Compound with AuroraB kinase degradation activity and application thereof

Abstract

The invention discloses a compound with Aurora B kinase degradation activity and application of the compound, the compound can efficiently and selectively degrade Aurora B protein and remarkably inhibit growth and proliferation of tumor cells, and the compound is expected to be used for preparing compounds for treating Aurora B related diseases including cancers.

Description

Technical Field

[0001] This invention relates to a compound with AuroraB kinase degradation activity and its applications, belonging to the field of medicinal chemistry technology. Background Technology

[0002] Aurora B kinase is a crucial mitogen-activated protein kinase in cell division. It plays a vital role in cell cycle regulation, chromosome division, spindle assembly, and cytokinesis. Research on Aurora B kinase has not only deepened our understanding of cell biology but also provided potential targets for the treatment of diseases such as cancer.

[0003] Aurora B kinase, a member of the Aurora kinase family, plays a crucial role in cell mitosis. Its main function is to regulate chromosome condensation, centromere-microtubule binding, spindle formation, and cytoskeleton reorganization during cytokinesis by phosphorylating its target proteins. Aurora B kinase forms the chromosome passenger complex (CPC) with proteins such as INCENP, Borealin, and Survivin, locating at the center of the chromosome and translocating to the middle of the spindle and the cell division groove during late cell division, thus ensuring normal cell division.

[0004] Given the importance of Aurora B kinase in cell division and proliferation, inhibiting Aurora B activity can be used to combat the pathological state of rapid cell proliferation, especially in cancer. Aurora B kinase inhibitors are considered promising anticancer drugs for treating various types of cancer, such as breast cancer, colorectal cancer, lung cancer, and leukemia. Furthermore, abnormal Aurora B activity is also associated with other proliferative diseases such as psoriasis and atherosclerosis. With in-depth research into Aurora B kinase function, its potential as a cancer therapeutic target is increasingly evident. The development of selective Aurora B inhibitors offers opportunities for precision medicine, enabling effective control of pathological cell proliferation. In addition, combining them with other targeted therapies (such as immunotherapy) can improve treatment efficiency and reduce drug resistance. In the future, with the development of personalized medicine, Aurora B inhibitors are expected to achieve wider clinical application.

[0005] Currently, several Aurora B inhibitors have entered clinical trials. Among them, AZD1152 (Barasertib), developed by AstraZeneca, is a highly selective Aurora B inhibitor; however, due to significant side effects and a narrow therapeutic window, some clinical trials have been temporarily suspended. Other small molecule inhibitors, such as GSK1070916 and CCT137690, are also demonstrating their anti-cancer activity in different stages of trials. Studies have also shown that the combined use of Aurora B inhibitors and chemotherapy drugs can produce a synergistic effect, improving treatment efficacy. Currently, no drugs targeting Aurora B have received market approval.

[0006] In recent years, PROTAC technology has attracted much attention due to its ability to induce protein degradation. Unlike traditional small molecule inhibitors, PROTACs form a ternary complex by binding to the target protein and E3 ligase, thereby labeling and degrading specific proteins. This mechanism provides a new approach for the drug development of Aurora B kinase. Designing PROTACs targeting Aurora B can achieve more efficient protein degradation, which not only improves drug selectivity and efficacy but also reduces drug resistance problems caused by simple inhibition. These PROTAC molecules are expected to play an important role in the treatment of various cancers, with potentially lower dosage requirements and fewer side effects. As an important target for cell division regulation, Aurora B kinase, combined with the development of modern biotechnology, especially the application of technologies such as PROTAC, has a very promising future in cancer treatment. Through further research and development, Aurora B-targeted degradation drugs are expected to provide new options for clinical treatment.

[0007] Chinese patent CN202010699560.6 discloses a compound based on the recruitment of ligands by three E3 ubiquitin ligases—CRBN, cIAP, and VHL—and degradation by known Aurora-A inhibitors. However, its anti-tumor activity is not high enough. Summary of the Invention

[0008] The purpose of this invention is to provide a compound with Aurora B kinase degradation activity and its application. This compound can efficiently and selectively degrade Aurora B protein and significantly inhibit the growth and proliferation of tumor cells, and is expected to be used to prepare compounds for the treatment of Aurora B-related diseases, including cancer.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] Compounds possessing AuroraB kinase degradation activity have one or more of the following structural formulas:

[0011]

[0012]

[0013] In the formula, R is H or Me; m = 1 or 2; n = 0 or 1.

[0014] Preferably, it also includes pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, prodrugs, isotope derivatives, or mixtures thereof.

[0015] The use of any of the above-mentioned compounds having AuroraB kinase degradation activity in the preparation of drugs that degrade AuroraB kinase.

[0016] The use of any of the above-mentioned compounds with Aurora kinase degradation activity in the preparation of antitumor drugs.

[0017] Preferably, the tumor is a tumor that highly expresses Aurora B or a tumor that is sensitive to Aurora B degrading agents.

[0018] The beneficial effects of this invention are as follows:

[0019] The prepared compounds can efficiently and selectively degrade Aurora B protein and significantly inhibit the growth and proliferation of tumor cells. This efficient and selective degradation reduces toxic side effects and minimizes drug resistance issues caused by simple inhibition, making them promising candidates for developing compounds to treat Aurora B-related diseases, including cancer. Attached Figure Description

[0020] Figure 1 After treating different cells with different concentrations of compound 1 for 24 h, the expression levels of Aurora B and its main substrate, phosphorylated histone H3 serine 10 (pH3S10), and the expression levels of internal references, histone H3 (H3) and β-actin, were determined.

[0021] Figure 2 The result is for the selectivity of compound 1;

[0022] Figure 3 Results of cell colony formation assay;

[0023] Figure 4 The inhibitory effect of different concentrations of compound 1 in HL-60 suspension cells. Detailed Implementation

[0024] Example 1: Synthesis of intermediate Frag-1:

[0025]

[0026] a. 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (1 g, 3.62 mmol), piperazine-1-carboxylic acid tert-butyl ester (1 g, 5.43 mmol), and DIPEA (1.4 g, 10.86 mmol) were dissolved in DMSO (20 mL) and stirred at 130 °C under nitrogen protection for 2 h. The reaction was monitored by TLC until completion was observed. The reaction mixture was poured into water (150 mL), filtered, and the solid precipitate was collected and dried to give 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazine-1-carboxylic acid tert-butyl ester (1.5 g, 93.7% yield) as a yellow solid. LC_MS: (ES+): m / z 443.14 [M+H] + .

[0027] b. 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazine-1-carboxylic acid tert-butyl ester (3.4 g, 7.4 mmol) was mixed with HCl / EA solution (4 M, 34 mL) and stirred overnight under nitrogen protection. TLC was monitored until the reaction was complete. The precipitate was collected by filtration and dried to give Frag-1:2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindoline-1,3-dione hydrochloride (2.5 g, yellow solid, 92% yield). LC-MS:(ES) + ):m / z343.15[M+H] + .

[0028] Example 2: Synthesis of intermediate Frag-3:

[0029]

[0030] The starting material was 5-fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindole-1,3-dione. The synthesis method was the same as that of Frag-1, and the final product was 2-(1-methyl-2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindole-1,3-dione hydrochloride (215 mg, yield 85%), which was a yellow solid. 1HNMR (400MHz, DMSO-d6): δ9.27 (s, 2H), 7.76-7.74 (d, J=8Hz, 1H), 7.46-7.45 (d, J=4Hz, 1H), 7.35-7.33 (d, J=8Hz, 1H), 5.19-5.14 (m, 1H ), 3.71 (s, 4H), 3.22 (s, 4H), 3.02 (s, 3H), 2.96-2.91 (t, J=20Hz, 1H), 2.79-2.75 (d, J=16Hz, 1H), 2.61-2.55 (m, 1H), 2.07-2.03 (m, 1H).

[0031] Example 3: Synthesis of intermediate Frag-2:

[0032]

[0033] a. To an ethanol solution (30 mL) containing 2-amino-4-fluorobenzoic acid (5 g, 32.26 mmol), formamidine acetate (6.7 g, 64.51 mmol) was added, and the mixture was refluxed overnight under a nitrogen atmosphere. TLC showed that the reaction was complete. The reaction mixture was cooled to room temperature and filtered. The filtrate was washed with 70% ethanol (50 mL), filtered, and dried under vacuum to give 7-fluoroquinazoline-4-ol (4.1 g, 77% yield) as a gray powder. 1 HNMR (400MHz, DMSO-d6): δ7.66-7.41 (m, 1H), 7.43-7.47 (m, 1H), 8.13 (s, 1H), 8.16-8.20 (m, 1H), 12.34 (s, 1H).

[0034] b. Under nitrogen protection at 0°C, sodium hydride (60% mineral, 7.3 g, 182 mmol) was added to a stirred solution of propane-1,3-diol (14 g, 182 mmol) in DMF (50 mL). After stirring the resulting solution at 0°C for 20 min, 7-fluoroquinazoline-4-ol (5 g, 30.5 mmol) was added in portions to the reaction mixture at 0°C. The resulting mixture was stirred overnight at 110°C under N2 atmosphere. TLC was monitored until the reaction was complete. The reaction mixture was concentrated to remove most of the DMF and then quenched with water (100 mL). The resulting mixture was adjusted to pH 6–7 with 1 N hydrochloric acid and filtered. The filter cake was washed with methyl tert-butyl ether (20 mL) and dried under vacuum to give 7-(3-hydroxypropoxy)quinazoline-4-ol (6.5 g, 96.7% yield) as a yellow solid.

[0035] LC_MS:(ES + ):m / z331.2[M+H] + .

[0036] c. Under nitrogen-protected stirring, N,N-dimethylformamide (33 mg, 0.45 mmol) was added to a solution of 7-(3-hydroxypropoxy)quinazoline-4-ol (2 g, 9.08 mmol) in thionyl chloride (20 mL). The resulting reaction mixture was stirred overnight at 80 °C. TLC was monitored until the reaction was complete. The reaction mixture was concentrated, and the residue was quenched with an aqueous sodium bicarbonate solution (1 M), extracted with ethyl acetate (50 mL × 2), the combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude residue, which was purified by rapid silica gel column chromatography (eluting with 20% ethyl acetate in a hexane gradient) to give 4-chloro-7-(3-chloropropoxy)quinazoline (1.1 g, 47% yield) as a white solid. LC-MS: (ES + ):m / z256.95[M+H] + .

[0037] d. Under nitrogen protection and stirring at 0°C, pentafluorophenyl trifluoroacetate (5.6 g, 20 mmol) was added to a mixed solution of 2-(3-amino-1H-pyrazol-5-yl)acetic acid (1.41 g, 10 mmol) in DMF (14 mL) and pyridine (1.8 mL, 22 mmol). The resulting mixture was stirred at 0°C for 10 min, then raised to room temperature and stirred for 90 min. 3-fluoroaniline (2.22 g, 20 mmol) was then added to the reaction mixture, and the resulting reaction mixture was stirred overnight at room temperature. TLC monitoring showed the reaction was complete. The reaction mixture was quenched in H₂O (30 mL) and neutralized with hydrochloric acid (1 N, 40 mL). The obtained compound was extracted with ethyl acetate (50 ml × 3), washed with brine (100 ml), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude residue, which was purified by rapid silica gel column chromatography (eluting with 0-50% ethyl acetate-hexane) to give 2,2,2-trifluoro-N-(5-(2-((3-fluorophenyl)amino)-2-oxoethyl)-1H-pyrazol-3-yl)acetamide (2.2 g, yield 67%) as a white solid.

[0038] LC_MS:(ES + ):m / z331.2[M+H] + .

[0039] 1HNMR (400MHz, DMSO-d6): δ3.74 (s, 2H), 6.47 (s, 1H), 6.86-6.91 (m, 1H), 7.28-7.32 (m, 1 H), 7.33-7.38 (m, 1H), 7.57 (d, J=11.6Hz, 1H), 10.4 (s, 1H), 11.91 (s, 1H), 12.6 (br, 1H).

[0040] e. 2,2,2-Trifluoro-N-(5-(2-((3-fluorophenyl)amino)-2-oxoethyl)-1H-pyrazol-3-yl)acetamide (2.4 g, 7.2 mmol) was dissolved in a mixture of dilute hydrochloric acid (2N, 18 mL) and methanol (18 mL), and stirred at 50 °C under nitrogen protection for 5 hours. TLC was monitored until the reaction was complete. The reaction mixture was concentrated to remove MeOH, and then neutralized with a saturated aqueous solution of NaHCO3. The resulting mixture was extracted with ethyl acetate (25 mL × 2), washed with water (25 mL × 2) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was pulped with DCM and filtered to give 2-(3-amino-1H-pyrazol-5-yl)-N-(3-fluorophenyl)acetamide (1.0 g, yield 58%) as a yellow solid. LC_MS:(ES+):m / z235.1[M+H] + .

[0041] 1 HNMR (400MHz, DMSO-d6): δ3.48 (s, 2H), 5.30 (s, 1H), 6.83-6.88 (m, 1H), 7.28-7.36 (m, 2H), 7.57-7.61 (m, 1H), 10.26 (s, 1H).

[0042] f. 2-(3-amino-1H-pyrazol-5-yl)-N-(3-fluorophenyl)acetamide (2 g, 7.78 mmol) and 4-chloro-7-(3-chloropropoxy)quinazoline (1.8 g, 7.78 mmol) were added to isopropanol (40 mL), and the resulting solution was stirred at 80 °C under nitrogen protection for 2 hours. TLC monitoring showed that the reaction was complete. The solid precipitate was collected by filtration and dried to give 2-(3-((7-(3-chloropropoxy)quinazoline-4-yl)amino)-1H-pyrazol-5-yl)-N-(3-fluorophenyl)acetamide (3 g, yield 77%) as a yellow solid.

[0043] LC_MS:(ES+):m / z455.10[M+H] + .

[0044] 1HNMR (400MHz, DMSO-d6): δ11.89 (s, 1H), 10.72 (s, 1H), 8.93 (s, 1H), 8.80 (d, J=9.3Hz, 1H), 7.64 (d, J=11.7Hz, 1H), 7.47 (dd, J=9. 3, 2.4Hz, 1H), 7.42-7.31 (m, 3H), 6.96-6.84 (m, 1H), 6.74 (s, 1H), 4.32 (t, J=6.0Hz, 2H), 3.92-3.81 (m, 4H), 2.27 (p, J=6.2Hz, 2H).

[0045] Example 4: Synthesis of intermediate Frag-4:

[0046]

[0047] a. Under nitrogen protection, tert-butylpiperazine-1-carboxylate (10 g, 53.8 mmol) and cesium carbonate (21 g, 64.5 mmol) were dissolved in acetonitrile (150 ml), and 1-bromo-2-chloroethane (15.4 g, 108 mmol) was added dropwise in an ice bath. After the addition was complete, the reaction temperature was brought back to room temperature and stirred overnight. Thin-layer chromatography (TLC) showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography (EA:DCM = 1:30 v / v) to give tert-butyl-4-(2-chloroethyl)piperazine-1-carboxylate (2.3 g, yield 17%), a pale yellow solid at room temperature. LC-MS: (ES + ):m / z250.10[M+2H] + .t R =1.195min.

[0048] b. In dimethyl sulfoxide (DMSO, 10 mL), tert-butyl 4-(2-chloroethyl)piperazine-1-carboxylate (500 mg, 2.016 mmol) and diisopropylethylamine (1.04 g, 8.063 mmol) were dissolved, and Frag-1 (764 mg, 2.016 mmol) was added. The mixture was heated to 85 °C and reacted for 16 hours. LCMS showed that the reaction was complete. After cooling, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (MeOH:DCM = 1:25 v / v) yielded 4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazine-1-yl)ethyl)piperazine-1-carboxylate (325 mg, yield 29%).

[0049] c. In an ice bath, tert-butyl 4-(2-(4-(2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)ethyl)piperazin-1-carboxylic acid ester (325 mg, 0.587 mmol) was gradually added to a dioxane / hydrochloric acid solution (20 mL, 4 M). The resulting mixture was stirred at 0–10 °C for 30 min, then brought to room temperature and stirred for another 2 h. Liquid chromatography-mass spectrometry (LC-MS) showed that the reaction was complete. The resulting mixture was concentrated under reduced pressure to give 2-(2,6-dioxadiazin-3-yl)-5-{4-[2-(piperazin-1-yl)ethyl]piperazin-1-yl}isoindoline-1,3-dione hydrochloride (293 mg, 95% yield), which was of high purity and required no further purification. LC-MS:(ES + ):m / z455.55[M+H] + .t R =0.759min.

[0050] Example 5: Synthesis of Compound 1:

[0051]

[0052] Under nitrogen protection, Frag-2 (100 mg, 0.219 mmol) was added to a DMF (2.5 mL) solution of Frag-4 (138 mg, 0.263 mmol), tripotassium phosphate (278 mg, 1.314 mmol), and sodium iodide (49 mg, 0.328 mmol). The resulting mixture was heated to 55 °C and stirred for 16 hours. TLC showed that the reaction was complete. The reaction solution was cooled and filtered, then concentrated under reduced pressure to obtain a crude product. This crude product was purified by preparative high-performance liquid chromatography (HPLC) to give compound 1 (28 mg, yield 15%) as a yellow solid.

[0053] LC_MS:(ES + ):m / z873.90[M+H] + .t R =2.766min.

[0054] 1HNMR (400MHz, DMSO-d6) δ2.02 (t, J=9.6Hz, 1H), 2.23 (s, 2H), 2.40 (s, 1H), 2.49 (s, 2H), 2.51 (s, 5H), 2.62 (t, J=37.2Hz, 1H), 2. 79 (s, 2H), 3.04 (d, J = 26.8Hz, 4H), 3.40 (s, 3H), 3.58 (s, 3H), 3.76 (s, 3H), 3.81 (s, 3H), 4.28 (t, J = 11.2Hz, 2H), 5.09 (m, 1H), 6.7 3 (s, 1H), 6.90 (t, J = 19.2Hz, 1H), 7.26 (s, 1H), 7.32 (t, J = 16.2Hz, 1H), 7.37 (t, J = 16.2Hz, 3H), 7.49 (d, J = 0.8Hz, 1H), 7.61 (t, J =16Hz, 1H), 7.77 (d, J = 8.4Hz, 1H), 8.72 (d, J = 13.2Hz, 1H), 8.88 (s, 1H), 9.82 (s, 1H), 10.51 (s, 1H), 11.10 (s, 1H), 11.62 (s, 1H).

[0055] Example 6: Synthesis of compound 11:

[0056]

[0057]

[0058] The starting main reactant was Frag-3, and the synthesis method was the same as in Example 5, yielding compound 12 (41.6 mg, yield 12.4%).

[0059] 1HNMR (400MHz, DMSO-d6): δ11.72 (s, 1H), 10.54 (s, 1H), 10.06 (s, 1H), 8.90 (s, 1H), 8.77-8.74 (d, J=12Hz, 1H), 7.79-7.77 (d, J=8Hz, 1H) , 7.64-7.61 (d, J=12Hz, 1H), 7.49-7.48 (d, J=4Hz, 1H), 7.42-7.36 (m, 3H), 7.32 (t, J=4Hz, 2H), 6.90 (t, J=16Hz, 1H), 6.74 (s, 1H), 5.19-5 .15 (m, 1H), 4.30 (t, J=8Hz, 2H), 3.82 (s, 2H), 3.77 (s, 5H), 3.58 (s, 2H), 3.42-3.28 (m, 5H), 3.10 (s, 3H), 3.04-3.00 (d, J=16Hz, 3H), 2.9 7-2.92 (m, 2H), 2.881-2.75 (m, 3H), 2.62-2.54 (m, 1H), 2.50-2.33 (d, J=68Hz, 3H), 2.24-2.23 (d, J=4Hz, 2H), 2.07-2.04 (m, J=12Hz, 2H).

[0060] Example 7: Synthesis of compound 2:

[0061]

[0062] a. Under nitrogen-protected stirring, tert-butyl 4-(2-oxoethyl)piperidin-1-carboxylate (827 mg, 3.64 mmol) and sodium triacetylborohydride (1.54 g, 7.28 mmol) were added to a 20 mL solution of Frag-1 (1 g, 3.64 mmol) in DMF. The resulting reaction mixture was stirred at room temperature for 2 h, and TLC was monitored until the reaction was complete. The reaction mixture was quenched in water (150 mL) and extracted with DCM (100 mL × 2). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude residue. This residue was purified by silica gel column chromatography (DCM:MeOH = 30:1) to give a yellow solid tert-butyl 4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)ethyl)piperidin-1-carboxylic acid (1.7 g, yield 75%). LC-MS: (ES) + ):m / z554.40[M+H] + .

[0063] b. 4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)ethyl)piperidin-1-carboxylic acid tert-butyl ester (553 mg, 1.0 mmol) was dissolved in HCl / EA solution (4 M, 10 mL), and the resulting mixture was stirred overnight at room temperature under nitrogen protection. TLC monitoring showed that the reaction was complete. The solid precipitate was collected by filtration and dried to give 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(piperidin-4-yl)ethyl)piperazin-1-yl)isoindoline-1,3-dione hydrochloride (490 mg, 100% yield) as a yellow solid. LC_MS: (ES + ):m / z454.10[M+H] + .

[0064] 1 HNMR (400MHz, Chloroform-d): δ8.13 (s, 1H), 7.69 (d, J=8.5Hz, 1H), 7.28 (d, J=2.3Hz, 1H) , 7.05 (dd, J=8.6, 2.4Hz, 1H), 4.93 (dd, J=12.3, 5.3Hz, 1H), 4.07 (s, 2H), 3.43 (t, J=5.1Hz , 4H), 2.92-2.64 (m, 4H), 2.59 (t, J=5.1Hz, 4H), 2.43 (t, J=7.3Hz, 2H), 2.16-2.08 (m, 1H), 1.67 (d, J=13.1Hz, 4H), 1.48 (d, J=7.0Hz, 2H), 1.45 (s, 9H), 1.15 (dd, J=12.8, 8.6Hz, 2H).

[0065] c. Under nitrogen protection at room temperature, sodium iodide (33 mg, 0.22 mmol) was added to a 5 mL solution of fragment Frag-2 (100 mg, 0.22 mmol) in acetone. The resulting mixture was stirred at 60 °C for 8 h, and then the reaction mixture was concentrated under reduced pressure to obtain a reaction mixture which was dissolved in DMF (5 mL). 2-(2,6-dioxadiazin-3-yl)-5-(4-(2-(piperidin-4-yl)ethyl)piperazin-1-yl)isoindoline-1,3-dione hydrochloride (108 mg, 0.22 mmol) and K₂CO₃ (91 mg, 0.66 mmol) were then mixed. The resulting mixture was stirred at 60 °C under nitrogen protection for 2 days. TLC and LCMS were monitored until the reaction was complete. The reaction mixture was filtered, and the filter cake was washed with DMF. The combined DMF organic phases were concentrated under reduced pressure. The crude product was first purified by thin-layer chromatography (DCM / MeOH / NH3·H2O=10 / 1 / 0.05), and then purified by HPLC to obtain compound 2 (90 mg, 46.9%) as a yellow solid. LC-MS: (ES + ):m / z872.30[M+H] + .tR=2.275min.

[0066] 1 HNMR (400MHz, DMSO-d6): δ11.13 (s, 1H), 10.53 (s, 1H), 9.55 (s, 1H), 8.87 (s, 1H), 8.73 (d, J=9.3Hz, 1H), 7.78 (d, J=8.4 Hz, 1H), 7.63 (dt, J=11.7, 2.2Hz, 1H), 7.51 (d, J=2.3Hz, 1H), 7.43-7.26 (m, 6H), 6.91 (td, J=8.6, 2.4Hz, 1H), 6.74 (s, 1 H), 5.11 (dd, J=12.9, 5.4Hz, 1H), 4.27 (q, J=11.1, 8.3Hz, 4H), 3.81 (s, 2H), 3.47 (s, 2H), 3.23 (d, J=18.5Hz, 8H), 2.97- 2.88 (m, 2H), 2.67-2.54 (m, 4H), 2.35-2.19 (m, 4H), 2.03 (dt, J=9.8, 4.1Hz, 1H), 1.92 (m, 2H), 1.64 (m, 2H), 1.41 (m, 2H).

[0067] Example 8: Synthesis of compound 4:

[0068]

[0069] The starting main reactant was 4-formylpiperidine-1-carboxylic acid tert-butyl ester, and the synthesis method was the same as in Example 7. The final product was...

[0070] 2-{5-[(7-{3-[4-({4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]piperazin-1-yl}-methyl)piperidin-1-yl]propoxy}-quinazolin-4-yl)amino]-2H-pyrazole-3-yl}-N-(3-fluorophenyl)acetamide (13 mg, yield 3.4%), is a yellow solid. LC-MS: (ES + ):m / z858.40[M+H] + .

[0071] 1 HNMR (400MHz, DMSO-d6): δ11.12 (s, 1H), 10.55 (s, 1H), 9.79 (s, 1H), 8.86 (s, 1H), 8.73 (d, J=9.2Hz, 1H), 7 .78 (d, J=8.4Hz, 1H), 7.67-7.60 (m, 1H), 7.51 (d, J=2.1Hz, 1H), 7.42-7.28 (m, 6H), 6.90 (ddd, J=9.4, 6.2, 2 .4Hz, 1H), 6.74 (s, 1H), 5.11 (dd, J=12.9, 5.4Hz, 1H), 4.29 (t, J=5.8Hz, 2H), 3.81 (s, 2H), 3.48 (s, 2H), 3.3 2 (m, 8H), 3.19-3.06 (m, 4H), 3.04-2.83 (m, 4H), 2.64-2.53 (m, 2H), 2.25 (m, 2H), 2.03 (m, 3H), 1.47 (m, 2H).

[0072] Example 9: Synthesis of compound 6:

[0073]

[0074] a. Under nitrogen-protected stirring, tert-butyl 3-formylazetane-1-carboxylate (490 mg, 2.64 mmol) and sodium triacetylborohydride (1.12 g, 5.28 mmol) were added to a DMF (20 mL) solution of Frag-1 (1 g, 2.64 mmol). The reaction mixture was stirred at room temperature for 2 hours, and TLC showed that the reaction was complete. The reaction mixture was poured into water (150 mL) and extracted with DCM (100 mL × 2). The organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by silica gel rapid column chromatography (DCM:MeOH = 30:1, v / v) to give 4-(2-(4-(2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)ethyl)piperidin-1-carboxylic acid tert-butyl ester (950 mg, yield 70%) as a yellow solid. LC-MS: (ES) + ):m / z512.25[M+H] + .

[0075] b. A solution of the above product (200 mg, 0.39 mmol) in TFA (2 ml) and DCM (10 ml) was stirred overnight under nitrogen protection. TLC showed the reaction was complete. The reaction mixture was concentrated to give 5-(4-(azacyclobutane-3-ylmethyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione trifluoroacetate (199 mg, 100% yield), as a yellow oil. No further purification was required; it was used directly in the next reaction. LC_MS:(ES + ):m / z412.10[M+H] + .

[0076] c. Under nitrogen protection at room temperature, a DMF (5 mL) solution of the intermediate fragment Frag-2 (213 mg, 0.39 mmol) was added with the product from step b (198 mg, 0.39 mmol) and K3PO4 (248 mg, 1.17 mmol). The resulting mixture was stirred at 60 °C for 2 days, and the reaction was monitored by TLC and LCMS until completion. The reaction mixture was filtered, and the filter cake was washed with DMF. The combined DMF organic phases were concentrated under reduced pressure. The resulting crude residue was first purified by preparative thin-layer chromatography (DCM:MeOH:NH3·H2O = 10:1:0.05, v / v / v) and then purified by preparative HPLC to obtain compound 6 (19 mg, yield 5.9%) as a yellow solid.

[0077] LC_MS:(ES + ):m / z830.10[M+H] + .

[0078] 1 HNMR (400MHz, DMSO-d6): δ11.09 (s, 1H), 10.82 (s, 1H), 8.59 (d, J=9.5Hz, 2H), 7.71 (d, J=8.4Hz, 1H), 7.65 (dd, J=11.7, 2. 3Hz, 1H), 7.50 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.37 (s, 1H), 7.32 (d, J=8.0Hz, 1H), 7.25 (d, J=4.4Hz, 2H), 7.21 (d, J=3.1 Hz, 1H), 6.87 (td, J=8.4, 2.5Hz, 1H), 5.08 (dd, J=12.9, 5.4Hz, 1H), 4.23 (h, J=7.4, 6.6Hz, 4H), 3.93 (s, 2H), 3.79 (s, 2H), 3.52-3.45(m, 4H), 3.16(s, 2H), 2.95-2.76(m, 3H), 2.64-2.52(m, 2H), 2.12-1.93(m, 4H), 1.36-1.24(m, 2H), 1.22(s, 2H).

[0079] Example 10: Synthesis of compound 3:

[0080]

[0081]

[0082] a. Under nitrogen protection and stirring, 2-(1-(tert-butyloxycarbonyl)piperidin-4-yl)acetic acid (353 mg, 1.45 mmol), HATU (753 mg, 1.98 mmol), and DIPEA (512 mg, 3.96 mmol) were added to 10 mL of Frag-1 (500 mg, 1.32 mmol) in DMF. The reaction mixture was stirred at room temperature for 2 hours, and TLC was monitored until the reaction was complete. The reaction mixture was poured into water (80 mL) and extracted with DCM (50 mL × 2). The organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude residue. The crude residue was purified by silica gel rapid column chromatography (DCM:MeOH = 30:1) to give 4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)-2-oxoethyl)piperidin-1-carboxylic acid tert-butyl ester (800 mg, yield 76%), as a yellow solid. LC-MS: (ES) + ):m / z568.20[M+H] + .

[0083] b. Dissolve the above product (200 mg, 0.35 mmol) in hydrogen chloride / ethyl acetate (4 N, 5 mL) solution and stir overnight under nitrogen protection. TLC was monitored until the reaction was complete. The precipitate was collected by filtration and dried to give 2-(2,6-dioxadiazin-3-yl)-5-(4-(2-(piperidin-4-yl)acetyl)piperazin-1-yl)isoindoline-1,3-dione hydrochloride (176 mg, 100% yield) as a yellow solid.

[0084] c. Under nitrogen protection, the product from step b (177 mg, 0.35 mmol) and K3PO4 (223 mg, 1.05 mmol) were added to a 5 mL solution of Frag-2 (191 mg, 0.35 mmol) in DMF. The resulting mixture was stirred at 60 °C for 2 days, and the reaction was monitored by TLC and LCMS until complete. After cooling, the reaction mixture was filtered, and the filter cake was washed with DMF. The combined DMF organic phases were concentrated under reduced pressure. The crude product was first purified by thin-layer chromatography (DCM:MeOH:NH3·H2O = 10:1:0.05), and then purified by preparative HPLC to give compound 3 (50 mg, yield 16%) as a yellow solid. LC-MS: (ES + ):m / z886.50[M+H] + .

[0085] 1 HNMR (400MHz, DMSO-d6): δ12.41 (s, 1H), 11.10 (s, 1H), 10.54 (s, 1H), 10.24 (s, 1H), 8.53 (s, 2H), 7.72 (d, J=8.5 Hz, 1H), 7.63 (dt, J=11.0, 1.9Hz, 1H), 7.43-7.30 (m, 3H), 7.30-7.09 (m, 3H), 6.89 (t, J=7.7Hz, 1H), 6.78 (s, 1H), 5.09 (dd, J=12.9, 5.4Hz, 1H), 4.24 (s, 2H), 3.78 (s, 2H), 3.64 (s, 4H), 3.58-3.43 (m, 6H), 3.26-3.15 (m, 2H), 3.05 -2.82 (m, 3H), 2.65-2.53 (m, 2H), 2.37 (d, J=5.8Hz, 2H), 2.23 (s, 2H), 2.09-1.82 (m, 4H), 1.49 (d, J=15.8Hz, 2H).

[0086] Example 11: Synthesis of compound 5:

[0087]

[0088] The starting main reactant was 1-(tert-butyloxycarbonyl)piperidine-4-carboxylic acid, synthesized using the same method as in Example 10. The final product was compound 5 (18 mg, yield 11.4%), a yellow solid. LC-MS: (ES) + ):m / z872.05[M+H] + .

[0089] 1 HNMR (400MHz, DMSO-d6): δ11.09 (s, 1H), 10.53 (s, 1H), 9.52 (s, 1H), 8.88 (s, 1H), 8.74 (d, J=9.3Hz, 1H), 7 .72 (d, J=8.4Hz, 1H), 7.62 (dt, J=11.7, 2.3Hz, 1H), 7.45-7.23 (m, 7H), 6.90 (td, J=7.9, 2.4Hz, 1H), 6.73 ( s, 1H), 5.08 (dd, J=12.9, 5.4Hz, 1H), 4.29 (t, J=6.0Hz, 2H), 3.81 (s, 2H), 3.71 (s, 2H), 3.63-3.61 (m, 2H), 3.27(d, J=5.1Hz, 2H), 3.10-2.95(m, 4H), 2.88(m, 2H), 2.78-2.53(m, 2H), 2.25(m, 4H), 2.07-1.79(m, 7H).

[0090] Example 12: Synthesis of compound 10:

[0091]

[0092]

[0093] A solution of tert-butyl piperazine-1-carboxylate (23 g, 123 mmol) and 7-bromoheptane-1-ol (24 g, 123 mmol) in MeCN (460 mL) was stirred overnight at 83 °C under nitrogen protection. TLC showed the reaction was complete. The reaction mixture was poured into water (150 mL), extracted with ethyl acetate (200 mL × 2), washed with brine, filtered, and concentrated. The crude product was subjected to rapid silica gel column chromatography to give 34.8 g of 4-(7-hydroxyheptyl)piperazine-1-carboxylate (93.7% yield), as a yellow liquid. 1 HNMR (400MHz, DMSO-d6): δ4.33 (t, J=5.1Hz, 1H), 3.40-3.35 (m, 2H), 3.28 (t, J=5.0Hz, 4H), 2.30-2.20 (m, 6H), 1.39 (s, 13H), 1.29-1.22 (m, 6H).

[0094] b. Under nitrogen protection and stirring, p-toluenesulfonyl chloride (8.6 g, 44.9 mmol) was added dropwise to a DCM solution of the product from step a (13.5 g, 44.9 mmol), triethylamine (13.6 g, 134.7 mmol), and DMAP (2.7 g, 22.5 mmol) at 0 °C, and the mixture was stirred at room temperature for 3 h. TLC showed that the reaction was complete. The reaction was quenched with water (100 mL), and the organic phase was dried over brine (100 mL) and anhydrous sodium sulfate. The organic layer was concentrated by filtration, and the crude residue was purified by silica gel column chromatography to give tert-butyl 4-(7-(toluenesulfonyloxy)heptyl)piperazine-1-carboxylic acid (17 g, yield 85%).

[0095] c. The product from step b (15 g, 33 mmol) was dissolved in an aqueous ethylamine solution (74 mL) and stirred at room temperature for 7 h under nitrogen protection. The mixture was concentrated to remove water, then diluted with DCM (200 mL) and washed with water (100 mL) and brine (100 mL), and dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated to give the crude residue 4-(7-(ethylamino)heptyl)piperazine-1-carboxylic acid tert-butyl ester (13.5 g, 100% yield). This crude product was used directly in the next step without further purification. LC-MS:(ES) + ):m / z328.30[M+H] + .

[0096] d. The product from step c (10 g, 30.5 mmol), triethylamine (3.4 g, 33.6 mmol), and DMAP (373 mg, 3.05 mmol) were dissolved in DCM (50 mL) and cooled to 0 °C. Then, benzyl chloroformate (5.7 g, 33.6 mmol) was added dropwise under nitrogen protection with stirring, and the mixture was stirred overnight at 0 °C. TLC showed the reaction was complete. The reaction mixture was quenched with saturated sodium bicarbonate solution, washed with water (100 mL) and brine (100 mL), and dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated, and the crude residue was purified by silica gel flash column chromatography to give 4-(7-(((benzyloxy)carbonyl)(ethyl)amino)heptyl)piperazine-1-carboxylic acid tert-butyl ester (10 g, 71% yield). LC-MS:(ES) + ):m / z462.85[M+H] + .

[0097] e. The product from step d (8 g, 17.3 mmol) was dissolved in HCl / EA (4 N, 40 mL) solution, and the reaction mixture was stirred overnight under nitrogen protection. TLC showed that the reaction was complete. The precipitate was collected by filtration and dried to give benzylethyl (7-(piperazin-1-yl)heptyl)carbamate (5.5 g, yield 87%) as a white solid.

[0098] f. Under nitrogen protection, 2-(2,6-dioxadiazine-3-yl)-5-fluoroisoindoline-1,3-dione (764 mg, 2.77 mmol) and DIPEA (1.07 g, 8.31 mmol) were added to a DMF (20 mL) solution of the product (1 g, 2.77 mmol) from step e at room temperature. The resulting mixture was stirred overnight at 90 °C. TLC showed that the reaction was complete. The reaction mixture was quenched with water (200 mL) and then extracted with DCM (100 mL first). All organic layers were combined and washed again with water (100 mL) and brine (100 mL), respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated under reduced pressure. The crude product was purified by silica gel rapid column chromatography (PE / EA = 1 / 1, v / v) to give benzyl(7-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindololin-5-yl)piperazin-1-yl)heptyl)(ethyl)carbamate (570 mg, yield 33.5%) as a yellow solid. LC-MS: (ES) + ):m / z618.35[M+H] + .

[0099] g. Add Pd(OH)₂ / C (55 mg, 10%) to a MeOH (6 mL) solution of the product from step f (550 mg, 0.89 mmol). Stir the mixture overnight at room temperature under hydrogen at 1 atm. TLC showed the reaction was complete. Filter the reaction mixture and wash with MeOH (50 mL). Combine the organic phases and concentrate to give 2-(2,6-dioxadiazin-3-yl)-5-(4-(7-(ethylamino)heptyl)piperazin-1-yl)isoindoline-1,3-dione (430 mg, 100% yield) as a yellow solid. This crude product was used directly in the next reaction without further purification. LC-MS:(ES) + ):m / z484.25[M+H] + .

[0100] h. Under nitrogen protection at room temperature, the product (400 mg, 0.83 mmol), K₂CO₃ (343 mg, 2.48 mmol), and KI (69 mg, 0.41 mmol) from step g were added to a DMF (10 mL) solution of fragment Frag-2 (376 mg, 0.83 mmol) in step g. The resulting mixture was stirred for 2 days at 60 °C under nitrogen protection, and the reaction was complete by TLC and LCMS. The reaction mixture was filtered, and the filter cake was washed with an appropriate amount of DMF. The DMF organic phases were combined and concentrated under reduced pressure. The crude product was first purified by preparative thin-layer chromatography (DCM:MeOH:NH₃·H₂O = 10:1:0.05), and then purified by preparative HPLC to give compound 11 (10 mg, yield 1.3%) as a yellow solid. LC_MS:(ES) + ):m / z902.80[M+H] + .

[0101] 1 HNMR (400MHz, DMSO-d6): δ11.11 (s, 1H), 10.50 (s, 1H), 8.81 (s, 1H), 8.69 (d, J = 9.3Hz, 1H), 7.77 (d, J = 8.4Hz, 1H), 7.62 (dt, J=11.7, 2.3Hz, 1H), 7.49 (d, J=2.2Hz, 1H), 7.40-7.28 (m, 5H), 7.25 (d, J=2.5Hz, 1H), 6.90 (td, J=8.3, 2.6 Hz, 1H), 6.73 (s, 1H), 5.10 (dd, J=12.9, 5.4Hz, 1H), 4.28 (m, 2H), 3.80 (s, 2H), 3.23-3.20 (m, 2H), 3.14-3.09 (m, 4H) , 2.89 (m, 2H), 2.70-2.53 (m, 4H), 2.19 (m, 2H), 2.09-1.94 (m, 2H), 1.65 (s, 5H), 1.33 (s, 8H), 1.24 (d, J=6.9Hz, 7H).

[0102] Example 13: Synthesis of compound 7:

[0103]

[0104] a. K₂CO₃ (32.9 g, 238.2 mmol) was added to a DMF (150 mL) solution of methyl 4-hydroxy-3-methoxybenzoate (14.5 g, 79.4 mmol) and 1-bromo-3-chloropropane (25 g, 158.8 mmol), and the mixture was stirred overnight at 50 °C under nitrogen protection. TLC showed that the reaction was complete. The reaction mixture was poured into water (1.2 L), the precipitate was filtered, collected, and dried to give methyl 4-(3-chloropropoxy)-3-methoxybenzoate (20 g, 97% yield) as a white solid. The product was very pure and required no further purification.

[0105] 1 HNMR (400MHz, DMSO-d6): δ7.57 (dd, J=8.4, 2.0Hz, 1H), 7.45 (d, J=2.0Hz, 1H), 7.10 (d, J=8. 5Hz, 1H), 4.16 (t, J=6.1Hz, 2H), 3.82 (s, 6H), 3.78 (t, J=6.5Hz, 2H), 2.19 (p, J=6.3Hz, 2H).

[0106] b. Under nitrogen protection and stirring, HNO3 (9 mL) was added dropwise to a solution of the product from step a (15 g, 58 mmol) and acetic anhydride (9 mL) in glacial acetic acid (100 mL) at 0 °C. The resulting compound was stirred at room temperature for 10 minutes, and then stirred overnight at 50 °C. TLC showed that the reaction was complete. The reaction mixture was diluted with EA (50 mL), and then adjusted to pH 7-8 with an aqueous sodium bicarbonate solution (1 M). The organic phase was separated, and the aqueous phase was extracted with EA (100 mL × 2). The organic phases were combined, washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The crude residue was slurried with PE / Et2O (1:1, v / v), filtered and dried to give the yellow product methyl 4-(3-chloropropoxy)-5-methoxy-2-nitrobenzoate (14.5 g, yield 77%).

[0107] 1 HNMR (400MHz, DMSO-d6): δ7.69 (s, 1H), 7.34 (s, 1H), 4.24 (t, J=6.1Hz, 2H), 3.93 (s, 3H), 3.83 (s, 3H), 3.78 (t, J=6.5Hz, 2H), 2.21 (p, J=6.3Hz, 2H).

[0108] c. Add Pd / C (1.2 g, 10%) to a MeOH (120 mL) solution of the product from step b (12 g, 39.5 mmol). Stir the reaction mixture overnight at room temperature under hydrogen atmosphere. TLC showed the reaction was complete. Filter the reaction mixture and wash with methanol (100 mL). Concentrate the combined organic layers to give crude methyl 2-amino-4-(3-chloropropoxy)-5-methoxybenzoate (11.4 g, 100% yield). The crude product was very pure and required no further purification. LC-MS:(ES) + ):m / z274.00[M+H] + .

[0109] d. Under nitrogen protection, a solution of the product from step c (11.4 g, 41.6 mmol), trimethyl orthoformate (11 g, 104.1 mmol), and ammonium acetate (8 g, 104.1 mmol) in MeOH (114 mL) was stirred overnight at 70 °C until TLC showed complete reaction. The reaction mixture was filtered, and the filter cake was washed with methanol (50 mL). The filter cake was dried to give 7-(3-chloropropoxy)-6-methoxyquinazoline-4-ol (11 g, 98% yield). It was a white solid, and the crude product was very pure and required no further purification. LC-MS: (ES) + ):m / z269.00[M+H] + .

[0110] e. Under nitrogen protection and stirring, phosphorus oxychloride (10 mL) was added dropwise to a toluene (50 mL) solution of product d (5 g, 18.6 mmol). The reaction mixture was then stirred at 110 °C for 4 h, and TLC showed that the reaction was complete. The reaction mixture was concentrated to remove toluene and phosphorus oxychloride. The concentrate was then slowly added to stirred ice water (50 mL) to quench the reaction. The precipitate was collected by filtration and dried to give 4-chloro-7-(3-chloropropoxy)-6-methoxyquinazoline acetamide (4.7 g, yield 88%) as a white solid. The crude product was very pure and required no further purification. LC-MS: (ES) + ):m / z287.00[M+H] + .

[0111] f. Under nitrogen-protected stirring, pentafluorophenyl trifluoroacetate (5.6 g, 20 mmol) was added to a mixed solution of 2-(3-amino-1H-pyrazol-5-yl)acetic acid (1.41 g, 10 mmol) in DMF (14 mL) and pyridine (1.8 mL, 22 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 10 min, then raised to room temperature and stirred for 90 min. 3-fluoroaniline (2.22 g, 20 mmol) was added to the reaction mixture, and the resulting reaction mixture was stirred overnight at room temperature. TLC monitoring showed that the reaction was complete. The reaction mixture was quenched in water (30 mL) and neutralized with dilute hydrochloric acid (1 N, 40 mL). The resulting reaction solution was extracted with ethyl acetate (50 mL × 3), washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude residue, which was purified by rapid silica gel column chromatography (eluting with 0-50% ethyl acetate-hexane) to give 2,2,2-trifluoro-N-(5-(2-((3-fluorophenyl)amino)-2-oxoethyl)-1H-pyrazol-3-yl)acetamide (2.2 g, yield 67%) as a white solid.

[0112] LC_MS:(ES+):m / z331.2[M+H] + .

[0113] 1 HNMR (400MHz, DMSO-d6): δ3.74 (s, 2H), 6.47 (s, 1H), 6.86-6.91 (m, 1H), 7.28-7.32 (m, 1 H), 7.33-7.38 (m, 1H), 7.57 (d, J=11.6Hz, 1H), 10.4 (s, 1H), 11.91 (s, 1H), 12.6 (br, 1H).

[0114] g. 2,2,2-Trifluoro-N-(5-(2-((3-fluorophenyl)amino)-2-oxoethyl)-1H-pyrazol-3-yl)acetamide (2.4 g, 7.2 mmol) was dissolved in a mixture of dilute hydrochloric acid (2N, 18 mL) and methanol (18 mL), and stirred at 50 °C under nitrogen protection for 5 hours. TLC was monitored until the reaction was complete. The reaction mixture was concentrated to remove MeOH, and then neutralized with saturated NaHCO3 aqueous solution. The resulting mixture was extracted with ethyl acetate (25 mL × 2), washed with water (25 mL × 2) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was pulped with DCM and filtered to give 2-(3-amino-1H-pyrazol-5-yl)-N-(3-fluorophenyl)acetamide (1.0 g, yield 58%) as a yellow solid. LC_MS:(ES+):m / z235.1[M+H] + .

[0115] 1 HNMR (400MHz, DMSO-d6): δ3.48 (s, 2H), 5.30 (s, 1H), 6.83-6.88 (m, 1H), 7.28-7.36 (m, 2H), 7.57-7.61 (m, 1H), 10.26 (s, 1H).

[0116] h. Under nitrogen protection, a mixture of the product from step e (800 mg, 3.17 mmol) and the product from step g (910 mg, 3.17 mmol) in isopropanol (20 mL) was stirred at 80 °C for 2 hours. TLC showed the reaction was complete. The precipitate was collected by filtration and dried to give 2-(3-amino-1H-pyrazol-5-yl)-N-(2,3-difluorophenyl)acetamide (1.47 g, 92% yield), abbreviated as Frag-5, as a yellow solid. The crude product was very pure and required no further purification. LC-MS (ES+): m / z 50 3.30 [M+H] + .

[0117] 1 HNMR (400MHz, DMSO-d6): δ11.84 (s, 1H), 10.38 (s, 1H), 8.99-8.81 (m, 1H), 8.29 (s, 1H), 7.70 (dd, J=8.6, 6.0Hz, 1H), 7.43 (s , 1H), 7.27-7.13 (m, 2H), 6.81 (s, 1H), 4.30 (s, 2H), 3.99 (s, 3H), 3.93 (s, 2H), 3.84 (t, J=6.4Hz, 2H), 2.30 (p, J=6.2Hz, 2H).

[0118] i. A solution of 2-(2-(piperazin-1-yl)ethoxy)ethanol-1-ol (1 g, 5.74 mmol) and (Boc)₂O (1.5 g, 6.88 mmol) in DCM (20 mL) was stirred at room temperature for 3 hours under nitrogen protection. TLC showed that the reaction was complete. The reaction mixture was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The crude residue was purified by rapid silica gel column chromatography to give 4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-carboxylic acid tert-butyl ester (1.19 g, yield 75.7%) as a colorless oil.

[0119] Under nitrogen protection at 0 °C, 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione (2 g, 7.29 mmol) and triphenylphosphine (2.87 g, 10.94 mmol) were added to DCM (40 mL) and stirred for 30 min. Then, the product from step i (2 g, 7.29 mmol) and diisopropyl azodicarboxylate (2 g, 7.29 mmol) were added to the solution, and the mixture was stirred overnight at 0 °C under nitrogen protection. TLC showed that the reaction was complete. The mixture was concentrated, and the crude residue was purified by silica gel rapid column chromatography (eluting with ethyl acetate) to give 4-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)ethoxy)ethyl)piperazine-1-carboxylate tert-butyl ester (1.8 g, yield 80%). LC-MS: (ES + ):m / z531.75[M+H] + .

[0120] k. A solution of the product from step j (1.8 g, 3.39 mmol) in HCl / EA (4 N, 18 mL) was stirred overnight under nitrogen protection. TLC showed that the reaction was complete. The precipitate was collected by filtration and dried to give Frag-6: 2-(2,6-dioxopiperidin-3-yl)-5-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)isoindoline-1,3-dione hydrochloride (1.44 g, 90% yield) as a yellow solid.

[0121] LC_MS:(ES + ):m / z431.66[M+H] + .

[0122] 1. Under nitrogen protection at room temperature, a solution of Frag-5 (215 mg, 0.43 mmol) in DMF (5 mL) was added with the product from step k (200 mg, 0.32 mmol), K₂CO₃ (178 mg, 1.29 mmol), and KI (37 mg, 0.22 mmol). The resulting mixture was stirred at 60 °C for 2 days, and the reaction was confirmed by TLC and LCMS. The reaction mixture was filtered, and the filter cake was washed with DMF. The combined DMF organic phases were concentrated under reduced pressure, and the crude residue was purified by preparative HPLC to give compound 7 (100 mg, 26% yield) as a yellow solid. LC-MS: (ES) + ):m / z897.30[M+H] + .

[0123] 1HNMR (400MHz, DMSO-d6): δ11.11 (s, 1H), 10.27 (s, 1H), 8.88 (s, 1H), 8.23 ​​(s, 1H), 7.85 (d, J=8.3Hz, 1H), 7.70 (t, J=7 .5Hz, 1H), 7.47 (d, J=2.2Hz, 1H), 7.37 (dd, J=8.3, 2.3Hz, 1H), 7.31 (s, 1H), 7.21 (dt, J=14.0, 5.1Hz, 2H), 6.80 (s, 1H ), 5.12 (dd, J=12.9, 5.4Hz, 1H), 4.38-4.34 (m, 2H), 4.25 (t, J=6.1Hz, 2H), 3.98 (s, 3H), 3.91 (s, 2H), 3.85-3.82 (m, 3 H), 3.78 (d, J=4.9Hz, 2H), 3.14 (s, 4H), 3.05-2.91 (m, 4H), 2.91-2.82 (m, 2H), 2.69-2.52 (m, 2H), 2.23-1.94 (m, 4H).

[0124] Example 14: Synthesis of compound 8:

[0125]

[0126] Under nitrogen protection at room temperature, Frag-4 (205 mg, 0.39 mmol) was dissolved in DMF (8 mL), followed by the addition of Frag-5 (254 mg, 0.51 mmol), K₂CO₃ (268 mg, 1.95 mmol), and potassium iodide (71 mg, 0.43 mmol). The resulting mixture was stirred overnight at 55 °C. Thin-layer chromatography (TLC) showed that the reaction was complete. The reaction mixture was cooled to below 0°C in an ice-salt bath, filtered, and concentrated under reduced pressure to give a crude product, which was purified by preparative thin-layer chromatography (TLC) to give compound 8: N-(2,3-difluorophenyl)-2-(5-((7-(3-(4-(2-(4-(2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindololin-5-yl)piperazin-1-yl)ethyl)piperazin-1-yl)propoxy)-6-methoxyquinazoline-4-yl)amino)-1H-pyrazole-3-yl)acetamide (10.3 mg, 2.8%) as a yellow solid. + ):m / z921.55[M+H] + .t R =2.458min.

[0127] 1HNMR (400MHz, DMSO-d6) δ12.32 (s, 1H), 11.08 (s, 1H), 10.20 (d, J=16.0Hz, 2H), 8.46 (s, 1H), 7.96 (s, 1H), 7.72 (t, J=6.4Hz, 1H), 7.67 (d, J=8.5Hz, 1H), 7.33 (d, J=1.7Hz, 1H), 7.27-7.24 (m, 1H), 7.22-7.1 7 (m, 2H), 7.14 (s, 1H), 5.07 (dd, J=12.9, 5.4Hz, 1H), 4.16 (t, J=6.2Hz, 2H), 3.93 (s, 3H), 3.84 (s, 2H) , 3.44 (d, J=17.1Hz, 5H), 2.95-2.80 (m, 1H), 2.54 (s, 5H), 2.44 (d, J=13.1Hz, 14H), 2.07-1.87 (m, 4H).

[0128] Example 15: Synthesis of compound 9:

[0129]

[0130] Frag-6 (150 mg, 0.32 mmol), K₂CO₃ (133 mg, 0.96 mmol), and potassium iodide (27 mg, 0.16 mmol) were added to a DMF (5 mL) solution of intermediate fragment Frag-2 (146 mg, 0.32 mmol) at room temperature under nitrogen protection. The resulting mixture was stirred at 60 °C for 2 days, and the reaction was complete by TLC and LCMS. The reaction mixture was filtered, and the filter cake was washed with DMF. The combined DMF organic phases were concentrated under reduced pressure to give a crude concentrate. This concentrate was purified first by preparative thin-layer chromatography (DCM:MeOH:NH₃·H₂O = 10:1:0.05) and then by preparative HPLC to give compound 9 (25 mg, yield 9.2%) as a yellow solid. LCMS: (ES) + ):m / z849.35[M+H] + .

[0131] 1HNMR (400MHz, DMSO-d6): δ11.15 (s, 1H), 10.55 (s, 1H), 8.93 (s, 1H), 8.76 (d, J=9.3Hz, 1H), 7.86 (d, J= 8.3Hz, 1H), 7.63 (dt, J=11.7, 2.3Hz, 1H), 7.49-7.25 (m, 7H), 6.91 (td, J=8.3, 2.5Hz, 1H), 6.75 (s, 1H), 5.13 (dd, J=12.8, 5.4Hz, 1H), 4.36 (dd, J=5.9, 3.0Hz, 2H), 4.27 (t, J=6.0Hz, 2H), 3.83 (d, J=6.5Hz, 6H) , 3.21 (s, 4H), 3.08 (s, 4H), 2.89 (ddd, J=16.7, 13.7, 5.3Hz, 2H), 2.69-2.53 (m, 3H), 2.20-1.93 (m, 4H).

[0132] Biological Experiment Section

[0133] 1. Bioactivity assay - Aurora B degradation activity assay.

[0134] Methods: Different concentrations of compound 1-11 (dissolved in DMSO) were added to HCT116 colon cancer cells, and the cells were cultured for 24 h (final DMSO concentration 0.2%). Cells were collected and lysed by sonication using RIPA lysis buffer (containing protease inhibitors). Cells were incubated on ice for 30 min, and quantification was performed using the BCA method. After complete denaturation of the protein samples, the intracellular Aurora B protein level was determined by Western blotting. The specific steps were as follows: Protein samples were separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% skim milk for 1 h, and incubated overnight at 4°C with primary antibody. After thorough washing with TBST, the membrane was incubated at room temperature for 30 min with secondary antibody, and then exposed for imaging in a chemiluminescence imaging system (Shanghai Tianneng).

[0135] Methods: ImageJ software was used to analyze the grayscale values ​​of the bands, and Graphpad Prism was used to calculate the half-maximal degradation concentration (DC) of the compound on Aurora B protein in HCT116 cells. 50 The values ​​were calculated, and the results are shown in Table 1 below.

[0136] Table 1. DCs of compounds that degrade Aurora B protein 50 value

[0137]

[0138] a comes from at least two independent tests

[0139] Table 1 shows that compounds 1, 2, 3, 4, and 5 exhibited more significant degradation effects, DC 50 All values ​​were below 20 nM. Based on this result, compound 1, which showed the best degradation effect, was selected, and the 24-hour DC value of Aurora B protein was further measured in the SW620 colon cancer cell line, A549 non-small cell lung cancer cell line, and HL-60 acute promyelocytic leukemia cell line. 50 value.

[0140] Figure 1 This study revealed the expression levels of Aurora B and its most important substrate, phosphorylated histone H3 serine 10 (pH3S10), in HCT116, SW620, A549, and HL-60 cells after treatment with different concentrations of compound 1 for 24 h, as well as the expression levels of internal references—histone H3 (H3) and β-actin. These results indicate that compound 1 degrades Aurora B protein in a concentration-dependent manner. Based on grayscale values, the concentrations of compound 1 in the dendritic cells (DCs) of cell lines were calculated. 50 The values ​​are shown in Table 2 below, indicating that compound 1 can efficiently degrade Aurora B protein in all four cell lines tested.

[0141] Table 2. DC effects of compound 1 on Aurora B protein in different cells. 50 value

[0142]

[0143] 'a' comes from at least two independent tests.

[0144] 2. Bioactivity assay—selectivity of compound 1.

[0145] The degradation effect of compound 1 on AuroraA in HCT116, SW620, A549, and HL-60 cell lines was determined by immunoblotting.

[0146] Methods: HCT116, SW620, A549, and HL-60 cells were seeded into six-well plates and cultured. After complete cell adhesion, different concentrations of Compound 1 (dissolved in DMSO solution) were added, and the cells were cultured for another 24 hours (final DMSO concentration: 0.2%). Cells were lysed using RIPA lysis buffer (containing protease inhibitors), and sample protein concentrations were measured. Western blotting was then performed. The experimental procedures were the same as above. Figure 2 Reveal the experimental results.

[0147] from Figure 2It can be seen that in all tested cells, low concentrations of compound 1 did not degrade Aurora A. When the compound concentration increased to 1 μM, Aurora A protein showed a slight decreasing trend, with weaker degradation activity compared to Aurora B.

[0148] 3. Bioactivity assay - cell activity experiment.

[0149] Methods: 10,000 (48 h) or 5,000 (96 h) cells in the logarithmic growth phase were seeded into 96-well plates and cultured. After complete cell adhesion, different concentrations of compound 1 were added, and the cells were cultured at 37°C and 5% CO2 for 48 h or 96 h, respectively. Cell counts were then performed, and the concentration of the compound that inhibited growth by 50% (GI) was calculated. 50 The results are shown in Table 3, indicating that compound 1 can significantly and efficiently inhibit the growth and proliferation of tumor cells.

[0150] Table 3 shows the effect of compound 1 on GI in tumor cells. 50 value

[0151]

[0152] 4. Bioactivity assay - cell clone formation assay.

[0153] 2000 HCT116 cells, 4000 SW620 cells, and 1000 A549 cells in logarithmic growth phase were seeded into 24-well plates and cultured. After complete cell adhesion, different concentrations of Compound 1 were added, and the cells were incubated at 37°C with 5% CO2 for 7 days. After colony formation, the cells were washed with PBS, fixed with anhydrous methanol, stained with crystal violet, destained, and then dried for photographing. The colony number was calculated using ImageJ software. The experimental results are shown below. Figure 3 As shown.

[0154] from Figure 3 As can be seen, in HCT116 cells, compound 1 at concentrations of 6.25 and 12.5 nM significantly inhibited cell colony formation, with inhibition rates of 25.96% (P<0.05) and 58.05% (P<0.01), respectively. In SW620 cells, the inhibition rates of compound 1 at concentrations of 12.5 and 25 nM were 26.28% (P<0.01) and 46.72% (P<0.01), respectively. In A549 cells, the inhibition rates of compound 1 at concentrations of 12.5 and 25 nM were 21.98% (P<0.05) and 55.11% (P<0.05), respectively. In these tumor adherent cell models, low concentrations of the compound significantly inhibited cell colony formation.

[0155] 0.6% agar was spread in 24-well plates as the lower gel and allowed to solidify. 5000 HL-60 cells in logarithmic growth phase were resuspended in complete medium and added at a 1:1 ratio with complete medium containing 0.7% agar (final agar concentration 0.35%). Different concentrations of Compound 1 were added simultaneously, and the mixture was thoroughly added to the 24-well plates with the lower gel. The plates were incubated at 4°C for 10 minutes until semi-solidified, then different concentrations of Compound 1 were added, and the plates were cultured for another 10 days. After fixation with 4% paraformaldehyde, the plates were stained with 0.05% crystal violet, rinsed with water, and air-dried.

[0156] from Figure 4 As can be seen, compared with the adherent cell model mentioned above, compound 1 showed a more significant growth inhibitory effect in HL-60 suspension cells. In HL-60 cells, when the concentration of compound 1 was 1.5625 and 3.125 nM, it significantly inhibited cell colony formation, with inhibition rates of 22.51% (P<0.01) and 29.93% (P<0.01), respectively.

Claims

1. A compound possessing Aurora B kinase degradation activity, characterized in that, The compound has one or more of the following structural formulas: In the formula, R is H or Me; m = 1 or 2; n = 0 or 1.

2. The compound with Aurora B kinase degradation activity according to claim 1, characterized in that, It also includes pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, prodrugs, isotope derivatives, or mixtures thereof.

3. The use of the compound having AuroraB kinase degradation activity as described in any one of claims 1-2 in the preparation of a medicament for degrading AuroraB kinase.

4. The use of the compound with AuroraB kinase degradation activity as described in any one of claims 1-2 in the preparation of antitumor drugs.

5. The application according to claim 4, characterized in that, The tumor is either a tumor with high expression of Aurora B or a tumor sensitive to Aurora B degrading agents.

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