Protein degradation targeting chimera compounds, methods of making and uses thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a protein degradation targeting chimera compound, a preparation method and application. The protein degradation targeting chimera compound or a pharmaceutically acceptable salt or solvate thereof provided by the application has good inhibitory effect on NEK2, and can be used for treating diseases mediated by NEK2 protein. Meanwhile, the protein degradation targeting chimera compound or the pharmaceutically acceptable salt or solvate thereof provided by the application has good inhibitory effect on K562 cell proliferation, and the compound can also synergistically enhance the inhibitory effect of known compounds such as ethyl, colchicine, thiomersal, orlistat, amitriptyline hydrochloride, clomipramine hydrochloride, fludarabine, etc. on K562 cell proliferation.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a protein degradation targeted chimera compound, a preparation method and an application thereof. Background Art

[0002] NEKS (NIMA-related kinase) kinases are serine / threonine protein kinases. The NEKS family comprises 11 protein kinases, from NEK1 to NEK11. NEK2 shares the highest homology with NIMA. NEK2 plays a central role in regulating the G2 / M phase of the cell cycle. NEK2 overexpression leads to chromosomal instability and aneuploidy in cancer cells. As a cellular protein kinase, NEK2 plays a crucial role in mitosis regulation and is closely associated with tumor initiation, progression, and metastasis. Overexpression of NEK2 in normal cells can create a tumorigenic environment.

[0003] In the prior art, patents such as WO2021155185A1, CN106496222A, and WO2018081719A1 disclose small molecule inhibitors of NEK2. Meanwhile, some literature reports on NEK2 inhibitors, such as J Med Chem. 2010 Nov 11; 53(21):7682-98; J Med Chem. 2011 Mar 24; 54(6):1626-39; J Med Chem. 2012 Apr 12; 55(7):3228-41; J Med Chem. 2011 Jun 23; 54(12):4133-46; and RSC Med Chem. 2020 May 22; 11(6):707-731. Compared with this type of NEK2 small molecule inhibitors, NEK2 protein degradation targeting chimeras can more effectively degrade NEK2 and inhibit the NEK2 signaling pathway, which may become a treatment for NEK2-related tumors.

[0004] Protein degradation targeted chimeric technology (PROTAC technology) has a structure containing two different ligands: one is a ubiquitin ligase E3 ligand, and the other is a target protein binding ligand, and the two ligands are connected by a linker. PROTAC brings the target protein and the intracellular ubiquitin ligase E3 closer to form a target protein-PROTAC-E3 ternary complex. The E3 ubiquitin ligase then tags the target protein with a ubiquitinated protein tag, subsequently initiating a powerful ubiquitination-proteasome system in the cell to specifically degrade the target protein, thereby inhibiting the corresponding protein signaling pathway. Compared with traditional small molecule inhibitors, PROTAC exhibits unique advantages: 1) PROTAC does not need to bind to the target protein for a long time and at high intensity. The process of degrading the target protein is similar to a catalytic reaction, which can cyclically bind and degrade the target protein, thereby reducing the systemic exposure of the drug and reducing the occurrence of toxicity and side effects; 2) After the target protein is degraded, it needs to be resynthesized to restore its function. Therefore, degradation of the target protein exhibits more effective and lasting anti-tumor effects than inhibiting its activity, and does not cause drug resistance due to target protein mutations.

[0005] Currently, there are relatively few studies on protein degradation-targeted chimeric technologies. In the prior art, patent WO2022006292A discloses a series of NEK2 PROTACs, but only discloses the degradation effect of this representative NEK2 PROTAC on NEK2 protein in diffuse large B-cell lymphoma (DLBCL) cell lines. It is well known in the art that DLBCL, as a hematological tumor, has different sensitivities to the same compound as solid tumors such as lung cancer.

[0006] Experiments have shown that the inhibitory effect of the representative NEK2 PROTAC in WO2022006292A on the proliferation of solid tumor cell lines such as lung cancer needs to be further improved. However, there is currently no research in the prior art on structurally modifying this type of compound to obtain NEK2 PROTACs with good anti-proliferative effects on solid tumor cells such as lung cancer. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide a protein degradation targeting chimera compound, which has a good inhibitory effect on NEK2 and A549 cell proliferation, providing technical support for the treatment of NEK2 protein-mediated diseases.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A protein degradation targeting chimeric compound, wherein the protein degradation targeting chimeric compound is a compound as shown in Formula I or a pharmaceutically acceptable salt or solvate thereof;

[0010]

[0011] in:

[0012] L is -(CH2) m O-(CH2CH2O) n -(CH2) w -; m is 1 or 2; n is 0, 1, 2, 3; w is 1 or 2.

[0013] Preferably, the protein degradation targeting chimera compound is any one or more of Compounds 1 to 6;

[0014]

[0015]

[0016] The second object of the present invention is to provide a method for preparing the protein degradation targeting chimera compound described in the first object.

[0017] To achieve the above object, the present invention adopts the following technical solutions:

[0018] The preparation method of protein degradation targeted chimera compounds comprises the following steps:

[0019] Step 1: According to Reaction Scheme 1, SM-1 and SM-2 are dissolved in dichloromethane, and triphenylphosphine and diethyl azodicarboxylate are added to react to obtain Intermediate 1.1;

[0020] Reaction 1:

[0021] Step 2: According to Reaction Scheme 2, the intermediate 1.1 obtained in Step 1 is dissolved in tetrahydrofuran, and trimethyl borate and lithium diisopropylamide are added to form a borate; SM-3 is dissolved in a mixed solution of tetrahydrofuran and water, 1,1'-bis(diphenylphosphino)ferrocenepalladium chloride and sodium carbonate are added, and the borate is added dropwise to react to obtain the intermediate 1.2;

[0022] Reaction 2:

[0023] Step 3: According to Reaction Scheme 3, 1,4-dioxane, bis(pinacolato)diboron, potassium acetate, tris(dibenzylideneacetone)dipalladium and tricyclohexylphosphine are sequentially added to the intermediate 1.2 obtained in Step 2 to react and obtain the intermediate 1.3;

[0024] Reaction 3:

[0025] Step 4 is carried out according to Reaction Scheme 4. The intermediate 1.3 obtained in step 3 is mixed with SM-4, and DMF, water, sodium carbonate, tris(dibenzylideneacetone)dipalladium and tricyclohexylphosphine are added in sequence to react to obtain intermediate 1;

[0026] Reaction 4:

[0027] Step 5: According to reaction formula 5, SM-5, NH2(CH2) m (CH2CH2O) n (CH2) w Cl and N,N-diisopropylethylamine were added to the DMF solution, heated and stirred, and the reaction was carried out to obtain compound A;

[0028] Reaction 5:

[0029] Step 6: According to Reaction Scheme 6, the intermediate 1 obtained in Step 4, the compound A obtained in Step 5, ethyldiisopropylamine and dimethylene sulfone of potassium iodide are mixed and reacted to obtain the compound of Formula I.

[0030] Reaction 6:

[0031]

[0032] wherein m, n, and w are defined the same as in Formula I above;

[0033] The reaction in step 4 is carried out under microwave irradiation conditions;

[0034] The NH2(CH2) m (CH2CH2O) n (CH2) w Cl is any one or more of SM-6, SM-7, SM-8, SM-9, SM-10 and SM-11 as shown below;

[0035]

[0036] Furthermore, step 1 is a dehydration reaction under Mitsunobu conditions; in step 2, the intermediate 1.1 obtained in step 1 is first subjected to a Miyaura Borylation reaction to obtain a boronic acid intermediate, which is then subjected to a Suzuki reaction with SM-3 to produce an intermediate 1.2; further, in step 3, the intermediate 1.2 is subjected to a Miyaura Borylation reaction to obtain an intermediate 1.3; in step 4, the intermediate 1.3 is subjected to a Suzuki coupling reaction with SM-4 to produce an intermediate 1; in step 5, SM-5 is reacted with NH2(CH2) m (CH2CH2O) n(CH2) w Cl undergoes an amino nucleophilic substitution reaction to prepare compound A; in step 6, the intermediate 1 obtained in step 4 and the compound A obtained in step 5 undergo an amino nucleophilic substitution reaction and an ester aminolysis reaction to obtain a compound of formula I.

[0037] Furthermore, the reagents used in the reaction of step 1 include triphenylphosphine, diethyl azodicarboxylate and dichloromethane; the reagents used in the reaction of step 2 include tetrahydrofuran, trimethyl borate and 1,1'-bis(diphenylphosphino)ferrocenepalladium chloride; the reagents used in the reaction of step 3 include 1,4-dioxane, bis(pinacolato)diboron, potassium acetate, tris(dibenzylideneacetone)dipalladium and tricyclohexylphosphine; the reagents used in the reaction of step 4 include sodium carbonate, tris(dibenzylideneacetone)dipalladium and tricyclohexylphosphine; the reagents used in the reaction of step 5 include N,N-diisopropylethylamine and N,N-dimethylformamide; the reagents used in the reaction of step 6 include ethyldiisopropylamine, potassium iodide, dimethylene sulfone and ammonia methanol.

[0038] Furthermore, the preparation method of the protein degradation targeted chimera compound is as follows:

[0039] Step 1: SM-1 and SM-2 were dissolved in dichloromethane and placed in an ice bath at -10°C. Triphenylphosphine was added, and diethyl azodicarboxylate was added dropwise. The reaction mixture was removed from the ice bath and stirred at room temperature for 15 hours to obtain a crude reaction mixture. The crude reaction mixture was adsorbed on silica and purified by hexane / EtOAc chromatography to obtain intermediate 1.1.

[0040] Step 2: Dissolve the intermediate 1.1 prepared in tetrahydrofuran and cool to -20°C. Add trimethyl borate and add lithium diisopropylamide dropwise to the reaction over 8 minutes. After about 30 minutes, the borate is generated. In a separate reaction vessel, dissolve SM-3 in 7:3 tetrahydrofuran / water and purge with argon. Add 1,1'-bis(diphenylphosphino)ferrocenepalladium chloride and sodium carbonate and heat the reaction to 65°C. Add the borate obtained above dropwise over 10 minutes and heat the reaction under reflux for 2 hours. Adsorb on silica and purify by dichloromethane / MeOH gradient chromatography. Isolate and dry to obtain intermediate 1.2.

[0041] Step 3: The solution of intermediate 1.2 prepared in step 2 was purged with nitrogen, and 1,4-dioxane, bis(pinacolato)diboron, potassium acetate, tris(dibenzylideneacetone)dipalladium, and tricyclohexylphosphine were added; the mixture was heated at 100°C for 2.5 hours; filtered through celite, and concentrated under vacuum to obtain the crude product of intermediate 1.3.

[0042] Step 4: mixing the intermediate 1.3 prepared in step 3 and SM-4, adding DMF and water, adding sodium carbonate, tris(dibenzylideneacetone)dipalladium, tricyclohexylphosphine; under microwave irradiation, stirring the reaction mixture at 120℃ for 30 minutes; filtering through celite, concentrating under vacuum; purifying by column chromatography with MeOH / dichloromethane to obtain the intermediate 1.

[0043] Step 5: mixing SM-5, NH2(CH2) m (CH2CH2O) n (CH2) w Cl, N,N-diisopropylethylamine in DMF solution, heating to 85℃, stirring for 18 hours; adding water to the reaction mixture and extracting with EtOAc; washing the organic phase with water once, washing with brine once, drying with Na2SO4, concentrating under vacuum; purifying the obtained mixture by hexane / EtOAC column chromatography to obtain compound A.

[0044] Step 6: mixing the compound A prepared in step 5, the intermediate 1 prepared in step 4, ethyldiisopropylamine and potassium iodide in dimethylsulfoxide at 80℃ for 18 hours; after cooling, adding methylammonium, extracting with dichloromethane; concentrating the organic phase under vacuum, purifying the residue by preparative HPLC to obtain the protein degradation targeting chimeric compound.

[0045] As a preferred, the compound A is any one or more of the following compounds 1A, compound 2A, compound 3A, compound 4A, compound 5A and compound 6A as shown below;

[0046]

[0047]

[0048] As a preferred, in step 1, the molar ratio of SM-1 and SM-2 is 1:1; in step 2, the molar ratio of intermediate 1.1 and SM-3 is 6.3:10; in step 3, the molar ratio of intermediate 1.2 and 1,4-dioxane is 1.7:10; in step 5, the molar ratio of SM-5 and NH2(CH2) m (CH2CH2O) n (CH2) w Cl is 0.5:2; in step 6, the molar ratio of compound A, intermediate 1, ethyldiisopropylamine and potassium iodide is 0.17:0.62:4:1.

[0049] The third object of the present application is to provide a pharmaceutical composition containing the protein degradation targeting chimeric compound of the above-mentioned object.

[0050] To achieve the above-mentioned object, the present application adopts the following technical scheme:

[0051] A pharmaceutical composition containing the protein degradation targeting chimera compound of the above purpose and a pharmaceutically acceptable excipient.

[0052] The fourth purpose of the present application is to provide an oral solid preparation containing the pharmaceutical composition of the third purpose.

[0053] Preferably, the oral solid preparation is composed of the compound 2, starch, starch paste, tartaric acid, talc, and light liquid paraffin; the mass ratio of the compound 2, the starch, the tartaric acid, and the talc is 5:4:0.2:2.6.

[0054] Further, the concentration of the talc is 5%.

[0055] Further, the concentration of the starch paste is 15%-17%.

[0056] Further, the preparation method of the oral solid preparation is as follows:

[0057] Mix the compound 2 with 1 / 3 of the starch (total amount is 4g) and add the starch paste (15%-17% containing tartaric acid) to prepare soft material for 10-15 minutes, pass the wet granules through a 14 or 16 mesh nylon screen to dry at 70°C, pass the dry granules through a 12 mesh nylon screen to size, then mix the granules with the remaining starch (previously dried at 100-105°C) and talc with adsorbed liquid paraffin together, and then pass through a 12 mesh nylon screen. The obtained granules are ① filled into capsules to obtain capsules; and ② compressed into tablets to obtain tablets.

[0058] The fifth purpose of the present application is to provide the use of the protein degradation targeting chimera compound of the first purpose and / or the pharmaceutical composition of the third purpose in the preparation of a drug for treating a NEK2 protein-mediated disease.

[0059] Further, the disease is cancer, and the cancer is leukemia, B-cell lymphoma, gastric cancer, colon cancer, breast cancer, pancreatic cancer, and / or liver cancer.

[0060] The sixth purpose of the present application is to provide the use of the protein degradation targeting chimera compound in the preparation of a drug for inhibiting the proliferation activity of tumor cells.

[0061] The seventh purpose of the present application is to provide the use of the protein degradation targeting chimera compound combined with compound B in the preparation of a drug for inhibiting the proliferation activity of tumor cells.

[0062] To achieve the above purposes, the present application adopts the following technical solutions:

[0063] The protein degradation targeted chimera compound is combined with compound B in a drug for inhibiting tumor cell proliferation activity, wherein compound B is any one or more of acetochlor, colchicine, thimerosal, orlistat, amitriptyline hydrochloride, clomipramine hydrochloride and fludarabine.

[0064] Furthermore, the tumor cells are A549 cells.

[0065] As a preference:

[0066] Use of compound 1 in combination with orlistat, amitriptyline hydrochloride, clomipramine hydrochloride and / or fludarabine in a drug for inhibiting tumor cell proliferation activity.

[0067] Use of compound 2 in combination with thimerosal and / or orlistat in a drug for inhibiting tumor cell proliferation activity.

[0068] Use of compound 3 in combination with orlistat and / or amitriptyline hydrochloride in a drug for inhibiting tumor cell proliferation activity.

[0069] Use of compound 4 and / or compound 6 in combination with amitriptyline hydrochloride and / or fludarabine in a drug for inhibiting tumor cell proliferation activity.

[0070] The use of compound 5 combined with amitriptyline hydrochloride in a drug for inhibiting tumor cell proliferation activity.

[0071] The beneficial effects of the present invention are:

[0072] 1. The protein degradation targeting chimeric compounds provided by the present invention have a good inhibitory effect on NEK2 and can be used to treat diseases mediated by NEK2 protein.

[0073] 2. The protein degradation targeted chimeric compounds provided by the present invention have a good inhibitory effect on A549 cell proliferation, and can also synergistically enhance the inhibitory effect of at least one known compound (acetochlor, colchicine, thimerosal, orlistat, amitriptyline hydrochloride, clomipramine hydrochloride, fludarabine, etc.) on A549 cell proliferation. DETAILED DESCRIPTION

[0074] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0075] In the examples of the present application, the intermediate 1 used to prepare compound 2-6 was prepared by the same method as in Example 1.

[0076] The structural formula of the compound involved in the embodiments of the present application is shown in Table 1:

[0077] Table 1. Structural formula of the compound

[0078]

[0079]

[0080]

[0081] In the embodiments of the present application, the reaction formula 1 for preparing intermediate 1.1 is as follows:

[0082] In the embodiments of the present application, the reaction formula 2 for preparing intermediate 1.2 is as follows:

[0083] In the embodiments of the present application, the reaction formula 3 for preparing intermediate 1.3 is as follows:

[0084] In the embodiments of the present application, the reaction formula 4 for preparing intermediate 1 is as follows:

[0085] In the embodiments of the present application, the reaction formula for preparing compound 1A is as follows:

[0086] In the embodiments of the present application, the reaction formula for preparing compound 1 is as follows:

[0087] In the embodiments of the present application, the reaction formula for preparing compound 2A is as follows:

[0088] In the embodiments of the present application, the reaction formula for preparing compound 2 is as follows:

[0089]

[0090] In the embodiments of the present application, the reaction formula for preparing compound 3A is as follows:

[0091] In the embodiments of the present application, the reaction formula for preparing compound 3 is as follows:

[0092]

[0093] In the examples of the present application, the reaction formula for preparing compound 4A is as follows:

[0094] In the examples of the present application, the reaction formula for preparing compound 4 is as follows:

[0095]

[0096] In the examples of the present application, the reaction formula for preparing compound 5A is as follows:

[0097] In the examples of the present application, the reaction formula for preparing compound 5 is as follows:

[0098]

[0099] In the examples of the present application, the reaction formula for preparing compound 6A is as follows:

[0100]

[0101] In the examples of the present application, the reaction formula for preparing compound 6 is as follows:

[0102]

[0103] Example 1. Preparation of Intermediate 1

[0104] 1. Preparation of Intermediate 1.1

[0105] Preparation of Intermediate 1.1: SM-1 (1421 mg, 10 mmol) and SM-2 (1 eq) were dissolved in dichloromethane (1000 ml) and placed in an ice bath at -10°C. Triphenylphosphine (1.4 eq) was added, and diethyl azodicarboxylate (1.4 eq) was added dropwise. The reaction was removed from the ice bath and stirred at room temperature for 15 hours. The crude reaction was adsorbed on silica and purified using hexane / EtOAc chromatography to afford 1980 mg of Intermediate 1.1, with a reaction yield of 63%.

[0106] result:

[0107] HRMS(ESI)C 15 H 14 F3O4 + [M+H] + : Calculated value 315.27, measured value 315.45.

[0108] Elemental analysis (theoretical value vs. measured value): C: 53.07 vs. 53.18; H: 5.67 vs. 5.68; O: 8.44 vs. 8.34.

[0109] 13 C-NMR (400Hz, DMSO-d) δ (ppm): 159.59, 158.76, 147.65, 135.81, 132.69, 131 .81, 128.28, 126.25, 126.20, 124.94, 124.29, 107.17, 79.26, 52.21, 20.15.

[0110] 2. Preparation of Intermediate 1.2

[0111] Intermediate 1.1 (1980 mg, 6.3 mmol) prepared in step 1 was dissolved in tetrahydrofuran and cooled to -20°C. Trimethyl borate (3 eq) was added, and lithium diisopropylamide (2 eq) was added dropwise to the reaction over 8 minutes. After approximately 30 minutes, the boronate was generated. In a separate reaction vessel, SM-3 (2315 mg, 10 mmol) was dissolved in a 7:3 tetrahydrofuran / water mixture and purged with argon. 1,1'-bis(diphenylphosphino)ferrocenepalladium chloride (0.2 eq) and sodium carbonate (2.5 eq) were added, and the reaction was heated to 65°C. The boronate obtained above was added dropwise over 10 minutes, and the reaction was heated to reflux for 2 hours. The product was adsorbed on silica and purified by dichloromethane / MeOH gradient chromatography. Isolation and drying gave 849 mg of intermediate 1.2, a reaction yield of 29%.

[0112] result:

[0113] HRMS(ESI)C 22 H 17 ClF3N2O4 + [M+H] + : Calculated value 465.83, measured value 465.75.

[0114] Elemental analysis: C:56.85 vs.57.22; H:3.47 vs.3.67; N:6.03 vs.5.61; O:13.77 vs.13.67.

[0115] 13C-NMR (400Hz, DMSO-d) δ (ppm): 161.79, 158.74, 154.88, 146.12, 141.09, 138.09, 135.75, 132.65, 131.6 7, 127.44, 126.13, 124.72, 124.69, 124.49, 123.95, 123.78, 120.17, 119.91, 108.88, 79.17, 51.80, 20.03.

[0116] 3. Preparation of Intermediate 1.3

[0117] Intermediate 1.2 (790 mg, 1.7 mmol) obtained in step 2 was dissolved in 1,4-dioxane (881 mg, 10 mmol) under nitrogen. Bis(pinacolato)diboron (1.2 eq), potassium acetate (3 eq), tris(dibenzylideneacetone)dipalladium (0.5 eq), and tricyclohexylphosphine (0.6 eq) were added. The mixture was heated at 100°C for 2.5 hours. Filtered through celite and concentrated under vacuum to afford the crude product of Intermediate 1.3, which was used in the next step without further purification.

[0118] 4. Preparation of Intermediate 1

[0119] Intermediate 1.3 (759 mg, 1.6 mmol) and SM-4 (1.2 eq) were mixed and dissolved in DMF-water (10:1, 100 mL). Sodium carbonate (3 eq), tris(dibenzylideneacetone)dipalladium (0.5 eq), and tricyclohexylphosphine (0.6 eq) were added. The reaction mixture was stirred at 120°C for 30 minutes under microwave irradiation. The mixture was filtered through celite, concentrated under vacuum, and purified by column chromatography using MeOH / dichloromethane to obtain 362 mg of Intermediate 1, with a reaction yield of 39%.

[0120] result:

[0121] HRMS(ESI)C 29 H 28 F3N6O4 + : Calculated value 581.58, measured value 581.96.

[0122] Elemental analysis (theoretical value vs. measured value): C: 60 vs. 60.33; H: 4.69 vs. 4.25; N: 14.48 vs. 14.83; O: 11.02 vs. 11.14.

[0123] 13C-NMR (400Hz, DMSO-d) δ (ppm): 158.59, 154.19, 145.37, 142.86, 137.32, 135.75, 132.54, 131.85, 131.61, 129.35, 128.27, 126.04, 124.7, 124.55, 124.46, 123.6, 121.65, 120.99, 120.17, 108.14, 106.31, 79.39, 57.42(2C), 51.68, 43.4(2C), 20.57.

[0124] Example 2. Preparation of Compound 1

[0125] 1. Preparation of Compound 1A

[0126] A mixture of SM-5 (138 mg, 0.5 mmol), SM-6 (307 mg, 2 mmol), N,N- diisopropylethylamine (DIPEA, 26 μL) in DMF (200 mL) was heated to 85 °C and stirred for 18 h. Water was added to the reaction mixture and extracted with EtOAc. The organic phase was washed once with water, once with brine, dried over Na2SO4, and concentrated in vacuo. The resulting mixture was purified by column chromatography with hexane / EtOAc to give 72 mg of compound 1A in 35% reaction yield.

[0127] Results:

[0128] HRMS (ESI) for C 18 H 21 ClN3O6 + [M+H] + Calcd 410.83, Found 411.02.

[0129] Elemental analysis: C: 52.75 vs. 52.64; H: 4.92 vs. 5.08; N: 10.25 vs. 9.82; O: 23.42 vs. 23.45.

[0130] 13C-NMR (400 Hz, DMSO-d) δ (ppm): 174.30, 169.30, 167.58 (2C), 147.61, 137.24, 132.90, 117.07, 116.22, 111.93, 70.51, 69.71, 67.98, 62.83, 48.30, 45.36, 29.51, 21.35.

[0131] 2. Preparation of compound 1

[0132] A mixture of compound 1A (70 mg, 0.17 mmol) prepared in Step 1, intermediate 1 (360 mg, 0.62 mmol) prepared in Example 1, ethyl diisopropylamine (517 mg, 4 mmol), and potassium iodide (166 mg, 1 mmol) in dimethylsulfoxide (500 mL) was heated at 80 °C for 18 h. After cooling, ammonium hydroxide (400 mL) was added and extracted with dichloromethane. The organic phase was concentrated in vacuo and the residue was purified by preparative HPLC to give 51 mg of compound 1 in 32% reaction yield.

[0133] Results:

[0134] HRMS (ESI) for C 46 H 46 F3N 10 O9 + [M+H] +: Calculated value 939.93, measured value 940.12.

[0135] Elemental analysis (predicted value vs. observed value): C: 58.84 vs. 58.37; H: 4.83 vs. 4.92; N: 14.92 vs. 15.26; O: 15.34 vs. 15.6.

[0136] 13C-NMR (400Hz, DMSO-d) δ (ppm): C, 174.24, 169.27, 167.45 (2C), 162.98, 156.31, 150.33, 147.7 ,145.93,142.73,138.13,137.44,139.05,132.61(2C),132.44,132.36,130.02,128.2,127.73, 126.26, 124.93, 124.05, 124.01, 124.96, 124.55, 119.48, 116.79, 116.6, 111.85, 108.62, 106.8 2, 78.51, 77.38, 70.05, 69.66, 68.13, 63.29, 55.71(2C), 48.99(2C), 45.26, 29.47, 21.42, 20.97.

[0137] Example 3. Preparation of Compound 2

[0138] 1. Preparation of Compound 2A

[0139] SM-5 (138 mg, 0.5 mmol), SM-7 (423 mg, 2 mmol), and N,N-diisopropylethylamine (DIPEA, 26 μL) were heated to 85° C. in a DMF (300 ml) solution and stirred for 18 hours. Water was added to the reaction mixture and extracted with EtOAc. The organic phase was washed once with water and once with brine, dried over Na2SO4, and concentrated in vacuo. The resulting mixture was purified by hexane / EtOAC column chromatography to obtain 77 mg of compound 2A, with a reaction yield of 34%.

[0140] result:

[0141] HRMS(ESI)C 20 H 25 ClN3O7 + [M+H] + : Calculated value 454.88, measured value 455.03.

[0142] Elemental analysis (predicted value vs. observed value): C: 52.93 vs. 52.66; H: 5.33 vs. 5.62; N: 9.26 vs. 9.13; O: 24.67 vs. 24.44.

[0143] 13C-NMR (400Hz, DMSO-d) δ (ppm): 173.53, 169.07, 168.11(2C), 168.11(2C), 147.11, 139.92, 132.8 1, 117.35, 115.68, 112.43, 70.28, 69.93, 69.72, 69.67, 67.93, 63.05, 48.64, 46.09, 29.02, 21.52.

[0144] 2. Preparation of Compound 2

[0145] A mixture of compound 2A (77 mg, 0.17 mmol) obtained in step 1, intermediate 1 (360 mg, 0.62 mmol), ethyldiisopropylamine (517 mg, 4 mmol) and potassium iodide (166 mg, 1 mmol) in dimethylene sulfone (500 mL) was heated at 80° C. for 18 hours. After cooling, ammonia methanol (400 mL) was added and the mixture was extracted with dichloromethane. The organic phase was concentrated in vacuo and the residue was purified by preparative HPLC to obtain 55 mg of compound 2 with a reaction yield of 33%.

[0146] result:

[0147] HRMS(ESI)C 48 H 50 F3N 10 O 10 + [M+H] + : Calculated value 983.98, measured value 984.06.

[0148] Elemental analysis (predicted value vs. observed value): C: 52.93 vs. 53.13; H: 5.33 vs. 5.21; N: 9.26 vs. 9.23; O: 24.67 vs. 24.73.

[0149] 13C-NMR (400Hz, DMSO-d) δ (ppm): 174.01, 169.17, 167.75 (2C), 163.07, 156.14, 151.01, 147.66, 145.67, 143.04, 137.8, 137.29, 139.12, 132.84, 132.34, 132.09, 131.96, 129.13, 128.11, 127.57 ,126.77,124.46,123.63,123.59,124.93,124.17,119.75,116.8,115.91,112.52,108.43,107.1 9, 78.34, 77.18, 70.48, 70.43, 70.24, 69.82, 68.26, 62.51, 55.27(2C), 49.32(2C), 45.62, 29.54.

[0150] Example 4. Preparation of Compound 3

[0151] 1. Preparation of Compound 3A

[0152] SM-5 (138 mg, 0.5 mmol), SM-8 (483 mg, 2 mmol), and N,N-diisopropylethylamine (DIPEA, 26 μL) were heated to 85° C. in a DMF (200 ml) solution and stirred for 18 hours. Water was added to the reaction mixture and extracted with EtOAc. The organic phase was washed once with water and once with brine, dried over Na2SO4, and concentrated in vacuo. The resulting mixture was purified by hexane / EtOAC column chromatography to obtain 87 mg of compound 3A, with a reaction yield of 35%.

[0153] result:

[0154] HRMS(ESI)C 22 H 29 ClN3O8 + [M+H] + : Calculated value 498.94, measured value 499.25.

[0155] Elemental analysis (predicted value vs. observed value): C: 53.07 vs. 53.72; H: 5.67 vs. 5.42; N: 8.44 vs. 8.53; O: 25.7 vs. 25.61.

[0156] 2. Preparation of Compound 3

[0157] A mixture of compound 3A (85 mg, 0.17 mmol) obtained in step 1, intermediate 1 (360 mg, 0.62 mmol), ethyldiisopropylamine (517 mg, 4 mmol) and potassium iodide (166 mg, 1 mmol) in dimethylene sulfone (500 mL) was heated at 80° C. for 18 hours. After cooling, ammonia methanol (400 mL) was added and the mixture was extracted with dichloromethane. The organic phase was concentrated in vacuo and the residue was purified by preparative HPLC to obtain 61 mg of compound 3 with a reaction yield of 35%.

[0158] result:

[0159] HRMS(ESI)C 50 H 54 F3N 10 O 11 + [M+H] + : Calculated value 1028.04, measured value 1027.95.

[0160] Elemental analysis (predicted value vs. observed value): C: 58.47 vs. 58.64; H: 5.2 vs. 5.4; N: 13.64 vs. 13.3; O: 17.14 vs. 17.45.

[0161] 13C-NMR (400Hz, DMSO-d) δ (ppm): 174.07, 168.86, 168.25 (2C), 162.82, 155.63, 150.74, 147.2 ,145.61,142.31,138,137.66,139.42,132.66,132.57,132.35,131.88,129.25,128.43,127.5 8, 126.33, 124.6, 123.91, 123.63, 125, 124.59, 119.45, 116.61, 116.52, 112.33, 108.44, 106. 35, 78.57, 77.8, 70.83, 70.22(2C), 70.15, 70.13, 69.79, 67.91, 63.07, 55.37(2C), 48.95(2C).

[0162] Example 5. Preparation of Compound 4

[0163] 1. Preparation of Compound 4A

[0164] A mixture of SM-5 (138 mg, 0.5 mmol), SM-9 (307 mg, 2 mmol), N,N- diisopropylethylamine (DIPEA, 26 μL) in DMF (200 mL) was heated to 85 °C and stirred for 18 h. Water was added to the reaction mixture and extracted with EtOAc. The organic phase was washed once with water, once with brine, dried over Na2SO4, and concentrated in vacuo. The resulting mixture was purified by column chromatography with hexane / EtOAc to give 87 mg of compound 4A in 36% reaction yield.

[0165] Results:

[0166] HRMS (ESI) for C 18 H 21 ClN3O6 + Calcd 410.83, Found 410.65.

[0167] Elemental analysis (predicted vs. found): C: 52.75 vs. 52.81; H: 4.92 vs. 4.53; N: 10.25 vs. 10.28; O: 23.42 vs. 23.78.

[0168] 2. Preparation of compound 4

[0169] A mixture of compound 4A (82 mg, 0.17 mmol) prepared in Step 1, intermediate 1 (360 mg, 0.62 mmol), ethyldiisopropylamine (517 mg, 4 mmol), and potassium iodide (166 mg, 1 mmol) in dimethylsulfoxide (500 mL) was heated at 80 °C for 18 h. After cooling, ammonium hydroxide (400 mL) was added and extracted with dichloromethane. The organic phase was concentrated in vacuo and the residue was purified by preparative HPLC to give 56 mg of compound 4 in 35% reaction yield.

[0170] Results:

[0171] HRMS (ESI) for C 46 H 46 F3N 10 O9 + [M+H] + Calcd 939.93, Found 940.25.

[0172] Elemental analysis (predicted vs. found): C: 58.84 vs. 58.38; H: 4.83 vs. 5.25; N: 14.92 vs. 15.16; O: 15.34 vs. 15.55.

[0173] 13C-NMR (400Hz, DMSO-d) δ (ppm): 174.18, 168.53, 167.61 (2C), 160.27, 155.89, 150.31, 147.83 ,145.32,143.24,137.27,137.06,139.67,132.85,132.47,132.14,132.08,129.96,128.4,128 .03, 126.06, 125, 124.41, 124.13, 122.02, 124.4, 119.53, 116.81, 115.86, 111.92, 108.79, 107 .02, 81.09, 78.51, 69.86(2C), 68.27, 62.5, 55.61(2C), 53.29(2C), 50.39, 29.54, 20.86, 20.83.

[0174] Example 6. Preparation of Compound 5

[0175] 1. Preparation of Compound 5A

[0176] SM-5 (138 mg, 0.5 mmol), SM-10 (395 mg, 2 mmol), and N,N-diisopropylethylamine (DIPEA, 26 μL) were heated to 85° C. in a DMF (200 ml) solution and stirred for 18 hours. Water was added to the reaction mixture and extracted with EtOAc. The organic phase was washed once with water and once with brine, dried over Na2SO4, and concentrated in vacuo. The resulting mixture was purified by hexane / EtOAc column chromatography to obtain 84 mg of compound 5A, with a reaction yield of 37%.

[0177] result:

[0178] HRMS(ESI)C 20 H 25 ClN3O7 + [M+H] + : Calculated value 454.88, measured value 454.65.

[0179] 2. Preparation of Compound 5

[0180] A mixture of compound 5A (77 mg, 0.17 mmol) obtained in step 1, intermediate 1 (360 mg, 0.62 mmol), ethyldiisopropylamine (517 mg, 4 mmol) and potassium iodide (166 mg, 1 mmol) in dimethylene sulfone (500 mL) was heated at 80° C. for 18 hours. After cooling, ammonia methanol (400 mL) was added and the mixture was extracted with dichloromethane. The organic phase was concentrated in vacuo and the residue was purified by preparative HPLC to obtain 57 mg of compound 5 with a reaction yield of 34%.

[0181] result:

[0182] HRMS(ESI)C 48 H 50 F3N 10 O 10 + [M+H] + : Calculated value 983.98, measured value 983.88.

[0183] 13C-NMR (400Hz, DMSO-d) δ (ppm): 173.87, 168.6, 167.65 (2C), 160.12, 156.02, 150.98, 147.53, 145. 52, 142.32, 137.84, 137.05, 139.55, 132.92, 132.35, 131.91, 131.8, 129.85, 127.74, 127.73, 126.77 , 124.72, 123.84, 123.68, 122.50, 124.16, 119.43, 116.51, 115.92, 111.79, 108.36, 106.55, 81.02, 78.57, 70.50, 69.87, 69.62, 69.36, 68.02, 63.00, 55.6(2C), 53.19(2C), 50.76, 29.30, 21.53, 20.20.

[0184] Example 7. Preparation of Compound 6

[0185] 1. Preparation of Compound 6A

[0186] SM-5 (138 mg, 0.5 mmol), SM-11 (455 mg, 2 mmol), and N,N-diisopropylethylamine (DIPEA, 26 μL) were heated to 85° C. in a DMF (200 ml) solution and stirred for 18 hours. Water was added to the reaction mixture and extracted with EtOAc. The organic phase was washed once with water and once with brine, dried over Na2SO4, and concentrated in vacuo. The resulting mixture was purified by hexane / EtOAc column chromatography to obtain 87 mg of compound 6A, with a reaction yield of 36%.

[0187] result:

[0188] HRMS(ESI)C 22 H 29 ClN3O8 + [M+H] + : Calculated value 498.94, measured value 499.12.

[0189] Elemental analysis (theoretical value vs. measured value): C: 53.07 vs. 53.06; H: 5.67 vs. 5.67; N: 8.44 vs. 8.59; O: 25.7 vs. 25.61.

[0190] 2. Preparation of Compound 6

[0191] A mixture of compound 6A (82 mg, 0.17 mmol) obtained in step 1, intermediate 1 (360 mg, 0.62 mmol), ethyldiisopropylamine (517 mg, 4 mmol) and potassium iodide (166 mg, 1 mmol) in dimethylene sulfone (500 mL) was heated at 80° C. for 18 hours. After cooling, ammonia methanol (400 mL) was added and the mixture was extracted with dichloromethane. The organic phase was concentrated in vacuo and the residue was purified by preparative HPLC to obtain 61 mg of compound 6 with a reaction yield of 35%.

[0192] result:

[0193] HRMS(ESI)C 50 H 54 F3N 10 O 11 + [M+H] + : Calculated value 1028.04, measured value 1027.95;

[0194] 13C-NMR (400Hz, DMSO-d) δ (ppm): 173.45, 169.31, 167.71 (2C), 161.81, 156.37, 150.45, 147.53, 145.52, 142.67, 138.13, 137.31, 139.13, 133.20, 132.58, 132.27, 132.26, 129.26, 128.27, 127.71, 126.65, 124. 90, 124.00, 123.84, 123.75, 123.31, 122.27, 116.78, 115.74, 112.27, 108.83, 107.13, 81.11, 78.29, 70. 77, 70.42, 70.40, 70.11, 69.96, 69.65, 67.78, 62.35, 55.65(2C), 53.03(2C), 50.50, 29.38, 21.76, 20.15.

[0195] Example 8. Biological Experiment - NanoBRET TM analyze

[0196] Using NanoBRET TMThe compounds were tested for their in vivo inhibitory activity against NEK2 as follows:

[0197] Step 1: HEK293 cells NEK-2 Transient transfection of fusion vector DNA. Culture HEK293 cells, trypsinize, and harvest the HEK293 cells. Prepare a lipid:DNA complex containing 9.0 μg / ml of transfection vector DNA, 1.0 μg / ml of NanoLuc fusion vector DNA, and 1 ml of Opti-MEM without phenol red. Mix the solutions thoroughly and add 30 μl of FuGENE HD Transfection Reagent per ml of DNA mixture to form the lipid:DNA complex. Mix the samples and incubate at ambient temperature for 20 minutes to allow the complex to form. Mix 1 part lipid:DNA complex with 20 parts suspended HEK293 cells. Place the HEK293 cells and lipid:DNA complex in a sterile culture dish and incubate for 22-24 hours.

[0198] Step 2: Add test compounds: Each test compound was added from the compound source plate to a 384-well white NBS plate via an Echo 550.

[0199] Step 3: Prepare NanoBRET TM Tracer K5 reagent. Aspirate the culture medium from the culture dish containing the transfected HEK293 cells and trypsinize to detach the cells. Neutralize the trypsin with serum-containing medium and pellet the cells by centrifugation at 200 × g for 5 minutes. Adjust the cell density to 2 × 105 cells / ml in Opti-MEM without phenol red. Prepare the complete 20X NanoBRET kit according to the manufacturer's instructions. TM Tracer K5 reagent. Add a portion of the complete 20X NanoBRET TM Add tracer K5 reagent to 16 aliquots of cells. After mixing, distribute the cell suspension into a white 384-well NBS plate to a final tracer K5 concentration of 2 μM. Incubate at 37°C, 5% CO₂ for 1 hour. Prepare a separate set of tracer-free samples.

[0200] Step 4: NanoBRET TM Analysis. Remove the NBS plate from the incubator and equilibrate at room temperature for 15 minutes. Before measuring BRET, prepare a 3X full substrate plus inhibitor assay medium solution (Opti-MEMR I reduced serum medium, no phenol red). Add the 3X full substrate plus inhibitor solution to each well of the 384-well plate and incubate at room temperature for 2-3 minutes. Use an Envision2104 plate reader to measure the donor emission wavelength (460nm) and the acceptor emission wavelength (600nm).

[0201] Step 5: BRET ratio determination. To generate the raw BRET ratio, divide the acceptor emission value (600 nm) by the donor emission value (460 nm) for each sample. To correct for background, subtract the BRET ratio without tracer (average of the no-tracer control samples) from the BRET ratio for each sample.

[0202] BRET ratio = [(Acceptor sample ÷ Donor sample) - (Acceptor no-tracer control ÷ Donor no-tracer control)]

[0203] The results of the in vitro assay are shown in Table 2, where + = IC 50 >100nM, ++=IC 50 10-100nM, +++=IC 50 <10nM.

[0204] Table 2. NanoBRET TM Analysis results

[0205] Compound number <![CDATA[NEK2抑制活性IC 50 (nM)]]> Compound 1 prepared in Example 2 ++ Compound 2 prepared in Example 3 +++ Compound 3 prepared in Example 4 ++ Compound 4 prepared in Example 5 ++ Compound 5 prepared in Example 6 +++ Compound 6 prepared in Example 7 ++ Comparative Compound 1 +

[0206] Example 9. Activity test of compounds in inhibiting tumor cell proliferation

[0207] 1. The inhibitory activity of tumor cell proliferation by single drug: CCK-8 method was used to detect cell proliferation activity. Specifically, A549 cells in the logarithmic growth phase were taken and 1×10 5 Cells were seeded in 96-well plates at a density of 100 μL per well. The experimental groups were treated with 50 μL of the test compound at a final concentration of 40 nM, with three replicates per well. A blank control group (A549 cells alone, without the test compound) was also established. The cells were cultured in a 37°C, 5% CO2 incubator for 24, 48, and 72 hours. 10 μL of CCK-8 solution was added and incubated for 2 hours. The absorbance (OD) at a wavelength of 450 nm was measured with a microplate reader, and the cell proliferation inhibition rate was calculated. The results are shown in Table 3.

[0208] Table 3. Inhibition rate

[0209]

[0210]

[0211] 2. Synergistic Inhibitory Effect on Cell Proliferation Assay: Using the method described in "1. Inhibitory Activity of Single Agents on Tumor Cell Proliferation," the inhibitory effects of the following test substances on A549 cell proliferation were determined: Test Substance A: A compound prepared in Example 2-7, at a final concentration of 5 nM. Test Substance B: A compound selected from acetochlor, colchicine, thimerosal, orlistat, amitriptyline hydrochloride, clomipramine hydrochloride, and fludarabine, at a final concentration of 500 nM. Test Substance C: Four randomly selected compounds from Test Substance B were combined with Test Substance A, with final concentrations of A and B of 5 nM and 500 nM, respectively.

[0212] The calculation formula of R is: R = IR 受试物C / (IR 受试物A +IR 受试物B ).

[0213] R is the ratio of the combined effect to the sum of the effects of each drug alone; when R > 1, it indicates that test substances A and B synergize to achieve a 1 + 1 > 2 inhibitory effect on K562 cell proliferation. The results are shown in Table 4.

[0214] Table 4

[0215] Test substance A Test substance B R of test substance C Compound 1 prepared in Example 2 Orlistat 1.08 Compound 1 prepared in Example 2 Amitriptyline hydrochloride 1.36 Compound 1 obtained in Example 2 Clomipramine hydrochloride 1.16 Compound 1 obtained in Example 2 Fludarabine 1.08 Compound 2 prepared in Example 3 colchicine 0.94 Compound 2 prepared in Example 3 Thimerosal 1.78 Compound 2 prepared in Example 3 Orlistat 1.03 Compound 2 prepared in Example 3 Amitriptyline hydrochloride 0.99 Compound 3 obtained in Example 4 Orlistat 1.88 Compound 3 obtained in Example 4 Amitriptyline hydrochloride 1.57 Compound 3 obtained in Example 4 Clomipramine hydrochloride 0.98 Compound 3 obtained in Example 4 Fludarabine 0.80 Compound 4 obtained in Example 5 Orlistat 0.93 Compound 4 obtained in Example 5 Amitriptyline hydrochloride 1.94 Compound 4 obtained in Example 5 Clomipramine hydrochloride 0.81 Compound 4 obtained in Example 5 Fludarabine 1.00 Compound 5 obtained in Example 6 colchicine 0.88 Compound 5 obtained in Example 6 Thimerosal 0.86 Compound 5 obtained in Example 6 Orlistat 0.86 Compound 5 obtained in Example 6 Amitriptyline hydrochloride 1.25 Compound 6 prepared in Example 7 Orlistat 0.84 Compound 6 prepared in Example 7 Amitriptyline hydrochloride 1.02 Compound 6 prepared in Example 7 Clomipramine hydrochloride 0.99 Compound 6 prepared in Example 7 Fludarabine 1.28

[0216] Example 10. Preparation of composition

[0217] 1. Prescription:

[0218] Compound 2 prepared in Example 3 5g starch 4g Starch slurry (15% to 17%) QS tartaric acid 0.2g 5% talc 2.6g Light liquid paraffin QS

[0219] 2. Preparation method

[0220] Compound 2 prepared in Example 3 was mixed with one-third of starch and a starch slurry (15%-17%, containing tartaric acid) to prepare a soft material for 10-15 minutes. The mixture was then passed through a 14- or 16-mesh nylon sieve to obtain wet granules. The granules were dried at 70°C and sieved through a 12-mesh nylon sieve. The granules were then mixed with the remaining starch (previously dried at 100-105°C) and talc adsorbed with liquid paraffin, and then passed through a 12-mesh nylon sieve. The resulting granules were then ① filled into capsules to obtain capsules; ② compressed into tablets to obtain tablets.

Claims

1. Protein degradation targeting chimera compound, characterized in that: The protein degradation targeting chimera compound is a compound as shown in Formula I or a pharmaceutically acceptable salt thereof; in: L is -(CH2) m O-(CH2CH2O) n -(CH2) w -; m is 1 or 2; n is 0, 1, 2, 3; w is 1 or 2.

2. The compound according to claim 1, characterized in that The protein degradation targeting chimera compound is any one or more of Compound 1 to Compound 6; 3. The method for preparing the protein degradation targeting chimera compound according to claim 1, characterized in that: The following steps are involved: Step 1: According to Reaction Scheme 1, SM-1 and SM-2 are dissolved in dichloromethane, and triphenylphosphine and diethyl azodicarboxylate are added to react to obtain Intermediate 1.1; Reaction 1: Step 2: According to Reaction Scheme 2, the intermediate 1.1 obtained in Step 1 is dissolved in tetrahydrofuran, and trimethyl borate and lithium diisopropylamide are added to form a borate; SM-3 is dissolved in a mixed solution of tetrahydrofuran and water, 1,1'-bis(diphenylphosphino)ferrocenepalladium chloride and sodium carbonate are added, and the borate is then added dropwise to react to obtain the intermediate 1.2; Reaction 2: Step 3: According to Reaction Scheme 3, 1,4-dioxane, bis(pinacolato)diboron, potassium acetate, tris(dibenzylideneacetone)dipalladium and tricyclohexylphosphine are sequentially added to the intermediate 1.2 obtained in Step 2 to react and obtain the intermediate 1.3; Reaction 3: Step 4 is carried out according to Reaction Scheme 4. The intermediate 1.3 obtained in step 3 is mixed with SM-4, and DMF, water, sodium carbonate, tris(dibenzylideneacetone)dipalladium and tricyclohexylphosphine are added in sequence to react to obtain intermediate 1; Reaction 4: Step 5: According to reaction formula 5, SM-5, NH2(CH2) m (CH2CH2O) n (CH2) w Cl and N,N-diisopropylethylamine were added to the DMF solution, heated and stirred, and the reaction was carried out to obtain compound A; Reaction 5: Step 6: According to Reaction Scheme 6, the intermediate 1 obtained in Step 4, the compound A obtained in Step 5, ethyldiisopropylamine and dimethylene sulfone solution of potassium iodide are mixed and reacted. The intermediate 1 and the compound A undergo amino nucleophilic substitution reaction and ester aminolysis reaction to obtain the compound of Formula I; Reaction 6: Wherein, the definitions of m, n, and w are the same as those of formula I in claim 1; The reaction in step 4 is carried out under microwave irradiation conditions; The NH2(CH2) m (CH2CH2O) n (CH2) w Cl is any one or more of SM-6, SM-7, SM-8, SM-9, SM-10 and SM-11 shown below; 4. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the protein degradation targeting chimera compound according to any one of claims 1 to 2, and a pharmaceutically acceptable excipient.

5. An oral solid preparation containing the pharmaceutical composition according to claim 4.

6. The oral solid preparation according to claim 5, characterized in that The oral solid preparation is composed of Compound 2, starch, starch slurry, tartaric acid, talc, and light liquid paraffin; the mass ratio of Compound 2, starch, tartaric acid, and talc is 5:4:0.2:2.6; the structural formula of Compound 2 is as follows:

7. Use of the protein degradation targeting chimera compound according to any one of claims 1 to 2 and / or the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating diseases mediated by NEK2 protein.

8. The use according to claim 7, characterized in that The disease is cancer, and the cancer is leukemia, B-cell lymphoma, gastric cancer, colon cancer, breast cancer, pancreatic cancer and / or liver cancer.

9. Use of the protein degradation targeted chimera compound according to any one of claims 1 to 2 in the preparation of a drug for inhibiting tumor cell proliferation activity.

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