Dissolve 120 mg of intermediate 14 and 103 mg of intermediate 10 in DMF (5 mL), add HATU (137 mg) and DIEA (200 µL), stir overnight at room temperature, concentrate the reaction solution, and purify by preparative high performance liquid chromatography to obtain the compound shown in Formula I-17. 1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.66 (s, 1H), 8.07 – 7.96 (m, 2H), 7.82 (d, J = 0.6 Hz, 1H), 7.80 – 7.72 (m, 2H), 7.63 – 7.57 (m, 2H),7.55 – 7.48 (m, 2H), 7.37 – 7.27 (m, 5H), 7.21 (d, J = 7.4 Hz, 1H), 7.10 –7.03 (m, 2H), 4.71 – 4.55 (m, 2H), 4.41 (td, J = 7.0, 0.8 Hz, 1H), 4.37 (ddt,J = 8.0, 4.9, 0.9 Hz, 3H), 4.13 (d, J = 7.4 Hz, 1H), 3.73 – 3.49 (m, 6H), 3.31 (td, J = 7.1, 2.0 Hz, 4H), 2.61 – 2.53 (m, 2H), 2.52 – 2.36 (m, 2H),2.37 (s, 2H), 2.19 – 2.04 (m, 2H), 1.56 (d, J = 6.8 Hz, 3H), 1.51 (d, J = 6.8Hz, 3H), 0.97 (s, 6H). HRMS (ESI-TOF) m / z calcd. for C 53 H 60 FN9O7S [M + H] + 986.43540, found 986.43416. The TNIK degrading agents with structures shown in Formulas I-2 to I-16 were prepared according to Example 1, and the TNIK degrading agents with structures shown in Formulas I-18 to I-32 were prepared according to Example 2. The characterization data are shown in Table 1.

[0047] Table 1 Characterization data of TNIK degradative agent

[0048] Experimental Example 1: Degradation of nLuc-TNIK in Jurkat Cells - Evaluation of the Protein Degrading Activity of TNIK Degrading Agents Jurkat cells expressing nLuc-TNIK were used at a rate of 1 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 99 μL of DMEM / FCS medium and placed in 96-well white plates for overnight incubation. Cells were then treated with DMSO or TNIK degrading agent as instructed. After 24 h, 100 μL of a Nano-Glo luciferase assay kit (Promega) was added to each well, and the cells were incubated at room temperature for 10 min. The luminescence intensity was then measured using a BioTek Synergy H1 plate, and the results were fitted to DC values. 50 Values. The results are shown in Table 2.

[0049] Table 2. Protein degradation activity of TNIK degraders

[0050] "++++" represents the half-maximal inhibitory concentration (MCC). 50 <10 μM; "+++" represents the half-maximal inhibitory concentration (MCC). 50 Between 10 μM and 30 μM; “++” represents the half-maximal inhibitory concentration (MCC). 50 Between 30 μM and 100 μM; "+" represents the half-maximal inhibitory concentration (DC). 50 Between 100 μM and 300 μM.

[0051] Experimental Example 2: The effect of the TNIK degrading agent of the present invention on inhibiting the proliferation of HCT116 In this experiment, DMEM medium was purchased from Gibco, penicillin and streptomycin were purchased from Hyclone, and CCK8 was purchased from Medchem Express.

[0052] HCT116 cells (colon cancer cells) were cultured in DMEM + 10% FBS + penicillin / streptomycin medium. During the experiment, cells in the logarithmic growth phase were collected and cultured at a specific number per well (2 × 10⁻⁶). 3Cells / wells were seeded in 96-well plates and cultured overnight at 37°C with 5% CO2. The next day, the TNIK degradation agent was diluted with culture medium and added to the corresponding wells of the 96-well plates, with three replicates per sample. A solvent control group and a blank control group containing only culture medium were also set up. The plates were cultured in a cell incubator for 72 h, and then 10 μL of CCK8 solution was added to each well and incubated for 1–3 h. The absorbance was then measured at 495 nm using a microplate reader, and the cell proliferation inhibition rate was calculated using the following formula: Cell proliferation inhibition rate = [(X-C0) / (C-C0)] × 100% In the formula, C, C0, and X represent the average absorbance values ​​of the solvent control group, blank control group, and TNIK degradation agent treatment group, respectively. Finally, the cell survival rate curve was fitted using Graphpad Prism 8.0 software, and the IC50 value of the TNIK degradation agent inhibiting cell proliferation was calculated. 50 The values ​​are shown in Table 3; the survival curve of HCT116 cells inhibited by the TNIK degrader I-18 is shown in Table 3. Figure 1 .

[0053] Table 3. Levels of TNIK degradative agents inhibiting HCT116 proliferation

[0054] "++++" represents the half-maximal inhibitory concentration (IC50). 50 <30 μM; "+++" represents the half-maximal inhibitory concentration (IC50). 50 Between 30 μM and 50 μM; "++" represents the half-maximal inhibitory concentration (IC50). 50 Between 50 μM and 100 μM; "+" represents the half-maximal inhibitory concentration (IC50). 50 Between 100 μM and 300 μM.

[0055] like Figure 1 As shown, the IC50 of TNIK degrader I-18 inhibits the proliferation of HCT116 cells. 50 The value was 25.06 μM. As can be seen from the results in Table 3, the TNIK degrading agent of this invention can inhibit the proliferation of HCT116 cells.

[0056] Experimental Example 3: The degradation effect of the TNIK degrading agent I-18 of the present invention on TNIK protein. In this experimental example, the RIPA lysis buffer was purchased from Beyotime Biotechnology Research Institute, the PMSF protease inhibitor was purchased from Sigma-Aldrich, and sodium dodecyl sulfate (SDS), glycine, acrylamide, tris(hydroxymethyl)aminomethane (Tris), ammonium persulfate (APS), N,N,N',N'-tetramethylethylenediamine (TEMED), and sodium carboxymethyl cellulose were all purchased from Sigma-Aldrich.

[0057] Extraction of total cellular protein: After treating cells with TNIK degradation agent I-18 or blank solvent for 24 h in the cell supernatant, discard the supernatant, wash three times with pre-cooled PBS or physiological saline, add RIPA lysis buffer (containing 1% cocktail and 1% PMSF protease inhibitor), and immediately place on ice for 15 min of lysis. After 15 min, scrape the cell lysate with a spatula and transfer to a 1.5 mL EP tube, and sonicate the cells. Then, centrifuge the tube in a low-temperature high-speed centrifuge (12000 rpm, 15 min) to remove cell debris. Quantify protein using the BCA method and construct a standard curve using protein standards. Calculate the protein concentration of each sample based on the standard curve, and then balance the concentrations of each group of protein samples. Add 5x protein loading buffer and incubate at 100℃ in a dry incubator for 10 min. Then, directly load the samples for electrophoresis or aliquot and store at -20℃ for later use. Avoid repeated freeze-thaw cycles for protein samples. After protein sample preparation, proteins were separated using polyacrylamide gel electrophoresis (SDS-PAGE). The polyacrylamide gel formulation is shown in Table 4, and a 10% separating gel was generally used. After electrophoretic separation, the proteins were fully transferred to a PVDF membrane using a wet transfer method. The PVDF membrane was then blocked at room temperature for at least 2 hours in 5% skim milk powder (prepared with TBS / T). PVDF membrane strips containing the desired protein molecular weight were obtained. The primary antibody was diluted according to the dilution ratio recommended in the antibody instructions, and the protein strips were incubated overnight at 4°C. The next day, each strip was removed, washed with TBS / T buffer (5 min, 3 times), and HRP-labeled secondary antibody diluted 1:5000 was added. The membrane was incubated at 37°C with shaking for 1 hour. Excess antibody was then eluted with TBS / T. HRP substrate was evenly added to the PVDF membrane, and the membrane was developed and photographed using a rapid gel imaging system. The results are shown in the table below. Figure 2 .

[0058] Table 4. Formulations of separating gel and stacking gel in SDS-PAGE

[0059] from Figure 2 As can be seen from this, the TNIK degrading agent I-18 of the present invention can degrade TNIK protein in a dose-dependent manner.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A TNIK degrading agent, characterized in that, The TNIK degrading agent has the structure shown in Formula I: In this context, R1 is an alkyl or alkoxy group, and R2 is an E3 ubiquitin ligand.

2. The TNIK degrading agent according to claim 1, characterized in that, In the structure shown in Equation I, R2 is... , , , , , , , , or .

3. The TNIK degrading agent according to claim 1 or 2, characterized in that, The specific structure is as follows: 。 4. The method for preparing the TNIK degrading agent according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) 4-(4-fluorophenyl)-1H-imidazolium and 2-bromopropane were subjected to a substitution reaction to obtain intermediate 3; (2) Intermediate 3 was subjected to an iodination reaction to obtain intermediate 4; (3) Under an inert atmosphere, intermediate 4 and (5-formyl-2-furanyl)boronic acid were coupled to obtain intermediate 6; (4) Hydrolyze intermediate 6 to obtain intermediate 7; (5) Intermediate 7 and 4-(4-tert-butyloxycarbonylpiperazin-1-yl)aniline were subjected to a first amidation reaction to obtain intermediate 9; (6) Intermediate 9 was subjected to a first acid hydrolysis reaction to obtain intermediate 10; (7) The E3 ubiquitin ligase ligand is subjected to a nucleophilic substitution reaction or a second amidation reaction with compound 12 to obtain intermediate 13. (8) The intermediate 13 was subjected to a second acid hydrolysis reaction to obtain the intermediate 14; (9) Intermediate 10 and intermediate 14 were subjected to a third amidation reaction to obtain the compound shown in Formula I; the synthetic route is as follows: 。 5. The method for preparing the TNIK degrading agent according to claim 4, characterized in that, Step (1) includes the following steps: dissolving 4-(4-fluorophenyl)-1H-imidazolium in an organic solvent, adding sodium hydride, stirring at -2~2℃ for 20~40 min, adding 2-bromopropane, and then carrying out a substitution reaction at 20~30℃ for 2~4 h; the iodination reaction in step (2) is carried out under the action of N-iodosuccinimide and trifluoroacetic acid at a temperature of 8~12℃ for 10~14 h; the coupling reaction in step (3) is carried out under the action of K3PO4 and a catalyst at a temperature of 90~95℃ for 4~6 h; the hydrolysis reaction in step (4) is carried out under the action of an inorganic base at a temperature of 25~35℃ for 1.5~2.5 h; the first amidation reaction in step (5) is carried out under the action of a condensing agent and an organic base catalyst at a temperature of 20~30℃ for 1.5~2.5 h. h; The first acidolysis reaction in step (6) is carried out in a 1,4-dioxane solution of hydrogen chloride at room temperature for 1-2 h.

6. The method for preparing the TNIK degrading agent according to claim 5, characterized in that, In step (1), the molar ratio of 4-(4-fluorophenyl)-1H-imidazolium, 2-bromopropane, and sodium hydride is (100~130):(200~250):(130~150); in step (2), the molar ratio of intermediate 3, N-iodosuccinimide, and trifluoroacetic acid is (100~110):(300~310):(20~40); in step (3), the molar mass to volume ratio of intermediate 4, (5-formyl-2-furanyl)boronic acid, K3PO4, and catalyst is (1~2) mmol:(2~3) mmol:(3~4) mL:(0.1~0.2). mmol; the molar ratio of intermediate 6 and inorganic base in step (4) is (0.1~0.2):(1~2); the molar ratio of intermediate 7, 4-(4-tert-butyloxycarbonylpiperazin-1-yl)aniline, condensing agent and organic base catalyst in step (5) is (70~80):(150~160):(150~160):(200~240); the feed-liquid ratio of intermediate 9 and 1,4-dioxane solution of hydrogen chloride in step (6) is (40~60) mg:(5~15) mL.

7. The method for preparing the TNIK degrading agent according to claim 4, characterized in that, In step (7), when the E3 ubiquitin ligand contains a halogen group, R3 in compound 12 is -NH2, and the E3 ubiquitin ligand undergoes a nucleophilic substitution reaction with compound 12. The nucleophilic substitution reaction is carried out under the action of an organic base catalyst at a temperature of 85~95℃ for 1.5~2.5h. The molar ratio of the E3 ubiquitin ligand, compound 12, and organic base catalyst is (0.1~0.2):(0.2~0.3):(0.3~0.4). When the E3 ubiquitin ligand contains an amino group, R3 in compound 12 is -COOH, and the E3 ubiquitin ligand undergoes a second amidation reaction with compound 12. The second amidation reaction is carried out under the action of an organic base catalyst and a condensing agent at room temperature for 10~14h. The feed-to-liquid ratio of the E3 ubiquitin ligand, compound 12, organic base catalyst, and condensing agent is (50~60). mg: (120~160) mg: (300~350) μL: (200~210) mg.

8. The method for preparing the TNIK degrading agent according to claim 4, characterized in that, The second acidolysis reaction in step (8) is carried out under the action of trifluoroacetic acid at room temperature for 0.5-1.5 h; the third amidation reaction in step (9) is carried out under the action of an organic base catalyst and a condensing agent at room temperature for 2-14 h.

9. The method for preparing the TNIK degrading agent according to claim 8, characterized in that, In step (8), the ratio of intermediate 13 to trifluoroacetic acid is (40~80) mg:(0.2~2) mL; in step (9), the ratio of intermediate 10, intermediate 14, organic base catalyst and condensing agent is (60~120) mg:(40~150) mg:(40~300) μL:(70~150) mg.

10. The use of the TNIK degrading agent according to any one of claims 1 to 3 in the preparation of medicaments for treating and / or preventing cancer.

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