Micromolecular degradation agent taking FGFR2 as target point as well as pharmaceutical composition and application of micromolecular degradation agent

Abstract

The invention discloses a micromolecule degradation agent taking FGFR2 as a target spot as well as a pharmaceutical composition and application thereof, and belongs to the technical field of pharmaceutical chemistry. The compound provided by the invention can be used as an FGFR2 kinase inhibitor and also can be used as a bifunctional molecule of an FGFR2 protein degradation agent. According to the compound, an FGFR2 inhibitor TAS-120 is used as a target binding part of targeted protein of a protein degradation agent, a thalidomide derivative, a hydrophobic tag and a molecular film segment are coupled through different connectors, a bifunctional molecule which can strongly inhibit the activity of FGFR2 kinase and promote degradation of FGFR2 protein is obtained, proliferation of various cancer cells can be inhibited, and the compound has a good application prospect. The compound has good potential of being developed into anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a class of small molecule degraders targeting FGFR2, their pharmaceutical compositions and uses. Background Art

[0002] Cancer, as one of the major diseases seriously threatening human physiological health at present, in the long-term practice and exploration of cancer treatment, people have gradually discovered that the occurrence of cancer is closely related to specific pathogenic genes. Based on this, targeted treatment means have been developed, and numerous excellent small molecule inhibitors have been successfully developed. However, as the treatment time goes by, the target protein of the treatment mutates, resulting in acquired drug resistance of small molecule drugs, and the drug efficacy is significantly reduced or even lost. To solve this problem, researchers need to update and improve the original small molecule inhibitors that have developed drug resistance, and then design and develop second-generation, third-generation, fourth-generation and even newer-generation inhibitors. This drug iteration process is often accompanied by high human and time costs. Therefore, there is an urgent need for a new targeted drug design strategy to open up new ways for cancer treatment.

[0003] Targeted protein degradation technology (TPD) utilizes two major natural protein degradation systems in cells - the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system (ALS) - to induce the target protein to be degraded by the proteasome or lysosome. Compared with small molecule inhibitors, targeted protein degraders do not need to occupy the binding cavity of the target protein for a long time, thus reducing the risk of target protein mutation, and at the same time expanding the application of those "undruggable" targets. At present, degradation tools such as proteolysis-targeting chimeras (PROTACs), molecular glues and hydrophobic tags (HyTDDs) have become research hotspots, and a large number of excellent anti-tumor drugs have emerged one after another, such as the PROTAC drug ARV-471 targeting estrogen receptor (ER), the molecular glue drug thalidomide for the treatment of multiple myeloma, and the HyTDD drug MS1943 targeting EZH2. It can be seen that the TPD technology shows extremely broad treatment prospects.

[0004] Fibroblast growth factor receptors (FGFR1-4) belong to the transmembrane receptor tyrosine kinase family. As the main high-affinity binding sites for fibroblast growth factors (FGF), the FGF / FGFR signaling pathway mediated by them plays a key role in regulating physiological processes such as cell proliferation, differentiation, and survival. However, when this signaling pathway is abnormally activated, it may trigger human malignancies including various tumors. For example, FGFR2 amplification was detected in 7.7% of advanced gastric cancer patients, and fusion proteins such as FGFR2-PPHLN1 and FGFR2-CCDC6 were found in cholangiocarcinoma, indicating that FGFRs are important targets for developing new anticancer drugs. To date, two generations of inhibitors have been developed for FGFRs, from the first-generation multi-target inhibitor Derazantinib to the second-generation irreversible covalent targeting inhibitor TAS-120, and TAS-120 has been approved for the treatment of FGFR2 fusion cholangiocarcinoma. However, in the phase I clinical trial of TAS-120, it was found that most patients developed hyperphosphatemia after treatment, and in patients previously treated with irreversible RTK inhibitors, cancer cells developed drug resistance through cysteine residue mutations. To overcome the problem of acquired drug resistance of small molecule drugs and avoid the disadvantages brought by drug iteration, designing proteolysis-targeting chimeras (PROTACs) using TPD technology has become a promising therapeutic strategy. Summary of the Invention

[0005] The primary technical problem to be solved in this application is to provide a class of compounds that can effectively degrade FGFR2 protein, which uses Futibatinib (TAS-120) as the ligand of FGFR2 protein, selects thalidomide derivatives, hydrophobic tags, and molecular glue fragments as ligands for E3 ubiquitin ligases, and couples the former two with a covalent or non-covalent linker (Linker). The secondary technical problem to be solved in this application is to provide a drug or pharmaceutical composition for treating diseases containing the aforementioned compounds. Another technical problem to be solved in this application is to provide the specific application of the aforementioned compounds, which is used to prepare a degrader that can effectively degrade FGFR2 and has three different modes of action to promote FGFR2 degradation. The last technical problem to be solved in this application is to provide the application of the aforementioned compounds in preparing drugs for treating diseases, such as anti-tumor drugs.

[0006] To solve the above technical problems, the technical solutions of this application are as follows:

[0007] A class of small molecule degraders targeting FGFR2, wherein the small molecule degraders are compounds of the structure shown in Formula I or Formula II or their isomers, pharmaceutically acceptable salts, or mixtures thereof:

[0008]

[0009] Wherein:

[0010] The linker in Formula I is: , , , , or ,

[0011] X is: O or NH,

[0012] In Formula II, R is: , , or .

[0013] In some embodiments of the present application, the small molecule degrader targeting FGFR2 is any one of the following compounds NT-01 to 13:

[0014] NT-01:

[0015] .

[0016] NT-02:

[0017] .

[0018] NT-03:

[0019] .

[0020] NT-04:

[0021] .

[0022] NT-05:

[0023] .

[0024] NT-06:

[0025] .

[0026] NT-07:

[0027] .

[0028] NT-08:

[0029] .

[0030] NT-09:

[0031] .

[0032] NT-10:

[0033] 。

[0034] NT-11:

[0035] 。

[0036] NT-12:

[0037] 。

[0038] NT-13:

[0039] 。

[0040] The structural formulas of the representative compounds described in this application are shown above, but the scope of protection is not limited to these compounds only, and also includes their derivatives and analogs.

[0041] In some embodiments, the pharmaceutically acceptable salt is a salt formed by the compound having the structure shown in Formula I or Formula II and an acid selected from any one of the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

[0042] A pharmaceutical composition comprising the small molecule degrader targeting FGFR and a pharmaceutically acceptable carrier or excipient.

[0043] Use of the small molecule degrader compound targeting FGFR or the pharmaceutical composition in the preparation of a drug for disease prevention and / or treatment.

[0044] In some embodiments, the disease is a disease caused by overexpression of FGFR2.

[0045] In some embodiments, the disease caused by overexpression of FGFR is specifically a tumor.

[0046] In some embodiments, the tumor is liver cancer, cholangiocarcinoma, breast cancer, bladder cancer or esophageal cancer.

[0047] Use of the small molecule degrader targeting FGFR2 or the pharmaceutical composition in the preparation of an FGFR2 inhibitor and / or an FGFR2 degrader.

[0048] A method for degrading FGFR2 in cells is to contact the cells with the small molecule degrader targeting FGFR2 or the pharmaceutical composition.

[0049] The specific synthetic routes of the above target compounds NT-01 to 13 are shown in Equations I-IV as follows:

[0050]

[0051] Equation I

[0052] Reaction conditions and reagents: (a) PPh3, DIAD, THF, N2, 0 °C to r.t., 5 h; (b) Pd(PPh3)4, DIPEA, CuI, N2, DMF, 80 °C, 8 h; (c) HCl / 1,4-dioxane, r.t., 2 h.

[0053]

[0054] Equation II

[0055] Reaction conditions and reagents: (a) DCC, DMAP, DCM, r.t., 24 h; (b) Pd / C, H2, HOAC, MeOH, 50 °C, 15 h; (c) DIPEA, DMSO, 90 °C, 10 h; (d) TFA, DCM, r.t., 4 h; (e) NaHCO3, KI, DMF, 80 °C, 10 h; (f) HCl / 1,4-dioxane, DCM, r.t., 2 h; (g) 2 nd Grubbs, N2, THF, 40 °C, 10 h; (h) HATU, DIPEA, DMF, 45 °C, 6 h.

[0056]

[0057] Equation III

[0058] Reaction conditions and reagents: (a) Et2O, r.t., 2 h; (b) HATU, DIPEA, DMF, 45 °C, 6 h.

[0059]

[0060] Equation IV

[0061] Reaction conditions and reagents: (a) HATU, DIPEA, DMF, 45 °C, 6 h; (b) TEA, DCM, 0 °C to r.t., 10 min; (c) Fe, NH4Cl, EtOH, 70 °C, 8 h; (d) Et2O, r.t., 2 h.

[0062] Compared with the prior art, the beneficial effects of this application are as follows:

[0063] In this application, the POI (Protein of Interest) binding part of TAS-120 is used as a protein degrader. By selecting different linkers, an E3 ubiquitin ligase ligand, a hydrophobic tag, and an aromatic ring with a molecular glue effect are coupled to obtain a small molecule degrader that can promote the degradation of FGFR2 protein. In addition, this type of compound also retains the inhibitory activity of the parent compound against the target, and the inhibitory activity of most of these compounds against the FGFR2 enzyme is better than that of the positive control TAS-120. In addition, the inhibitory activity of most of the compounds in this application against tumor cell proliferation is better than that of the positive control, with excellent effects and good potential for development as anti-tumor drugs. Description of the Drawings

[0064] Figure 1 It is a detection result diagram of the degradation activity of the target compound against the FGFR2 protein. Detailed Description of the Invention

[0065] The following further elaborates on this application in conjunction with the examples, but the protection scope of this application is not limited to these examples. All reagents in the examples are of analytical grade. The nuclear magnetic spectra of the compounds were measured by a Bruker ARX-600 nuclear magnetic resonance spectrometer with TMS as the internal standard; high-resolution mass spectrometry was measured by an Agilent6224 TOF LC / MS instrument.

[0066] Example 1

[0067] Synthesis of Intermediate Compound IM-3

[0068] Weigh compound IM-1 (1.04 g, 4.0 mmol), compound IM-2 (1.12 g, 6.0 mmol) and PPh3 (1.57 g, 6.0 mmol) into a two-necked flask, add 25.0 mL of anhydrous tetrahydrofuran (THF) as the reaction solvent, and inject diisopropyl azodicarboxylate (DIAD, 1.21 g, 6.0 mmol) into the reaction flask under the protection of N2. After stirring in an ice bath for 10 min, slowly return to room temperature and continue stirring the reaction for 5 h. After the reaction is completed, concentrate the reaction solution under reduced pressure and purify it by column chromatography. The eluent is dichloromethane / methanol (DCM / MeOH) (v:v = 20:1) to obtain the white intermediate compound IM-3 (1.85 g, yield: 72%).

[0069] 1 H NMR (600 MHz, DMSO-d6) δ 8.21 (s, 1H), 7.85 (s, 1H), 6.59 (s, 1H), 5.34 (d, J = 5.2 Hz, 1H), 3.74 - 3.69 (m, 1H), 3.58 - 3.49 (m, 2H), 3.43 - 3.37 (m, 1H), 2.34 - 2.28 (m, 2H), 1.40 (d, J = 18.7 Hz, 9H) ppm.

[0070] Example 2

[0071] Synthesis of intermediate compound IM-5

[0072] Weigh compound IM-3 (1.29 g, 3.0 mmol), 1-ethynyl-3,5-dimethoxybenzene (IM-4, 0.58 g, 3.6 mmol), Pd(PPh3)4 (0.17 g, 0.2 mmol), DIPEA (0.58 g, 4.5 mmol) and CuI (0.17 g, 0.9 mmol) into a flask, add DMF (30.0 mL) to dissolve the mixture, and heat the reaction system to 80 °C and stir the reaction for 8 h under a nitrogen atmosphere. After the reaction is completed, cool the reaction system to room temperature, add water (50.0 mL) to the reaction flask, and then extract with ethyl acetate (EA) (30.0 mL × 3). Collect the organic phase, wash it again with saturated NaCl solution (30.0 mL), and then dry the organic phase over anhydrous sodium sulfate and concentrate it under reduced pressure. Finally, purify it by column chromatography (DCM:MeOH = 10:1) to obtain the pale yellow intermediate compound IM-5 (0.73 g, yield: 53%).

[0073] 11H NMR (600 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.99 (s, 1H), 6.92 (s, 2H), 6.75 (s, 1H), 6.61 (s, 1H), 5.42 (d, J = 5.5 Hz, 1H), 3.79 (s, 6H), 3.76 - 3.74 (m, 1H), 3.63 - 3.60 (m, 1H), 3.56 - 3.52 (m, 1H), 3.45 (d, J = 5.6 Hz, 1H), 2.40 - 2.34 (m, 1H), 2.31 - 2.29 (m, 1H), 1.41 (d, J = 17.4 Hz, 9H) ppm。

[0074] Example 3

[0075] Synthesis of Intermediate Compound IM-6

[0076] Weigh 0.70 g (1.5 mmol) of Compound IM-5 and dissolve it in 5.0 mL of DCM. Add 5.0 mL of 4.0 mol / L HCl / 1,4-dioxane solution and stir at room temperature for 2 h. Monitor the reaction process by thin-layer chromatography (TLC) until the reactants are completely consumed. After the reaction is completed, concentrate the reaction solvent under reduced pressure. Add a small amount of water (10.0 mL) to the reaction flask and adjust the pH of the solution to weakly basic with saturated NaHCO3 solution. A large amount of solid precipitates. Filter by suction to obtain the filter cake, and dry it under vacuum to obtain the light yellow target intermediate compound IM-6 (0.50 g, yield: 90%).

[0077] 1 1H NMR (600 MHz, DMSO-d6) δ 8.24 (s, 1H), 7.96 (s, 1H), 6.91 (d, J = 2.2 Hz, 2H), 6.74 (s, 1H), 6.61 (t, J = 2.1 Hz, 1H), 5.31 - 5.27 (m, 1H), 3.79 (s, 6H), 3.78 - 3.77 (m, 1H), 3.23 - 3.20 (m, 1H), 3.12 - 3.10 (m, 1H), 3.02 - 2.99 (m, 1H), 2.95 - 2.90 (m, 1H), 2.24 - 2.18 (m, 1H), 2.11 - 2.06 (m, 1H) ppm。

[0078] Example 4

[0079] Synthesis of Intermediate Compound IM-9

[0080] Weigh 0.80 g (3.0 mmol) of compound IM-7, 0.27 g (3.7 mmol) of tert-butanol (IM-8) and 0.04 g (0.3 mmol) of DMAP, dissolve them in 20.0 mL of DCM. After reacting for 10 min, add 0.74 g (3.6 mmol) of DCC to the system and react at room temperature for 24 h. After the reaction is completed, filter the reaction solution with a Buchner funnel and collect the filtrate. Concentrate the filtrate under reduced pressure to remove the excess solvent, and purify the crude product by column chromatography. The eluent is petroleum ether / ethyl acetate (PE / EA) (v : v = 1 : 1), and obtain the oily colorless intermediate compound IM-9 (0.63 g, yield: 65%).

[0081] 1 1H NMR (600 MHz, DMSO-d6) δ 7.37 - 7.29 (m, 5H), 7.23 (t, J = 5.5 Hz, 1H), 5.00 (s, 2H), 2.98 - 2.95 (m, 2H), 2.16 (t, J = 7.3 Hz, 2H), 1.49 - 1.44 (m, 2H), 1.39 (s, 9H), 1.37 - 1.35 (m, 2H), 1.26 - 1.21 (m, 2H) ppm.

[0082] Example 5

[0083] Synthesis of Intermediate Compound IM-10

[0084] Weigh 0.32 g (1.0 mmol) of the intermediate product IM-9, 0.03 g of Pd / C and an appropriate amount of acetic acid (3.0 mL) into a two-necked flask, add an appropriate amount of methanol (10.0 mL) to dissolve the mixture, and heat the system to 50 °C in a hydrogen atmosphere and react for 15 h. After detecting the completion of the reaction by TLC, cool the reaction solution to room temperature, filter it with diatomaceous earth, collect the filtrate, and concentrate it under reduced pressure to obtain the oily colorless target intermediate compound IM-10 (0.18 g, yield: 95%).

[0085] 11H NMR (600 MHz, DMSO-d6) δ 3.38 - 3.35 (m, 2H), 3.21 (d, J = 1.5 Hz, 2H), 2.88 (t, J = 7.3 Hz, 2H), 2.20 (d, J = 7.3 Hz, 2H), 2.16 (d, J = 7.5 Hz, 2H), 2.10 (s, 9H), 2.00 - 1.95 (m, 2H) ppm。

[0086] Example 6

[0087] Synthesis of Intermediate Compound IM-14

[0088] Weigh 0.38 g (2.3 mmol) of tert-butyl glycinate hydrochloride (IM-12) and 0.78 g (6.0 mmol) of DIPEA and dissolve them in 10.0 mL of DMSO. Heat the system to 60 °C and stir for 30 min, then add 0.41 g (1.5 mmol) of compound IM-11, and continue to heat to 90 °C for reaction for 10 h. After the reaction is completed, cool the reaction solution to room temperature, add 30.0 mL of water to the reaction flask, then extract with EA (20.0 mL × 3), collect the organic phase and wash it again with saturated NaCl solution (10.0 mL). Then dry the organic phase with anhydrous sodium sulfate and concentrate it under reduced pressure to remove the excess solvent. Finally, the crude product is purified by column chromatography (PE:EA = 3:1) to obtain the yellow intermediate compound IM-14 (0.35 g, yield: 60%).

[0089] 1 1H NMR (600 MHz, DMSO-d6) δ 11.12 (s, 1H), 7.60 - 7.57 (m, 1H), 7.08 (d, J = 7.0 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 5.09 - 5.06 (m, 1H), 4.09 (s, 2H), 2.92 - 2.86 (m, 1H), 2.61 - 2.56 (m, 2H), 2.54 - 2.52 (m, 1H), 2.05 - 2.03 (m, 1H), 1.43 (s, 9H) ppm。

[0090] Example 7

[0091] Synthesis of Intermediate Compound IM-15

[0092] Using IM-11 and tert-butyl 4-aminobutyrate hydrochloride (IM-13) as starting materials and referring to the synthesis method of intermediate IM-14, the yellow intermediate compound IM-15 was obtained (yield: 63%).

[0093] 1 H NMR (600 MHz, DMSO-d6) δ 11.10 (s, 1H), 7.60 - 7.57 (m, 1H), 7.11 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.66 (t, J = 5.8 Hz, 1H), 5.07 - 5.04 (m, 1H), 3.33 - 3.30 (m, 2H), 2.91 - 2.85 (m, 1H), 2.60 - 2.52 (m, 2H), 2.28 (t, J = 7.3 Hz, 2H), 2.04 - 2.00 (m, 1H), 1.79 - 1.75 (m, 2H), 1.39 (s, 9H) ppm。

[0094] Example 8

[0095] Synthesis of Intermediate Compound IM-16

[0096] Using IM-10 and IM-11 as starting materials and referring to the synthesis method of intermediate IM-14, the yellow intermediate compound IM-16 was obtained (yield: 66%).

[0097] 1 H NMR (600 MHz, DMSO-d6) δ 11.08 (d, J = 13.0 Hz, 1H), 7.62 - 7.56 (m, 1H), 7.09 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 6.9 Hz, 1H), 6.54 (t, J = 4.9 Hz, 1H), 5.10 - 5.03 (m, 1H), 3.46 - 3.43 (m, 1H), 3.31 - 3.28 (m, 1H), 2.91 - 2.85 (m, 1H), 2.60 - 2.53 (m, 2H), 2.20 - 2.14 (m, 2H), 2.03 - 2.01 (m, 1H), 1.58 - 1.56 (m, 2H), 1.54 - 1.47 (m, 2H), 1.37 (s, 9H), 1.33 (d, J = 6.6 Hz, 1H), 1.26 - 1.18 (m, 1H) ppm。

[0098] Example 9

[0099] Synthesis of Intermediate Compound IM-17

[0100] Weigh 0.35 g (0.9 mmol) of compound IM-14 and dissolve it in 10.0 mL of DCM. Then, add 1.0 mL of trifluoroacetic acid (TFA) dropwise and stir at room temperature for 2 h. After monitoring the reaction by TLC until completion, add saturated NaHCO3 to the reaction flask to adjust the pH of the solution to weakly basic. Extract it once with EA (10.0 mL), collect the aqueous phase, add 1.0 mol / L HCl to the aqueous phase to adjust the pH of the solution to weakly acidic, and then extract it three times with EA (10.0 mL). Collect the organic phase, wash it once with saturated NaCl solution (10.0 mL), then dry the organic phase with anhydrous sodium sulfate, and concentrate it under reduced pressure to remove the excess solvent to obtain the yellow target intermediate compound IM-17 (0.23 g, yield: 75%).

[0101] 1 H NMR (600 MHz, DMSO-d6) δ 12.90 (s, 1H), 11.12 (s, 1H), 7.58 (t, J =7.7 Hz, 1H), 7.08 (d, J = 7.0 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 6.86 (t, J =5.2 Hz, 1H), 5.09-5.06 (m, 1H), 4.11 (d, J = 5.6 Hz, 2H), 2.92-2.86 (m, 1H),2.61-2.54 (m, 2H), 2.05-2.03 (m, 1H) ppm.

[0102] Example 10

[0103] Synthesis of Intermediate Compound IM-18

[0104] Using IM-15 as the starting material and referring to the synthesis method of IM-17, the yellow target intermediate compound IM-18 was obtained (yield: 76%).

[0105] 11H NMR (600 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.11 (s, 1H), 7.59 - 7.57(m, 1H), 7.13 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.66 (t, J = 6.0Hz, 1H), 5.07 - 5.04 (m, 1H), 3.32 - 3.30 (m, 2H), 2.89 - 2.86 (m, 1H), 2.60 - 2.52(m, 2H), 2.31 (t, J = 7.2 Hz, 2H), 2.04 - 2.01 (m, 1H), 1.81 - 1.77 (m, 2H) ppm。

[0106] Example 11

[0107] Synthesis of Intermediate Compound IM-19

[0108] Using IM-16 as the starting material and referring to the synthesis method of IM-17, the yellow target intermediate compound IM-19 was obtained (yield: 73%), which could be directly used for the next step without further purification.

[0109] ESI-MS(m / z): [M + H] + = 388.1464。

[0110] Example 12

[0111] Synthesis of Intermediate Compound IM-21

[0112] Weigh 0.82 g (3.0 mmol) of compound IM-20, 0.50 g (6.0 mmol) of NaHCO3 and 0.05 g (0.3 mmol) of KI into a flask, add 10.0 mL of DMF to dissolve the mixture, heat the system to 60 °C and stir for 30 min, then add 0.70 g (3.6 mmol) of tert-butyl bromoacetate to the reaction flask, and continue to heat to 80 °C for reaction for 10 h. After the reaction is completed, cool the reaction solution to room temperature, add 30.0 mL of water to the reaction flask, extract with EA (20.0 mL × 3), collect the organic phase and wash it once with saturated NaCl solution (10.0 mL). Then dry the organic phase with anhydrous sodium sulfate and concentrate it under reduced pressure to remove the excess solvent. Finally, purify the crude product by column chromatography (DCM : EA = 1 : 1) to obtain the white intermediate compound IM-21 (0.73 g, yield: 63%).

[0113] 11H NMR (600 MHz, DMSO-d6) δ 11.12 (s, 1H), 7.81-7.79 (m, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 5.12-5.09 (m, 1H), 4.97 (s, 2H), 2.89 (s, 1H), 2.59 (d, J = 17.8 Hz, 1H), 2.56-2.53 (m, 1H), 2.06-2.03 (m, 1H), 1.43 (s, 9H) ppm。

[0114] Example 13

[0115] Synthesis of Intermediate Compound IM-22

[0116] Using IM-20 and tert-butyl 4-bromobutyrate as starting materials and referring to the synthesis method of compound IM-21, the yellow intermediate compound IM-22 was obtained (yield: 65%).

[0117] 1 1H NMR (600 MHz, DMSO-d6) δ 11.12 (s, 1H), 7.82-7.80 (m, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.10-5.07 (m, 1H), 4.21 (t, J = 6.3 Hz, 2H), 2.91-2.85 (m, 1H), 2.60-2.52 (m, 2H), 2.43 (t, J = 7.5 Hz, 2H), 2.04-2.01 (m, 1H), 1.98-1.94 (m, 2H), 1.39 (s, 9H) ppm。

[0118] Example 14:

[0119] Synthesis of Intermediate Compound IM-23

[0120] Using IM-20 and tert-butyl 6-bromohexanoate as starting materials and referring to the synthesis method of compound IM-21, the yellow intermediate compound IM-23 was obtained (yield: 60%).

[0121] 11H NMR (600 MHz, DMSO-d6) δ 11.12 (s, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.09 - 5.06 (m, 1H), 4.19 (t, J = 6.3 Hz, 2H), 2.91 - 2.85 (m, 1H), 2.58 (d, J = 17.5 Hz, 1H), 2.54 - 2.52 (m, 1H), 2.21 (t, J = 7.3 Hz, 2H), 2.04 - 2.00 (m, 1H), 1.78 - 1.73 (m, 2H), 1.59 - 1.54 (m, 2H), 1.48 - 1.42 (m, 2H), 1.38 (s, 9H) ppm。

[0122] Example 15

[0123] Synthesis of Intermediate Compound IM-24

[0124] Weigh 0.58 g (1.5 mmol) of Compound IM-21 and dissolve it in 10.0 mL of DCM. Add 3.0 mL of 4.0 mol / L HCl / 1,4-dioxane solution dropwise and react at room temperature for 2 h. After monitoring the reaction by TLC until completion, add saturated NaHCO3 to the reaction flask to adjust the pH of the solution to weakly basic, extract it once with 10.0 mL of EA, collect the aqueous phase, add 1.0 mol / L HCl to the aqueous phase to adjust the pH of the solution to weakly acidic, a large amount of solid precipitates, filter by suction to obtain the filter cake, and vacuum dry it to obtain the pale yellow target intermediate compound IM-24 (0.44 g, yield: 88%).

[0125] 1 1H NMR (600 MHz, DMSO-d6) δ 13.26 (s, 1H), 11.12 (s, 1H), 7.81 - 7.78 (m, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 5.12 - 5.09 (m, 1H), 4.99 (s, 2H), 2.92 - 2.86 (m, 1H), 2.61 - 2.58 (m, 1H), 2.56 - 2.53 (m, 1H), 2.06 - 2.02 (m, 1H) ppm。

[0126] Example 16

[0127] Synthesis of Intermediate Compound IM-25

[0128] Using IM-22 as the starting material and referring to the synthesis method of IM-24, the yellow target intermediate compound IM-25 was obtained (yield: 85%).

[0129] 1 H NMR (600 MHz, DMSO-d6) δ 12.20 (s, 1H), 11.12 (s, 1H), 7.82 - 7.80(m, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.10 - 5.07 (m,1H), 4.23 (t, J = 6.3 Hz, 2H), 2.91 - 2.87 (m, 1H), 2.60 - 2.52 (m, 2H), 2.45 (t,J = 7.4 Hz, 2H), 2.04 - 2.00 (m, 1H), 1.99 - 1.95 (m, 2H) ppm.

[0130] Example 17

[0131] Synthesis of Intermediate Compound IM-26

[0132] Using IM-23 as the starting material and referring to the synthesis method of compound IM-24, the yellow target intermediate compound IM-26 was obtained (yield: 90%), which can be directly used for the next step without further purification.

[0133] ESI-MS(m / z): [M + H] + = 389.1304.

[0134] Example 18

[0135] Synthesis of Intermediate Compound IM-27

[0136] Using IM-20 and 4-bromo-1-butene as the starting materials and referring to the synthesis method of compound IM-21, the light yellow intermediate compound 2-27 was obtained (yield: 53%).

[0137] 11H NMR (600 MHz, DMSO-d6) δ 11.12 (s, 1H), 7.81 (t, J = 7.6 Hz, 1H),7.53 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 6.9 Hz, 1H), 5.94 - 5.90 (m, 1H), 5.21(d, J = 17.0 Hz, 1H), 5.11 - 5.07 (m, 2H), 4.26 (t, J = 5.6 Hz, 2H), 2.87 (t, J= 15.2 Hz, 1H), 2.58 (d, J = 18.1 Hz, 2H), 2.53 (d, J = 5.9 Hz, 2H), 2.03 - 2.01 (m, 1H) ppm。

[0138] Example 19

[0139] Synthesis of Intermediate Compound IM-28

[0140] Using IM-20 and 5-bromo-1-pentene as starting materials and referring to the synthesis method of compound IM-21, a pale yellow intermediate compound IM-28 (yield: 50%) was obtained.

[0141] 1 1H NMR (600 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.82 - 7.99 (m, 1H), 7.51 (d,J = 8.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.92 - 5.86 (m, 1H), 5.10 - 5.07 (m,2H), 4.99 (d, J = 10.2 Hz, 1H), 4.21 (t, J = 6.3 Hz, 2H), 2.91 - 2.85 (m, 1H),2.60 - 2.50 (m, 2H), 2.25 - 2.22 (m, 2H), 2.05 - 2.01 (m, 1H), 1.87 - 1.83 (m, 2H)ppm. Then the purple-gray target intermediate compound 2-32 (yield: 52%) was obtained.

[0142] Example 20

[0143] Synthesis of Intermediate Compound IM-29

[0144] Using IM-20 and 6-bromo-1-hexene as starting materials and referring to the synthesis method of compound IM-27, a pale yellow intermediate compound IM-29 (yield: 55%) was obtained.

[0145] 1 1H NMR (600 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.86 - 5.79 (m, 1H), 5.10 - 5.03 (m, 2H), 4.97 (d, J = 10.2 Hz, 1H), 4.21 (t, J = 6.3 Hz, 2H), 2.91 - 2.85 (m, 1H), 2.60 - 2.52 (m, 2H), 2.13 - 2.09 (m, 2H), 2.04 - 1.99 (m, 1H), 1.79 - 1.74 (m, 2H), 1.58 - 1.53 (m, 2H) ppm。

[0146] Example 21

[0147] Synthesis of Intermediate Compound IM-31

[0148] Weigh 0.66 g (2.0 mmol) of Compound IM-27 and 6.49 g (90.0 mmol) of acrylic acid (IM-30) into a two-necked flask. Add 10.0 mL of anhydrous THF to the reaction flask to dissolve the mixture. Add 0.76 g (0.3 mmol) of Grubbs second-generation catalyst under the atmosphere of N2. Heat the system to 40 °C and react for 10 h. After the reaction is completed, cool the reaction solution to room temperature. Add 30.0 mL of water to the reaction flask and extract with EA (20.0 mL × 3). Collect the organic phase and wash it once with saturated NaCl solution (10.0 mL). Dry the organic phase with anhydrous sodium sulfate and concentrate it under reduced pressure to remove the excess solvent. The obtained crude product is slurried with EA / PE and DCM / PE systems respectively, filtered by suction, collect the filter cake, and vacuum dry it to obtain the target intermediate compound IM-31 as an off-white solid (0.37 g, yield: 53%).

[0149] 11H NMR (600 MHz, DMSO-d6) δ 12.30 (s, 1H), 11.11 (s, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.46 (d, J = 7.1 Hz, 1H), 6.94 - 6.89 (m, 1H), 5.95 (d, J = 15.7 Hz, 1H), 5.10 - 5.07 (m, 1H), 4.34 (t, J = 6.1 Hz, 2H), 2.91 - 2.85 (m, 1H), 2.71 - 2.69 (m, 2H), 2.60 - 2.52 (m, 2H), 2.03 - 2.02 (m, 1H) ppm。

[0150] Example 22

[0151] Synthesis of Intermediate Compound IM-32

[0152] Using IM-28 as the starting material and referring to the synthesis method of compound IM-31, the purple-gray target intermediate compound IM-32 was obtained (yield: 52%).

[0153] 1 1H NMR (600 MHz, DMSO-d6) δ 12.24 (s, 1H), 11.11 (s, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 6.94 - 6.83 (m, 1H), 5.87 - 5.75 (m, 1H), 5.10 - 5.07 (m, 1H), 4.22 (t, J = 6.0 Hz, 2H), 2.91 - 2.85 (m, 1H), 2.60 - 2.52 (m, 2H), 2.41 - 2.37 (m, 2H), 2.04 - 2.02 (m, 1H), 1.94 - 1.90 (m, 2H) ppm。

[0154] Example 23

[0155] Synthesis of Intermediate Compound IM-33

[0156] Using IM-29 as the starting material and referring to the synthesis method of compound IM-31, the gray target intermediate compound IM-33 was obtained (yield: 48%).

[0157] 11H NMR (600 MHz, DMSO-d6) δ 12.20 (s, 1H), 11.10 (s, 1H), 7.84 - 7.76 (m, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 6.86 - 6.81 (m, 1H), 5.80 (d, J = 15.6 Hz, 1H), 5.10 - 5.07 (m, 1H), 4.22 (t, J = 6.1 Hz, 2H), 2.91 - 2.85 (m, 1H), 2.60 - 2.50 (m, 2H), 2.29 - 2.26 (m, 2H), 2.04 - 2.01 (m, 1H), 1.79 - 1.75 (m, 2H), 1.64 - 1.60 (m, 2H) ppm。

[0158] Example 24

[0159] Synthesis of Intermediate Compound IM-37

[0160] Weigh 0.37 g (3.0 mmol) of 5-norbornene-2-methanamine (IM-35) into a flask, add an appropriate amount of diethyl ether (10 mL), stir at room temperature, add 1.47 g (15.0 mmol) of maleic anhydride (IM-34) to the reaction flask, and stir at room temperature for 2 h. After the reaction is completed, a white solid precipitates. Filter by suction, collect the filter cake, and dry it under vacuum to obtain the white intermediate compound IM-37 (0.57 g, yield 85%).

[0161] 1 1H NMR (600 MHz, DMSO-d6) δ 15.14 (s, 1H), 9.14 (d, J = 48.9 Hz, 1H), 6.43 (d, J = 12.5 Hz, 1H), 6.24 (d, J = 12.5 Hz, 1H), 6.19 - 6.09 (m, 1H), 6.08 - 5.88 (m, 1H), 3.62 - 3.04 (m, 1H), 2.97 - 2.93 (m, 1H), 2.82 (s, 1H), 2.81 - 2.77 (m, 2H), 2.27 - 2.21 (m, 1H), 1.83 - 1.79 (m, 1H), 1.33 (d, J = 6.4 Hz, 1H), 1.22 - 1.21 (m, 1H) ppm。

[0162] Example 25

[0163] Synthesis of Intermediate Compound IM-38

[0164] Using 1-adamantanamine (IM-36) and Compound 2-34 as reaction raw materials, referring to the synthesis route of Compound IM-37, the white target intermediate compound IM-38 (yield: 86%) was obtained and could be directly used for the next step without further purification. ESI-MS (m / z): [M + H] + = 250.1398.

[0165] Example 26

[0166] Synthesis of Intermediate Compound IM-41

[0167] Weigh 1.0 mL (40.0 mmol) of dimethylamine and 2.0 mL (40.0 mmol) of triethylamine (TEA) into a flask. Add an appropriate amount of DCM (10.0 mL) under a nitrogen atmosphere and react for 45 min in an ice bath. Then add 5.0 g (20.0 mmol) of 4-chloro-3-nitrobenzenesulfonyl chloride (IM-40), and restore the system to room temperature and react for 10 min. After the reaction is completed, add 30.0 mL of water to the reaction flask, extract with EA (20.0 mL × 3), collect the organic phase, wash it once with saturated NaCl solution (10.0 mL), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the excess solvent. The obtained crude product was purified by column chromatography (PE:EA = 3:1) to obtain the white intermediate compound IM-41 (1.06 g, yield: 20%).

[0168] 1 1H NMR (600 MHz, CDCl3) δ 8.25 (d, J = 1.9 Hz, 1H), 7.91 - 7.89 (m, 1H), 7.76 (d, J = 8.4 Hz, 1H), 2.79 (s, 6H) ppm.

[0169] Example 27

[0170] Synthesis of Intermediate Compound IM-42

[0171] Weigh 0.80 g (3.0 mmol) of compound IM-41 into a reaction flask, add 30.0 mL of ethanol (EtOH), heat the system to 50 °C and stir until the compound is completely dissolved. Then add an aqueous solution (10.0 mL) of 1.60 g (30.0 mmol) of NH4Cl to the reaction flask and stir the reaction for 30 min. Then add 1.0 g of iron powder to the reaction flask, continue to heat to 70 °C and stir for 8 h. After the reaction is completed, cool the reaction solution to room temperature, filter it through diatomaceous earth, collect the filtrate, wash the crude product after concentration under reduced pressure with water (20 mL×3), filter by suction, collect the filter cake, and dry it under vacuum to obtain the white intermediate compound IM-42 (0.51 g, yield: 73%), which can be directly used for the next step without further purification. ESI-MS(m / z): [M + H] + = 236.0200。

[0172] Example 28

[0173] Synthesis of intermediate compound IM-43

[0174] Using compounds IM-42 and IM-34 as starting materials and referring to the synthetic route of intermediate IM-37, the white target intermediate compound IM-43 (yield: 80%) was obtained.

[0175] 1 H NMR (600 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.30 (s, 1H), 8.25 (s,1H), 7.80 (d, J = 8.3 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 6.62 (d, J = 11.9Hz, 1H), 6.39 (d, J = 11.8 Hz, 1H), 2.63 (s, 6H) ppm.

[0176] Example 29

[0177] Synthesis of target compound NT-01

[0178] Weigh 0.37 g (1.1 mmol) of compound IM-24 and 0.57 g (1.5 mmol) of HATU into a flask, add 5.0 mL of DMF to dissolve the mixture, and stir for 30 min at room temperature. Then add 0.36 g (1.0 mmol) of compound IM-6 to the reaction flask, dropwise add 0.52 g (4.0 mmol) of DIPEA, and heat the system to 45 °C for reaction for 6 h. After detecting the completion of the reaction by TLC, cool the reaction solution to room temperature, add 30.0 mL of water to the reaction flask, extract with EA (20.0 mL × 3), collect the organic phase and wash it once with saturated NaCl solution (10.0 mL). Dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure to remove the excess solvent, and purify the obtained crude product by column chromatography. The eluent is DCM / MeOH (v : v = 20 : 1) to obtain the white target compound NT-01 (0.44 g, yield: 65%).

[0179] 1 H NMR (600 MHz, DMSO-d6) δ 11.11 (s, 1H), 8.28 (d, J = 5.0 Hz, 1H),8.04 (s, 1H), 7.77 (d, J = 6.9 Hz, 1H), 7.42-7.37 (m, 2H), 6.92 (s, 2H), 6.72(s, 1H), 6.62 (s, 1H), 5.52 (d, J = 56.3 Hz, 1H), 5.17 (s, 1H), 5.09 (s, 2H),4.09-4.06 (m, 1H), 3.94-3.84 (m, 2H), 3.79 (s, 6H), 3.72-3.68 (m, 1H), 3.57(d, J = 9.4 Hz, 1H), 2.89 (t, J = 14.7 Hz, 1H), 2.60 (s, 2H), 2.39 (d, J =5.7 Hz, 1H), 2.04 (d, J = 5.4 Hz, 1H) ppm. 1313C NMR (150 MHz, DMSO-d6) δ173.27, 170.41, 167.26, 165.72, 165.50, 160.84, 158.18, 156.95, 156.05, 153.65, 136.95, 133.53, 126.25, 123.04, 120.68, 116.60, 115.87, 110.13, 102.98, 101.24, 93.84, 80.99, 66.70, 55.97, 54.62, 50.57, 49.61, 49.23, 45.11, 44.11, 31.69, 31.43, 29.32, 22.46 ppm. HR-MS (m / z) (ESI): calcd for C 34 H 30 N8O8 [M + H] + : 679.2265; found: 679.2257。

[0180] Example 30

[0181] Synthesis of the target compound NT-02

[0182] Using compounds IM-25 and IM-6 as starting materials and referring to the synthetic route of compound NT-01, the white target compound NT-02 was obtained (yield: 63%).

[0183] 11H NMR (600 MHz, DMSO-d6) δ 11.09 (s, 1H), 8.25 (d, J = 14.2 Hz, 1H), 8.02 (s, 1H), 7.81 - 7.77 (m, 1H), 7.56 - 7.51 (m, 1H), 7.44 - 7.39 (m, 1H), 6.90 (s, 2H), 6.75 (s, 1H), 6.61 (d, J = 2.0 Hz, 1H), 5.51 - 5.40 (m, 1H), 5.10 - 5.02 (m, 1H), 4.29 - 4.18 (m, 2H), 4.01 - 3.98 (m, 1H), 3.87 - 3.82 (m, 2H), 3.78 (d, J = 4.2 Hz, 6H), 3.73 - 3.63 (m, 2H), 3.56 - 3.52 (m, 1H), 2.89 - 2.83 (m, 1H), 2.59 - 2.52 (m, 2H), 2.45 - 2.37 (m, 2H), 2.36 - 2.28 (m, 1H), 2.04 - 1.98 (m, 2H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 173.23, 170.54, 170.39, 167.32, 165.85, 160.83, 158.20, 156.94, 156.34, 153.57, 137.50, 133.71, 133.61, 126.10, 123.04, 120.23, 116.81, 115.70, 110.12, 102.97, 101.24, 93.77, 81.02, 68.43, 56.29, 55.96, 55.05, 51.00, 50.32, 49.23, 45.30, 44.74, 31.57, 30.00, 24.29, 22.47 ppm. HR-MS (m / z) (ESI): calcd for C 36 H 34 N8O8 [M + H] + : 707.2578; found: 707.2559。

[0184] Example 31

[0185] Synthesis of the target compound NT-03

[0186] Using compounds IM-26 and IM-6 as starting materials and referring to the synthetic route of compound NT-01, the white target compound NT-03 was obtained (yield: 60%).

[0187] 1 H NMR (600 MHz, DMSO-d6) δ 11.11 (s, 1H), 8.26 (s, 1H), 8.02 (s, 1H), 7.80 - 7.76 (m, 1H), 7.49 - 7.47 (m, 1H), 7.42 (t, J = 7.9 Hz, 1H), 6.90 (s, 2H), 6.75 (s, 1H), 6.61 - 6.57 (m, 1H), 5.50 - 5.40 (m, 1H), 5.10 - 5.03 (m, 1H), 4.21 - 4.16 (m, 2H), 4.01 - 3.98 (m, 1H), 3.85 - 3.79 (m, 2H), 3.77 (d, J = 3.4 Hz, 6H), 3.73 - 3.60 (m, 2H), 3.53 - 3.49 (m, 1H), 2.90 - 2.83 (m, 1H), 2.58 (d, J = 17.4 Hz, 1H), 2.35 - 2.31 (m, 2H), 2.26 (t, J = 7.2 Hz, 1H), 2.03 - 2.00 (m, 1H), 1.81 - 1.73 (m, 2H), 1.64 - 1.55 (m, 2H), 1.52 - 1.44 (m, 2H) ppm. 13 C NMR (150 MHz, DMSO-d6) δ 173.25, 171.10, 170.42, 167.32, 165.76, 160.81, 158.21, 156.93, 156.46, 153.54, 137.44, 133.69, 126.01, 123.04, 120.22, 116.68, 115.57, 110.09, 102.96, 101.23, 93.79, 81.03, 69.22, 55.95, 55.04, 50.88, 50.34, 49.21, 45.35, 44.60, 34.14, 33.84, 31.43, 29.90, 28.78, 25.54, 24.41, 22.48 ppm. HR-MS (m / z) (ESI): calcd for C 38 H 38 N8O8 [M + H]+ : 735.2891; found: 735.2874。

[0188] Example 32

[0189] Synthesis of the target compound NT-04

[0190] Using compounds IM-17 and IM-6 as starting materials and referring to the synthetic route of compound NT-01, the yellow target compound NT-04 (yield: 61%) was obtained.

[0191] 1 H NMR (600 MHz, DMSO-d6) δ 11.11 (s, 1H), 8.29 (d, J = 9.8 Hz, 1H), 8.03 (s, 1H), 7.62 - 7.57 (m, 1H), 7.10 - 7.06 (m, 1H), 7.05 (d, J = 8.9 Hz, 1H), 7.04 - 7.00 (m, 1H), 6.91 (s, 2H), 6.76 (s, 1H), 6.61 (s, 1H), 5.61 - 5.46 (m, 1H), 5.09 - 5.06 (m, 1H), 4.17 (d, J = 45.9 Hz, 2H), 4.08 - 4.05 (m, 1H), 3.95 - 3.89 (m, 1H), 3.84 - 3.80 (m, 1H), 3.78 (d, J = 2.7 Hz, 6H), 3.73 (d, J = 7.1 Hz, 1H), 3.64 - 3.60 (m, 1H), 2.93 - 2.84 (m, 1H), 2.61 - 2.54 (m, 2H), 2.43 - 2.38 (m, 1H), 2.05 - 2.03 (m, 1H) ppm. 1313C NMR (150 MHz, DMSO-d6) δ 173.27, 170.53, 169.27, 166.81, 160.84, 158.24, 156.97, 153.67, 153.61, 146.00, 136.58, 132.49, 126.24, 123.04, 118.60, 111.30, 110.12, 102.99, 101.22, 93.86, 81.03, 56.25, 55.96, 54.85, 50.70, 49.87, 49.08, 45.14, 44.84, 44.63, 44.32, 31.48, 29.62, 22.63 ppm. HR-MS (m / z) (ESI): calcd for C 34 H 31 N9O7 [M + H] + : 678.2425; found: 678.2407。

[0192] Example 33

[0193] Synthesis of the target compound NT-05

[0194] Using compounds IM-18 and IM-6 as starting materials and referring to the synthetic route of compound NT-01, the yellow target compound NT-05 (yield: 58%) was obtained.

[0195] 11H NMR (600 MHz, DMSO-d6) δ 11.11 (d, J = 3.3 Hz, 1H), 8.27 (d, J =1.1 Hz, 1H), 8.05 (s, 1H), 7.59-7.56 (m, 1H), 7.20-7.17 (m, 1H), 7.00 (t, J =6.0 Hz, 1H), 6.91-6.90 (m, 2H), 6.78 (s, 1H), 6.72-6.65 (m, 1H), 6.61-6.60(m, 1H), 5.53-5.41 (m, 1H), 5.07-5.03 (m, 1H), 3.88-3.80 (m, 2H), 3.78 (d, J= 3.3 Hz, 6H), 3.75-3.71 (m, 1H), 3.67-3.62 (m, 1H), 3.58-3.52 (m, 1H), 3.33-3.30 (m, 1H), 2.91-2.85 (m, 1H), 2.61-2.52 (m, 2H), 2.44-2.38 (m, 2H), 2.34-2.32 (m, 2H), 2.05-2.00 (m, 1H), 1.86-1.78 (m, 2H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 173.26, 170.55, 169.30, 167.79, 160.83, 158.23, 156.96, 153.60,146.86, 136.65, 132.68, 126.15, 126.10, 123.04, 117.72, 110.83, 110.11,109.57, 102.96, 101.24, 93.79, 81.02, 55.96, 55.06, 50.83, 50.39, 49.01,45.32, 44.69, 42.04, 31.58, 31.46, 31.30, 29.92, 24.35, 22.63 ppm. HR-MS (m / z) (ESI): calcd for C 36 H 35 N9O7 [M + H] + : 706.2738; found: 706.2723。

[0196] Example 34

[0197] Synthesis of the target compound NT-06

[0198] Using compounds IM-19 and IM-6 as starting materials and referring to the synthetic route of compound NT-01, the yellow target compound NT-06 was obtained (yield: 68%).

[0199] 1 H NMR (600 MHz, DMSO-d6) δ 11.09 - 11.06 (m, 1H), 8.39 (s, 1H), 8.35 (s, 1H), 7.60 - 7.53 (m, 1H), 7.24 - 7.18 (m, 1H), 7.07 - 7.05 (m, 1H), 7.03 - 6.97 (m, 1H), 6.91 - 6.90 (m, 2H), 6.62 - 6.58 (m, 1H), 6.52 (s, 1H), 5.54 - 5.40 (m, 1H), 5.09 - 5.02 (m, 1H), 3.84 - 3.80 (m, 2H), 3.77 (d, J = 3.7 Hz, 6H), 3.71 - 3.59 (m, 2H), 3.53 - 3.42 (m, 1H), 2.98 (d, J = 3.6 Hz, 1H), 2.87 (t, J = 14.8 Hz, 1H), 2.59 (t, J = 14.6 Hz, 2H), 2.36 - 2.30 (m, 2H), 2.27 - 2.22 (m, 1H), 2.05 - 1.97 (m, 1H), 1.62 - 1.57 (m, 2H), 1.55 - 1.53 (m, 2H), 1.42 - 1.33 (m, 2H), 1.24 (t, J = 11.2 Hz, 1H) ppm. 1313C NMR (150 MHz, DMSO-d6) δ 173.31, 171.12, 170.58, 169.39, 167.78, 160.80, 152.71, 146.85, 136.72, 132.63, 127.00, 122.84, 110.81, 110.21, 109.43, 103.05, 101.00, 94.43, 80.35, 56.68, 55.98, 55.33, 50.92, 50.44, 48.99, 45.35, 44.60, 42.23, 34.10, 33.83, 31.63, 31.43, 29.91, 29.08, 26.50, 24.57, 22.54, 14.44 ppm. HR-MS (m / z) (ESI): calcd for C 38 H 39 N9O7 [M + H] + : 734.3051; found: 734.3030。

[0200] Example 35

[0201] Synthesis of the target compound NT-07

[0202] Using compounds IM-31 and IM-6 as starting materials and referring to the synthetic route of compound NT-01, the white target compound NT-07 was obtained (yield: 52%).

[0203] 11H NMR (600 MHz, DMSO-d6) δ 11.09 (s, 1H), 8.27 (s, 1H), 8.04 (s, 1H), 7.83 - 7.79 (m, 1H), 7.56 - 7.52 (m, 1H), 7.46 - 7.43 (m, 1H), 6.91 (t, J = 4.7 Hz, 2H), 6.84 - 6.78 (m, 1H), 6.69 (s, 1H), 6.61 (d, J = 1.6 Hz, 1H), 6.54 - 6.42 (m, 1H), 5.56 - 5.43 (m, 1H), 5.10 - 5.03 (m, 1H), 4.36 - 4.31 (m, 2H), 4.12 - 4.08 (m, 1H), 3.94 - 3.82 (m, 2H), 3.78 (d, J = 2.1 Hz, 6H), 3.70 - 3.57 (m, 1H), 2.89 - 2.82 (m, 1H), 2.73 - 2.66 (m, 2H), 2.59 - 2.54 (m, 2H), 2.42 - 2.28 (m, 2H), 2.05 - 1.96 (m, 1H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 173.28, 170.42, 167.29, 165.76, 163.94, 160.81, 158.22, 156.96, 156.13, 153.53, 140.81, 137.52, 133.71, 126.16, 124.88, 123.02, 120.31, 117.15, 116.75, 115.84, 110.09, 102.99, 101.22, 93.85, 81.00, 67.92, 56.34, 55.95, 54.87, 50.94, 50.57, 49.22, 45.40, 44.90, 31.42, 29.89, 22.48 ppm. HR-MS (m / z) (ESI): calcd for C 37 H 34 N8O8 [M + H] + : 719.2578; found: 719.2563。

[0204] Example 36

[0205] Synthesis of the target compound NT-08

[0206] Using compounds IM-32 and IM-6 as starting materials and referring to the synthetic route of compound NT-01, the white target compound NT-08 (yield: 56%) was obtained.

[0207] 1 H NMR (600 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.27 (s, 1H), 7.94 (d, J =116.3 Hz, 1H), 7.78 (d, J = 14.9 Hz, 1H), 7.52 - 7.38 (m, 2H), 6.91 (s, 2H),6.77 (s, 1H), 6.64 (s, 1H), 6.60 (s, 1H), 6.34 - 6.24 (m, 1H), 5.47 (d, J =40.2 Hz, 1H), 5.08 (s, 1H), 4.21 (d, J = 15.1 Hz, 2H), 4.03 (s, 1H), 3.83 (s,2H), 3.78 (s, 6H), 3.62 (d, J = 49.8 Hz, 2H), 2.87 (d, J = 10.8 Hz, 1H),2.64 - 2.55 (m, 2H), 2.42 - 2.39 (m, 2H), 2.32 (s, 1H), 2.02 (s, 1H), 1.94 (s,2H) ppm. 13 C NMR (150 MHz, DMSO-d6) δ 173.28, 170.43, 167.31, 165.79, 163.99,160.81, 158.22, 156.96, 156.35, 153.53, 144.22, 137.44, 133.69, 126.15,123.04, 120.32, 116.75, 115.74, 110.10, 102.98, 101.23, 93.78, 81.02, 68.50,65.40, 56.28, 55.96, 54.86, 50.57, 49.22, 45.33, 44.85, 31.42, 29.84, 28.32,27.66, 22.48, 15.63 ppm. HR-MS (m / z) (ESI): calcd for C 38 H 36 N8O8 [M + H] + :733.2734; found: 733.2727。

[0208] Example 37

[0209] Synthesis of the target compound NT-09

[0210] Using compounds IM-33 and IM-6 as raw materials and referring to the synthetic route of compound NT-01, the white target compound NT-09 was obtained (yield: 50%).

[0211] 1 H NMR (600 MHz, DMSO-d6) δ 11.10 (s, 1H), 8.27 (s, 1H), 8.01 (s, 1H), 7.83 - 7.77 (m, 1H), 7.52 - 7.41 (m, 2H), 6.91 (d, J = 1.9 Hz, 2H), 6.86 - 6.73 (m, 1H), 6.72 - 6.69 (m, 1H), 6.60 (d, J = 1.8 Hz, 1H), 6.37 - 6.26 (m, 1H), 5.53 - 5.43 (m, 1H), 5.09 - 5.05 (m, 1H), 4.24 - 4.19 (m, 2H), 3.92 - 3.86 (m, 1H), 3.82 (d, J = 7.7 Hz, 1H), 3.78 (d, J = 1.9 Hz, 6H), 3.69 - 3.58 (m, 2H), 2.87 (t, J = 17.4 Hz, 1H), 2.57 (d, J = 17.2 Hz, 2H), 2.47 - 2.44 (m, 1H), 2.41 - 2.36 (m, 1H), 2.33 - 2.25 (m, 2H), 2.03 - 2.00 (m, 1H), 1.81 - 1.73 (m, 2H), 1.67 - 1.60 (m, 2H) ppm. 1313C NMR (150 MHz, DMSO-d6) δ 173.26, 170.44, 167.32, 165.80, 164.02, 160.81, 158.22, 156.96, 156.42, 153.56, 145.14, 137.47, 133.71, 126.03, 123.05, 122.87, 122.58, 120.24, 116.70, 115.65, 110.10, 103.00, 101.22, 93.77, 81.01, 69.01, 55.96, 54.86, 50.91, 50.57, 49.19, 45.35, 44.84, 31.64, 31.55, 31.42, 28.34, 24.61, 22.46 ppm. HR-MS (m / z) (ESI): calcd for C 39 H 38 N8O8[M + H] + : 747.2891; found: 747.2880。

[0212] Example 38

[0213] Synthesis of the target compound NT-10

[0214] Using compounds IM-37 and IM-6 as starting materials and referring to the synthetic route of compound NT-01, the white target compound NT-10 was obtained (yield: 72%).

[0215] 11H NMR (600 MHz, DMSO-d6) δ 8.42 - 8.32 (m, 1H), 8.26 (d, J = 15.1 Hz, 1H), 8.02 (s, 1H), 6.93 - 6.92 (m, 2H), 6.74 (s, 1H), 6.61 (s, 1H), 6.42 - 6.32 (m, 1H), 6.14 - 6.12 (m, 1H), 6.09 - 6.00 (m, 1H), 5.94 - 5.90 (m, 1H), 5.46 - 5.40 (m, 1H), 3.95 - 3.85 (m, 1H), 3.79 (s, 6H), 3.72 - 3.63 (m, 2H), 3.60 - 3.53 (m, 1H), 2.92 - 2.82 (m, 1H), 2.79 - 2.72 (m, 2H), 2.69 - 2.58 (m, 1H), 2.43 - 2.39 (m, 2H), 2.17 (d, J = 23.5 Hz, 1H), 1.79 - 1.72 (m, 1H), 1.30 - 1.27 (m, 1H), 1.19 - 1.16 (m, 1H), 0.46 - 0.42 (m, 1H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 166.20, 163.96, 160.81, 158.22, 156.91, 153.64, 137.53, 133.37, 132.85, 128.26, 127.64, 126.05, 123.09, 110.12, 102.97, 101.21, 93.74, 81.02, 55.96, 54.98, 51.18, 49.86, 49.40, 45.84, 44.16, 43.17, 42.38, 38.70, 31.31, 30.26, 29.46 ppm. HR-MS (m / z) (ESI): calcd for C 31 H 33 N7O4 [M + H] + : 568.2672; found: 568.2665。

[0216] Example 39

[0217] Synthesis of the target compound NT-11

[0218] Using compounds IM-38 and IM-6 as raw materials, referring to the synthetic route of compound NT-01, the white target compound NT-11 was obtained (yield: 70%).

[0219] 1 H NMR (600 MHz, DMSO-d6) δ 8.26 (d, J = 17.2 Hz, 1H), 8.00 (s, 1H), 7.87 (s, 1H), 6.91 (s, 2H), 6.74 (s, 1H), 6.61 (s, 1H), 6.33 - 6.23 (m, 1H), 6.06 - 5.99 (m, 1H), 5.43 (d, J = 21.6 Hz, 1H), 3.91 (s, 1H), 3.78 (s, 6H), 3.66 (s, 2H), 3.56 (d, J = 25.5 Hz, 1H), 2.42 (d, J = 22.6 Hz, 2H), 1.98 (s, 3H), 1.92 (s, 3H), 1.85 (s, 3H), 1.58 (s, 6H) ppm. 13 C NMR (150 MHz, DMSO-d6) δ 166.41, 163.43, 160.81, 158.22, 156.94, 153.63, 132.82, 132.35, 129.41, 128.71, 126.10, 125.95, 123.09, 110.08, 102.95, 101.23, 93.71, 93.64, 81.14, 55.95, 55.05, 51.43, 51.30, 51.24, 49.90, 45.79, 44.28, 41.23, 41.15, 36.45, 31.30, 29.34, 29.22 ppm. HR-MS (m / z) (ESI): calcd for C 33 H 37 N7O4 [M + H] + : 596.2985; found: 596.2977。

[0220] Example 40

[0221] Synthesis of the target compound NT-12

[0222] Using compounds IM-39 and IM-6 as starting materials, referring to the synthetic route of the target compound NT-01, the white target compound NT-12 was obtained (yield: 68%).

[0223] 1 1H NMR (600 MHz, DMSO-d6) δ 8.27 (s, 1H), 8.12 (s, 1H), 8.03 (s, 2H), 7.89 - 7.78 (m, 1H), 7.29 - 7.18 (m, 1H), 7.08 (s, 2H), 6.90 (s, 2H), 6.74 (s, 1H), 6.59 (s, 1H), 5.53 (d, J = 27.2 Hz, 1H), 4.22 - 4.07 (m, 1H), 4.07 - 3.93 (m, 2H), 3.85 (s, 3H), 3.77 (s, 6H), 3.71 - 3.65 (m, 1H), 2.40 (d, J = 36.3 Hz, 2H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 188.10, 164.22, 162.91, 160.80, 158.22, 156.97, 153.60, 133.94, 133.80, 133.62, 133.24, 131.65, 129.93, 126.13, 123.03, 114.79, 110.10, 102.99, 101.22, 93.81, 81.00, 56.12, 55.95, 54.83, 51.14, 50.95, 45.72, 45.36, 31.71, 29.86 ppm. HR-MS (m / z) (ESI): calcd for C 30 H 28 N6O5 [M + H] + : 553.2199; found: 553.2191。

[0224] Example 41

[0225] Synthesis of the target compound NT-13

[0226] Using compounds IM-43 and IM-6 as starting materials and referring to the synthetic route of the target compound NT-01, the white target compound NT-13 (yield: 63%) was obtained.

[0227] 11H NMR (600 MHz, DMSO-d6) δ 10.78 (d, J = 61.8 Hz, 1H), 8.34 (d, J = 16.3 Hz, 1H), 8.26 (d, J = 10.4 Hz, 1H), 8.04 (s, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 6.91 (s, 2H), 6.81 (s, 1H), 6.75 - 6.65 (m, 1H), 6.61 (s, 1H), 6.49 - 6.41 (m, 1H), 5.52 - 5.42 (m, 1H), 4.01 - 3.94 (m, 1H), 3.84 - 3.81 (m, 1H), 3.78 (s, 6H), 3.71 - 3.61 (m, 2H), 2.63 (s, 6H), 2.45 - 2.40 (m, 2H) ppm. 13 13C NMR (150 MHz, DMSO-d6) δ 165.26, 163.78, 160.82, 158.20, 156.98, 153.66, 136.01, 134.25, 131.10, 129.44, 128.87, 126.16, 125.01, 123.83, 123.06, 110.11, 103.00, 101.19, 93.79, 80.97, 65.39, 55.96, 54.92, 51.22, 50.10, 46.02, 44.52, 38.03, 31.44, 29.59, 15.64 ppm. HR-MS (m / z) (ESI): calcd for C 31 H 31 N8O6S [M + H] + : 679.1854; found: 679.1849。

[0228] Example 42

[0229] Test on the inhibitory effect of the target compounds NT-01~13 on the activity of exogenous FGFR2 enzyme

[0230] Experimental method: For the detection of the inhibitory effect of all target compounds in this application on the exogenous FGFR2 enzyme activity, a commercial FGFR2 enzyme-linked immunosorbent assay (ELISA) kit provided by Shanghai Fusheng Industrial Co., Ltd. was used, and the operation was carried out strictly in accordance with the kit instructions. The experimental design included a blank group, a test group, and a positive control group. After adding the target protein diluent to each well, the test group and the positive control group were respectively added with 5 gradient concentrations (500.0, 100.0, 20.0, 4.0, and 0.8 nM) of the test compound solution, and the positive control drug was TAS-120. After sealing the plate, it was incubated in an incubator at 37 °C for 2 h. After washing 5 times with the washing solution, except for the blank group, 50 μL of the enzyme-labeled reagent was added to each well, the plate was sealed again and incubated at 37 °C for another 30 min. Subsequently, 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B were added successively, shaken gently and evenly, and incubated at 37 °C in the dark for 10 min. Finally, 50 μL of the termination solution was added to each well. Using the blank well to zero, the absorbance (OD value) of each well was measured successively at a wavelength of 490 nm using an enzyme-linked immunosorbent assay reader. Each compound was subjected to three parallel independent experiments, and the results were averaged to calculate the enzyme activity inhibition rate and IC 50 value, and the specific data are shown in Table 1.

[0231] Table 1. Inhibitory effect of target compounds on exogenous FGFR2 enzyme activity

[0232]

[0233] In this application, a commercial human FGFR2 ELISA enzyme activity detection kit was used to determine the inhibitory effect of all target compounds on the FGFE2 enzyme activity, with TAS-120 as the positive control. The experimental results are shown in Table 1. The positive reference TAS-120 showed a relatively high inhibitory effect on FGFR2, and the IC 50 value was 451.23 nM. In addition, the designed and synthesized PROTACs molecules all retained the inhibitory activity of the original inhibitors on FGFR2, and the inhibitory effects of all target compounds were better than the positive reference. The detailed analysis results are as follows:

[0234] The inhibitory activities of compounds NT-01~03 with 4-hydroxy thalidomide as the E3 ubiquitin ligase ligand were generally better than those of the traditional thalidomide ligands NT-04~06. Especially when the carbon chain of the Linker was extended, 4-hydroxy thalidomide showed obvious advantages. When the carbon chain length was 2 C, the inhibitory activity of NT-01 (IC 50 = 410.55 nM) was lower than that of compound NT-04 with the same carbon chain length (IC 50 = 33.67 nM); when the carbon chain was 4 C, the result was exactly the opposite, and the inhibitory activity of compound NT-02 (IC 50= 79.26 nM) is 1.8 times that of NT-05 (IC 50 = 140.04 nM); when the carbon chain has 6 carbons, the inhibitory activity (IC 50 = 116.61 nM) of compound NT-03 is about 1.3 times that of NT-06 (IC 50 = 151.63 nM). Thus, it can be seen that a medium-length Linker with a carbon chain of 4 - 6 carbons can effectively improve the inhibitory activity of the proteolysis targeting chimera against kinases. It has been reported that introducing a covalent bond into the Linker part of the proteolysis targeting chimera can promote the interaction between the compound and cysteine residues, enhancing the affinity of the degrader for the target protein. The target compounds NT-07 - 09 showed inhibitory activity superior to that of the positive reference TAS-120. Compared with the compounds described above, although the activity of the compound containing a 5-carbon-length Linker (compound NT-07) decreased slightly (IC 50 = 329.63 nM), the compound NT-08 with a 6-carbon length showed excellent enzyme inhibitory activity, with an inhibitory activity against FGFR2 kinase of 17.16 nM, which was approximately 4.6 times, 2.0 times, and 26.2 times higher than those of compounds NT-02, NT-04, and TAS-120, respectively. Meanwhile, the inhibitory activity of compound NT-09 containing an irreversible covalent bond with a 7-carbon length was comparable to that of NT-03 and NT-06 with similar lengths. Therefore, the type and length of the Linker play an important role in the enzyme inhibitory activity of the proteolysis targeting chimera. In addition, it can also be found that the steric bridged-ring structure did not affect the inhibitory activity of the compound against FGFR2 kinase, and the inhibitory activities of compounds NT-10 and NT-11 were 1.9 times and 2.9 times that of TAS-120, respectively. Similarly, NT-12 with a methoxyphenyl as the molecular glue fragment also showed good inhibitory activity (IC 50 = 166.27 nM), showing a certain degree of improvement compared with NT-10 and NT-11 containing hydrophobic tags and NT-7 with 4-hydroxythalidomide as the ligand. When an arylsulfonamide molecular glue fragment was introduced, NT-13 showed superior inhibitory activity, with an IC 50 of 19.14 nM, which was comparable to the inhibitory activity of NT-8 (IC 50 = 17.16 nM). In summary, through preliminary enzyme inhibitory activity tests, it was shown that compounds containing hydrophobic tags and molecular glue fragments have the ability of targeted inhibition.

[0235] Example 43

[0236] Antiproliferative activity test of target compounds NT-01 - 13 against various different types of cancer cells

[0237] Experimental method: The MTT method was used to test the cytotoxic activity of all compounds in this application. Cells in the logarithmic growth phase were counted and inoculated into a 96-well culture plate, with an inoculation amount of 8,000 - 10,000 cells per well. After culturing overnight until the cells adhered, the drug administration group and the control group were set. The test compound was first formulated into a stock solution with DMSO and diluted into gradient concentrations with cell culture medium before use (the final concentration of DMSO ≤ 0.4%, the same below). Three replicate wells were set for each concentration. After adding the drug, the cells were cultured for another 72 h, and then 20 μL of MTT solution with a concentration of 5 mg / mL was added to each well and incubated at 37 °C for 4 h. The supernatant was carefully aspirated, and 150 μL of DMSO was added to each well to dissolve the crystals. The optical density (OD value) of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell inhibition rate was calculated and a concentration-inhibition rate curve was plotted to obtain the IC 50 value. The experimental results are shown in Table 2.

[0238] Table 2 IC 50 values of the target compounds against various different types of cancer cells

[0239]

[0240] In this application, the human hepatocellular carcinoma cell lines BEL-7402 and SMMC-7721, the cholangiocarcinoma cell lines CCLP-1 and HCCC-9810, the human triple-negative breast cancer cell line MDA-MB-231, and the human esophageal cancer cell line KYSE-150 were respectively selected to evaluate the inhibitory activity of the target compounds on cancer cell proliferation, with TAS-120 as the positive control. The results are shown in Table 2. The target compounds basically retained the original cytotoxicity of TAS-120. For example, the IC 50 values of compound NT-01 against these six cancer cell lines were all maintained between 9.19 - 18.11 μM, while the IC 50 values of TAS-120 against the tested cancer cells were also all in the range of 11.88 - 21.15 μM. However, different from the enzyme inhibition activity, the shorter the Linker length of the degrader molecule, the better the anti-proliferative activity on cells. The IC 50They were 13.09, 20.74, and 21.53 μM, respectively. This trend may be related to the molecular weight of the compound. Relevant literature has pointed out that the large molecular weight of PROTACs will affect their cell permeability and oral bioavailability. At the same time, the PROTACs molecules with 4-hydroxythalidomide as the E3 ubiquitin ligase ligand are mostly more cytotoxic to these 6 cancer cell lines than the similar molecules with pomalidomide as the ligand, especially in CCLP-1, KYSE-150, and BEL-7402 cells. For example, the anti-proliferative activity of compound NT-01 against CCLP-1 cells is about 2.7 times that of NT-04; the inhibitory activities against BEL-7402 and KYSE-150 cells are 2.0 times and 1.9 times that of NT-04, respectively. When the length of the Linker increases, the gap between the two gradually narrows. The inhibitory activity of compound NT-02 with a 5-carbon Linker length against BEL-7402 cells is slightly better than that of the similar NT-05 (IC 50 : 12.75 vs 20.08 μM), and the activities of compound NT-03 with a 6-carbon Linker length and the similar NT-06 are equivalent (IC 50 : 11.17 vs 11.96 μM). This trend is also very obvious in CCPL-1, HCCC-9810, and MDA-MB-231 cell lines. It shows that TAS-120 conjugated with 4-hydroxythalidomide retains its inhibitory activity against FGFR2 kinase in cells.

[0241] The PROTACs molecules NT-07~09 with irreversible covalent bonds as the Linker show certain selectivity for these 6 cancer cell lines. From the data in Table 2, it can be found that the inhibitory activities of compound NT-07 against CCLP-1, HCCC-9810, and KYSE-150 are 9.99, 8.93, and 14.94 μM, respectively, all better than the corresponding positive references (11.88, 15.19, and 21.15 μM). For MDA-MB-231, the inhibitory activities of compounds NT-07~09 are greater than 16 μM. In addition, in SMMC-7721, NT-07 and NT-08 retained the cytotoxicity of the original TAS-120 (IC 50 : 10.37 vs 15.48 μM and 14.85 vs 15.48 μM). Therefore, compared with PROTACs with non-covalent aliphatic chains as the Linker, introducing covalent bonds as the Linker can effectively improve the selectivity of the drug. However, the latter does not have a significant improvement in the anti-proliferative activity against cells.

[0242] The cytotoxicities of NT-10 and NT-11 constructed from two HyTDDs molecules against 6 cancer cell lines were basically consistent with those of TAS-120. However, in KYSE-150 cells, the cytotoxicities of both compounds were superior to the positive reference (IC 50 = 21.15 μM), and the inhibitory activities were 12.90 and 13.46 μM, respectively. At the same time, compared with NT-7~09 (IC 50 = 14.94, 24.52 and 23.55 μM), there was a significant improvement. NT-12 containing the molecular glue fragment showed good cytotoxicity, and the inhibitory activities against 6 cancer cells were 4.81, 4.64, 5.22, 7.18, 4.16 and 5.09 μM, respectively. Especially in KYSE-150 cells, the activity of NT-12 was about 4.2 times higher than that of the positive reference TAS-120 (5.09 vs 21.15 μM). Probably due to the influence of molecular weight, compound NT-13 did not maintain the inhibitory effect of NT-12, and its anti-proliferative activity against cells was comparable to that of HyTDDs (compounds NT-10, NT-11). In summary, through two rounds of compound design and enzyme inhibition activity and cytotoxicity screening, the optimized compound NT-12 was initially obtained. Using methoxyphenyl as the molecular glue fragment not only had high enzyme inhibition activity but also had excellent anti-proliferative ability against cancer cells, especially against KYSE-150 cells.

[0243] Example 44

[0244] Detection of the ability of target compounds NT-01~13 to degrade FGFR2 protein in KYSE-150 cells

[0245] Seed KYSE-150 cells in a 6-well plate and culture them. After they adhere, replace the medium with fresh medium. Treat the cancer cells with different target compounds NT-01~13 at a concentration of 10.0 μM for 6 h. Lyse the cells using a cell lysis buffer containing protease inhibitors and phosphatase inhibitors. The protein concentration is detected using a BCA protein assay kit (Shanghai Beyotime Biotechnology Co., Ltd.). Subsequently, boil the samples at 100 °C for 15 min for denaturation. Take equal amounts of protein, separate them by 10%-15% SDS-PAGE, and then electrotransfer them to an NC membrane. Block the membrane with 5% skim milk at room temperature for 1 h. After blocking, incubate with the corresponding primary antibody overnight at 4 °C. Wash away the unbound antibody with PBST, and then incubate with the corresponding secondary antibody in the dark for 1 h. Add chemiluminescent reagent and use an enhanced chemiluminescence system BeyECL Plus (Shanghai Beyotime Biotechnology Co., Ltd.) to detect protein bands.

[0246] To evaluate whether the target compound can effectively promote the degradation of FGFR2 protein in cancer cells, Western blot was used in this application to detect the expression level of FGFR2 protein in cancer cells (using β-actin as an internal reference) after co-incubating the target compound with FGFR2 cells for 6 hours. As Figure 1 shown, after treating KYSE-150 cells with each compound at a dosage of 10.0 μM for 6 hours, different degrees of FGFR2 protein degradation were induced. Among the compounds (NT-01~09) with 4-hydroxythalidomide or pomalidomide derivatives as ligands, the compounds with medium-length Linkers (NT-02, 03, 05, 06, 08, and 09) could effectively degrade endogenous FGFR2 protein. Notably, the degradation ability of NT-8 and NT-9 containing irreversible covalent bonds on FGFR2 did not show a significant improvement compared to NT-02 and NT-03 without covalent bonds, and even decreased slightly. The possible reason is that the irreversible covalent interaction hinders the process of PROTACs molecules' re-release and recycling. The norbornene-containing HyTDD molecule NT-10 has excellent degradation activity, with more than 80% degradation of FGFR2 protein at a concentration of 10.0 μM; the similar adamantane-containing NT-11 has a moderate effect on FGFR2 level, with only about 50% degradation of FGFR2 protein. The molecular glue-containing NT-12 and NT-13 can also cause the degradation of FGFR2 protein. Especially the degrader NT-12, which degrades more than 85% of FGFR2 protein at a concentration of 10.0 μM and is not affected by the covalent irreversible effect of the Linker. Thus, it can be seen that the compound NT-12 has excellent enzyme inhibition activity, cytotoxicity, and protein degradation ability.

Claims

1. A small molecule degradation agent targeting FGFR2, characterized by: The small molecule degradation agent is a structural compound represented by Formula 1 or Formula 2 or its isomer, pharmaceutically acceptable salt or mixture thereof: 、 ； in: The linker in formula 1 is: , , , , or , In formula 1, X is: O or NH, R in Formula 2 is: , , or .

2. The small molecule degradation agent targeting FGFR2 according to claim 1, characterized in that: A compound selected from any of the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 。 3. The small molecule degradation agent targeting FGFR2 according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by the compound of the structure represented by Formula 1 or Formula 2 and any one of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and ferulic acid.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the small molecule degrader targeting FGFR2 according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier or excipient.

5. Use of the small molecule degradation agent targeting FGFR2 according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 in the preparation of a drug for disease prevention and / or treatment.

6. The use according to claim 5, characterized in that: The disease is a disease caused by overexpression of FGFR2.

7. The use according to claim 6, characterized in that: The disease caused by overexpression of FGFR2 is specifically a tumor.

8. The use according to claim 7, characterized in that: The tumor is liver cancer, bile duct cancer, breast cancer, bladder cancer or esophageal cancer.

9. Use of the small molecule degradation agent targeting FGFR2 according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 in the preparation of a FGFR2 inhibitor or a FGFR2 degradation agent.

10. A method for degrading FGFR2 in cells, characterized in that: The cell is contacted with the small molecule degrader targeting FGFR2 as described in any one of claims 1 to 3 or the pharmaceutical composition as described in claim 4, and the method is for non-disease diagnosis and non-disease treatment purposes.

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