FLT3 covalent inhibitor containing isoxazolo [5, 4-b] pyridine structure as well as preparation method and application of FLT3 covalent inhibitor

Abstract

The invention belongs to the technical field of medicines, and relates to an FLT3 covalent inhibitor containing an isoxazolo [5, 4-b] pyridine structure as well as a preparation method and application of the FLT3 covalent inhibitor. The invention provides a novel isoxazolo [5, 4-b] pyridine covalent inhibitor targeting FLT3 protein, the novel isoxazolo [5, 4-b] pyridine covalent inhibitor contains an acryloyl warhead, and the warhead can covalently target cysteine and is covalently bound with 807-site Cys in FLT3, so that the activity of the FLT3 is effectively inhibited. The inhibitor has remarkable anti-tumor activity in in-vitro and in-vivo models, especially for acute myeloid leukemia caused by FLT3 mutation. The FLT3 inhibitor provided by the invention has good research and development prospects and clinical value.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to an FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure, a preparation method thereof and uses thereof. Specifically, it relates to an FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure, a preparation method thereof and its application in the prevention and treatment of acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant blood disease characterized by abnormal proliferation of hematopoietic cells in peripheral blood, bone marrow or other tissues, resulting in reduced erythropoiesis, excessive leukocyte proliferation, organ dysfunction and increased risk of infection. The occurrence of AML is closely related to the abnormal phosphorylation of Fms-like tyrosine kinase 3 (FLT3) (Daver N, Schlenk R F, Russell N H, et al. Targeting FLT3 mutations in AML: review of current knowledge and evidence[J]. Leukemia, 2019, 33(2):299-312).

[0003] In the past decade, targeted therapy has significantly improved the prognosis of patients with FLT3-mutated positive AML. However, the first-generation FLT3 inhibitors such as Sunitinib, Sorafenib, Midostaurin, Lestaurtinib and Tandutinib, as broad-spectrum and multi-target inhibitors, lack selectivity for FLT3. This non-selectivity not only limits the therapeutic effect but also increases the risk of adverse reactions in patients. In particular, the non-specific inhibition of other kinases such as JAK2 and VEGFR may interfere with key biological pathways, leading to unpredictable side effects, including cardiovascular problems, bleeding and immunosuppression. Therefore, the development of more selective FLT3 inhibitors is crucial for reducing the side effects of clinical treatment of AML.

[0004] The inhibitory potency and selectivity of second-generation FLT3 inhibitors have been significantly improved. For example, Quizartinib, Gilteritinib, and Crenolanib have shown better efficacy in FLT3-ITD positive cases. However, there are still some problems in the clinical application of second-generation FLT3 inhibitors. For example, drug resistance appears rapidly (Abdel-Aziz AK, Dokla EME, Saadeldin MK, et al. FLT3 inhibitors and novel therapeutic strategies to reverse AML resistance: An updated comprehensive review [J]. Crit Rev Oncol Hematol 2023, 191, 104139). Therefore, there is an urgent need to develop new FLT3 inhibitors to overcome drug-resistant mutations.

[0005] In recent years, covalent inhibitors, as an emerging type of inhibitor, have important development potential in overcoming drug resistance. Different from traditional reversible inhibitors, covalent inhibitors can form irreversible covalent bonds with specific active sites of target proteins (such as cysteine residues), so they can provide persistent and potent inhibitory effects. FF-10101 is the first reported FLT3 covalent inhibitor and is currently in the clinical trial stage (Yamaura T, Nakatani T, Uda K, et al. A novel irreversible FLT3 inhibitor, FF-10101, shows excellent efficacy against AML cells with FLT3 mutations [J]. Blood, 2018, 131(4): 426-438). Studies have shown that FF-10101 can effectively bind to the active and inactive conformations of FLT3 and exhibits excellent affinity for FLT3 by forming a covalent bond with the thiol group of Cys695 in the hinge region. In addition, the IC 50 values of FF-10101 for wild-type FLT3 and FLT3 D835Y mutants are 0.2 nM and 0.16 nM, respectively. At the same time, it also has good inhibitory activity against cells expressing amino acid mutations such as FLT3-ITD, FLT3-ITD-F691L, and FLT3-ITD-D835Y. The GI 50 values are 1.9 nM, 10 nM, and 0.81 nM, respectively.

[0006] Therefore, the development of covalent inhibitors against FLT3, by irreversibly covalently binding to key amino acid residues of the FLT3 kinase, is expected to overcome drug resistance caused by traditional inhibitors and provide a new option for the drug treatment of AML. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure for the treatment of AML in view of the deficiencies of the prior art.

[0008] The further technical problem to be solved by the present invention is to provide a preparation method of the above-mentioned FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure.

[0009] The further technical problem to be solved by the present invention is to provide an application of the above-mentioned FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure.

[0010] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0011] The present invention discloses a FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure, and the FLT3 covalent inhibitor has the structure shown in Formula I:

[0012]

[0013] Wherein,

[0014] R is selected from hydrogen, halogen, cyano, halo-C 1 -C 4 alkyl, C 1 -C 4 alkoxy or C 1 -C 4 alkyl;

[0015] L is selected from Wherein, R 1 is selected from hydrogen or C 1 -C 4 alkyl.

[0016] In some embodiments, preferably, R is selected from hydrogen, fluorine, chlorine, cyano, trifluoromethyl, methoxy or methyl;

[0017] L is selected from Wherein, R 1 is selected from hydrogen or methyl.

[0018] In some embodiments, further preferably, the FLT3 covalent inhibitor is selected from any of the following structures:

[0019]

[0020] In some embodiments, most preferably, the FLT3 covalent inhibitor is:

[0021]

[0022] Furthermore, the present invention discloses the tautomers, mesomers, racemates, enantiomers, diastereomers or pharmaceutically acceptable salts of the above-mentioned FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure.

[0023] Furthermore, the present invention discloses a preparation method of the above-mentioned FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure, comprising the following steps:

[0024] (1) Compound 1-6-a and compound 1-6-b are subjected to a first reaction under the action of a first base to obtain compound 1-6; compound 1-6 is subjected to a second reaction under the action of a reducing agent and ammonium chloride to obtain compound 1-7;

[0025]

[0026] (2) Compound 1-7 and triphosgene are subjected to a third reaction, and the obtained isocyanate intermediate 1-8 and compound 1-5 continue to be subjected to a fourth reaction under the action of a second base to obtain the FLT3 covalent inhibitor I containing an isoxazolo[5,4-b]pyridine structure;

[0027]

[0028] Wherein,

[0029] R is selected from hydrogen, halogen, cyano, halo-C 1 -C 4 alkyl, C 1 -C 4 alkoxy or C 1 -C 4 alkyl;

[0030] L is selected from Wherein, R 1 is selected from hydrogen or C 1 -C 4 alkyl;

[0031] X is Cl or Br.

[0032] In some embodiments, in the first reaction, the first base is triethylamine; the molar ratio of compound 1-6-a to compound 1-6-b is 1.0:(0.9-3.0); the first reaction is carried out at room temperature; the solvent used in the first reaction is N,N-dimethylformamide.

[0033] In some embodiments, preferably, in the first reaction, the first base is triethylamine; the molar ratio of compound 1-6-a to compound 1-6-b is 1.0:(0.9 - 1.8); the first reaction is carried out at room temperature; the solvent used in the first reaction is N,N-dimethylformamide.

[0034] Among them, in the first reaction, the amount of the first base used is small, preferably the molar ratio of compound 1-6-a to the first base is 1:(0.02 - 0.10), and more preferably 1:(0.02 - 0.05).

[0035] Among them, in the first reaction, there is no special requirement for the amount of the solvent used, and it is only necessary to dissolve or disperse the raw materials evenly.

[0036] In some embodiments, in the second reaction, the reducing agent is iron powder; the molar ratio of compound 1-6 to the reducing agent is 1.0:(2.0 - 6.0); the reaction temperature of the second reaction is 60 - 100 °C; the solvent used in the second reaction is a mixture of ethanol and water in any proportion.

[0037] In some embodiments, preferably, in the second reaction, the reducing agent is iron powder; the molar ratio of compound 1-6 to the reducing agent is 1.0:2.0; the reaction temperature of the second reaction is 80 °C; the solvent used in the second reaction is a mixture of ethanol and water with a volume ratio of (5 - 10):1, and more preferably a mixture of 5:1.

[0038] Among them, in the second reaction, the ammonium chloride exists in the form of an aqueous solution, and the addition form of the ammonium chloride is a saturated ammonium chloride aqueous solution; the ratio of the molar amount of compound 1-6 to the volume amount of the saturated ammonium chloride aqueous solution is (1.58 mmol - 2.20 mmol):1 mL.

[0039] Among them, in the second reaction, there is no special requirement for the amount of the solvent used, and it is only necessary to dissolve or disperse the raw materials evenly.

[0040] In some embodiments, in the third reaction, the molar ratio of compound 1-7 to triphosgene is (2.0 - 6.0):1.0; the third reaction is carried out under the protection of an inert gas; for the third reaction, after stirring in an ice bath for 0.5 - 1.0 h, then reacting at room temperature for 1 - 3 h, and finally reacting at 60 - 100 °C for 3 - 6 h; the solvent used in the third reaction is ethyl acetate.

[0041] In some embodiments, preferably, in the third reaction, the molar ratio of the compound 1-7 to triphosgene is (2.0 to 4.0):1.0; the third reaction is carried out under the protection of an inert gas; in the third reaction, after stirring in an ice bath for 0.5 to 1.0 h, the reaction is carried out at room temperature for 1 to 2 h, and finally the reaction is carried out at 70 to 90 °C for 3 to 5 h; the solvent used in the third reaction is ethyl acetate.

[0042] In some embodiments, further preferably, in the third reaction, the molar ratio of the compound 1-7 to triphosgene is 2.0:1.0; the third reaction is carried out under the protection of nitrogen gas; in the third reaction, after stirring in an ice bath for 0.5 h, the reaction is carried out at room temperature for 1 h, and finally the reaction is carried out at 82 °C for 4 h; the solvent used in the third reaction is ethyl acetate.

[0043] Among them, in the third reaction, there is no special requirement for the amount of the solvent used, and it is only necessary to dissolve or disperse the raw materials evenly.

[0044] In some embodiments, in the fourth reaction, the second base is triethylamine; the molar ratio of the compound 1-7 used in the third reaction to the compound 1-5 used in the fourth reaction is (4.0 to 8.0):(0.005 to 0.500); the fourth reaction is carried out at a reaction temperature of -20 °C to 10 °C; the solvent used in the fourth reaction is dichloromethane.

[0045] In some embodiments, preferably, in the fourth reaction, the second base is triethylamine; the molar ratio of the compound 1-7 used in the third reaction to the compound 1-5 used in the fourth reaction is (5.0 to 8.0):(0.100 to 0.300); the fourth reaction is carried out at a reaction temperature of -5 °C to 5 °C; the solvent used in the fourth reaction is dichloromethane.

[0046] In some embodiments, further preferably, in the fourth reaction, the second base is triethylamine; the molar ratio of the compound 1-7 used in the third reaction to the compound 1-5 used in the fourth reaction is 6.0:0.125; the fourth reaction is carried out at a reaction temperature of 0 °C; the solvent used in the fourth reaction is dichloromethane.

[0047] Among them, in the fourth reaction, the amount of the second base used is small. Preferably, the molar ratio of the compound 1-5 to the second base is 1.0:(0.2 to 1.2), and more preferably 1.0:(0.4 to 0.8).

[0048] Among them, in the fourth reaction, there is no special requirement for the amount of the solvent used, and it is only necessary to dissolve or disperse the raw materials evenly.

[0049] In some embodiments, Compounds 1-5 are commercially available or can be prepared according to the following steps:

[0050] (a) p-Nitrobenzaldehyde and acetone undergo an aldol condensation reaction under the action of a third base to obtain Compound 1-1;

[0051]

[0052] (b) Compound 1-1 and cyanoacetamide undergo a cyclization reaction under the action of a fourth base to obtain Compound 1-2;

[0053]

[0054] (c) Compound 1-2 and phosphorus oxychloride undergo a chlorination reaction to obtain Compound 1-3;

[0055]

[0056] (d) Compound 1-3 undergoes a reduction reaction under the action of a reducing agent and an acid to obtain Compound 1-4;

[0057]

[0058] (e) Compound 1-4 and acetohydroxamic acid undergo a cyclization reaction under the action of a fifth base to obtain Compound 1-5;

[0059]

[0060] In some embodiments, in step (a), the molar amount of p-nitrobenzaldehyde and the volume amount of acetone have a ratio of 1 mol: 500 mL to 3750 mL; the third base is potassium carbonate; the molar ratio of p-nitrobenzaldehyde to the third base is 1: (0.2 to 1.0); for the aldol condensation reaction, the reaction is carried out at room temperature.

[0061] In some embodiments, preferably, in step (a), the molar amount of p-nitrobenzaldehyde and the volume amount of acetone have a ratio of 1 mol: 1000 mL to 1500 mL; the third base is potassium carbonate; the molar ratio of p-nitrobenzaldehyde to the third base is 1: (0.2 to 0.6); for the aldol condensation reaction, the reaction is carried out at room temperature.

[0062] In some embodiments, further preferably, in step (a), the molar amount of p-nitrobenzaldehyde and the volume amount of acetone have a ratio of 1 mol: 1250 mL; the third base is potassium carbonate; the molar ratio of p-nitrobenzaldehyde to the third base is 1: 0.2; for the aldol condensation reaction, the reaction is carried out at room temperature.

[0063] In some embodiments, in step (b), the fourth base is potassium tert-butoxide; the molar ratio of compound 1-1 to cyanoacetamide is 1.0: (0.8 - 3.0); the molar ratio of compound 1-1 to the fourth base is 1.0: (1.0 - 4.0); for the cyclization reaction, the reaction temperature is 40°C to 70°C; the solvent used in the cyclization reaction is dimethyl sulfoxide; there is no special requirement for the amount of the solvent used in the cyclization reaction, and it is only necessary to dissolve or disperse the raw materials evenly.

[0064] In some embodiments, preferably, in step (b), the fourth base is potassium tert-butoxide; the molar ratio of compound 1-1 to cyanoacetamide is 1.0: (0.8 - 1.5); the molar ratio of compound 1-1 to the fourth base is 1.0: (1.0 - 1.5); for the cyclization reaction, the reaction temperature is 40°C to 70°C; the solvent used in the cyclization reaction is dimethyl sulfoxide; there is no special requirement for the amount of the solvent used in the cyclization reaction, and it is only necessary to dissolve or disperse the raw materials evenly.

[0065] In some embodiments, further preferably, in step (b), the fourth base is potassium tert-butoxide; the molar ratio of compound 1-1 to cyanoacetamide is 1.0: 1.0; the molar ratio of compound 1-1 to the fourth base is 1.0: 1.1; for the cyclization reaction, the reaction temperature is 40°C to 70°C; the solvent used in the cyclization reaction is dimethyl sulfoxide; there is no special requirement for the amount of the solvent used in the cyclization reaction, and it is only necessary to dissolve or disperse the raw materials evenly.

[0066] In some embodiments, in step (c), the ratio of the molar amount of compound 1-2 to the volume amount of phosphorus oxychloride is (2.0 - 6.0) mmol: (5 - 20) mL; for the chlorination reaction, the reaction temperature is 50 - 120°C.

[0067] In some embodiments, preferably, in step (c), the ratio of the molar amount of compound 1-2 to the volume amount of phosphorus oxychloride is (3.0 - 5.0) mmol: (8 - 12) mL; for the chlorination reaction, the reaction temperature is 80 - 120°C.

[0068] In some embodiments, further preferably, in step (c), the molar dosage of the compound 1-2 and the volume dosage of the phosphorus oxychloride are in a ratio of 3.9 mmol: 10 mL; for the chlorination reaction, the reaction temperature is 100 °C.

[0069] In some embodiments, in step (d), the reducing agent is iron powder; the acid is acetic acid; the molar ratio of the compound 1-3 to the reducing agent is 1.0: (1.0 - 6.0); the molar ratio of the compound 1-3 to the acid is 1.0: (2.0 - 12.0); for the reduction reaction, the reaction temperature is 60 - 100 °C; the solvent used in the reduction reaction is absolute ethanol, and there is no special requirement for the amount of the solvent, as long as the raw materials can be dissolved or dispersed evenly.

[0070] In some embodiments, preferably, in step (d), the reducing agent is iron powder; the acid is acetic acid; the molar ratio of the compound 1-3 to the reducing agent is 1.0: (1.0 - 3.0); the molar ratio of the compound 1-3 to the acid is 1.0: (2.0 - 5.0); for the reduction reaction, the reaction temperature is 70 - 90 °C; the solvent used in the reduction reaction is absolute ethanol, and there is no special requirement for the amount of the solvent, as long as the raw materials can be dissolved or dispersed evenly.

[0071] In some embodiments, further preferably, in step (d), the reducing agent is iron powder; the acid is acetic acid; the molar ratio of the compound 1-3 to the reducing agent is 1.0: 2.0; the molar ratio of the compound 1-3 to the acid is 1.0: 3.9; for the reduction reaction, the reaction temperature is 80 °C; the solvent used in the reduction reaction is absolute ethanol, and there is no special requirement for the amount of the solvent, as long as the raw materials can be dissolved or dispersed evenly.

[0072] In some embodiments, in step (e), the fifth base is potassium carbonate; the molar ratio of the compound 1-4 to acetohydroxamic acid is 1.0: (0.8 - 3.0); the molar ratio of the compound 1-4 to the fifth base is 1.0: (1.0 - 8.0); for the cyclization reaction, the reaction temperature is 60 - 100 °C; the solvent used in the cyclization reaction is N,N-dimethylformamide, and there is no special requirement for the amount of the solvent, as long as the raw materials can be dissolved or dispersed evenly.

[0073] In some embodiments, preferably, in step (e), the fifth base is potassium carbonate; the molar ratio of the compound 1-4 to acetohydroxamic acid is 1.0:(0.8 - 2.0); the molar ratio of the compound 1-4 to the fifth base is 1.0:(1.0 - 4.0); for the cyclization reaction, the reaction temperature is 60 - 90 °C; the solvent used in the cyclization reaction is N,N-dimethylformamide, and there is no special requirement for the amount of the solvent, as long as the raw materials can be dissolved or dispersed evenly.

[0074] In some embodiments, more preferably, in step (e), the fifth base is potassium carbonate; the molar ratio of the compound 1-4 to acetohydroxamic acid is 1.0:1.0; the molar ratio of the compound 1-4 to the fifth base is 1.0:2.0; for the cyclization reaction, the reaction temperature is 70 °C; the solvent used in the cyclization reaction is N,N-dimethylformamide, and there is no special requirement for the amount of the solvent, as long as the raw materials can be dissolved or dispersed evenly.

[0075] Furthermore, the present invention discloses a pharmaceutical composition comprising (i) the FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure shown in the above formula (I), or a tautomer, or a meso form, or a racemate, or an enantiomer, or a diastereoisomer, or a pharmaceutically acceptable salt thereof, and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0076] The use of the above-mentioned FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure, or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and treating tumors is also within the protection scope of the present invention.

[0077] The use of the above-mentioned FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure, or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and treating acute myeloid leukemia is also within the protection scope of the present invention.

[0078] The use of a tautomer, meso form, racemate, enantiomer, diastereoisomer or pharmaceutically acceptable salt of the above-mentioned FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure in the preparation of a drug for preventing and treating tumors is also within the protection scope of the present invention.

[0079] The use of a tautomer, meso form, racemate, enantiomer, diastereoisomer or pharmaceutically acceptable salt of the above-mentioned FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure in the preparation of a drug for preventing and treating acute myeloid leukemia is also within the protection scope of the present invention.

[0080] Beneficial effects:

[0081] (1) The present invention provides a novel isoxazolo[5,4-b]pyridine-based covalent inhibitor targeting the FLT3 protein. Such inhibitors contain an acryloyl warhead that can covalently target cysteine and covalently bind to Cys at position 807 in FLT3, thereby effectively inhibiting its activity. The inhibitor has significant anti-tumor activity in vitro and in vivo models, especially against acute myeloid leukemia caused by FLT3 mutations. The FLT3 inhibitor provided by the present invention has good R & D prospects and clinical value.

[0082] (2) By adopting the above technical solutions, the present invention is based on the isoxazolo[5,4-b]pyridine structure to synthesize a series of FLT3 covalent inhibitors containing the isoxazolo[5,4-b]pyridine structure, and it is found that the compound has a good inhibitory effect on acute myeloid leukemia caused by the malignant proliferation of hematopoietic cells and lymphocytes, providing a scientific basis and research direction for the R & D of new FLT3 drugs. The FLT3 inhibitor provided by the present invention has a novel structure, mild preparation conditions, abundant and easily available raw materials, and simple operation and post-treatment.

[0083] (3) Compared with the prior art or with the positive drug Quizaitinib, a) the compound W-3 provided by the present invention has significant inhibitory activity against the human leukemia cell lines MOLM-13 and MV4-11 expressing FLT3, especially has strong inhibitory activity against MV4-11; b) at the same concentration (100 nM), the total apoptosis rate of the compound W-3 against MOLM-13 is better than that of Quizartinib; c) at the same concentration (30 nM), the inhibitory effect of the compound W-3 on the phosphorylation of the FLT3-mediated signaling pathway is almost the same as that of the positive control drug Quizartinib, and even better than Quizartinib; d) compared with Quizartinib, almost no obvious decrease in the body weight of mice was observed after administration of the compound W-3, and the body weight remained stable and slowly increased, indicating that mice have good tolerance to the compound W-3; e) the compound W-3 did not show significant blood toxicity in the zebrafish model and has reliable safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0085] Figure 1 It is a structural diagram of the general formula (Ⅰ) of the FLT3 inhibitor of the present invention.

[0086] Figure 2 It is the nuclear magnetic resonance 1 1H NMR spectrum of the compound W-3.

[0087] Figure 3 1H NMR of compound W-3 13 13C NMR spectrum.

[0088] Figure 4 1H NMR spectrum of FLT3 protein, 1H NMR spectrum of covalent binding of FLT3 protein and W-3, and data graph of the binding ratio of FLT3 protein and compound W-3; among them, Figure a is the 1H NMR spectrum of FLT3 protein; Figure b is the 1H NMR spectrum of covalent binding of FLT3 protein and W-3; Figure c is the data graph of the binding ratio of FLT3 protein and compound W-3.

[0089] Figure 5 Peptide map of covalent linkage between compound W-3 and FLT3 based on PEAKS analysis.

[0090] Figure 6 Test result graph of apoptosis-promoting activity of different compounds on MV4-11 cells for 24 h; among them, Figure A is the flow cytometry graph of the effects of compound W-3 and Quizartinib on MV4-11 cells for 24 h respectively; Figure B is the bar graph of the total apoptosis rate of MV4-11 cells measured by flow cytometry for 24 h.

[0091] Figure 7 Test result graph of apoptosis-promoting activity of different compounds on MV4-11 cells for 72 h; among them, Figure A is the flow cytometry graph of the effects of compound W-3 and Quizartinib on MV4-11 cells for 72 h respectively; Figure B is the bar graph of the total apoptosis rate of MV4-11 cells measured by flow cytometry for 72 h.

[0092] Figure 8 Test result graph of apoptosis-promoting activity of different compounds on MOLM-13 cells for 24 h; among them, Figure A is the flow cytometry graph of the effects of compound W-3 and Quizartinib on MOLM-13 cells for 24 h respectively; Figure B is the bar graph of the total apoptosis rate of MOLM-13 cells measured by flow cytometry for 24 h.

[0093] Figure 9 Test result graph of apoptosis-promoting activity of different compounds on MOLM-13 cells for 72 h; among them, Figure A is the flow cytometry graph of the effects of compound W-3 and Quizartinib on MOLM-13 cells for 72 h respectively; Figure B is the bar graph of the total apoptosis rate of MOLM-13 cells measured by flow cytometry for 72 h.

[0094] Figure 10Graph showing the effects of different compounds on the cell cycle of MOLM-13 cells and bar graph of the percentage of cell populations in MOLM-13; among them, Panel A is the graph showing the effects of blank DMSO, compound W-3, and Quizartinib on the cell cycle of MOLM-13 cells; Panel B is the bar graph of the percentage of cell populations in MOLM-13.

[0095] Figure 11 Graph showing the phosphorylation inhibition levels of FLT3 and its mediated signaling pathways and the relative expression levels of proteins in MV4-11 cells by compound W-3; among them, Panel A is the graph showing the phosphorylation inhibition levels of FLT3 and its mediated signaling pathways in MV4-11 cells by compound W-3; Panel B is the relative expression levels of P-FLT3, P-STAT5, P-AKT, and P-ERK compared to the normal proteins; where ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001.

[0096] Figure 12 In vivo anti-tumor results of different drugs in the BALB / c nude mouse xenograft tumor model; among them, Panel A is the graph of the change in tumor volume of nude mice during drug administration; Panel B is the graph of the change in body weight of nude mice during drug administration; Panel C is the graph comparing the weights of tumor tissues obtained by decapitating and sacrificing mice 14 days after drug administration; Panel D is the photo of tumor tissues; where ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001.

[0097] Figure 13 Micrographs of tumor tissues stained with hematoxylin-eosin (HE), Ki-67, TUNEL, and P-STAT5.

[0098] Figure 14 Micrographs of HE staining of the heart, liver, spleen, lungs, and kidneys taken from dissected nude mice.

[0099] Figure 15 Graph showing the results of toxicity studies based on the zebrafish model; among them, Panel A is the representative graph of neutrophils (green fluorescence) in zebrafish treated with DMSO, compound W-3, and Quizartinib at 1 μM and 10 μM for 24 h respectively; Panel B is the count graph of zebrafish neutrophils (n = 3). Detailed implementation methods

[0100] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0101] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0102] In this application, unless otherwise defined, the abbreviations used have the following meanings:

[0103] min means minute; h means hour; d means day; ℃ means degree Celsius; V:V means volume ratio.

[0104] DCM refers to dichloromethane; EA or EtOAc refers to ethyl acetate; PE refers to petroleum ether; MeOH refers to methanol; THF refers to tetrahydrofuran; DMF refers to N,N-dimethylformamide; DMSO refers to dimethyl sulfoxide; TEA refers to triethylamine; Pd(OAc) 2 Refers to palladium acetate; Pd(dppf)Cl 2 Refers to 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride.

[0105] The starting materials known in the present invention can be synthesized by methods known in the art, or purchased from companies such as Anergy and Aladdin.

[0106] Unless otherwise specified in the examples, the reactions can be carried out under an argon atmosphere or a nitrogen atmosphere. Argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L. Hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1 L. The hydrogenation reaction is usually evacuated, filled with hydrogen, and the operation is repeated 3 times.

[0107] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0108] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0109] The reaction progress in the embodiment is monitored by thin layer chromatography (TLC), the developing solvent used in the reaction, the system of the eluent for column chromatography used for purifying the compound and the developing solvent system for thin layer chromatography include: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system, C: petroleum ether / ethyl acetate system, D: acetone, E: dichloromethane / acetone system, F: ethyl acetate / dichloromethane system, G: ethyl acetate / dichloromethane / n-hexane, H: ethyl acetate / dichloromethane / acetone, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

[0110] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shifts were multiplied by 10 -6Given in the unit of (ppm). The NMR measurement was performed using an Ascend nuclear magnetic resonance spectrometer, and the solvent for the measurement was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS).

[0111] The MS measurement was performed using an Agilent 1100 liquid chromatography-mass spectrometry.

[0112] Column chromatography generally uses silica gel with 300 - 400 mesh from Yantai Huanghai or acidic or basic alumina with 100 - 200 mesh as the carrier.

[0113] The structural formula of the FLT3 inhibitor of the general formula (Ⅰ) of the present invention is as shown in Figure 1 the figure.

[0114] Example 1: Preparation of 4-(4-nitrophenyl)but-3-en-2-one (Compound 1-1)

[0115]

[0116] Dissolve p-nitrobenzaldehyde (15.2 g, 0.1 mol) in 125 mL of acetone solution, add an aqueous solution of K 2 CO 3 (10 mL, 0.02 mol), stir at room temperature for 24 h, then slowly add concentrated hydrochloric acid (40 mL), and continue to stir at room temperature for 12 h. After monitoring the reaction by TLC and completion, add a large amount of water. Yellow solid precipitates, filter by suction, and dry the filter cake to obtain yellow solid 4-(4-nitrophenyl)but-3-en-2-one, denoted as Compound 1-1 (16.3 g, 84.76%).

[0117] The NMR data of Compound 1-1 are as follows: 1 H NMR (500 MHz, CDCl 3 ) δ (ppm): 8.27 (d, J = 8.7 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 16.1 Hz, 1H), 6.84 (d, J = 16.1 Hz, 1H), 2.43 (s, 3H).

[0118] Example 2: Preparation of 6-methyl-4-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (Compound 1-2)

[0119]

[0120] Take the above-prepared compound 1-1 (5 g, 0.026 mol) and cyanoacetamide (2.2 g, 0.026 mol) and dissolve them in DMSO (10 mL). Dissolve potassium tert-butoxide (2.8 g, 0.029 mol) in DMSO (25 mL) and slowly add it dropwise to the DMSO solution containing compound 1-1 and cyanoacetamide at 0 °C. After the addition is complete, transfer the reaction solution to react at 50 °C - 55 °C for 4 hours while bubbling with oxygen. After the reaction is completed, let it cool to room temperature, add water for dilution, slowly add 4N HCl dropwise at 0 °C to adjust the pH of the reaction solution to about 7, filter by suction, and wash with water multiple times. The filter cake is dried to obtain the crude product. Use dichloromethane and methanol (DCM:MeOH = 60:1) as the mobile phase to carry out column chromatography separation and purification of the crude product to obtain 6-methyl-4-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile as a brown oily liquid, denoted as compound 1-2 (5.60 g, 83.90%).

[0121] The NMR data of compound 1-2 are as follows: 1 H NMR (500 MHz, DMSO-d 6 ) δ (ppm): 12.79 (s, 1H), 8.35 (d, J = 8.7 Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 6.37 (s, 1H), 2.32 (s, 3H).

[0122] Example 3: Preparation of 2-chloro-6-methyl-4-(4-nitrophenyl)nicotinonitrile (compound 1-3)

[0123]

[0124] Take the above-prepared intermediate 1-2 (1 g, 3.9 mmol) and dissolve it in POCl 3 (10 mL), and reflux and stir the reaction at 100 °C for 3 hours. After the reaction is completed, let it cool to room temperature, quench the reaction solution with ice cubes, let it stand for 30 min, and then a solid will precipitate. Filter by suction, wash with water, collect the filter cake, and dry it to obtain a brown solid as the crude product. Use petroleum ether and ethyl acetate (PE:EA = 4:1) as the mobile phase to carry out column chromatography separation and purification of the crude product to obtain 2-chloro-6-methyl-4-(4-nitrophenyl)nicotinonitrile as a pale yellow solid, denoted as compound 1-3 (786 mg, 73.82%).

[0125] The NMR data of compound 1-3 are as follows: 1 H NMR (500 MHz, DMSO-d 6 ) δ (ppm): 8.54 (d, J = 8.7 Hz, 2H), 7.99 (d, J = 8.7 Hz, 2H), 7.72 (s, 1H), 2.62 (s, 3H).

[0126] Example 4: Preparation of 2-chloro-6-methyl-4-(4-aminophenyl)nicotinonitrile (Compound 1-4)

[0127]

[0128] Take the above-prepared Intermediate 1-3 (432 mg, 1.58 mmol), iron powder (177 mg, 3.16 mmol), and glacial acetic acid (356 μL) and dissolve them in absolute ethanol (15 mL). React under reflux at 80 °C for 2 hours. After monitoring the reaction by TLC until completion, filter while hot, wash with ethyl acetate, collect the filtrate, and rotary evaporate to obtain the crude product. Perform column chromatography separation and purification of the crude product using petroleum ether and ethyl acetate (PE:EA = 2:1) as the mobile phase to obtain the yellow solid 2-chloro-6-methyl-4-(4-aminophenyl)nicotinonitrile, denoted as Compound 1-4 (330 mg, 85.96%).

[0129] The NMR data of Compound 1-4 are as follows: 1 H NMR (500 MHz, DMSO-d 6 ) δ (ppm): 7.52 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 6.63 (d, J = 8.5 Hz, 2H), 5.86 (s, 2H), 2.54 (s, 3H).

[0130] Example 5: Preparation of 4-(4-aminophenyl)-6-methylisoxazolo[5,4-b]pyridin-3-amine (Compound 1-5)

[0131]

[0132] Take the above-prepared Compound 1-4 (242 mg, 1.0 mmol) and dissolve it in 3 mL of DMF. Dropwise add an aqueous solution (3 mL) containing acetohydroxamic acid (75 mg, 1.0 mmol) and potassium carbonate (276 mg, 2.0 mmol). Stir at 70 °C for 16 h. After the reaction is complete, dilute the system with 50 mL of water and extract with ethyl acetate (100 mL × 3). Combine the organic layers and dry over anhydrous Na 2 SO 4 Dry, and perform column chromatography separation and purification of the crude product using petroleum ether and ethyl acetate (PE:EA = 1:1) as the mobile phase to obtain the yellow solid 4-(4-aminophenyl)-6-methylisoxazolo[5,4-b]pyridin-3-amine, denoted as Compound 1-5 (63 mg, 26.25%).

[0133] The NMR data of Compound 1-5 are as follows: 1 H NMR (500 MHz, DMSO-d 6) δ (ppm): 7.38 (d, J = 8.5 Hz, 2H), 7.10 (s, 1H), 6.74 (d, J = 8.5 Hz, 2H), 5.65 (s, 2H), 5.46 (s, 2H), 2.55 (s, 3H).

[0134] Example 6: Preparation of N-(2-Fluoro-4-nitrophenyl)acrylamide (Compound 1-6-1)

[0135]

[0136] Weigh 2-fluoro-4-nitroaniline (412 mg, 2.64 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), then transfer the reaction mixture to room temperature and react for 2 h. After monitoring the reaction by TLC until it is completed, slowly pour the reaction solution into a large amount of water. A solid precipitates out. After suction filtration, wash it three times with water to obtain the synthesis of light yellow solid N-(2-fluoro-4-nitrophenyl)acrylamide, denoted as Compound 1-6-1 (451 mg, 81.31%).

[0137] The NMR data of Compound 1-6-1 are as follows: 1 H NMR (500 MHz, CDCl 3 ) δ (ppm): 8.28 (d, J = 8.8 Hz, 1H), 7.59 (s, 1H), 6.78 (d, J = 2.7 Hz, 1H), 6.46 (d, J = 2.7 Hz, 1H), 6.53 (d, J = 16.8 Hz, 1H), 6.37 (d, J = 16.8, 10.2 Hz, 1H), 5.61 (d, J = 10.2 Hz, 1H).

[0138] Example 7: Preparation of N-(4-Amino-2-fluorophenyl)acrylamide (Compound 1-7-1)

[0139]

[0140] Weigh the above-prepared Compound 1-6-1 (412 mg, 1.96 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, successively add iron powder (219.52 mg, 3.92 mmol) and 1 mL of saturated ammonium chloride solution, and finally add a magnetic stir bar. Heat the reaction mixture to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC until it is completed, filter off the iron powder in the reaction solution while it is hot, concentrate the solution under reduced pressure to remove the solvent, and purify it by column chromatography to obtain the synthesis of white solid N-(4-amino-2-fluorophenyl)acrylamide, denoted as Compound 1-7-1 (280 mg, 79.37%).

[0141] The NMR data of Compound 1-7-1 are as follows: 1 H NMR(500MHz,CDCl 3 )δ(ppm):8.20(d,J=8.8Hz,1H),7.52(s,1H),6.68(d,J=2.7Hz,1H),6.42(d,J=2.7Hz,1H),6.58(d,J=16.8Hz,1H),6.36(d,J=16.8,10.2Hz,1H),5.58(d,J=10.2Hz,1H),3.69(s,2H).

[0142] Example 8: Preparation of N-(3-Fluoro-4-nitrophenyl)acrylamide (Compound 1-6-2)

[0143]

[0144] Weigh 3-fluoro-4-nitroaniline (412 mg, 2.64 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), then transfer the mixture to room temperature and react for 2 h. After monitoring the reaction by TLC until it is completed, slowly pour the reaction solution into a large amount of water. A solid precipitates out. After filtration by suction, wash it three times with water to obtain the synthesis of light yellow solid N-(3-fluoro-4-nitrophenyl)acrylamide, denoted as Compound 1-6-2 (446 mg, 79.28%).

[0145] The NMR data of Compound 1-6-2 are as follows: 1 H NMR(500MHz,CDCl 3 )δ(ppm):8.24(d,J=8.8Hz,1H),7.55(s,1H),6.68(d,J=2.7Hz,1H),6.40(d,J=2.6Hz,1H),6.52(d,J=16.9Hz,1H),6.34(d,J=16.8,10.2Hz,1H),5.56(d,J=10.1Hz,1H).

[0146] Example 9: Preparation of N-(4-Amino-3-fluorophenyl)acrylamide (Compound 1-7-2)

[0147]

[0148] Weigh the above-prepared compound 1-6-2 (412 mg, 1.96 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, add reduced iron powder (219.52 mg, 3.92 mmol) and 1 mL of saturated ammonium chloride solution in sequence. Finally, add a magnetic stir bar, heat up to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC until it is completed, filter off the iron powder in the reaction solution while it is hot, concentrate under reduced pressure to remove the solvent, and purify by column chromatography to obtain the synthesis of light yellow solid N-(4-amino-3-fluorophenyl)acrylamide, denoted as compound 1-7-2 (276 mg, 78.23%).

[0149] The NMR data of compound 1-7-2 are as follows: 1 H NMR(500MHz,CDCl 3 )δ(ppm):8.22(d,J=8.8Hz,1H),7.53(s,1H),6.64(d,J=2.7Hz,1H),6.40(d,J=2.7Hz,1H),6.52(d,J=16.8Hz,1H),6.34(d,J=16.8,10.2Hz,1H),5.56(d,J=10.2Hz,1H),3.59(s,2H).

[0150] Example 10: Preparation of N-(4-nitro-2-(trifluoromethyl)phenyl)acrylamide (compound 1-6-3)

[0151]

[0152] Weigh 4-nitro-2-trifluoromethylaniline (412 mg, 2.00 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), and transfer to room temperature for reaction for 2 h. After monitoring the reaction by TLC until it is completed, slowly pour the reaction solution into a large amount of water. A solid precipitates out. After suction filtration, wash it three times with water to obtain light yellow solid N-(4-nitro-2-(trifluoromethyl)phenyl)acrylamide, denoted as compound 1-6-3 (483 mg, 92.88%).

[0153] The NMR data of compound 1-6-3 are as follows: 1 H NMR(500MHz,DMSO-d 6)δ(ppm): 10.09 (s, 1H), 7.94 (d, J = 2.5 Hz, 1H), 7.88 (d, J = 8.8, 2.5 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.68 (d, J = 16.9, 10.1 Hz, 1H), 6.27 (d, J = 17.0, 2.1 Hz, 1H), 5.74 (d, J = 10.1, 2.1 Hz, 1H).

[0154] Example 11: Synthesis of N-(4-amino-2-(trifluoromethyl)phenyl)acrylamide (Preparation of Compound 1-7-3)

[0155]

[0156] Weigh the above-prepared Compound 1-6-3 (412 mg, 1.58 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, add iron powder (176.96 mg, 3.16 mmol) and 1 mL of saturated ammonium chloride solution in sequence, and finally add a magnetic stir bar. Heat up to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC until it is completed, filter off the iron powder in the reaction solution while it is hot, concentrate the solvent under reduced pressure, and purify by column chromatography to obtain pale yellow oily N-(4-amino-2-(trifluoromethyl)phenyl)acrylamide, denoted as Compound 1-7-3 (260 mg, 71.55%).

[0157] The NMR data of Compound 1-7-3 are as follows: 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm): 9.98 (s, 1H), 7.81 (d, J = 2.5 Hz, 1H), 7.49 (d, J = 8.8, 2.5 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 6.37 (d, J = 16.9, 10.1 Hz, 1H), 6.21 (d, J = 17.0, 2.1 Hz, 1H), 5.71 (d, J = 10.1, 2.1 Hz, 1H), 5.42 (s, 2H).

[0158] Example 12: Synthesis of N-(4-nitro-3-(trifluoromethyl)phenyl)acrylamide (Preparation of Compound 1-6-4)

[0159]

[0160] Weigh 4-nitro-3-(trifluoromethyl)aniline (412 mg, 2.00 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), then transfer the reaction mixture to room temperature and react for 2 h. After monitoring the reaction by TLC until it is completed, slowly pour the reaction solution into a large amount of water. A solid precipitates out. After filtration by suction, wash it three times with water to obtain a pale yellow solid, N-(4-nitro-3-(trifluoromethyl)phenyl)acrylamide, denoted as Compound 1-6-4 (452 mg, 86.92%).

[0161] The NMR data of Compound 1-6-4 are as follows: 1 H NMR (500 MHz, DMSO-d 6 ) δ (ppm): 10.04 (s, 1H), 7.98 (d, J = 2.5 Hz, 1H), 7.69 (d, J = 8.8, 2.5 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 6.36 (d, J = 16.9, 10.1 Hz, 1H), 6.29 (d, J = 16.8, 2.1 Hz, 1H), 5.88 (d, J = 10.1, 2.1 Hz, 1H).

[0162] Example 13: Preparation of N-(4-amino-3-(trifluoromethyl)phenyl)acrylamide (Compound 1-7-4)

[0163]

[0164] Weigh the above-prepared Compound 1-6-4 (412 mg, 1.58 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, successively add reduced iron powder (176.96 mg, 3.16 mmol) and 1 mL of saturated ammonium chloride solution. Finally, add a magnetic stir bar and heat the reaction mixture to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC until it is completed, filter off the iron powder in the reaction solution while it is hot, concentrate the solution under reduced pressure to remove the solvent, and purify it by column chromatography to obtain a pale yellow oil: N-(4-amino-3-(trifluoromethyl)phenyl)acrylamide, denoted as Compound 1-7-4 (260 mg, 71.55%).

[0165] The NMR data of Compound 1-7-4 are as follows: 1 H NMR (500 MHz, DMSO-d 6)δ(ppm): 9.96 (s, 1H), 7.82 (d, J = 2.5 Hz, 1H), 7.47 (d, J = 8.8, 2.5 Hz, 1H), 6.68 (d, J = 8.8 Hz, 1H), 6.32 (d, J = 16.9, 10.1 Hz, 1H), 6.25 (d, J = 16.8, 2.1 Hz, 1H), 5.72 (d, J = 10.1, 2.1 Hz, 1H), 5.44 (s, 2H).

[0166] Example 14: Synthesis of N-(2-methoxy-4-nitrophenyl)acrylamide (Preparation of Compound 1-6-5)

[0167]

[0168] Weigh 2-methoxy-4-nitroaniline (412 mg, 2.45 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), then transfer to room temperature and react for 2 h. After monitoring the reaction by TLC until it is completed, slowly pour the reaction solution into a large amount of water. A solid precipitates out. After filtration by suction, wash it three times with water to obtain a pale white solid, N-(2-methoxy-4-nitrophenyl)acrylamide, denoted as Compound 1-6-5 (484 mg, 89.00%).

[0169] The NMR data of Compound 1-6-5 are as follows: 1 H NMR (500 MHz, CDCl 3 )δ(ppm): 8.17 (d, J = 8.6 Hz, 1H), 7.69 (s, 1H), 6.71 (d, J = 2.7 Hz, 1H), 6.53 (d, J = 2.7 Hz, 1H), 6.52 (d, J = 16.2 Hz, 1H), 6.46 (d, J = 16.2, 10.2 Hz, 1H), 5.64 (d, J = 10.1 Hz, 1H), 1.72 (s, 3H).

[0170] Example 15: Synthesis of N-(4-amino-2-methoxyphenyl)acrylamide (Preparation of Compound 1-7-5)

[0171]

[0172] Weigh the above-prepared compound 1-6-5 (412 mg, 1.86 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, add reduced iron powder (208.32 mg, 3.72 mmol) and 1 mL of saturated ammonium chloride solution in sequence. Finally, add a magnetic stir bar, heat up to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC until completion, filter off the iron powder in the reaction solution while it is hot, concentrate under reduced pressure to remove the solvent, and separate and purify by column chromatography to obtain the white solid N-(4-amino-2-methoxyphenyl)acrylamide, denoted as compound 1-7-5 (239 mg, 66.92%).

[0173] The NMR data of compound 1-7-5 are as follows: 1 H NMR(500MHz,CDCl 3 )δ(ppm):8.05(d,J=8.6Hz,1H),7.53(s,1H),6.64(d,J=2.7Hz,1H),6.40(d,J=2.7Hz,1H),6.52(d,J=16.2Hz,1H),6.34(d,J=16.2,10.2Hz,1H),5.56(d,J=10.1Hz,1H),3.59(s,2H),1.72(s,3H).

[0174] Example 16: Synthesis of N-(2-chloro-4-nitrophenyl)acrylamide (Preparation of compound 1-6-6)

[0175]

[0176] Weigh 2-chloro-4-nitroaniline (412 mg, 2.40 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), and transfer to room temperature for reaction for 2 h. After monitoring the reaction by TLC until completion, slowly pour the reaction solution into a large amount of water. A solid precipitates. After suction filtration, wash it three times with water to obtain the white solid N-(2-chloro-4-nitrophenyl)acrylamide, denoted as compound 1-6-6 (457 mg, 84.26%).

[0177] The NMR data of compound 1-6-6 are as follows: 1 H NMR(500MHz,CDCl 3 )δ(ppm):8.28(d,J=8.8Hz,1H),7.60(s,1H),6.89(d,J=2.7Hz,1H),6.68(d,J=8.8,2.7Hz,1H),6.46(d,J=16.8Hz,1H),6.36(d,J=16.9,10.2Hz,1H),5.72(d,J=10.2Hz,1H).

[0178] Example 17: Synthesis of N-(4-amino-2-chlorophenyl)acrylamide (Preparation of Compound 1-7-6)

[0179]

[0180] Weigh the above-prepared Compound 1-6-6 (412 mg, 1.82 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, add iron powder (203.84 mg, 3.64 mmol) and 1 mL of saturated ammonium chloride solution in sequence. Finally, add a magnetic stir bar and heat the mixture to 80 °C for reflux reaction for 2.5 h. After monitoring the reaction by TLC until completion, filter off the iron powder in the reaction solution while it is still hot, concentrate the solvent under reduced pressure, and purify by column chromatography to obtain white solid N-(4-amino-2-chlorophenyl)acrylamide, denoted as Compound 1-7-6 (227 mg, 63.64%).

[0181] The NMR data of Compound 1-7-6 are as follows: 1 H NMR (500 MHz, CDCl 3 ) δ (ppm): 8.14 (d, J = 8.8 Hz, 1H), 7.49 (s, 1H), 6.74 (d, J = 2.7 Hz, 1H), 6.62 (d, J = 8.8, 2.7 Hz, 1H), 6.42 (d, J = 16.8 Hz, 1H), 6.29 (d, J = 16.9, 10.2 Hz, 1H), 5.78 (d, J = 10.2 Hz, 1H), 3.69 (s, 2H).

[0182] Example 18: Synthesis of N-(3-chloro-4-nitrophenyl)acrylamide (Preparation of Compound 1-6-7)

[0183]

[0184] Weigh 3-chloro-4-nitroaniline (412 mg, 2.40 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), and transfer the mixture to room temperature for reaction for 2 h. After monitoring the reaction by TLC until completion, slowly pour the reaction solution into a large amount of water. A solid will precipitate. Filter it and wash it three times with water to obtain white solid N-(3-chloro-4-nitrophenyl)acrylamide, denoted as Compound 1-6-7 (463 mg, 85.36%).

[0185] The NMR data of Compound 1-6-7 are as follows: 1 H NMR (500 MHz, CDCl 3)δ(ppm): 8.12 (d, J = 8.7 Hz, 1H), 7.47 (s, 1H), 6.74 (d, J = 2.7 Hz, 1H), 6.68 (d, J = 8.7, 2.7 Hz, 1H), 6.42 (d, J = 16.8 Hz, 1H), 6.27 (d, J = 16.7, 10.2 Hz, 1H), 5.78 (d, J = 10.2 Hz, 1H).

[0186] Example 19: Synthesis of N-(4-Amino-3-chlorophenyl)acrylamide (Preparation of Compound 1-7-7)

[0187]

[0188] Weigh the above-prepared Compound 1-6-7 (412 mg, 1.80 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, add reduced iron powder (201.60 mg, 3.60 mmol) and 1 mL of saturated ammonium chloride solution in sequence. Finally, add a magnetic stir bar, heat up to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC until it is completed, filter off the iron powder in the reaction solution while it is hot, concentrate the solvent under reduced pressure, and purify by column chromatography to obtain light yellow oily N-(4-Amino-3-chlorophenyl)acrylamide, denoted as Compound 1-7-7 (246 mg, 69.73%).

[0189] The NMR data of Compound 1-7-7 are as follows: 1 H NMR (500 MHz, CDCl 3 )δ(ppm): 8.12 (d, J = 8.7 Hz, 1H), 7.47 (s, 1H), 6.74 (d, J = 2.7 Hz, 1H), 6.68 (d, J = 8.7, 2.7 Hz, 1H), 6.42 (d, J = 16.8 Hz, 1H), 6.27 (d, J = 16.7, 10.2 Hz, 1H), 5.78 (d, J = 10.2 Hz, 1H), 3.58 (s, 2H).

[0190] Example 20: Synthesis of N-(2-Methyl-4-nitrophenyl)acrylamide (Preparation of Compound 1-6-8)

[0191]

[0192] Weigh 2-methyl-4-nitroaniline (412 mg, 2.71 mmol) and dissolve it in DMF (3 mL). Add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol) at 0°C and react at room temperature for 2 h. After the reaction is completed by TLC monitoring, the reaction solution is slowly poured into a large amount of water. Solids precipitate, filter and wash three times with water to obtain white solid N-(2-methyl-4-nitrophenyl) acrylamide, recorded as compound 1-6-8 (445 mg, 79.71%).

[0193] The NMR data of compound 1-6-8 are: 1 H NMR (500 MHz, CDCl 3 )δ(ppm):8.28(d,J=8.6Hz,1H),7.70(s,1H),6.69(d,J=2.7Hz,1H),6.60(d,J=2.7Hz,1H), 6.54(d,J=16.2Hz,1H), 6.38(d,J=16.2,10.2Hz,1H), 5.57(d,J=10.1Hz,1H), 2.52(s,3H).

[0194] Example 21: Synthesis of N-(4-amino-2-methylphenyl)acrylamide (Compound 1-7-8)

[0195]

[0196] The compound 1-6-8 prepared above (412 mg, 2 mmol) was weighed and dissolved in a mixed solution of ethanol and water (ethanol: water = 5: 1, 6 mL), and reduced iron powder (224.00 mg, 4.00 mmol) and 1 mL of saturated ammonium chloride solution were added in sequence at room temperature, and finally a magnetic bar was added, and the temperature was raised to 80 ° C and refluxed for 2.5 h. After the reaction was completed by TLC monitoring, the iron powder in the reaction solution was removed by hot filtration, and the solvent was removed by vacuum concentration, and the reaction solution was separated and purified by column chromatography to obtain a white solid N-(4-amino-2-methylphenyl) acrylamide, which was recorded as compound 1-7-8 (248 mg, 70.45%).

[0197] The NMR data of compound 1-7-8 are: 1 H NMR (500 MHz, CDCl 3 )δ(ppm):8.15(d,J=8.6Hz,1H),7.66(s,1H),6.64(d,J=2.7Hz,1H),6.56(d,J=2.7Hz,1H),6.49( d,J=16.2Hz,1H),6.38(d,J=16.2,10.2Hz,1H),5.62(d,J=10.1Hz,1H),3.48(s,2H),2.42(s,3H).

[0198] Example 22: Synthesis of N-(2-cyano-4-nitrophenyl)acrylamide (Compound 1-6-9)

[0199]

[0200] Weigh 2-cyano-4-nitroaniline (412 mg, 2.53 mmol) and dissolve it in DMF (3 mL). Add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol) at 0°C and react at room temperature for 2 h. After the reaction is completed by TLC monitoring, the reaction solution is slowly poured into a large amount of water. Solids precipitate, filter and wash three times with water to obtain white solid N-(2-cyano-4-nitrophenyl) acrylamide, recorded as compound 1-6-9 (429 mg, 78.65%).

[0201] The NMR data of compound 1-6-9 are: 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm):9.88(s,1H),7.22–7.09(m,1H),6.92–6.75(m,2H),6.57(d,J=17 .1,10.2Hz,1H),6.38(d,J=17.1,2.0Hz,1H),5.86(d,J=10.2,2.0Hz,1H).

[0202] Example 23: Synthesis of N-(4-amino-2-cyanophenyl)acrylamide (Compound 1-7-9)

[0203]

[0204] The compound 1-6-9 prepared above (412 mg, 1.90 mmol) was weighed and dissolved in a mixed solution of ethanol and water (ethanol: water = 5: 1, 6 mL), and reduced iron powder (212.80 mg, 3.80 mmol) and 1 mL of saturated ammonium chloride solution were added in sequence at room temperature, and finally a magnetic bar was added, and the temperature was raised to 80 ° C and refluxed for 2.5 h. After the reaction was completed by TLC monitoring, the iron powder in the reaction solution was removed by hot filtration, and the solvent was removed by vacuum concentration, and the reaction solution was separated and purified by column chromatography to obtain a white solid N-(4-amino-2-cyanophenyl) acrylamide, which was recorded as compound 1-7-9 (236 mg, 66.42%).

[0205] The NMR data of compound 1-7-9 are: 1 H NMR (500 MHz, DMSO-d 6) δ (ppm): 9.88 (s, 1H), 7.22–7.09 (m, 1H), 6.92–6.75 (m, 2H), 6.43 (d, J = 17.1, 10.2 Hz, 1H), 6.24 (d, J = 17.1, 2.0 Hz, 1H), 5.76 (d, J = 10.2, 2.0 Hz, 1H), 5.53 (s, 2H).

[0206] Example 24: Synthesis of N-(4-nitrophenyl)acrylamide (Preparation of Compound 1-6-10)

[0207]

[0208] Weigh 4-nitroaniline (412 mg, 3.00 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), then transfer the reaction mixture to room temperature and react for 2 h. After monitoring the reaction by TLC until it is completed, slowly pour the reaction solution into a large amount of water. A solid precipitates out. After filtration, wash it three times with water to obtain the white solid of the synthesis of N-(4-nitrophenyl)acrylamide, denoted as Compound 1-6-10 (454 mg, 78.82%).

[0209] The NMR data of Compound 1-6-10 are as follows: 1 H NMR (500 MHz, DMSO-d 6 ) δ (ppm): 9.88 (s, 1H), 7.46–7.38 (m, 2H), 6.68–6.56 (m, 2H), 6.47 (d, J = 17.0, 10.1 Hz, 1H), 6.18 (d, J = 17.0, 2.2 Hz, 1H), 5.75 (d, J = 10.1, 2.2 Hz, 1H).

[0210] Example 25: Synthesis of N-(4-aminophenyl)acrylamide (Preparation of Compound 1-7-10)

[0211]

[0212] Weigh the above-prepared Compound 1-6-10 (412 mg, 2.15 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, successively add iron powder (240.80 mg, 4.30 mmol) and 1 mL of saturated ammonium chloride solution, and finally add a magnetic stir bar. Heat the reaction mixture to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC until it is completed, filter off the iron powder in the reaction solution while it is hot, concentrate the solution under reduced pressure to remove the solvent, and purify it by column chromatography to obtain the white solid of N-(4-aminophenyl)acrylamide, denoted as Compound 1-7-10 (265 mg, 76.08%).

[0213] The NMR data of Compound 1-7-10 are as follows: 1 H NMR(500MHz,DMSO-d 6 )δ(ppm):9.73(s,1H),7.37–7.26(m,2H),6.60–6.46(m,2H),6.37(d,J=17.0,10.1Hz,1H),6.17(d,J=17.0,2.2Hz,1H),5.65(d,J=10.1,2.2Hz,1H),4.92(s,2H).

[0214] Example 26: Preparation of N-(2-Fluoro-5-nitrophenyl)acrylamide (Compound 1-6-11)

[0215]

[0216] Weigh 2-fluoro-5-nitroaniline (412 mg, 2.64 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), then transfer the mixture to room temperature and react for 2 h. After monitoring the reaction by TLC until it is completed, slowly pour the reaction solution into a large amount of water. A solid precipitates out. After suction filtration, wash it three times with water to obtain white solid N-(2-fluoro-5-nitrophenyl)acrylamide, denoted as Compound 1-6-11 (437 mg, 78.82%).

[0217] The NMR data of Compound 1-6-11 are as follows: 1 H NMR(500MHz,CDCl3)δ(m):9.79(s,1H),7.48(s,1H),7.29(t,J=8.0Hz,1H),6.83(d,J=7.6Hz,1H),6.46(d,J=16.9Hz,1H),6.39(d,J=16.8,10.2Hz,1H),5.66(d,J=10.2Hz,1H).

[0218] Example 27: Preparation of N-(5-Amino-2-fluorophenyl)acrylamide (Compound 1-7-11)

[0219]

[0220] Weigh the above-prepared compound 1-6-11 (412 mg, 2 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, add iron powder (224.00 mg, 4.00 mmol) and 1 mL of saturated ammonium chloride solution in sequence. Finally, add a magnetic stir bar and heat up to 80 °C for reflux reaction for 2.5 h. After monitoring the reaction by TLC until it is completed, filter off the iron powder in the reaction solution while it is hot, concentrate the solution under reduced pressure to remove the solvent, and purify it by column chromatography to obtain the white solid N-(5-amino-2-fluorophenyl)acrylamide, denoted as compound 1-7-11 (237 mg, 65.83%).

[0221] The NMR data of compound 1-7-11 are as follows: 1 H NMR(500MHz,CDCl3)δ(ppm):7.48(s,1H),7.27(s,1H),7.10(t,J=8.0Hz,1H),6.76(d,J=7.6Hz,1H),6.46(d,J=7.6Hz,1H),6.43(d,J=16.9Hz,1H),6.38(d,J=16.8,10.2Hz,1H),5.67(d,J=10.2Hz,2H)

[0222] Example 28: Synthesis of N-(2-chloro-5-nitrophenyl)acrylamide (preparation of compound 1-6-12)

[0223]

[0224] Weigh 2-chloro-5-nitroaniline (412 mg, 2.40 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and acryloyl chloride (299 μL, 3.3 mmol), and transfer to room temperature for reaction for 2 h. After monitoring the reaction by TLC until it is completed, slowly pour the reaction solution into a large amount of water. A solid precipitates. After suction filtration, wash it three times with water to obtain the white solid N-(2-chloro-5-nitrophenyl)acrylamide, denoted as compound 1-6-12 (426 mg, 78.35%).

[0225] The NMR data of compound 1-6-12 are as follows: 1 H NMR(500MHz,CDCl 3 )δ(ppm):9.52(s,1H),7.27(s,1H),7.14(t,J=8.0Hz,1H),6.76(d,J=7.6Hz,1H),6.45(d,J=16.9Hz,1H),6.38(d,J=16.8,10.2Hz,1H),5.64(d,J=10.2Hz,1H).

[0226] Example 29: Synthesis of N-(5-amino-2-chlorophenyl)acrylamide (Preparation of Compound 1-7-12)

[0227]

[0228] Weigh 1-6-12 (412 mg, 1.82 mmol) prepared above and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, add iron powder (203.84 mg, 3.64 mmol) and 1 mL of saturated ammonium chloride solution in sequence. Finally, add a magnetic stir bar, heat up to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC until completion, filter off the iron powder in the reaction solution while it is hot, concentrate the solution under reduced pressure to remove the solvent, and purify by column chromatography to obtain white solid N-(5-amino-2-chlorophenyl)acrylamide, denoted as Compound 1-7-12 (252 mg, 70.64%).

[0229] The NMR data of Compound 1-7-12 are as follows: 1 H NMR(500MHz,CDCl 3 )δ(ppm):7.27(s,1H),7.10(t,J=8.0Hz,1H),6.76(d,J=7.6Hz,1H),6.46(d,J=7.6Hz,1H),6.43(d,J=16.9Hz,1H),6.38(d,J=16.8,10.2Hz,1H),5.67(d,J=10.2Hz,1H),5.54(s,2H).

[0230] Example 30: Synthesis of (E)-N-(4-nitrophenyl)but-2-enamide (Preparation of Compound 1-6-13)

[0231]

[0232] Weigh 4-nitroaniline (412 mg, 3.00 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and crotonyl chloride (299 μL, 2.90 mmol), and transfer to room temperature for reaction for 2 h. After monitoring the reaction by TLC until completion, slowly pour the reaction solution into a large amount of water. A solid precipitates out. After filtration, wash it three times with water to obtain white solid (E)-N-(4-nitrophenyl)but-2-enamide, denoted as Compound 1-6-13 (463 mg, 74.92%).

[0233] The NMR data of Compound 1-6-13 are as follows: 1 H NMR(500MHz,DMSO-d 6) δ (ppm): 9.86 (s, 1H), 7.37–7.26 (m, 2H), 6.66 (m, 1H), 6.37 (d, J = 17.0, 10.2 Hz, 1H), 6.28 (d, J = 17.0, 2.2 Hz, 1H), 5.86 (d, J = 10.1, 2.2 Hz, 1H), 2.39 (s, 3H).

[0234] Example 31: Synthesis of (E)-N-(4-aminophenyl)but-2-enamide (Preparation of Compound 1-7-13)

[0235]

[0236] Weigh the above-prepared Compound 1-6-13 (412 mg, 2.00 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, add iron powder (224.00 mg, 4.00 mmol) and 1 mL of saturated ammonium chloride solution in sequence. Finally, add a magnetic stir bar, heat up to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC and it is completed, filter off the iron powder in the reaction solution while it is hot, concentrate the solution under reduced pressure to remove the solvent, and purify by column chromatography to obtain the white solid (E)-N-(4-aminophenyl)but-2-enamide, denoted as Compound 1-7-13 (239 mg, 67.90%).

[0237] The NMR data of Compound 1-7-13 are as follows: 1 H NMR (500 MHz, DMSO-d 6 ) δ (ppm): 9.71 (s, 1H), 7.37–7.26 (m, 2H), 6.66 (m, 1H), 6.37 (d, J = 17.0, 10.2 Hz, 1H), 6.18 (d, J = 17.0, 2.2 Hz, 1H), 5.68 (d, J = 10.1, 2.2 Hz, 1H), 4.85 (s, 2H), 2.24 (s, 3H).

[0238] Example 32: Synthesis of (E)-N-(2-fluoro-4-nitrophenyl)but-2-enamide (Preparation of Compound 1-6-14)

[0239]

[0240] Weigh 2-fluoro-4-nitroaniline (412 mg, 2.64 mmol) and dissolve it in DMF (3 mL). At 0 °C, add triethylamine (10 μL) and crotonyl chloride (299 μL, 2.90 mmol), then transfer the reaction mixture to room temperature and react for 2 h. After monitoring the reaction by TLC until it is completed, slowly pour the reaction solution into a large amount of water. A solid precipitates out. After suction filtration, wash it three times with water to obtain the white solid (E)-N-(2-fluoro-4-nitrophenyl)but-2-enamide, denoted as compound 1-6-14 (449 mg, 75.93%).

[0241] The NMR data of compound 1-6-14 are as follows: 1 H NMR (500 MHz, CDCl 3 ) δ (ppm): 8.25 (d, J = 8.8 Hz, 1H), 7.57 (s, 1H), 6.66 (d, J = 2.7 Hz, 1H), 6.40 (d, J = 2.7 Hz, 1H), 6.52 (d, J = 16.8 Hz, 1H), 6.39 (d, J = 16.8, 10.2 Hz, 1H), 2.01 (s, 3H).

[0242] Example 33: Synthesis of (E)-N-(4-amino-2-fluorophenyl)but-2-enamide (preparation of compound 1-7-14)

[0243]

[0244] Weigh the above-prepared compound 1-6-14 (412 mg, 1.84 mmol) and dissolve it in a mixed solution of ethanol and water (ethanol: water = 5:1, 6 mL). At room temperature, successively add iron powder (206.08 mg, 3.68 mmol) and 1 mL of saturated ammonium chloride solution. Finally, add a magnetic stir bar, heat up to 80 °C and reflux for 2.5 h. After monitoring the reaction by TLC until it is completed, filter off the iron powder in the reaction solution while it is hot, concentrate the solution under reduced pressure to remove the solvent, and purify it by column chromatography to obtain the white solid (E)-N-(4-amino-2-fluorophenyl)but-2-enamide, denoted as compound 1-7-14 (241 mg, 67.51%).

[0245] The NMR data of compound 1-7-14 are as follows: 1 H NMR (500 MHz, CDCl 3 ) δ (ppm): 8.22 (d, J = 8.8 Hz, 1H), 7.54 (s, 1H), 6.64 (d, J = 2.7 Hz, 1H), 6.40 (d, J = 2.7 Hz, 1H), 6.52 (d, J = 16.8 Hz, 1H), 6.34 (d, J = 16.8, 10.2 Hz, 1H), 4.08 (s, 2H), 1.91 (s, 3H).

[0246] Example 34: Preparation of Isocyanate Compounds (Compounds 1-8)

[0247]

[0248] Take a 25 mL round-bottom flask, dissolve triphosgene (0.3 mmol, 89 mg) in 5 mL of ethyl acetate, stir and cool in an ice bath to 0 °C. Take the substituted aniline compound (0.6 mmol) prepared above, dissolve it in 5 mL of ethyl acetate and slowly add it dropwise to the reaction solution. Under N 2 protection, after stirring in an ice bath for 0.5 h, remove the ice bath and react at room temperature for 1 h, then reflux and react at 82 °C for 4 h. After the reaction is completed, remove the solvent under reduced pressure to obtain the product isocyanate compounds 1-8 (this synthesis step only has different R 1 substituents, and multiple compounds conforming to the structural formulas of 1-8 can be synthesized according to this method), which can be directly used for the next reaction without further treatment.

[0249] Specifically, the structural formulas of Compounds 1-7 and the corresponding Compounds 1-8 are shown in Table 1.

[0250] Table 1

[0251]

[0252]

[0253] Example 35: Preparation of N-(4-(3-(4-(3-Amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-fluorophenyl)acrylamide (Compound W-1)

[0254]

[0255] Take the above-prepared Compounds 1-5 (30 mg, 0.125 mmol), dissolve them in anhydrous DCM (5 mL), add them to the above-prepared N-(2-Fluoro-4-isocyanatophenyl)acrylamide (Compound 1-8-1), and finally add 10 μL of triethylamine. Stir at 0 °C for 20 min for an addition reaction. After the reaction is completed, purify the crude product by column chromatography using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(4-(3-(4-(3-Amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-fluorophenyl)acrylamide, denoted as Compound W-1 (calculated based on Compound 1-5, the yield is 56%).

[0256] Compound W-1: mp: 264.3-266.1 °C; 11H NMR (500 MHz, DMSO-d 6 ) δ (ppm): 9.83 (s, 1H), 9.04 (s, 1H), 8.98 (s, 1H), 7.80 (t, J = 8.9 Hz, 1H), 7.70–7.65 (m, 2H), 7.60 (d, J = 13.3, 2.4 Hz, 1H), 7.58–7.55 (m, 2H), 7.19 (s, 1H), 7.14–7.09 (m, 1H), 6.57 (d, J = 17.0, 10.2 Hz, 1H), 6.26 (d, J = 17.0, 2.0 Hz, 1H), 5.76 (d, J = 10.1, 2.0 Hz, 1H), 5.46 (s, 2H), 2.60 (s, 3H). 13 13C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 163.83, 160.68, 158.43, 155.36, 153.43, 152.78, 147.49, 137.77, 131.87, 129.81, 129.02, 127.44, 125.42, 120.24, 119.45, 118.92, 114.27, 106.05, 105.86, 102.53, 24.52.

[0257] Example 36: Preparation of N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-3-fluorophenyl)acrylamide (Compound W-2)

[0258]

[0259] The above-prepared Compound 1-5 (30 mg, 0.125 mmol) was dissolved in anhydrous DCM (5 mL), added to the above-prepared N-(3-fluoro-4-isocyanatophenyl)acrylamide (Compound 1-8-2), and finally 10 μL of triethylamine was added. The addition reaction was carried out by stirring at 0 °C for 20 min. After the reaction was completed, the crude product was separated and purified by column chromatography using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-3-fluorophenyl)acrylamide, denoted as Compound W-2 (yield: 61% based on Compound 1-5).

[0260] Compound W-2: mp: 266.1~267.8 °C; 1 1H NMR (500 MHz, DMSO-d 6) δ (ppm): 10.29 (s, 1H), 9.30 (s, 1H), 8.58 (d, J = 2.4 Hz, 1H), 8.05 (t, J = 9.0 Hz, 1H), 7.80 (d, J = 13.6, 2.3 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.33–7.24 (m, 1H), 7.19 (s, 1H), 6.42 (d, J = 16.9, 10.1 Hz, 1H), 6.27 (d, J = 16.9, 2.1 Hz, 1H), 5.78 (d, J = 10.0, 2.0 Hz, 1H), 5.48 (s, 2H), 2.60 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.90, 163.58, 160.68, 158.43, 152.64, 147.48, 141.38, 134.75, 132.09, 129.87, 128.99, 127.56, 123.04, 121.82, 121.80, 119.45, 118.68, 115.59, 115.56, 107.09, 106.89, 102.53, 24.52.

[0261] Example 37: Preparation of N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-(trifluoromethyl)phenyl)acrylamide (Compound W-3)

[0262]

[0263] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL). Add it to the above-prepared N-(4-isocyanato-2-(trifluoromethyl)phenyl)acrylamide (Compound 1-8-3). Finally, add 10 μL of triethylamine and stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, column chromatography separation and purification of the crude product are carried out using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-(trifluoromethyl)phenyl)acrylamide, denoted as Compound W-3 (yield: 59.7% based on Compound 1-5).

[0264] Compound W-3: mp: 275.0–276.8 °C; 1 H NMR (500 MHz, DMSO-d 6) δ (ppm): 9.69 (s, 1H), 9.18 (s, 1H), 9.12 (s, 1H), 8.05 (d, J = 2.5 Hz, 1H), 7.72–7.67 (m, 2H), 7.63 (d, J = 8.7, 2.5 Hz, 1H), 7.60–7.55 (m, 2H), 7.42 (d, J = 8.6 Hz, 1H), 7.20 (s, 1H), 6.51 (d, J = 17.1, 10.2 Hz, 1H), 6.24 (d, J = 17.1, 2.0 Hz, 1H), 5.77 (d, J = 10.3, 1.9 Hz, 1H), 5.49 (s, 2H), 2.60 (s, 3H). 13 13C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.90, 164.89, 160.70, 158.44, 152.90, 147.46, 141.25, 138.78, 131.63, 131.61, 129.82, 129.17, 129.08, 127.51, 124.97, 122.80, 122.70, 119.47, 119.09, 115.88, 102.54, 24.53. 13C NMR of compound W-3 1 The 1H NMR spectrum of compound W-3 is as shown in Figure 2 Figure [Figure number not provided], and the 13C NMR spectrum of compound W-3 is as shown in 13 Figure [Figure number not provided]. Figure 3 as shown below.

[0265] Example 38: Preparation of N-(4-(3-(4-(3-Amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-3-(trifluoromethyl)phenyl)acrylamide (Compound W-4)

[0266]

[0267] The above-prepared compound 1-5 (30 mg, 0.125 mmol) was dissolved in anhydrous DCM (5 mL), added to the above-prepared N-(4-Isocyanato-3-(trifluoromethyl)phenyl)acrylamide (Compound 1-8-4), and finally 10 μL of triethylamine was added. The addition reaction was carried out by stirring at 0 °C for 20 min. After the reaction was completed, the crude product was separated and purified by column chromatography using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(4-(3-(4-(3-Amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-3-(trifluoromethyl)phenyl)acrylamide, denoted as Compound W-4.

[0268] Compound W-4: mp: 272.3–274.2 °C;1 1H NMR (500 MHz, DMSO-d 6 ) δ (ppm): 10.48 (s, 1H), 9.65 (s, 1H), 8.25 (s, 1H), 8.17 (d, J = 2.2 Hz, 1H), 7.84–7.80 (m, 2H), 7.69–7.65 (m, 2H), 7.58–7.54 (m, 2H), 7.19 (s, 1H), 6.44 (d, J = 17.0, 10.1 Hz, 1H), 6.30 (d, J = 17.0, 1.9 Hz, 1H), 5.81 (d, J = 10.1, 2.0 Hz, 1H), 5.48 (s, 2H), 2.59 (s, 3H). 13 13C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.89, 163.89, 160.71, 158.42, 153.25, 147.50, 141.50, 135.92, 131.93, 131.59, 129.85, 128.97, 128.16, 127.99, 125.25, 123.77, 123.08, 121.98, 121.75, 119.44, 118.73, 116.77, 102.52, 24.51.

[0269] Example 39: Preparation of N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-methoxyphenyl)acrylamide (Compound W-5)

[0270]

[0271] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL). Add it to the above-prepared N-(4-isocyanato-2-methoxyphenyl)acrylamide (Compound 1-8-5). Finally, add 10 μL of triethylamine and stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, column chromatographically separate and purify the crude product using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-methoxyphenyl)acrylamide, denoted as Compound W-5.

[0272] Compound W-5: mp: 281.0–282.8 °C; 1 1H NMR (500 MHz, DMSO-d 6) δ (ppm): 9.31 (s, 1H), 8.98 (s, 1H), 8.82 (s, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.70–7.63 (m, 2H), 7.59–7.52 (m, 2H), 7.42 (d, J = 2.3 Hz, 1H), 7.20 (s, 1H), 6.89 (d, J = 8.7, 2.3 Hz, 1H), 6.66 (d, J = 17.0, 10.2 Hz, 1H), 6.21 (d, J = 17.0, 2.1 Hz, 1H), 5.69 (d, J = 10.2, 2.1 Hz, 1H), 5.48 (s, 2H), 3.84 (s, 3H), 2.60 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.90, 163.57, 160.69, 158.44, 152.89, 150.80, 147.53, 141.56, 137.15, 132.58, 129.80, 128.82, 126.65, 123.45, 121.84, 119.44, 118.83, 110.20, 102.51, 102.43, 56.01, 24.53.

[0273] Example 40: Preparation of N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-chlorophenyl)acrylamide (Compound W-6)

[0274]

[0275] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL), add it to the above-prepared N-(2-chloro-4-isocyanatophenyl)acrylamide (Compound 1-8-6), and finally add 10 μL of triethylamine. Stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, column chromatography separation and purification of the crude product are carried out using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-chlorophenyl)acrylamide, denoted as Compound W-6.

[0276] Compound W-6: mp: 230.4~232.0 °C; 1 H NMR (500 MHz, DMSO-d 6) δ (ppm): 9.67 (s, 1H), 9.07 (s, 1H), 9.00 (s, 1H), 7.83 (d, J = 2.4 Hz, 1H), 7.71–7.66 (m, 2H), 7.63 (d, J = 8.7 Hz, 1H), 7.60–7.54 (m, 2H), 7.30 (d, J = 8.7, 2.5 Hz, 1H), 7.20 (s, 1H), 6.58 (d, J = 17.0, 10.2 Hz, 1H), 6.26 (d, J = 17.1, 2.0 Hz, 1H), 5.77 (d, J = 10.2, 1.9 Hz, 1H), 5.48 (s, 2H), 2.60 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.91, 164.00, 160.68, 158.43, 152.80, 147.48, 141.34, 138.31, 131.89, 129.81, 129.14, 129.07, 127.80, 127.55, 119.46, 118.98, 117.73, 102.54, 24.53.

[0277] Example 41: Preparation of N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-3-chlorophenyl)acrylamide (Compound W-7)

[0278]

[0279] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL), add it to the above-prepared N-(3-chloro-4-isocyanatophenyl)acrylamide (Compound 1-8-7), and finally add 10 μL of triethylamine. Stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, use dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to carry out column chromatography separation and purification of the crude product to obtain white solid N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-3-chlorophenyl)acrylamide, denoted as Compound W-7.

[0280] Compound W-7: mp: 232.0~234.7 °C; 1 H NMR (500 MHz, DMSO-d 6) δ (ppm): 10.26 (s, 1H), 9.60 (s, 1H), 8.36 (s, 1H), 8.07 (d, J = 9.0 Hz, 1H), 8.00 (d, J = 2.4 Hz, 1H), 7.71–7.65 (m, 2H), 7.61–7.54 (m, 2H), 7.46 (d, J = 8.9, 2.4 Hz, 1H), 7.20 (s, 1H), 6.41 (d, J = 17.0, 10.1 Hz, 1H), 6.27 (d, J = 17.0, 2.0 Hz, 1H), 5.78 (d, J = 10.0, 2.0 Hz, 1H), 5.47 (s, 2H), 2.60 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.91, 163.61, 160.68, 158.42, 152.64, 147.47, 141.38, 135.19, 132.05, 131.75, 129.88, 129.06, 127.61, 123.00, 122.70, 120.17, 119.45, 118.99, 118.76, 102.54, 24.52.

[0281] Example 42: Preparation of N-(4-(3-(4-(3-Amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-methylphenyl)acrylamide (Compound W-8)

[0282]

[0283] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL), add it to the above-prepared N-(4-isocyanato-2-methylphenyl)acrylamide (Compound 1-8-8), and finally add 10 μL of triethylamine. Stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, column chromatographically separate and purify the crude product using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-methylphenyl)acrylamide, denoted as Compound W-8.

[0284] Compound W-8: mp: 231.2~233.5 °C; 11 H NMR (500 MHz, DMSO-d 6) δ (ppm): 9.43 (s, 1H), 8.98 (s, 1H), 8.74 (s, 1H), 7.69–7.64 (m, 2H), 7.59–7.53 (m, 2H), 7.39–7.34 (m, 2H), 7.27 (d, J = 8.6, 2.5 Hz, 1H), 7.20 (s, 1H), 6.51 (d, J = 17.1, 10.2 Hz, 1H), 6.23 (d, J = 17.0, 2.1 Hz, 1H), 5.73 (d, J = 10.1, 2.2 Hz, 1H), 5.48 (s, 2H), 2.60 (s, 3H), 2.19 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.91, 163.75, 160.67, 158.44, 152.88, 147.53, 141.64, 137.26, 132.99, 132.29, 130.88, 129.80, 128.76, 126.73, 126.17, 120.48, 119.44, 118.75, 116.55, 102.51, 24.53, 18.62.

[0285] Example 43: Preparation of N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-cyanophenyl)acrylamide (Compound W-9)

[0286]

[0287] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL), add it to the above-prepared N-(4-isocyanato-2-cyanophenyl)acrylamide (Compound 1-8-9), and finally add 10 μL of triethylamine. Stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, column chromatographic separation and purification of the crude product are carried out using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-cyanophenyl)acrylamide, denoted as Compound W-9.

[0288] Compound W-9: mp: 257.6~258.4 °C; 1 H NMR (500 MHz, DMSO-d 6)δ(ppm): 10.27 (s, 1H), 9.19 (d, J = 16.1 Hz, 2H), 8.00 (d, J = 2.5 Hz, 1H), 7.69 (d, J = 8.9, 2.5 Hz, 3H), 7.56 (d, J = 15.4, 8.6 Hz, 3H), 7.20 (s, 1H), 6.50 (d, J = 17.0, 10.2 Hz, 1H), 6.30 (d, J = 17.1, 1.9 Hz, 1H), 5.83 (d, J = 10.1, 1.9 Hz, 1H), 5.48 (s, 2H), 2.60 (s, 3H). 13 C NMR(126 MHz, DMSO-d 6 )δ(ppm): 169.90, 164.16, 160.69, 158.43, 152.87, 147.46, 141.25, 137.91, 134.34, 131.41, 129.82, 129.17, 128.27, 127.29, 124.17, 122.03, 119.47, 119.05, 117.28, 108.49, 102.54, 24.52.

[0289] Example 44: Preparation of N-(4-(3-(4-(3-Amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)phenyl)acrylamide (Compound W-10)

[0290]

[0291] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL), add it to the above-prepared N-(4-isocyanatophenyl)acrylamide (Compound 1-8-10), and finally add 10 μL of triethylamine. Stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, purify the crude product by column chromatography using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)phenyl)acrylamide, denoted as Compound W-10.

[0292] Compound W-10: mp: 243.0 - 245.1 °C; 1 H NMR(500 MHz, DMSO-d 6) δ (ppm): 10.10 (s, 1H), 9.18 (s, 1H), 8.96 (s, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.62–7.58 (m, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.46–7.42 (m, 2H), 7.19 (s, 1H), 6.43 (d, J = 17.0, 10.1 Hz, 1H), 6.24 (d, J = 17.0, 2.1 Hz, 1H), 5.73 (d, J = 10.1, 2.1 Hz, 1H), 5.48 (s, 2H), 2.59 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.89, 163.29, 160.70, 158.43, 152.98, 147.58, 141.77, 135.75, 133.96, 132.40, 129.78, 128.65, 126.92, 120.45, 119.44, 119.22, 118.72, 102.50, 24.51.

[0293] Example 45: Preparation of N-(5-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-fluorophenyl)acrylamide (Compound W-11)

[0294]

[0295] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL), add it to the above-prepared N-(2-fluoro-5-isocyanatophenyl)acrylamide (Compound 1-8-11), and finally add 10 μL of triethylamine. Stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, purify the crude product by column chromatography using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(5-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-fluorophenyl)acrylamide, denoted as Compound W-11.

[0296] Compound W-11: mp: 240.3~241.9 °C; 1 H NMR (500 MHz, DMSO-d 6) δ (ppm): 9.92 (s, 1H), 8.91 (d, J = 3.8 Hz, 2H), 8.15 (d, J = 7.1, 2.7 Hz, 1H), 7.69–7.65 (m, 2H), 7.58–7.54 (m, 2H), 7.33 (ddd, J = 8.9, 4.2, 2.7 Hz, 1H), 7.23–7.18 (m, 2H), 6.64 (d, J = 17.0, 10.3 Hz, 1H), 6.29 (d, J = 17.0, 1.9 Hz, 1H), 5.79 (d, J = 10.1, 2.0 Hz, 1H), 5.46 (s, 2H), 2.60 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.91, 163.95, 160.67, 158.43, 152.89, 147.51, 141.54, 136.10, 131.88, 129.79, 129.13, 128.86, 127.87, 126.51, 126.41, 119.44, 118.82, 115.91, 115.75, 115.43, 114.36, 102.53, 24.52.

[0297] Example 46: Preparation of N-(5-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-chlorophenyl)acrylamide (Compound W-12)

[0298]

[0299] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL), add it to the above-prepared N-(2-chloro-5-isocyanatophenyl)acrylamide (Compound 1-8-12), and finally add 10 μL of triethylamine. Stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, column chromatographic separation and purification of the crude product are carried out using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain white solid N-(5-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-chlorophenyl)acrylamide, denoted as Compound W-12.

[0300] Compound W-12: mp: 275.0~276.8 °C; 1 H NMR (500 MHz, DMSO-d 6) δ (ppm): 9.69 (s, 1H), 9.02 (s, 1H), 8.98 (s, 1H), 7.99 (d, J = 2.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 7.44–7.35 (m, 2H), 7.20 (s, 1H), 6.64 (d, J = 17.0, 10.2 Hz, 1H), 6.30 (d, J = 17.1, 2.0 Hz, 1H), 5.80 (d, J = 10.2, 1.9 Hz, 1H), 5.48 (s, 2H), 2.60 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.90, 163.95, 160.69, 158.43, 152.73, 147.48, 141.37, 139.19, 135.29, 131.86, 129.95, 129.81, 129.01, 127.96, 119.46, 118.90, 116.68, 115.98, 102.52, 24.52.

[0301] Example 47: Preparation of (E)-N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)phenyl)but-2-enamide (Compound W-13)

[0302]

[0303] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL). Add it to the above-prepared (E)-N-(4-isocyanatophenyl)but-2-enamide (Compound 1-8-13). Finally, add 10 μL of triethylamine and stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, purify the crude product by column chromatography using dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain the white solid (E)-N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)phenyl)but-2-enamide, denoted as Compound W-13.

[0304] Compound W-13: mp: 231.6~233.7 °C; 1 H NMR (500 MHz, DMSO-d 6)δ(ppm): 9.87 (s, 1H), 8.94 (s, 1H), 8.71 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.57 (t, J = 9.2 Hz, 4H), 7.41 (d, J = 8.6 Hz, 2H), 7.20 (s, 1H), 6.77 (d, J = 15.0, 7.1 Hz, 1H), 6.17–6.06 (m, 1H), 5.48 (s, 2H), 2.60 (s, 3H), 1.87 (d, J = 6.9 Hz, 3H). 13 C NMR (126 MHz, DMSO-d 6 )δ(ppm): 169.90, 163.60, 160.68, 158.45, 152.89, 147.55, 141.69, 139.82, 135.34, 134.36, 129.82, 128.70, 126.54, 120.31, 119.45, 119.24, 118.70, 102.50, 24.52, 17.99.

[0305] Example 48: Preparation of (E)-N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-fluorophenyl)but-2-enamide (Compound W-14)

[0306]

[0307] Take the above-prepared Compound 1-5 (30 mg, 0.125 mmol) and dissolve it in anhydrous DCM (5 mL), add it to the above-prepared (E)-N-(2-fluoro-4-isocyanatophenyl)but-2-enamide (Compound 1-8-14), and finally add 10 μL of triethylamine. Stir at 0 °C for 20 min for the addition reaction. After the reaction is completed, column chromatograph and purify the crude product with dichloromethane and methanol (DCM:MeOH = 80:1) as the mobile phase to obtain the white solid (E)-N-(4-(3-(4-(3-amino-6-methylisoxazolo[5,4-b]pyridin-4-yl)phenyl)ureido)-2-fluorophenyl)but-2-enamide, denoted as Compound W-14.

[0308] Compound W-14: mp: 235.1~236.8 °C; 1 H NMR (500 MHz, DMSO-d 6) δ (ppm): 9.63 (d, J = 6.4 Hz, 1H), 9.07 (s, 1H), 9.01 (s, 1H), 7.78 (t, J = 8.8 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.63–7.52 (m, 3H), 7.20 (s, 1H), 7.13–7.06 (m, 1H), 6.84–6.73 (m, 1H), 6.26 (d, J = 15.0, 2.4 Hz, 1H), 5.50 (s, 2H), 2.60 (s, 3H), 1.86 (d, J = 6.8, 1.7 Hz, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ (ppm): 169.90, 164.11, 160.68, 158.45, 155.35, 153.41, 152.78, 147.49, 141.38, 140.46, 137.53, 129.83, 128.98, 125.97, 125.47, 119.47, 118.89, 114.21, 114.19, 106.02, 105.82, 102.52, 24.52, 18.03.

[0309] Example 49: Pharmacodynamic Experiment of FLT3 Covalent Inhibitor (I) on FLT3 Kinase

[0310] (1) Experimental Method:

[0311] The ADP-Glo TM kinase assay (Promega, Madison, WI) was used to test the FLT3 inhibitory activity. The optimal concentrations were selected as follows: FLT3 10 ng / μL, substrate 0.1 mg / mL, and the amount of ATP used was 50 μM (for FLT3). The specific operation was as follows: According to the operation procedure of the kit instruction manual, prepare 5×DMSO test compound (compounds W-1 to W-14 prepared in the above examples) drug solution, 2.5×ATP / substrate mixed solution, 2.5×kinase solution, and 1×kinase buffer in advance. Add 2 μl of 2.5×kinase solution, 1 μl of 5×DMSO drug solution, and 2 μl of 2.5×ATP / substrate mixed solution into a 384-well plate in sequence, and set duplicate wells (2 wells), blank group (without adding enzyme and drug), and control group (adding enzyme but not adding drug). Mix at room temperature for 10 minutes, then add 2 μL of ATP / substrate mixture and incubate at 37°C for 1 hour. After the reaction is completed, the ADP-Glo TMKinase assay. Subsequently, 5 μL of ADP-Glo reagent was added to each well to stop the kinase reaction and deplete the unconsumed ATP. Then the mixture was incubated at room temperature for 40 minutes. 10 μL of kinase detection reagent was added to convert ADP to ATP and introduce luciferase and luciferin to detect ATP. Finally, the mixture was incubated at room temperature for 30 - 60 minutes, and the chemiluminescence was measured. The luminescence signal is related to the content of ATP in the reaction system and is inversely proportional to the kinase activity. Quizartinib was used as a positive control drug for the assay. According to the formula: Inhibition rate % = (RLU control - RLU experiment) / (RLU control - RLU blank) × 100% (RLU refers to relative luminescence intensity), the inhibition rate of the compound on FLT3 kinase at a concentration of 1 μM was calculated.

[0312] (2) The experimental results of the assay are shown in Table 2 below:

[0313] Table 2

[0314]

[0315] The experimental results in Table 2 show that compounds such as W-2, W-3, W-6, and W-8 have significant inhibitory activity against FLT3 kinase. In particular, compounds W-3 and W-6 have the strongest activity.

[0316] Example 50: Pharmacodynamic experiment of FLT3 covalent inhibitor (I) on cells expressing FLT3

[0317] (1) Experimental method:

[0318] Human leukemia cell lines (MV4-11 / MOLM-13) were purchased from the American Type Culture Collection (ATCC, USA). MV4-11 cells were cultured in Dulbecco's medium (Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. MOLM-13 cells were cultured in RPMI 1640 medium (BI) containing 10% FBS and 1% penicillin / streptomycin. All cells were cultured at 37 °C, 5% CO 2Cultured under the conditions. The cells were seeded into 96-well plates (2000 - 5000 cells / well). The compounds (Compound W-1 to Compound W-14 prepared in the above examples) were made into a 10 mM concentration with solvent DMSO and diluted to 9 concentrations. 0.5% DMSO was used as the blank control. Starting from the next day, the cells were treated with the drug for 72 h. The final concentrations used in the measurement were 10, 3.3, 1.1, 0.37, 0.122, 0.041, 0.0136, 0.0045, and 0.0015 μM, and three wells were set for each concentration for measurement. The CCK8 assay method (Apexbio) was used to measure cell viability, and the absorbance was measured at 450 nm using an iMark microplate reader (Bio-Rad). The data was normalized to the control group (DMSO) and expressed as the mean of three independent measurements, with the standard error < 20%. GraphPad Prism 8.0.1 (GraphPad Software, San Diego, CA, USA) was used to calculate the IC 50 value.

[0319] (2) The experimental results are shown in Table 3 below:

[0320] Table 3 (The SD values in the table represent standard deviations)

[0321]

[0322] The experimental results in Table 2 show that compounds W-1, W-3, W-5, etc. all have significant inhibitory activities against the human leukemia cell lines MOLM-13 and MV4-11 expressing FLT3. In particular, Compound W-3 has the strongest activity. According to the IC 50 value, it can be seen that W-3 has in vitro anti-tumor activity comparable to Quizartinib.

[0323] Example 51: Determination of the covalent binding of Compound W-3 to FLT3

[0324] (1) Experimental method:

[0325] Mix FLT3 protein and compound W-3 (prepared in the above examples) at a molar ratio of 1:10 ([Protein]final = 5 μM, [W-3]final = 50 μM), and then incubate at 4 °C for 24 hours to ensure sufficient reaction between the compound and the protein. Use a liquid chromatography-mass spectrometry (LC-MS, Waters QTOF) to perform a complete mass analysis on the incubated sample. Detect different binding states of the FLT3 protein by mass spectrometry, with a focus on changes in the protein mass after covalent binding to W-3. Precipitate the incubated protein sample with acetylacetone, followed by reduction with dithiothreitol (DTT), alkylation with iodoacetamide (IAA), and digestion with trypsin at 37 °C for 4 hours (the weight ratio of the enzyme to the protein is 1:50). Subsequently, perform LC-MS / MS analysis using an Orbitrap Exploris mass spectrometer. Use PEAKS software for data processing to identify the binding sites and perform manual checks to verify the accuracy of the results.

[0326] (2) Experimental results are as Figure 4 and Figure 5 shown (provided by WuXi AppTec Detection and Analysis). It can be seen from Figure 4 that in the reaction between FLT3 protein and W-3, three sites of covalent binding between FLT3 protein and W-3 were detected by mass spectrometry, as shown in Figure 4 Figure b. Specifically, the mass spectrometry results showed that after incubation, the protein mass increased by 497 Da, which is consistent with the theoretical molecular weight of covalent binding to W-3. The specific binding ratio of FLT3 protein to compound W-3 is shown in Figure 4 Figure c. In the experiment, the binding ratio of FLT3 protein to compound W-3 was approximately 76.88%, indicating that most of the proteins were bound to at least one molecule of W-3.

[0327] Figure 5Peptide map of the covalent linkage between compound W-3 and FLT3 based on PEAKS analysis. Through LC-MS / MS peptide map analysis, 85% of the FLT3 protein sequence was covered, and all peptide segments containing cysteine (Cys) were detected. Confirmed by the PEAKS software and manual inspection, the Cys residue at position 194 (Cys194) of the FLT3 protein is the main covalent binding site of compound W-3. To ensure consistency with the standard database, sequence alignment showed that the FLT3 protein structure number of Cys194 in the PDB database is Cys807. This difference is due to the different sequence numbering systems used in the experiment and the PDB database, but both are confirmed to be the same amino acid residue through sequence alignment. Therefore, both Cys194 (experimental number) and Cys807 (PDB number) point to the same binding site, which is the main covalent binding position of compound W-3. Mass spectrometry results further showed that under the incubation condition of a molar ratio of 1:10 between the FLT3 protein and compound W-3, the covalent modification of compound W-3 to Cys807 reached a binding rate of 76.88%( Figure 4 ).

[0328] The above covalent binding experimental results indicate that the compound W-3 screened in the present invention covalently binds to the FLT3 protein at the Cys807 site, and the protein can bind compound W-3 at a relatively high ratio. Compound W-3 can be developed into a new anti-tumor drug.

[0329] Example 52: Effects of Compound W-3 on Cell Cycle and Apoptosis

[0330] (1) Experimental method:

[0331] Cell cycle detection: Spread the MOLM-13 cell / MV4-11 cell suspension in a 6-well plate. Using Quizartinib as the control group, add different concentrations of compound W-3 (prepared in the above example) (the solvent is DMSO) to the 6-well plate. After mixing, incubate at 37 °C and 5% CO 2 for 24 h. Subsequently, centrifuge to collect the cell precipitate, fix it overnight at -20 °C with pre-cooled 70% EtOH; centrifuge to collect the cells, stain, incubate at 37 °C for 1 h, and detect the cell cycle status with a Gallios flow cytometer (Beckman Coulter). Use FlowJo 7.6.1 for data analysis.

[0332] Cell apoptosis detection: Spread the MOLM-13 cell / MV4-11 cell suspension in a 6-well plate. Using Quizartinib as the control group, add different concentrations of compound W-3 (prepared in the above example) (the solvent is DMSO) to the 6-well plate. After mixing, incubate at 37 °C and 5% CO2 After incubation for 24 h / 72 h under the given conditions, the cells were collected by centrifugation, and apoptosis detection was performed according to the instructions of the Annexin V-FITC / PI apoptosis detection kit. Data analysis was carried out using GuavaSoft 3.1.1.

[0333] (2) The experimental results are as Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 shown. Generally speaking, compound W-3 induced apoptosis in MOLM-13 and MV4-11 cells in a concentration-dependent manner. The total apoptosis rates of compound W-3 on MOLM-13 at 24 h and 72 h were 7.91% - 21.60% and 3.69% - 73.90% respectively in the range of 0.1 nM to 100 nM; the total apoptosis rates of Quizartinib on MOLM-13 at 24 h and 72 h were 10.66% - 18.95% and 4.75% - 70.40% respectively in the range of 0.1 nM to 100 nM. Compound W-3 was generally comparable to Quizartinib; the total apoptosis rates of compound W-3 on MV4-11 at 24 h and 72 h were 7.69% - 19.11% and 7.61% - 71.20% respectively in the range of 0.1 nM to 100 nM, and the total apoptosis rates of Quizartinib on MV4-11 at 24 h and 72 h were 8.74% - 20.92% and 9.69% - 70.90% respectively in the range of 0.1 nM to 100 nM. Compound W-3 was generally comparable to Quizartinib. At the same time, it can be seen that at the same concentration (100 nM), the total apoptosis rate of compound W-3 on MOLM-13 was better than that of Quizartinib and showed time-dependence. Therefore, it was shown that the effect of compound W-3 in inducing apoptosis was almost the same as that of Quizartinib, and the longer the administration time at a certain concentration, the better the efficacy compared to Quizartinib. This may be attributed to the fact that compound W-3 is a covalent inhibitor and exerts an inhibitory effect for a longer time after binding to the FLT3 protein.

[0334] To study the anti-proliferation mechanism of compound W-3 on leukemia cells, the effect of compound W-3 on the cell cycle of MOLM-13 cells was investigated, as specifically shown in Figure 10 . The results showed that compound W-3 mainly blocked cell proliferation in the G0-G1 phase and inhibited cell proliferation in a concentration-dependent manner; the potencies of compound W-3 and Quizartinib were almost the same, effectively arresting cell growth in the G0-G1 phase.

[0335] Example 53: Effects of Compound W-3 on Cell Cycle and Apoptosis

[0336] (1) Experimental method:

[0337] The MV4-11 cell suspension was plated in a 6-well plate, and different concentrations of compound W-3 and the control Quizartinib (the solvent was DMSO, and the concentrations were 0.1 nM, 0.3 nM, 1.0 nM, 3.0 nM, 10 nM, and 30 nM respectively) were added. Incubate at 37 °C for 12 h; then centrifuge to collect the cells, lyse the cells on ice to extract protein samples for standby. Prepare an SDS-PAGE gel, add 2 mL of marker and 10 mL of the sample into the loading wells. After adding, electrophorese at a voltage of 80 V for about 20 min until the marker reaches the separation layer; increase the voltage to 120 V and electrophorese for 1 h. After electrophoresis, transfer the gel to a PVDF membrane; incubate the PVDF membrane with the corresponding primary antibody on a shaker at 4 °C overnight, and then incubate with the secondary antibody at room temperature for 1 h; finally, use the ECL Western Blotting kit to detect the specific protein level by chemiluminescence method.

[0338] (2) The experimental results are shown in Figure 11 , and it can be seen from the figure that compound W-3 can significantly inhibit FLT3 autophosphorylation in MV4-11 cells at a concentration of 1 nM, and inhibit the phosphorylation levels of FLT3 and its downstream pathway signals STAT5, AKT, and ERK in a concentration-dependent manner. It is worth noting that at the same concentration (30 nM), the inhibitory effect of compound W-3 on the phosphorylation of the FLT3-mediated signaling pathway is almost the same as that of the positive control drug Quizartinib, and even better than Quizartinib.

[0339] Example 54: Pharmacodynamics and immunohistochemistry experiments of compound W-3

[0340] (1) Method for in vivo anti-tumor pharmacodynamics experiment of compound W-3:

[0341] After evaluating the in vitro anti-tumor activity of compound W-3, using Quizartinib as the positive control, the in vivo anti-tumor pharmacodynamics of compound W-3 was evaluated through the in vivo subcutaneous xenograft tumor experiment of compound W-3 in BALB / c nude mice.

[0342] Experimental cells: MV4-11 cells.

[0343] Experimental drugs to be tested: Compound W-3 and the positive drug Quizartinib were weighed in corresponding amounts and dissolved in a small amount of DMSO, and prepared with a mixed solution of cyclodextrin aqueous solution and PEG400.

[0344] Experimental animals: Female BALB / c nude mice (SPF grade, 20 g - 22 g) were used in this experiment, purchased from Jiangsu Jicui Yakang Co., Ltd. Breeding environment: SPF grade, breeding temperature: 22 - 25 °C.

[0345] When the experimental cells are in the logarithmic growth phase, collect the cells, wash them once with PBS (pH = 7.4), resuspend them in a mixture of serum-free medium IMDM and Matrigel (volume ratio 1:1), and inject the cell suspension into the subcutaneous tissue of the right abdomen of mice using a 1 mL syringe. Observe the status of the mice and the growth of subcutaneous tumors every other day. When the average tumor volume grows to approximately 180 mm 3 , eliminate mice with too large or too small volumes, randomly divide the mice into 5 groups, namely the blank group (cyclodextrin + PEG400 aqueous solution), high-dose compound W-3 (50 mg / kg / d), medium-dose compound W-3 (25 mg / kg / d), low-dose compound W-3 (10 mg / kg / d), Quizartinib (10 mg / kg / d), with 4 mice in each group. Administer the drug once a day for a dosing period of 14 days. During the dosing period, measure the tumor diameter and weigh the mice every other day, observe the living status of the mice, and record any abnormal conditions. After the experiment, sacrifice the mice by cervical dislocation, dissect the subcutaneous tumors and weigh and record them, dissect the heart, liver, spleen, lungs, and kidneys and preserve them at room temperature with tissue fixative for immunohistochemical staining experiments. The immunohistochemical experiments were mainly completed by Jiangsu Jichu Biotechnology Co., Ltd.

[0346] Data processing: The formula for calculating the tumor volume (TV) is: TV = 1 / 2 × a × b 2 (where a represents the long diameter of the tumor; b represents the short diameter of the tumor); Tumor growth inhibition (TGI): The formula is: TGI = [1 - (TVt - TVinitial) / (CVt - CVinitial)] × 100% (where TVt represents the tumor volume at each measurement in the treatment group; TVinitial represents the tumor volume of the treatment group at the time of grouping and dosing; CVt represents the tumor volume at each measurement in the control group; CVinitial represents the tumor volume of the control group at the time of grouping and dosing); Tumor weight inhibition rate (IR): The formula is: IR = (WC - WT) / WC × 100% (where WC represents the tumor weight of the control group; WT represents the tumor weight of the treatment group). The experimental data were calculated and statistically processed using Microsoft Office Excel 2016 and GraphPad Prism 9.0.0 software.

[0347] The experimental results are as Figure 12As shown, in the dosing group, the compound W-3 was administered once a day, and it could significantly inhibit tumor growth at doses of 10 mg / kg, 25 mg / kg, and 50 mg / kg, respectively. At the same time, the anti-tumor activity of compound W-3 was dose-dependent. The TGI values at 10 mg / kg, 25 mg / kg, and 50 mg / kg were 136%, 163%, and 178% respectively, and the IR values were 25.0%, 48.2%, and 78.6% respectively. The TGI and IR values of Quizartinib at a dose of 10 mg / kg were 152% and 50.0% respectively, indicating that compound W-3 had good in vivo anti-tumor activity. In addition, compared with Quizartinib, almost no significant decrease in the body weight of mice was observed after the administration of compound W-3, and the body weight remained stable and increased slowly, indicating that mice had good tolerance to compound W-3. Compared with the blank group, compound W-3 could inhibit the tumors of mice to varying degrees after administration. Thus, compound W-3 had good in vivo pharmacodynamic properties in the subcutaneous xenograft tumor model of BALB / c nude mice.

[0348] (2) To further evaluate the in vivo pharmacodynamics of compound W-3, immunohistochemical staining experiments were performed on the treated tumor tissues to evaluate the effects of W-3 on the histological morphology, apoptosis induction, proliferation inhibition, phosphorylation and other pharmacological functions of tumor tissues. The results were as Figure 13 shown. The HE staining results showed that, morphologically, the tumor cells in the blank group were diffusely distributed. With the increase of the dosing dose, compound W-3 gradually induced large areas of necrosis in the tumor, showing a dose-dependence. Compared with Quizaitinib, the efficacy of inducing apoptosis was comparable at the same dose. The results of Ki67 and TUNEL staining showed that the tumor cells in the blank group had a high proliferation index. W-3 inhibited tumor cell proliferation and induced tumor cell apoptosis in a dose-dependent manner respectively. In addition, the phosphorylation of the downstream signal STAT5 mediated by FLT3 in the tumor tissue gradually decreased with the increase of the dosing dose. At 25 mg / kg, the phosphorylation of STAT5 in the tissue was almost absent, indicating that compound W-3 had a targeted inhibitory effect on FLT3 phosphorylation in vivo.

[0349] In addition, by performing HE staining on the main organs of animals such as the heart, liver, spleen, lungs, and kidneys to analyze the histological morphological differences ( Figure 14) The toxicity damage of W-3 to the main organs of animals was evaluated. The staining results showed that after administration, the cross striations of cardiomyocytes in the hearts of mice in all groups were clear, the nuclei were centered, and there were abundant blood vessels and a small amount of connective tissue in the interstitium; there was no obvious hypertrophy or pyknosis of liver cells; the white pulp of the spleen was obvious and the splenic corpuscles had clear structures; the alveolar cavities were clear and the alveolar wall thickness was normal; the renal cortex was obvious and the renal glomerular cavities were clearly visible. No obvious abnormalities were found compared with the organ staining of the blank solvent group. Therefore, it is considered that compound W-3 has no obvious toxicity to the important organs such as the heart, liver, spleen, lungs and kidneys of nude mice.

[0350] Example 55: Safety evaluation experiment of compound W-3 in zebrafish model

[0351] (1) Experimental method:

[0352] The zebrafish were divided into three groups: DMSO, compound W-3 and Quizartinib, with three replicate wells in each group. 30 zebrafish were incubated in each well, the volume of the solution in each well was 3 mL, and they were incubated at 28 °C for 24 h after administration; after the administration ended, the activity status of the fish was observed, and 10 fish were randomly selected from each well for photographing under a fluorescence microscope and the number of neutrophils was calculated using Image J software.

[0353] (2) The experimental results are as Figure 15 shown. Compared with the blank group (DMSO group), compound W-3 did not cause obvious changes in the number of neutrophils in zebrafish at both high and low concentrations, indicating that the compound has no obvious inhibitory effect on the bone marrow hematopoietic function of zebrafish and has not caused obvious negative effects on the generation of neutrophils. Therefore, compound W-3 did not show significant blood toxicity in the zebrafish model and has reliable safety.

[0354] The present invention provides an idea and method for a FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure, its preparation method and use. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure, characterized in that: The FLT3 covalent inhibitor has a structure shown in Formula I: in, R is selected from hydrogen, halogen, cyano, halogenated C1-C4 alkyl, C1-C4 alkoxy or C1-C4 alkyl; L is selected from Wherein, R1 is selected from hydrogen or C1-C4 alkyl.

2. The FLT3 covalent inhibitor according to claim 1, characterized in that R is selected from hydrogen, fluorine, chlorine, cyano, trifluoromethyl, methoxy or methyl; L is selected from Wherein, R1 is selected from hydrogen or methyl.

3. The FLT3 covalent inhibitor according to claim 1, characterized in that The FLT3 covalent inhibitor is selected from any of the following structures:

4. The tautomer, mesomer, racemate, enantiomer, diastereomer or pharmaceutically acceptable salt thereof of the FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure according to claim 1 or 2.

5. The method for preparing the FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure according to any one of claims 1 to 3, characterized in that: The steps include: (1) Compound 1-6-a and compound 1-6-b undergo a first reaction under the action of a first base to obtain compound 1-6; compound 1-6 undergoes a second reaction under the action of a reducing agent and ammonium chloride to obtain compound 1-7; (2) Compound 1-7 and triphosgene undergo a third reaction, and the obtained isocyanate intermediate 1-8 and compound 1-5 continue to undergo a fourth reaction under the action of a second base to obtain a FLT3 covalent inhibitor I containing an isoxazolo[5,4-b]pyridine structure; in, R is selected from hydrogen, halogen, cyano, halogenated C1-C4 alkyl, C1-C4 alkoxy or C1-C4 alkyl; L is selected from Wherein, R1 is selected from hydrogen or C1-C4 alkyl; X is Cl or Br.

6. The preparation method according to claim 5, characterized in that: In the first reaction, the first base is triethylamine; the molar ratio of the compound 1-6-a to the compound 1-6-b is 1.0:(0.9-3.0); the first reaction is carried out at room temperature; the solvent used in the first reaction is N,N-dimethylformamide; In the second reaction, the reducing agent is iron powder; the molar ratio of the compound 1-6 to the reducing agent is 1.0:(2.0-6.0); the reaction temperature of the second reaction is 60-100° C.; the solvent used in the second reaction is a mixture of ethanol and water in any proportion; In the third reaction, the molar ratio of the compound 1-7 to triphosgene is (2.0-6.0):1.0; the third reaction is carried out under the protection of an inert gas; the third reaction is stirred in an ice bath for 0.5-1.0 h, then reacted at room temperature for 1-3 h, and finally reacted at 60-100° C. for 3-6 h; the solvent used in the third reaction is ethyl acetate; In the fourth reaction, the second base is triethylamine; the molar ratio of the compound 1-7 used in the third reaction to the compound 1-5 used in the fourth reaction is (4.0-8.0): (0.005-0.500); the reaction temperature of the fourth reaction is -20°C to 10°C; the solvent used in the fourth reaction is dichloromethane.

7. A pharmaceutical composition, characterized in that The invention comprises (i) a FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure represented by formula (I) as described in any one of claims 1 to 3, or a tautomer thereof, or a mesoform thereof, or a racemate thereof, or an enantiomer thereof, or a diastereomer thereof, or a pharmaceutically acceptable salt thereof, and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

8. Use of the FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 7 in the preparation of drugs for preventing and treating tumors.

9. Use of the FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 7 in the preparation of a drug for preventing and treating acute myeloid leukemia.

10. Use of the tautomer, mesomer, racemate, enantiomer, diastereomer or pharmaceutically acceptable salt of the FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure according to claim 1 or 2 in the preparation of a drug for preventing and treating tumors.

11. Use of the tautomer, mesomer, racemate, enantiomer, diastereomer or pharmaceutically acceptable salt of the FLT3 covalent inhibitor containing an isoxazolo[5,4-b]pyridine structure according to claim 1 or 2 in the preparation of a drug for preventing and treating acute myeloid leukemia.

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