An isoxazole compound, a preparation method and application thereof

Abstract

The application relates to the technical field of pharmaceutical compounds, and particularly discloses an isoxazole compound, a preparation method and application thereof. The isoxazole compound is designed by introducing a substituted aromatic ring, piperazine, morpholine and an isoxazole group into an imidazopyridine skeleton. The specific aromatic and isoxazole groups can enhance the P-II or Pi-Pi conjugated system of the compound and the PI3K related receptor, enhance the inhibition of the PI3K signal path by the small molecule, and improve the antitumor activity. The piperazine group can increase the hydrogen bond force between the compound and the receptor, improve the biological affinity and the anticancer effect. In addition, the terminal ring-opened ethylenediamine structure can allow more conformations of the compound when the compound is docked with the target, and the targeting property of the compound can be improved. The isoxazole compound provided by the application has excellent antitumor activity and high potential application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical compound technology, and in particular to a series of isoxazole compounds, their preparation methods, and applications. Background Technology

[0002] In recent years, the incidence of cancer has been on the rise, becoming a major killer threatening human health. Chemotherapy is one of the most effective treatments for cancer. However, the drawbacks of traditional chemotherapy drugs, such as severe toxicity in solid tumors and the tendency to develop drug resistance, are becoming increasingly prominent, highlighting the urgent need for the development of novel anticancer drugs in clinical practice. Small molecule targeted inhibitors target key regulatory molecules in the molecular pathological processes of tumors, selectively preventing the excessive proliferation, invasion, and metastasis of cancer cells, and have the advantages of high efficacy and few side effects.

[0003] Phosphainositide-3 kinases (PI3K) play a crucial role in cell proliferation, differentiation, and apoptosis. Studies have shown that overexpression and abnormal activation of the PI3K signaling pathway are closely related to the development and progression of various tumors. Therefore, the development of PI3K inhibitors has become a hot topic in targeted cancer drug research both domestically and internationally. Literature reports that the imidazopyridine parent ring is an effective structure for targeting PI3K inhibitors. Although these compounds show certain anticancer activity, they are still far from being drug-grade and require further optimization. Therefore, developing a series of novel isoxazole compounds is of great significance for overcoming the current bottlenecks in cancer treatment. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a series of isoxazole compounds, their preparation methods, and applications.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: A series of isoxazole compounds, the structures of which are shown in formula (Ⅰ): Equation (I) Where R is , or ; R' is , , , , or .

[0006] This invention uses imidazopyridine as a backbone and, by introducing substituted aromatic rings, piperazine, morpholine, and isoxazole groups, designs a series of novel isoxazole compounds. The introduced specific aromatic and isoxazole groups enhance the P-π or π-π conjugation system between the compound and PI3K-related receptors, strengthening the inhibition of the PI3K signaling pathway by small molecules and thus improving antitumor activity. The introduced piperazine group increases the hydrogen bonding between the compound and the receptor, improving biocompatibility and anticancer effects. Furthermore, the terminally open ethylenediamine structure allows for more conformations when the compound docks with the target, potentially improving the compound's targeting specificity. The novel isoxazole compounds provided by this invention exhibit excellent antitumor activity, offering new drug compound structures for the development of novel antitumor drugs, enriching the database of imidazopyridine compounds, and possessing high potential application value.

[0007] This invention also provides a method for preparing the above-mentioned isoxazole compounds, comprising the following steps: S1, using 2-amino-5-bromopyridine and ethyl 2-chloro-3-oxypropionate as raw materials, undergoes a cyclization reaction to obtain the compound shown in formula (II); S2, using the compound shown in formula (II) and hydrazine hydrate as raw materials, undergoes hydrazolysis to obtain the compound shown in formula (III); S3, using the compound shown in formula (III), acrolein and iodophenyl diacetic acid as raw materials, undergoes a cyclization reaction to obtain the compound shown in formula (IV); S4, using the compound shown in formula (Ⅳ) and R'-H as raw materials, undergoes an addition reaction to obtain the compound shown in formula (Ⅴ); S5, using 3-amino-5-bromo-2-methoxypyridine and pinacol diboronic acid ester as raw materials, was reacted via the Miyaura reaction to obtain the compound shown in formula (VI); S6, using the compound shown in formula (VI) and R-Cl as raw materials, undergoes nucleophilic addition and elimination reactions to obtain the compound shown in formula (VII); S7, using the compounds shown in formula (VII) and formula (V) as raw materials, the isoxazole compounds shown in formula (I) are obtained by the Suzuki reaction; .

[0008] Compared with existing technologies, the method for preparing isoxazole compounds provided by this invention uses readily available raw materials, is simple to operate, has mild reaction conditions, and produces high-purity target products, thus possessing good industrial utilization and application value.

[0009] The structural formula of the R'-H compound in this invention is as follows: It should be noted that when R'-H is To avoid both imine groups reacting, one of the imine groups needs to be protected with BOC before the reaction, or a commercially available 1-tert-butyloxycarbonylpiperazine can be directly selected as the reactant. Correspondingly, after step S7, a further deprotection reaction is required, namely the following chemical reaction: The structural formula of the R-Cl compound in this invention is as follows: Furthermore, the preparation method of the isoxazole compound specifically includes the following steps: S1, 2-amino-5-bromopyridine and ethyl 2-chloro-3-oxypropionate were added to anhydrous ethanol and refluxed for 15 h to 30 h. After post-treatment, the compound shown in formula (II) was obtained. S2, the compound shown in formula (II) and hydrazine hydrate were added to anhydrous ethanol and refluxed for 10 h to 15 h. After post-treatment, the compound shown in formula (III) was obtained. S3, add the compound shown in formula (Ⅲ), acrolein and glacial acetic acid to dichloromethane, stir and react for 8 h to 12 h, then add iodophenyl diacetic acid, stir and react for 10 h to 14 h, and then perform post-treatment to obtain the compound shown in formula (Ⅳ); S4, the compound shown in formula (Ⅳ) was dissolved in dichloromethane, R'-H and 1,8-diazacyclo[5,4,0]undecene-7 were added, the reaction was stirred for 10 h to 15 h, and then post-treated to obtain the compound shown in formula (Ⅴ); S5, 3-amino-5-bromo-2-methoxypyridine, pinacol diboronic acid, potassium acetate and PdCl2(dppf)-CH2Cl2 were dispersed in anhydrous dioxane and reacted at 50 ℃~100 ℃ for 15 h~20 h under an inert atmosphere, followed by post-treatment to obtain the compound shown in formula (VI); S6, Dissolve the compound shown in formula (VI) in anhydrous tetrahydrofuran, add triethylamine, mix well, add R-Cl, stir for 8 h to 12 h, and then perform post-treatment to obtain the compound shown in formula (VII); S7, the compound shown in formula (VII), the compound shown in formula (V), potassium carbonate, and PdCl2(dppf)-CH2Cl2 were dispersed in an aqueous solution of dioxane and reacted at 50 ℃~100 ℃ for 1 h~5 h under an inert atmosphere. After post-treatment, the isoxazole compound shown in formula (I) was obtained. The specific synthetic route is shown below: Further, in S1, the molar ratio of 2-amino-5-bromopyridine to ethyl 2-chloro-3-oxypropionate is 1:(1~3).

[0010] Further, in S1, the molar volume ratio of 2-amino-5-bromopyridine to anhydrous ethanol is 1 mmol : (1~20) mL.

[0011] Furthermore, in S2, the molar ratio of the compound represented by formula (II) to hydrazine hydrate is 1:(10~30).

[0012] Furthermore, in S2, the volume molar ratio of the anhydrous ethanol to the compound represented by formula (II) is (1~30) mL : 1 mmol.

[0013] Furthermore, in S3, the molar ratio of the compound represented by formula (Ⅲ) to acrolein is 1:(1~10).

[0014] Furthermore, in S3, the volume molar ratio of the dichloromethane to the compound represented by formula (Ⅲ) is (10~50) mL : 1 mmol.

[0015] Furthermore, in S3, the ratio of the iodophenylacetic acid to the compound shown in formula (Ⅲ) is (1~5): 1.

[0016] Furthermore, in S3, the amount of glacial acetic acid added is (1~5)% of the compound shown in formula (Ⅲ).

[0017] Furthermore, in S4, the molar ratio of the compound shown in formula (Ⅳ) to R'-H is 1:(1~5).

[0018] Furthermore, in S4, the volume molar ratio of the dichloromethane to the compound shown in formula (Ⅳ) is (1~50) mL : 1 mmol.

[0019] Furthermore, in S4, the molar ratio of the 1,8-diazacyclo[5,4,0]undecene-7 to the compound shown in formula (Ⅳ) is (1~3):1.

[0020] Further, in S5, the molar ratio of 3-amino-5-bromo-2-methoxypyridine, pinacol diboronate, potassium acetate, and PdCl2(dppf)-CH2Cl2 is 1 : (1~5) : (1~10) : (0.01~0.5).

[0021] Further, in S5, the volume molar ratio of anhydrous dioxane to 3-amino-5-bromo-2-methoxypyridine is (0.1~20) mL : 1 mmol.

[0022] Further, in S6, the molar ratio of the compound represented by formula (VI), triethylamine and R-Cl is 1 : (0.5~10) : (1~5).

[0023] In S6, the volume molar ratio of the anhydrous tetrahydrofuran to the compound shown in formula (VI) is (1~40) mL : 1 mmol.

[0024] Furthermore, in S7, the molar ratio of the compound represented by formula (V), the compound represented by formula (VII), potassium carbonate, and PdCl2(dppf)-CH2Cl2 is 1 : (1~5) : (1~10) : (0.01~1).

[0025] Furthermore, in S7, the volume molar ratio of dioxane to the compound shown in formula (V) is (1~30) mL : 1 mmol, and the molar ratio of dioxane to water is (1~10) : 1.

[0026] The above-mentioned optimized reaction conditions are beneficial to improving the yield and purity of the target product.

[0027] It should be noted that after the reaction in S1 to S7, conventional post-processing procedures in this field are required, such as filtration, extraction, and purification, to obtain the product of the corresponding step.

[0028] Double-line synthesis offers a simpler and more efficient synthetic route. Therefore, the target compound was divided into two parts, and the compound shown in formula (VII) and the compound shown in formula (V) were synthesized separately. Finally, they were coupled by the Suzuki reaction to obtain the target product.

[0029] This invention also provides the application of the above-mentioned isoxazole compounds in the preparation of antitumor drugs.

[0030] Furthermore, the present invention also provides the use of the above-mentioned isoxazole compounds in the preparation of drugs for treating colon cancer, cervical cancer, liver cancer, non-small cell lung cancer, or melanoma.

[0031] The isoxazole compounds provided by this invention have significant in vitro inhibitory activity against tumor cell proliferation and can be used to prepare drugs for the treatment and prevention of tumors. At the same time, the raw materials for these compounds are abundant and inexpensive, the preparation process is simple, they are easy to industrialize, and they have broad potential applications. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] To better illustrate the present invention, further examples are provided below.

[0034] Example 1 Preparation of compound A1: Compound A1 was prepared according to the following route: At room temperature, ethyl 2-chloro-3-oxypropionate (0.96 g, 6.36 mmol) was added to a solution of 2-amino-5-bromopyridine (1.00 g, 5.78 mmol) in anhydrous ethanol (20 mL). The mixture was heated to reflux and stirred for 24 h. The reaction solution was then concentrated to dryness under reduced pressure. Dichloromethane (20 mL) was added, and the mixture was washed with pure water (2 × 30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate 10:1) to give a white solid compound A1 (1.40 g, yield 89.7%). mp = (116.6-117.4) ℃; 1 H NMR (500 MHz, DMSO- d 6 ) δ 9.34 (d, J = 1.7 Hz, 1H), 8.31 (s, 1H), 7.82 (d, J = 9.5 Hz, 1H), 7.73(dd, J = 9.5, 1.9 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H); LC-MS (ESI, m / z ): Calcd for C 10 H9BrN2O2[M+H] + 269.0, found: 269.1. Preparation of compound A2: Compound A2 was prepared according to the following route: Compound A1 (0.12 g, 0.446 mmol) was dissolved in 4 mL of anhydrous ethanol, and 0.44 mL of hydrazine hydrate (0.45 g, 8.92 mmol) was added. The mixture was heated to reflux and stirred for 13 h. After filtration, the filter cake was washed with 95% ethanol and dried to give a white solid compound A2 (0.10 g, yield 87.6%). mp = (221.4-222.0) ℃; 1 H NMR (500MHz, DMSO-d6) δ 9.87 (s, 1H), 9.61 (d, J = 1.2 Hz, 1H), 8.29 (s, 1H), 7.72(dd, J = 9.5, 1.2 Hz, 1H), 7.60 (dd, J = 9.5, 2.0 Hz, 1H), 4.52 (s, 2H); LC-MS (ESI, m / z): Calcd for C8H7BrN4O [M+H] + 254.9, found: 254.9. Preparation of compound A3: Compound A3 was prepared according to the following route: At room temperature, 0.22 mL of acetic acid and acrolein (65.6 mg, 1.17 mmol) were added sequentially to a solution of A2 (0.10 g, 0.39 mmol) in 10 mL of dichloromethane. After reacting at room temperature for 10 h, iodophenyl diacetic acid (118.4 mg, 0.585 mmol) was added under ice bath conditions. After stirring at room temperature for 12 h, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 50:1) and dried to give a white solid A3 (63.6 mg, yield 56.0%). mp = (176.4-177.3)℃; 1 H NMR (500 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.49 (s, 1H), 7.88 (d, J = 9.5Hz, 1H), 7.75 (dd, J = 9.5, 1.7 Hz, 1H), 6.95 (dd, J = 17.6, 11.3 Hz, 1H), 6.42 (d, J = 17.6 Hz, 1H), 6.03 (d, J = 11.3 Hz, 1H); LC-MS (ESI, m / z): Calcdfor C 11 H7BrN4O [M+H] +293.0, found: 292.9. Preparation of A4-1 to A4-6: Compound A4-1 was prepared according to the following route: N-methylpiperazine (79.0 mg, 0.79 mmol) and 1,8-diazacyclo[5,4,0]undecene-7 (DBU) (120.0 mg, 0.79 mmol) were added sequentially to a solution of A3 (210.0 mg, 0.72 mmol) in 10 mL of dichloromethane. After reacting at room temperature for 12 h, the mixture was washed with water (2 × 20 mL) and saturated brine (20 mL), respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 50:1) and dried to give a white solid A4-1 (89.0 mg, yield 67%). mp = (146.2-147.2) ℃; 1 H NMR (500MHz, CDCl3) δ 9.62 (s, 1H), 8.23 ​​(s, 1H), 7.68 (d, J = 9.5 Hz, 1H), 7.51 (dd, J =9.5, 1.5 Hz, 1H), 3.16 (t, J = 7.4 Hz, 2H), 2.92 (t, J = 7.4 Hz, 2H), 2.60 (m,4H), 2.45 (m, 4H), 2.29 (s, 3H); LC-MS (ESI, m / z ): Calcd for C 16 H 19 BrN6O [M+H] + 392.3, found: 392.8. Compound A4-2 was prepared according to the following route: Morpholine (1.17 mL, 13.44 mmol) and 1,8-diazacyclo[5,4,0]undecene-7 (DBU) (2.02 mL, 13.44 mmol) were added sequentially to a solution of A3 (3.26 g, 11.20 mmol) in dichloromethane (70 mL). After stirring at room temperature for 12 h, the mixture was washed with water (2 × 20 mL) and saturated brine (20 mL), respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 40:1). The purified product was dried to give a white solid A4-2 (3.01 g, yield 71.16%). mp = (194.5–195.4) ℃; 1 H NMR (500MHz, Chloroform- d ) δ 9.60 (d, J = 2.0 Hz, 1H), 8.22 (s, 1H), 7.67 (d, J = 9.5 Hz, 1H), 7.51 (dd, J = 9.5, 2.0 Hz, 1H), 3.70 (t, J = 4.7 Hz, 4H), 3.17 (t, J = 7.3 Hz, 2H), 2.91 (t, J = 7.3 Hz, 2H), 2.56 (t, J = 4.7 Hz, 4H); LC-MS (ESI, m / z ): Calcdfor C 15 H 16 BrN5O2[M+H] + 378.1, found: 378.3. Compound A4-3 was prepared according to the following route: Piperidine (0.31 ml, 3.10 mmol) and 1,8-diazacyclo[5,4,0]undecene-7 (DBU) (0.47 ml, 3.10 mmol) were added sequentially to a solution of A3 (750 mg, 2.58 mmol) in 20 mL of dichloromethane. After stirring at room temperature for 12 h, the mixture was washed with water (2 × 20 mL) and saturated brine (20 mL), respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 50:1). The purified product was dried to give a white solid A4-3 (450 mg, yield 46.44%). mp = (265.7–266.1) ℃; 1 H NMR (500 MHz, Chloroform- d ) δ 9.60 (dd, J = 1.9, 0.9 Hz, 1H), 8.23 ​​(s, 1H), 7.67 (dd, J = 9.5, 0.9 Hz, 1H), 7.50 (dd, J = 9.5, 1.9 Hz, 1H), 3.17 (t, J = 8.1, 7.0 Hz, 2H), 2.89(t, J = 8.2, 7.0 Hz, 2H), 2.50 (t, J = 5.5 Hz, 4H), 1.59 (q, J = 5.6 Hz, 4H), 1.45(tt, J = 8.1, 4.9 Hz, 2H); LC-MS (ESI, m / z ): Calcd for C 16 H 18 BrN5O [M+H] + 376.1, found: 376.5. Compound A4-4 was prepared according to the following route: 4-Methylpiperidine (0.32 ml, 3.10 mmol) and 1,8-diazacyclo[5,4,0]undecene-7 (DBU) (0.47 ml, 3.10 mmol) were added sequentially to a solution of A3 (750 mg, 2.58 mmol) in 30 mL of dichloromethane. After stirring at room temperature for 12 h, the mixture was washed with water (2 × 20 mL) and saturated brine (20 mL), respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 60:1). The purified product was dried to give a white solid A4-4 (423 mg, yield 42.09%). mp = (136.5–137.5) ℃; 1 H NMR (500MHz, Chloroform- d ) δ 9.62 (dd, J = 1.9, 0.9 Hz, 1H), 8.24 (s, 1H), 7.68 (dd, J =9.5, 0.9 Hz, 1H), 7.52 (dd, J = 9.5, 1.9 Hz, 1H), 3.18 (t, J = 8.1, 6.9 Hz, 2H), 3.00 – 2.87 (m, 4H), 2.09 (td, J = 11.6, 2.5 Hz, 2H), 1.66 (ddd, J = 13.2, 4.1,2.1 Hz, 2H), 1.44 – 1.32 (m, 1H), 1.32 – 1.18 (m, 2H), 0.93 (d, J = 6.5 Hz, 3H); LC-MS (ESI, m / z ): Calcd for C 17 H 20 BrN5O [M+H] + 390.1, found: 390.7. Compound A4-5 was prepared according to the following route: Diethylamine (0.30 ml, 3.10 mmol) and 1,8-diazacyclo[5,4,0]undecene-7 (DBU) (0.47 ml, 3.10 mmol) were added sequentially to a solution of A3 (750 mg, 2.58 mmol) in dichloromethane (30 mL). After stirring at room temperature for 12 h, the mixture was washed with water (2 × 20 mL) and saturated brine (20 mL), respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 80:1). The purified product was dried to give a yellow solid A4-5 (357 mg, yield 38.51%). mp = (112.3–113.1) ℃; 1 H NMR (500MHz, Chloroform- d ) δ 9.61 (dd, J = 1.9, 0.9 Hz, 1H), 8.22 (s, 1H), 7.67 (dd, J =9.5, 0.9 Hz, 1H), 7.50 (dd, J = 9.5, 1.9 Hz, 1H), 3.14 – 3.09 (m, 2H), 3.01(dd, J = 7.8, 6.3 Hz, 2H), 2.61 (q, J = 7.1 Hz, 4H), 1.06 (t, J = 7.1 Hz, 6H); LC-MS (ESI, m / z ): Calcd for C 15 H 18 BrN5O [M+H] + 365.1, found: 365.2. Compound A4-6 was prepared according to the following route: 1-tert-butyloxycarbonylpiperazine (576 mg, 3.10 mmol) and 1,8-diazacyclo[5,4,0]undecene-7 (DBU) (0.47 mL, 3.10 mmol) were added sequentially to a solution of A3 (750 mg, 2.58 mmol) in dichloromethane (30 mL). After stirring at room temperature for 12 h, the mixture was washed with water (2 × 20 mL) and saturated brine (20 mL), respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1) to give a white solid A4-6 (6.47 mg, yield 52.43%). mp = (153.5–154.3) ℃; 1 H NMR (500MHz, Chloroform- d ) δ 9.49 (dd, J = 1.9, 0.9 Hz, 1H), 8.14 (s, 1H), 7.58 (dd, J =9.5, 0.9 Hz, 1H), 7.43 (dd, J = 9.5, 1.9 Hz, 1H), 3.37 (t, J = 5.0 Hz, 4H), 3.11(t, J = 7.3 Hz, 2H), 2.87 (t, J = 7.3 Hz, 2H), 2.44 (t, J = 5.0 Hz, 4H), 1.39 (s,9H); LC-MS (ESI, m / z ): Calcd for C 20 H 25 BrN6O3[M+H] + 477.1, found: 477.8. Example 2 Preparation of compound R1: Compound R1 was prepared according to the following route: 3-Amino-5-bromo-2-methoxypyridine (10.00 g, 49.26 mmol), pinacol diboronate (18.76 g, 73.89 mmol), potassium acetate (14.50 g, 147.78 mmol), and PdCl2(dppf)-CH2Cl2 (0.80 g, 0.98 mmol) were dissolved sequentially in anhydrous dioxane (100 mL). Under nitrogen protection, the mixture was gradually heated to 80 °C and kept at this temperature for 18 h. The reaction solution was then concentrated, diluted with ethyl acetate, filtered, and the filtrate was washed sequentially with water (50 mL) and saturated saline solution (50 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate 10:1~2:1) to give a pale yellow solid R1 (10.16 g, yield 82.60%). mp=(195.7-196.4) ℃; 1H NMR (500 MHz, Chloroform-d) δ 8.00 (d, J = 1.6 Hz, 1H), 7.25 (d, J = 1.6 Hz, 1H), 4.03 (s, 3H), 3.74 (br, 2H), 1.35 (s, 12H); LC-MS(ESI, m / z):Calcd for C 12 H 19 BN2O3[M+H] + 251.1, found: 251.2. Preparation of compounds BR1~BR7: Compound BR1 was synthesized according to the following route: Under ice-water bath conditions, triethylamine (1.66 mL) was added to a solution of R1 (3.00 g, 12 mmol) in anhydrous tetrahydrofuran (30 mL). After stirring for 5 min, 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carbonyl chloride (3.84 g, 13.2 mmol) was added in portions. After the addition was complete, the mixture was kept in an ice bath for 10 min, then brought to room temperature and stirred for 10 h. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate 10:1~4:1) to give a white solid BR1 (4.12 g, yield 68.10%). mp = (227.6-228.6) ℃; 1 H NMR (500 MHz, Chloroform-d )δ 8.92 (d, J = 1.7 Hz, 1H), 8.20 (d, J = 1.7 Hz, 1H), 7.58 – 7.50 (m, 4H), 3.76(s, 3H), 2.91 (s, 3H), 1.33 (s, 12H); LC-MS (ESI, m / z): Calcd for C 23 H 24 BCl2N3O5[M+H] + 504.1, found: 504.5. Compound BR2 was prepared according to the following route: Triethylamine (1.66 mL) was added to an anhydrous tetrahydrofuran (30 mL) solution of R1 (3.00 mg, 12.00 mmol) under ice-water bath conditions. After stirring for 5 min, 3-(2-chloro-6-fluorophenyl)-5-methylisoxazole-4-carbonyl chloride (3.62 g, 13.2 mmol) was added in portions. After the addition was complete, the mixture was kept in an ice bath for 10 min, then brought to room temperature and stirred for 10 h. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate 10:1~4:1) to give a white solid BR2 (3.78 g, yield 64.73%). mp = (223.4-224.4) ℃; 1 H NMR (500 MHz, Chloroform-d) δ 8.93 (d, J = 1.7 Hz, 1H), 8.21 (d, J = 1.7 Hz, 1H), 7.60 -7.55 (m, 2H), 7.46 (d, J = 8.1 Hz, 1H), 7.30 – 7.24 (m, 1H), 3.76 (s, 3H),2.91 (s, 3H), 1.32 (s, 12H); LC-MS (ESI, m / z): Calcd for C 23 H 24 BClFN3O5[M+H] + 488.1, found: 488.2. Compound BR3 was prepared according to the following route: Under ice-water bath conditions, triethylamine (1.66 mL) was added to an anhydrous tetrahydrofuran (30 mL) solution of R1 (3.00 g, 12.00 mmol). After stirring for 5 min, 3,5-dimethylisoxazole-4-carbonyl chloride (2.11 g, 13.2 mmol) was added in portions. After the addition was complete, the mixture was kept in an ice bath for 10 min, then brought to room temperature and stirred for 10 h. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate 10:1~4:1) to give a white solid BR3 (3.02 g, yield 67.53%). 1 H NMR (500 MHz, Chloroform- d ) δ 8.95 (d, J = 1.7 Hz, 1H), 8.31 (d, J = 1.7 Hz, 1H), 7.91 (s, 1H), 4.11 (s, 3H), 2.75 (s, 3H), 2.58 (s, 3H), 1.37 (s, 12H); (HRMS, m / z ): Calcd for C 18 H 24 BN3O5[M+H] + 374.18090, found: 374.18787. Example 3 Compound E1 was synthesized according to the following route: At room temperature, A4-1 (0.72 g, 1.84 mmol), BR1 (1.03 g, 2.05 mmol), potassium carbonate (0.85 g, 6.15 mmol), and PdCl2(dppf)-CH2Cl2 (0.14 g, 0.17 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E1 (0.39 g, yield 34.21%, purity 99.43%). mp = (218.7-219.7) ℃; 1H NMR (500 MHz, Chloroform-d) δ9.56 (d, J = 1.9 Hz, 1H), 8.97 (d, J = 2.3 Hz, 1H), 8.27 (s, 1H), 8.10 (d, J= 2.3 Hz, 1H), 7.83 (d, J = 9.3 Hz, 1H), 7.66 – 7.52 (m, 5H), 3.79 (s, 3H), 3.16 (t, J = 7.4 Hz, 2H), 2.93 (t, J = 7.4 Hz, 2H), 2.90 (s, 3H), 2.70 – 2.40(m, 8H), 2.31 (s, 3H); LC-MS (ESI, m / z): Calcd for C 33 H 31 Cl2N9O4[M+H] + 689.2, found: 689.1. Example 4 Compound E2 was synthesized using the following route: At room temperature, A4-2 (0.70 g, 1.84 mmol), BR1 (1.03 g, 2.05 mmol), potassium carbonate (0.85 g, 6.15 mmol), and PdCl2(dppf)-CH2Cl2 (0.14 g, 0.17 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1), and dried to give a white solid E2 (0.35 g, yield 28.18%, purity 98.44%). mp = (254.6-255.6) ℃; 1H NMR (500 MHz, Chloroform-d)δ 9.59 (dd, J = 1.9, 1.0 Hz, 1H), 9.00 (d, J = 2.3 Hz, 1H), 8.30 (s, 1H),8.13 (d, J = 2.3 Hz, 1H), 7.87 (dd, J = 9.3, 1.0 Hz, 1H), 7.72 – 7.53 (m,5H), 3.74 (s, 3H), 3.14 – 3.12 (m, 2H), 2.96 – 2.94 (m, 2H), 2.93 (s, 3H),2.67 – 2.41 (m, 8H); LC-MS (ESI, m / z):Calcd for C 32 H 28 Cl2N8O5[M+H] + 675.2, found: 675.3. Example 5 Compound E3 was prepared according to the following route: At room temperature, A4-3 (0.25 g, 0.66 mmol), BR1 (0.40 g, 0.79 mmol), potassium carbonate (0.27 g, 1.98 mmol), and PdCl2(dppf)-CH2Cl2 (0.03 g, 0.03 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (10 mL) and H2O (2 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E3 (0.16 g, yield 35.96%, purity 98.22%). mp = (230.8-231.8) ℃; 1H NMR (500 MHz, Chloroform-d) δ9.56 (s, 1H), 8.98 (d, J = 2.3 Hz, 1H), 8.28 (s, 1H), 8.11 (d, J = 2.3 Hz, 1H), 7.84 (d, J = 9.3 Hz, 1H), 7.65 (dd, J = 9.3, 1.8 Hz, 1H), 7.60 (d, J =9.4 Hz, 3H), 7.58 – 7.54 (m, 1H), 3.80 (s, 3H), 3.21 (t, J = 7.5 Hz, 2H), 2.94 (t, J = 7.5 Hz, 2H), 2.91 (s, 3H), 2.58 – 2.52 (m, 4H), 1.65 – 1.61 (m,4H), 1.48 – 1.45 (m, 2H); LC-MS (ESI, m / z): Calcd for C 33 H 30 Cl2N8O4[M+H] + 673.2, found: 673.8. Example 6 Compound E4 was prepared according to the following route: At room temperature, A4-4 (0.25 g, 0.66 mmol), BR1 (0.40 g, 0.79 mmol), potassium carbonate (0.27 g, 1.98 mmol), and PdCl2(dppf)-CH2Cl2 (0.03 g, 0.03 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (10 mL) and H2O (2 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E4 (0.22 g, yield 48.46%, purity 98.91%). mp = (205.7-206.7) ℃; 1H NMR (500 MHz, Chloroform-d) δ9.56 (s, 1H), 8.98 (d, J = 2.3 Hz, 1H), 8.28 (s, 1H), 8.11 (d, J = 2.3 Hz,1H), 7.84 (d, J = 9.3 Hz, 1H), 7.65 (dd, J = 9.3, 1.8 Hz, 1H), 7.62 - 7.58(m, 3H), 7.58 - 7.54 (m, 1H), 3.80 (s, 3H), 3.21 (t, J = 7.5 Hz, 2H), 2.94(t, J = 7.5 Hz, 2H), 2.91 (s, 3H), 2.11 – 2.08 (m, 2H), 1.65 – 1.62 (m, 2H), 1.44 – 1.22 (m, 5H), 0.91 – 0.89 (m, 3H); LC-MS (ESI, m / z): Calcd forC 34 H 32 Cl2N8O4[M+H] + 688.2, found: 688.4. Example 7 Compound E5 was prepared according to the following route: At room temperature, A4-5 (0.47 g, 1.32 mmol), BR1 (0.80 g, 1.59 mmol), potassium carbonate (0.55 g, 3.97 mmol), and PdCl2(dppf)-CH2Cl2 (0.10 g, 0.12 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (10 mL) and H2O (2 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E5 (0.34 g, yield 39.04%, purity 98.38%). mp = (202.4-203.4) ℃; 1 H NMR (500 MHz, Chloroform- d ) δ9.57 (t, J = 1.3 Hz, 1H), 8.96 (d, J = 2.3 Hz, 1H), 8.27 (s, 1H), 8.10 (d, J= 2.3Hz, 1H), 7.83 (dd, J = 9.2, 1.0 Hz, 1H), 7.66 – 7.51 (m, 5H), 3.79 (s, 3H), 3.12 (t, J = 7.3 Hz, 2H), 3.02 (t, J = 7.3 Hz, 2H), 2.90 (s, 3H), 2.62 (q, J = 7.1Hz, 4H), 1.06 (t, J = 7.1 Hz, 6H); LC-MS (ESI, m / z): Calcd for C 32 H 30 Cl2N8O4[M+H] + 661.2, found: 661.2. Example 8 Compound E6 was prepared according to the following route: At room temperature, A4-6 (0.63 g, 1.32 mmol), BR1 (0.80 g, 1.59 mmol), potassium carbonate (0.55 g, 3.97 mmol), and PdCl2(dppf)-CH2Cl2 (0.10 g, 0.12 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (10 mL) and H2O (2 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1). After drying, a white solid E6-1 (0.23 g, yield 22.51%) was obtained. 1 H NMR (500 MHz, Chloroform- d ) δ 9.59 (dd, J = 1.8, 1.0 Hz, 1H), 8.99 (d, J = 2.3 Hz, 1H), 8.30 (s, 1H), 8.13 (d, J = 2.3 Hz, 1H), 7.86 (dd, J =9.2, 1.0 Hz, 1H), 7.68 – 7.54 (m, 5H), 3.81 (s, 3H), 3.46 (t, J = 5.1 Hz, 4H), 3.20 (t, J= 7.3 Hz, 2H), 2.96 (t, J = 7.3 Hz, 2H), 2.93 (s, 3H), 2.53 (t, J = 5.1Hz, 4H), 1.48 (s, 9H); LC-MS (ESI, m / z): Calcd for C 37 H 37 Cl2N9O6[M+H] + 774.2, found: 774.3. At room temperature, trifluoroacetic acid (2 mL) was slowly added dropwise to a solution of E6-1 (757 mg, 0.98 mmol) in dichloromethane (6 mL). After the addition was complete, the mixture was stirred at room temperature for 12 h, concentrated under reduced pressure, diluted with water (10 mL), and the pH of the aqueous phase was adjusted to 7-8 with 3N NaOH. The mixture was extracted with ethyl acetate (20 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 70:1-20:1), and dried to give a pale yellow solid E6 (460 mg, yield 69.64%, purity 98.01%). mp = (228.4-229.4) ℃; 1 H NMR (500 MHz, DMSO-) d 6) δ 9.43 – 9.39 (m, 1H), 8.75 (s, 1H), 8.68 (d, J = 2.3 Hz,1H), 8.40 (s, 1H), 8.31 (d, J = 2.3 Hz, 1H), 7.97 (dd, J = 9.3, 1.0 Hz, 1H), 7.86(dd, J = 9.3, 1.9 Hz, 1H), 7.80 – 7.74 (m, 2H), 7.70 (dd, J = 9.1, 7.1 Hz, 1H),3.89 (s, 3H), 3.16 (t, J = 7.2 Hz, 2H), 2.87 (s, 3H), 2.78 (t, J = 7.2 Hz, 2H), 2.67 (t, J= 4.7 Hz, 4H), 2.50 – 2.53 (m, 2H) 2.44 –2.42 (m, 2H), 2.40 (br,1H); LC-MS (ESI, m / z): Calcd for C 32 H 29 Cl2N9O4[M+H] + 674.2, found: 674.5. Example 9 Compound E7 was prepared according to the following route: At room temperature, A4-1 (0.72 g, 1.84 mmol), BR2 (1.00 g, 2.04 mmol), potassium carbonate (0.85 g, 6.15 mmol), and PdCl2(dppf)-CH2Cl2 (0.14 g, 0.17 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E7 (0.32 g, yield 25.81%, purity 98.86%). mp = (207.6-208.6) ℃; 1 H NMR (500 MHz, Chloroform-d) δ9.51 (d, J = 1.9 Hz, 1H), 8.92 (d, J = 2.3 Hz, 1H), 8.22 (s, 1H), 8.06 (d, J= 2.3 Hz, 1H), 7.79 (d, J = 9.3 Hz, 1H), 7.65 – 7.54 (m, 3H), 7.44 (d, J =8.2 Hz, 1H), 7.25 (t, J = 8.4 Hz, 1H), 3.74 (s, 3H), 3.13 (t, J = 7.3 Hz,2H), 2.90 (t, J = 7.3 Hz, 2H), 2.86 (s, 3H), 2.76 – 2.37 (m, 8H), 2.28 (s,3H); LC-MS (ESI, m / z): Calcd for C 33 H 31 ClFN9O4[M+H] + 672.2, found: 672.8. Example 10 Compound E8 was prepared according to the following route: At room temperature, A4-2 (0.56 g, 1.49 mmol), BR2 (0.80 g, 1.63 mmol), potassium carbonate (0.62 g, 4.47 mmol), and PdCl2(dppf)-CH2Cl2 (0.11 g, 0.13 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E8 (0.54 g, yield 55.00%, purity 98.12%). mp = (245.2-246.2) ℃; 1 H NMR (500 MHz, Chloroform- d ) δ9.61 – 9.57 (m, 1H), 9.00 (d, J = 2.3 Hz, 1H), 8.31 (s, 1H), 8.14 (d, J = 2.3 Hz, 1H), 7.87 (d, J = 9.3 Hz, 1H), 7.73 – 7.60 (m, 3H), 7.52 (dd, J = 8.1, 1.2 Hz, 1H), 7.33 (dd, J LC-MS (ESI, m / z): Calcd forC 33 H 28 ClFN8O5[M+H] + 659.2, found: 659.4. Example 11 Compound E9 was prepared according to the following route: At room temperature, A4-3 (0.51 g, 1.37 mmol), BR2 (0.80 g, 1.64 mmol), potassium carbonate (0.57 g, 4.10 mmol), and PdCl2(dppf)-CH2Cl2 (0.10 g, 0.12 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E9 (0.37 g, yield 41.17%, purity 98.69%). mp = (209.6-210.6) ℃; 1 H NMR (500 MHz, Chloroform- d ) δ9.55 (s, 1H), 8.96 (d, J = 2.3 Hz, 1H), 8.27 (s, 1H), 8.09 (d, J = 2.3 Hz, 1H), 7.82 (d, J = 9.3 Hz, 1H), 7.68 - 7.56 (m, 3H), 7.47 (d, J = 8.1 Hz, 1H), 7.28 (t, J = 8.4 Hz, 1H), 3.77 (s, 3H), 3.18 (t, J = 7.4 Hz, 2H), 2.90 (t, J = 7.4 Hz, 2H),2.85 (s, 3H), 2.52 (t, J = 5.8 Hz, 4H), 1.62 – 1.59 (m, 4H), 1.47 – 1.43 (m,2H); LC-MS (ESI, m / z): Calcd for C 33 H 30 ClFN8O4[M+H] + 657.2, found: 657.5. Example 12 Compound E10 was prepared according to the following route: At room temperature, A4-4 (0.53 g, 1.37 mmol), BR2 (0.80 g, 1.64 mmol), potassium carbonate (0.57 g, 4.10 mmol), and PdCl2(dppf)-CH2Cl2 (0.10 g, 0.12 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E10 (0.41 g, yield 44.78%, purity 98.29%). mp = (192.6-193.6) ℃; 1 H NMR (500 MHz, Chloroform- d ) δ9.59 – 9.54 (m, 1H), 8.98 (d, J = 2.3 Hz, 1H), 8.28 (s, 1H), 8.11 (d, J = 2.3 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H), 7.71 – 7.58 (m, 3H), 7.49 (dd, J = 8.2, 1.2 Hz,1H), 7.33 – 7.26 (m, 1H), 3.79 (s, 3H), 3.22 (t, J = 7.5 Hz, 2H), 2.92 (t, J LC-MS (ESI, m / z): Calcd for C 34 H 32 ClFN8O4[M+H] + 671.2, found: 671.7. Example 13 Compound E11 was prepared according to the following route: At room temperature, A4-5 (0.49 g, 1.37 mmol), BR2 (0.80 g, 1.64 mmol), potassium carbonate (0.57 g, 4.10 mmol), and PdCl2(dppf)-CH2Cl2 (0.10 g, 0.12 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E11 (0.61 g, yield 69.32%, purity 98.14%). mp = (175.7-176.7) ℃; 1 H NMR (500 MHz, Chloroform- d ) δ9.60 – 9.58 (m, 1H), 8.99 (d, J = 2.3 Hz, 1H), 8.30 (s, 1H), 8.13 (d, J = 2.3 Hz, 1H), 7.86 (dd, J = 9.3, 1.0 Hz, 1H), 7.71 – 7.60 (m, 3H), 7.51 (dt, J = 8.2, 1.0Hz, 1H), 7.33 – 7.30 (m, 1H), 3.80 (s, 3H), 3.16 (t, J = 7.2 Hz, 2H), 3.06(t, J = 7.2 Hz, 2H), 2.92 (s, 3H), 2.66 (q, J = 7.1 Hz, 4H), 1.10 (t, J = 7.1 Hz, 6H); LC-MS (ESI, m / z): Calcd for C 32 H 30 ClFN8O4[M+H] + 645.2, found: 645.5. Example 14 Compound E12 was prepared according to the following route: At room temperature, A4-6 (0.65 g, 1.37 mmol), BR2 (0.80 g, 1.64 mmol), potassium carbonate (0.57 g, 4.10 mmol), and PdCl2(dppf)-CH2Cl2 (0.10 g, 0.12 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E12-1 (0.82 g, yield 79.00%). 1 H NMR (500 MHz, Chloroform- d ) δ 9.58 (dd, J = 1.9, 1.0 Hz, 1H), 8.98 (d, J = 2.3 Hz, 1H), 8.29 (s, 1H), 8.12 (d, J = 2.3 Hz, 1H), 7.86 (dd, J =9.3, 1.0 Hz, 1H), 7.70 – 7.64 (m, 2H), 7.64 – 7.59 (m, 1H), 7.50 (dt, J = 8.2,1.0 Hz, 1H), 7.33 – 7.29 (m, 1H), 3.80 (s, 3H), 3.45 (t, J = 5.0 Hz, 4H), 3.20(t, J = 7.3 Hz, 2H), 2.96 (t, J = 7.3 Hz, 2H), 2.91 (s, 3H), 2.53 (t, J = 5.0 Hz, 4H), 1.48 (s, 9H); LC-MS (ESI, m / z): Calcd for C 37 H 37 ClFN9O6[M+Na] + 780.3, found: 780.3. At room temperature, trifluoroacetic acid (2 mL) was slowly added dropwise to a solution of E12-1 (702 mg, 0.90 mmol) in dichloromethane (6 mL). After the addition was complete, the mixture was stirred at room temperature for 12 h, concentrated under reduced pressure, diluted with water (10 mL), and the pH of the aqueous phase was adjusted to 7-8 with 3N NaOH. The mixture was extracted with ethyl acetate (20 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 70:1-20:1) to give a pale yellow solid E12 (436 mg, yield 73.67%, purity 98.16%). mp = (198.3-199.3) ℃; 1 H NMR (500 MHz, DMSO-) d 6) δ 9.91 (br, 1H), 9.41 (d, J = 1.9 Hz, 1H), 9.20 (s, 1H), 8.66 – 8.56 (m, 1H), 8.44 (s, 1H), 8.32 (d, J = 2.3 Hz, 1H), 7.98 (d, J = 9.3 Hz, 1H), 7.87 (dd, J = 9.3, 1.9 Hz, 1H), 7.71 (td, J = 8.3, 6.0 Hz, 1H), 7.60 (d, J =8.2 Hz, 1H), 7.51 (t, J = 8.8 Hz, 1H), 3.90 (s, 3H), 3.68 – 3.23 (m, 12H), 2.84(s, 3H); LC-MS (ESI, m / z): Calcd for C 32 H 29 ClFN9O4[M+H] + 658.2, found: 658.6. Example 15 Compound E13 was prepared according to the following route: At room temperature, A4-2 (0.76 g, 2.00 mmol), BR3 (0.82 g, 2.20 mmol), potassium carbonate (0.83 g, 6.00 mmol), and PdCl2(dppf)-CH2Cl2 (0.15 g, 0.18 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E13 (0.56 g, yield 51.47%, purity 99.93%). mp = (251.5-252.5) ​​℃; 1 H NMR (500 MHz, Chloroform-d) δ9.66 – 9.62 (m, 1H), 9.01 (d, J = 2.3 Hz, 1H), 8.33 (s, 1H), 8.22 (d, J = 2.3Hz, 1H), 8.05 (s, 1H), 7.88 (d, J = 9.3 Hz, 1H), 7.70 (dd, J = 9.1, 1.8 Hz,1H), 4.16 (s, 3H), 3.75 (m, 4H), 3.22 (t, J = 7.4 Hz, 2H), 2.96 (t, J = 7.4Hz, 2H), 2.77 (m, 4H), 2.61 (s, 6H); (HRMS, m / z ): Calcd for C 27 H 28 N8O5[M+H] + 545.2183, found: 545.2239. Example 16 Compound E14 was prepared according to the following route: At room temperature, A4-3 (0.75 g, 2.00 mmol), BR3 (0.82 g, 2.20 mmol), potassium carbonate (0.83 g, 6.00 mmol), and PdCl2(dppf)-CH2Cl2 (0.15 g, 0.18 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (20 mL) and H2O (4 mL), and the solution was heated to 80°C. oAfter reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E14 (0.52 g, yield 47.97%, purity 99.47%). mp = (226.5-227.5) ℃; 1 H NMR (500 MHz, Chloroform-d) δ9.64 (dd, J = 1.9, 1.0 Hz, 1H), 9.00 (d, J = 2.3 Hz, 1H), 8.31 (s, 1H), 8.22(d, J = 2.3 Hz, 1H), 8.04 (s, 1H), 7.88 (dd, J = 9.3, 1.0 Hz, 1H), 7.70 (dd,J = 9.3, 1.8 Hz, 1H), 4.16 (s, 3H), 3.21 (t, J = 7.5 Hz, 2H), 2.94 (t, J =7.5 Hz, 2H), 2.77 (s, 3H), 2.61 (s, 3H), 2.55 (m, 4H), 1.65 (m, 4H), 1.49 (q,J = 6.0 Hz, 2H); (HRMS, m / z ): Calcd for C 28 H 30 N8O4[M+H] + 543.2390, found: 543.2439. Example 17 Compound E15 was prepared according to the following route: At room temperature, A4-6 (2.15 g, 4.49 mmol), BR3 (1.84 g, 4.93 mmol), potassium carbonate (1.86 g, 13.47 mmol), and PdCl2(dppf)-CH2Cl2 (0.33 g, 0.40 mmol) were dissolved sequentially in a mixed solvent of 1,4-dioxane (45 mL) and H2O (9 mL), and the solution was heated to 80°C. o After reaction C for 3 h, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 100:1~20:1) to give a white solid E15-1 (1.40 g, yield 48.44%). (HRMS) m / z ): Calcd for C 32 H 37 N9O6[M+H]+ 644.2867, found: 644.2899. At room temperature, trifluoroacetic acid (2 mL) was slowly added dropwise to a solution of E15-1 (612 mg, 0.95 mmol) in dichloromethane (6 mL). After the addition was complete, the mixture was stirred at room temperature for 12 h, concentrated under reduced pressure, diluted with water (10 mL), and the pH of the aqueous phase was adjusted to 7-8 with 3N NaOH. The mixture was extracted with ethyl acetate (20 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 70:1-20:1) to give a pale yellow solid E15 (398 mg, yield 76.98%, purity 98.75%). mp = (210.5-211.5) ℃; 1 H NMR (500 MHz, DMSO-d6) δ 9.53 (s, 1H), 9.46 (s, 1H), 8.70 (d, J = 2.4 Hz, 1H), 8.43 – 8.36 (m, 2H), 8.00 (d, J = 9.3 Hz, 1H), 7.91 (dd, J = 2.42 (m, 4H), 2.40 (s, 3H); (HRMS, m / z ): Calcd for C 27 H 29 N9O4[M+H] + 544.2343, found: 544.2388. The reaction raw materials in the above embodiments, as well as the amount of each reaction raw material added, can all be prepared using other reaction conditions defined by this invention. As long as they are within the range defined by this invention, the corresponding target product with high purity can be obtained.

[0035] Activity test 1. Inhibition rate 1.1 Preparation before measurement (1) The 15 synthesized compounds were prepared as follows: a certain amount of the test compound was accurately weighed in sequence, and DMSO was added to completely dissolve it to prepare a test solution with a concentration of 25 μM.

[0036] (2) Prepare a 5 mg / mL MTT solution. The preparation and storage method is as follows: Weigh 0.25 g of MTT and dissolve it completely in 50 mL of PBS to reach the required concentration. Then filter it through a microporous membrane (0.22 mm) and dispense it into EP tubes (1.5 mL). Wrap the tubes in aluminum foil to protect them from light and store them at -20 ℃ for later use.

[0037] (3) Preparation of complete culture medium: Fetal bovine serum (FBS, approximately 10% of total volume) and penicillin and streptomycin (both at a concentration of 100 U / mL, approximately 1% of total volume) were added to the incomplete culture medium (approximately 90% of total volume). Mouse melanoma cells B16, human liver cancer cells HepG2, and human non-small cell lung cancer cells A549 were prepared using RPMI1640, MEM, and F12, respectively, while human colon cancer cells HCT116 and human malignant melanoma cells A375 were prepared using DMEM.

[0038] 1.2 Cell proliferation inhibition experiment (1) Use 0.25% trypsin to remove glycoproteins and mucins between the test cells in the logarithmic growth phase, thereby affecting the cytoskeleton, causing cytoplasmic retraction, increasing intercellular spaces, and enabling cell separation. Control the digestion time of the trypsin solution to (2-5) min, then aspirate the trypsin, and terminate the digestion with the required complete culture medium (containing 10% FBS), mix the cell suspension well, and adjust the culture density to (2 × 10⁻⁶). 4 (units / mL).

[0039] (2) Add the above cell suspension to a 96-well plate (200 μL per well), incubate at 37 °C in a 5% CO2 incubator for 24 h, and then continue to maintain the same external environment as above. Add the test compound at a concentration of 25 μM, set up 3 replicate wells, and continue to incubate for 48 h.

[0040] (3) MTT colorimetric method. MTT can be reduced to blue-purple formazan crystals by succinate dehydrogenase (present in the mitochondria of living cells). These crystals, after being dissolved in DMSO, can be measured at a wavelength of 490 nm. This principle applies only to living cells. Using this principle, 20 μL of MTT reagent was added to each well, and the cells were cultured for 4 h under the aforementioned external conditions. The 96-well plate was then removed, and 150 μL of DMSO was added simultaneously with 150 μL of culture medium at a 1:1 volume ratio to dissolve the formazan. Finally, the absorbance was measured at 490 nm using a microplate reader to calculate the inhibitory rate of the compound on tumor cells. Compound 15A, as shown in the following formula, was used as a control.

[0041] Inhibition rate = (Absorbance value of control well - Absorbance value of drug well - Absorbance value of 0.5% DMSO) / Absorbance value of control well × 100% Table 1. Inhibition rate (%) of compounds E1-E15 on different tumor cells Note: "-" indicates that activity test data are not yet available. "15A" refers to compounds with excellent activity reported in the literature.

[0042] 2. Effects of certain compounds on the proliferation of human cervical cancer cells HeLa. Cell Counting Kit-8 (CCK8) is a colorimetric assay for measuring cell proliferation inhibition, replacing the complex MTT assay. Under the catalysis of dehydrogenase, WST-8 contained in CCK8 is reduced to an orange-yellow product, and the amount of this product is related to cell activity. The better the cell activity, the more product and the darker the color. It is widely used due to its ease of operation.

[0043] The specific steps are as follows: (1) Tumor cells in the logarithmic growth phase were seeded in 96-well plates at a density of 2500 cells / 100 μL / well and cultured overnight.

[0044] (2) The stock solutions of the test compounds (E5, E6, E10, E11, E12) were diluted with complete culture medium to make a final concentration of 100 μM. Six concentrations (1 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM) were added to a 96-well plate with three replicate wells, a blank control group, and positive control groups for 15A and doxorubicin. The 96-well plate was placed in an incubator and incubated at 37 °C for 24 h.

[0045] (3) Remove the remaining culture medium from the 96-well plate, add the CCK8 assay solution, incubate the 96-well plate in an incubator for 4 hours, and then measure the cell viability and inhibition rate after treatment with different concentrations of the test compound. The IC50 of the compound is calculated by plotting the results using GraphPad Prism 6. 50 The values ​​are shown in Table 2.

[0046] Table 2 shows the effects of some compounds on IC50 in HeLa cells. 50 value Note: "15A" refers to compounds with excellent activity reported in the literature.

[0047] Table 2 shows that the target compounds (E5, E6, E10, E11, E12) exhibited good anti-tumor cell proliferation activity against HeLa cells, with an IC50 score of 100%. 50 The range was mostly less than 15 and all below 20. The tested compounds E5, E6, E10, E11, and E12 all showed stronger activity against HeLa cells than the control compound 15A (IC50). 50 = 27.860 μM). In particular, E5 had the strongest inhibitory effect on this cell, IC50 = 27.860 μM. 50 The value was 4.030, which is nearly 7 times different from 15A, showing an anti-tumor effect similar to that of the positive control drug DOX.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An isoxazole compound, characterized in that, Its structure is shown in equation (Ⅰ): Equation (I) Where R is , or ; R' is , , , , or .

2. A method for preparing the isoxazole compound according to claim 1, characterized in that, Includes the following steps: S1, using 2-amino-5-bromopyridine and ethyl 2-chloro-3-oxypropionate as raw materials, undergoes a cyclization reaction to obtain the compound shown in formula (II); S2, using the compound shown in formula (II) and hydrazine hydrate as raw materials, undergoes hydrazolysis to obtain the compound shown in formula (III); S3, using the compound shown in formula (III), acrolein and iodophenyl diacetic acid as raw materials, undergoes a cyclization reaction to obtain the compound shown in formula (IV); S4, using the compound shown in formula (Ⅳ) and R'-H as raw materials, undergoes an addition reaction to obtain the compound shown in formula (Ⅴ); S5, using 3-amino-5-bromo-2-methoxypyridine and pinacol diboronic acid ester as raw materials, was reacted via the Miyaura reaction to obtain the compound shown in formula (VI); S6, using the compound shown in formula (VI) and R-Cl as starting materials, undergoes nucleophilic addition and elimination reactions to obtain the compound shown in formula (VII); S7, using the compounds shown in formula (Ⅶ) and (Ⅴ) as raw materials, the isoxazole compounds shown in formula (Ⅰ) are obtained by the Suzuki reaction; 。 3. The method for preparing isoxazole compounds as described in claim 2, characterized in that, Specifically, the following steps are included: S1, 2-amino-5-bromopyridine and ethyl 2-chloro-3-oxypropionate were added to anhydrous ethanol and refluxed for 15 h to 30 h. After post-treatment, the compound shown in formula (II) was obtained. S2, the compound shown in formula (II) and hydrazine hydrate were added to anhydrous ethanol and refluxed for 10 h to 15 h. After post-treatment, the compound shown in formula (III) was obtained. S3, add the compound shown in formula (Ⅲ), acrolein and glacial acetic acid to dichloromethane, stir and react for 8 h to 12 h, then add iodophenyl diacetic acid, stir and react for 10 h to 14 h, and then perform post-treatment to obtain the compound shown in formula (Ⅳ); S4, the compound shown in formula (Ⅳ) was dissolved in dichloromethane, R'-H and 1,8-diazacyclo[5,4,0]undecene-7 were added, the reaction was stirred for 10 h to 15 h, and then post-treated to obtain the compound shown in formula (Ⅴ); S5, 3-amino-5-bromo-2-methoxypyridine, pinacol diboronic acid, potassium acetate and PdCl2(dppf)-CH2Cl2 were dispersed in anhydrous dioxane and reacted at 50 ℃~100 ℃ for 15 h~20 h under an inert atmosphere, followed by post-treatment to obtain the compound shown in formula (VI); S6, Dissolve the compound shown in formula (VI) in anhydrous tetrahydrofuran, add triethylamine, mix well, add R-Cl, stir for 8 h to 12 h, and then perform post-treatment to obtain the compound shown in formula (VII); S7. The compound shown in formula (VII), the compound shown in formula (V), potassium carbonate and PdCl2(dppf)-CH2Cl2 are dispersed in a mixed solution of dioxane and water. The mixture is reacted at 50 ℃~100 ℃ for 1 h~5 h under an inert atmosphere. After post-treatment, the isoxazole compound shown in formula (I) is obtained.

4. The method for preparing isoxazole compounds as described in claim 3, characterized in that, In S1, the molar ratio of 2-amino-5-bromopyridine to ethyl 2-chloro-3-oxypropionate is 1:(1~3); and / or In S1, the molar volume ratio of 2-amino-5-bromopyridine to anhydrous ethanol is 1 mmol : (1~20) mL; and / or In S2, the molar ratio of the compound represented by formula (II) to hydrazine hydrate is 1:(10~30); and / or In S2, the volume molar ratio of the anhydrous ethanol to the compound represented by formula (II) is (1~30) mL : 1 mmol.

5. The method for preparing isoxazole compounds as described in claim 3, characterized in that, In S3, the molar ratio of the compound represented by formula (Ⅲ) to acrolein is 1:(1~10); and / or In S3, the volume molar ratio of the dichloromethane to the compound represented by formula (III) is (10~50) mL : 1 mmol; and / or In S3, the molar ratio of the iodophenyl diacetic acid to the compound represented by formula (Ⅲ) is (1~5):

1.

6. The method for preparing isoxazole compounds as described in claim 3, characterized in that, In S4, the molar ratio of the compound represented by formula (Ⅳ) to R'-H is 1 : (1~5); and / or In S4, the volume molar ratio of the dichloromethane to the compound shown in formula (Ⅳ) is (1~50) mL : 1 mmol; and / or In S4, the molar ratio of 1,8-diazacyclic [5,4,0]undecene-7 to the compound shown in formula (Ⅳ) is (1~3):

1.

7. The method for preparing isoxazole compounds as described in claim 3, characterized in that, In S5, the molar ratio of 3-amino-5-bromo-2-methoxypyridine, pinacol diboronate, potassium acetate, and PdCl2(dppf)-CH2Cl2 is 1 : (1~5) : (1~10) : (0.01~0.5); and / or In S5, the volume molar ratio of anhydrous dioxane to 3-amino-5-bromo-2-methoxypyridine is (0.1~20) mL: 1 mmol.

8. The method for preparing isoxazole compounds as described in claim 3, characterized in that, In S6, the molar ratio of the compound represented by formula (VI), triethylamine, and R-Cl is 1 : (0.5~10) : (1~5); In S6, the volume molar ratio of the anhydrous tetrahydrofuran to the compound shown in formula (VI) is (1~40) mL : 1 mmol.

9. The method for preparing isoxazole compounds as described in claim 3, characterized in that, In S7, the molar ratio of the compound represented by formula (V), the compound represented by formula (VII), potassium carbonate, and PdCl2(dppf)-CH2Cl2 is 1 : (1~5) : (1~10) : (0.01~1).

10. The use of the isoxazole compound of claim 1 in the preparation of a medicament for treating melanoma, non-small cell lung cancer, colon cancer, or liver cancer.

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