Use of FLT3 kinase inhibitor F-50 in the preparation of a medicament for treating acute myeloid leukemia with FLT3 mutation

Abstract

The application discloses application of FLT3 kinase inhibitor F-50 in preparation of a medicine for treating acute myeloid leukemia with FLT3 mutation, and belongs to the technical field of biological medicines. A chemical structural formula of the FLT3 kinase inhibitor F-50 is shown in the following, and it is found through experiments that the FLT3 kinase inhibitor F-50 can be used as an inhibitor of FLT3 kinase, and can play a role in resisting FLT3-ITD and drug-resistant mutant acute myeloid leukemia; in vitro, the FLT3 kinase inhibitor F-50 can inhibit the growth of leukemia cells with FLT3-ITD mutation, inhibit the phosphorylation level of FLT3, promote cell apoptosis and cause cycle arrest, and the FLT3 kinase inhibitor F-50 has a certain degree of inhibiting effect on FLT3 protein kinase activity at a biochemical level. The application brings a new scientific thought for development of a new FLT3 small-molecule targeted inhibitor, and is expected to bring a better choice for treatment of FLT3 mutant AML patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the FLT3 kinase inhibitor F-50 in the preparation of drugs for treating acute myeloid leukemia with FLT3 mutations. Background Technology

[0002] Acute myeloid leukemia (AML) accounts for approximately 80% of adult acute leukemia cases and is a significant type of leukemia. Over the past 40 years, apart from acute promyelocytic leukemia (AML) where the introduction of targeted therapy has significantly improved cure rates, progress in the treatment of other AML types has been slow. Treatment primarily relies on the classic "7+3" regimen of induction chemotherapy followed by risk-stratified consolidation therapy—high-dose cytarabine chemotherapy or allogeneic hematopoietic stem cell transplantation. Under this strategy, the 5-year survival rate for AML patients under 60 years of age is only around 40%, while the 5-year survival rate for AML patients over 60 years of age is less than 10%. In the era of precision medicine, actively exploring targeted therapies holds promise for improving the prognosis of this type of AML.

[0003] FLT3 is a member of the receptor tyrosine kinase class III subfamily and is mutated in up to 30% of adult AML cases. There are two main types of FLT3 mutations in AML: internal tandem repeat (ITD) mutations, accounting for approximately two-thirds, and kinase domain (TKD) point mutations, accounting for approximately one-third. Both types of mutations are gain-of-function mutations, leading to persistent autophosphorylation of the FLT3 receptor independent of FLT3 ligands, abnormal activation of signaling pathways, participation in the development and progression of leukemia, and maintenance of abnormal cell proliferation. Furthermore, FLT3-ITD-positive AML patients have a poorer response to induction chemotherapy, a higher relapse rate, and shorter relapse-free survival and overall survival compared to mutation-negative patients. The prognostic impact of FLT3-TKD mutations on AML patients remains controversial, but some FLT3-ITD-positive AML patients develop secondary FLT3-TKD mutations after initial targeted therapy, leading to acquired resistance and treatment failure, posing a significant challenge to subsequent AML treatment. Since FLT3 mutation is the most common molecular mutation in AML, and patients with this mutation have a poor prognosis under traditional treatment strategies, FLT3 has naturally become one of the most attractive therapeutic targets for AML, with the hope of improving the prognosis of this type of AML.

[0004] Currently, there are more than ten small molecule inhibitors targeting FLT3 receptor kinase that have entered clinical trials or have been approved. Although they have all achieved encouraging clinical efficacy to varying degrees, each inhibitor has its own problems. In addition to changes in pharmacokinetics after human application, important problems include targeting nonspecificity, off-target toxicity, and secondary mutational resistance. The core issue is deficiencies in their design. This is because these targeted inhibitors were mostly obtained through biochemical or cellular level specific experiments or designed, screened, and optimized using other tyrosine kinase proteins as structural models. Even recent designs specifically targeting FLT3 kinase proteins often rely solely on the wild-type, self-inhibiting FLT3 protein crystal structure (1RJB) as their structural biology basis.

[0005] Therefore, based on the latest understanding of the function and structure of FLT3 kinase protein and its mutants, this invention re-analyzes the structure of FLT3 in its self-inhibitory and activated states, especially the structural characteristics of the mutants themselves. Based on the unique structural characteristics of FLT3, new small molecule binding sites are sought and specific small molecule inhibitors are screened and optimized, bringing new scientific ideas to the development of novel FLT3 small molecule targeted inhibitors and potentially providing better treatment options for FLT3-mutant AML patients. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the FLT3 kinase inhibitor F-50 in the preparation of drugs for treating acute myeloid leukemia with FLT3 mutations, so as to solve the problems existing in the prior art. Through experimental verification, it has been found that the small molecule compound FLT3 kinase inhibitor F-50 is promising for the treatment of acute myeloid leukemia with FLT3 mutations.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides the use of the FLT3 kinase inhibitor F-50 in the preparation of a drug for treating acute myeloid leukemia with FLT3 mutations, the chemical structural formula of which is shown below:

[0009]

[0010] Preferably, the acute myeloid leukemia with FLT3 mutation includes acute myeloid leukemia with FLT3-ITD mutation, FLT3-TKD mutation, FLT3-ITD-TKD mutation or FLT3-ITD-F691L mutation.

[0011] Preferably, the FLT3 kinase inhibitor F-50 achieves the treatment of acute myeloid leukemia with FLT3 mutation by inhibiting the growth of acute myeloid leukemia cells with FLT3 mutation, inhibiting the phosphorylation level of FLT3 kinase in the cells, promoting apoptosis of the cells, or arresting the cell cycle of the cells.

[0012] This invention also provides the use of the FLT3 kinase inhibitor F-50 in the preparation of a medicament for treating FLT3-ITD resistant mutant acute myeloid leukemia, the chemical structural formula of which is shown below:

[0013]

[0014] Preferably, the FLT3 kinase inhibitor F-50 achieves the treatment of FLT3-ITD resistant mutant acute myeloid leukemia by inhibiting the growth of FLT3-ITD resistant mutant acute myeloid leukemia cells, inhibiting the phosphorylation level of FLT3 kinase in the cells, promoting apoptosis of the cells, or arresting the cell cycle of the cells.

[0015] The present invention discloses the following technical effects:

[0016] This invention discovers that the small molecule compound F-50 can act as an inhibitor of FLT3 kinase, exerting anti-FLT3-ITD and anti-drug-resistant mutant acute myeloid leukemia effects. Experiments have shown that F-50 can inhibit the growth of FLT3-ITD mutant leukemia cells in vitro, suppress FLT3 phosphorylation, promote apoptosis, and induce cell cycle arrest. Furthermore, at the biochemical level, F-50 has a certain degree of inhibitory effect on FLT3 protein kinase activity, with an IC50 value of [missing information]. 50 The values ​​can reach the micromolar level. This invention offers new hope for precise targeted therapy for patients with FLT3 mutation-positive acute myeloid leukemia. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The chemical structural formula of F-50 of this invention;

[0019] Figure 2 This invention provides molecular dynamics models for the activated states of wild-type FLT3, FLT3-ITD, FLT3-ITD with D835Y, and FLT3-ITD with F691L.

[0020] Figure 3 To investigate the inhibitory effect of a single 100 μM concentration of small molecule drug 26-51 on stable 32D-ITD mutant cells and normal 32D cells after 72 hours of treatment; in the figure, "50" represents F-50, "32D" represents parental 32D cells, and "8#" represents stable 32D-ITD mutant cells.

[0021] Figure 4 The results of cell viability assays using CCK8 were obtained after treating normal 32D cells without FLT3 mutation and stable 32D-ITD mutant cells with different concentrations of F-50 for 72 hours. All experimental results were repeated three times and yielded similar results. The figure shows the statistical results of three replicates from one experiment. In the figure, 32D represents normal 32D cells and 8# represents stable 32D-ITD mutant cells.

[0022] Figure 5 To determine the half-maximal inhibitory concentration (WMC) of F-50 in FLT3-free normal 32D cells, 32D-ITD mutant stable cell lines, FLT3 mutant leukemia cell lines (MV-4-11, Molm13), FLT3-free leukemia cell lines (THP-1, HL60, K562), and hematopoietic stem cells (HSCs); the results are obtained from three replicates, and ** indicates P < 0.01;

[0023] Figure 6 This diagram illustrates the binding of F-50 to the FLT3 protein; solid lines represent hydrogen bonding, and dashed lines represent hydrophobic interactions.

[0024] Figure 7 After 6 hours of treatment with F-50 on the stable transgenic 293T-FLT3-ITD strain, the phosphorylation level of FLT3 was detected by Western blotting; A: Effect of F-50 on FLT3 phosphorylation; B: Effect of F-50 on FLT3 phosphorylation and activation of downstream STAT5, AKT, and ERK pathways in 293T-ITD / 32D-ITD.

[0025] Figure 8 To detect apoptosis in cells after treatment with F-50 and AC220 on the 32D parental strain and the 32D-ITD stable transgenic strain, flow cytometry was used.

[0026] Figure 9 Results on the drug sensitivity of cell lines resistant to AC220 or sorafenib to F-50. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] The inventive concept of this invention is as follows: Through extensive and in-depth research, the inventors, for the first time, utilized the "Sunway TaihuLight" supercomputer to perform molecular dynamics simulations of FLT3 wild-type, FLT3-ITD, FLT3-ITD with D835Y double mutant, and FLT3-ITD with F691L double mutant proteins, exploring novel small molecule binding sites unique to FLT3 (see...). Figure 2Unlike the current mainstream design approach that uses the crystal structure of wild-type FLT3 protein in a self-inhibited state as the basis for structural biology, the inventors utilized the structural characteristics of the intermediate inactive state of newly discovered mutant and wild-type FLT3 proteins. Based on homologous molecular models, they constructed activated state structural models of mutant and wild-type FLT3, as well as existing self-inhibition models of wild-type FLT3. This led to the discovery of novel small molecule binding sites unique to FLT3. Through high-throughput screening using computer virtual screening software in databases such as the National Cancer Institute Public Database (NCI) and the China National Compound Library, dozens of lead small molecule compounds were obtained. After screening and optimization at the cellular and biochemical levels, a highly sensitive and specific FLT3 small molecule inhibitor, 2-(2,4-Dinitrophenyl)sulfanyl-4-(1-methylimidazol-2-yl)sulfanylpyrimidine (F-50), was obtained, with the chemical structure shown below. Figure 1 (As shown). Then, the efficacy of the optimized drug F-50 was verified.

[0033] The following specific examples will further illustrate the effectiveness of the F-50 described above.

[0034] The following examples involve the main biomaterials and reagents:

[0035] (1) Experimental cells

[0036] Human Molm-13, MV4-11, THP-1, K562, HL60, 293T cells and mouse 32D cells were all purchased from Beina Biotechnology Co., Ltd. Mouse 32D-FLT3-ITD stable cell lines and 293T-FLT3-ITD stable cell lines were constructed internally.

[0037] (2) Experimental reagents

[0038] FLT3 inhibitors AC220 and Sorafenib were purchased from Shanghai Taosu Biochemical Technology Co., Ltd.; F-50 was purchased from ABIChem (Germany). Recombinant Murine IL-3 was purchased from PEPROTECH (USA); CCK8 reagent was purchased from Tongren Chemical Co., Ltd. (Japan); Annexin V-APC apoptosis detection kit was purchased from Biogems (USA); Anti-pFLT3, p-STAT5, STAT5, p-AKT, and AKT antibodies were purchased from Cell Signaling Technology (USA); p-ERK and ERK antibodies were purchased from Santa Cruz (USA); HA-tag mouse mAb was purchased from Genscript (China); and horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) was purchased from Beyotime (China).

[0039] The following examples illustrate the construction and detection methods of the main mutants:

[0040] 1. Construction of FLT3-WT / ITD plasmid

[0041] The plasmid was constructed using pLenti-III-EF1α(LV043) as the plasmid vector by Applied Biological Materials (abm) Inc. (Canada). The constructed plasmid was verified by gene sequencing to be the successfully constructed target FLT3 plasmid.

[0042] 2. Construction of stable 293T and 32D cell lines

[0043] 2.1 FLT3-WT / ITD Lentiviral Packaging

[0044] 1) Select 293T cells, seed them in 10cm cell culture dishes, and passage them to the logarithmic growth phase. After adjusting the cell line condition to the optimal level, wait for the cells to grow evenly to occupy about 70-80% of the bottom volume of the dish (the cell count is approximately 5-7 × 10⁶ cells / cm²). 6 (pieces / dish).

[0045] 2) Two hours before the virus begins packaging, discard the original culture medium and replace it with 5 mL of fresh DMEM complete culture medium.

[0046] 3) The lentivirus packaging system is shown in Table 1 below. Take two 1.5 mL EP tubes, label tubes A and B, and add 500 μL of serum-free DMEM culture medium to each tube. First, add 40 μL of Lipo 2000 to tube B and gently pipette to mix. Take tube A and add the packaging system shown in Table 1 below, and gently pipette to mix.

[0047] Table 1 Lentiviral Packaging Systems

[0048]

[0049] 4) Pipette the liquid from tube B and slowly add it dropwise to tube A until all the liquid has been added. Gently rinse and mix three times, then incubate at 37°C for 20–30 minutes. Gently and slowly add the mixture from tubes A and B dropwise to a cell culture dish, mix thoroughly using the cross-hatching method, and incubate in an incubator.

[0050] 5) After culturing for 6-8 hours, discard all the original culture medium into a waste liquid processor containing disinfectant, and add 5 mL of fresh DMEM complete culture medium to complete the cell culture medium replacement.

[0051] 6) After 24 hours of incubation, the culture medium containing lentivirus was collected for the first time in 15 mL centrifuge tubes, sealed and stored at 4°C. 5 mL of fresh DMEM complete medium was added.

[0052] 7) After culturing for 48 hours and 72 hours, collect the virus for the second and third time. Centrifuge all collected virus solutions at 1500 rpm at 4°C for 15 minutes. Filter the supernatant through a 0.22 μm filter to remove cell debris and other impurities. Aliquot and store at -80°C.

[0053] 2.2 Infection of 293T and 32D cells with FLT3-WT / ITD lentivirus and screening of stable transfected cells

[0054] 1) After completing the packaging of the FLT3 mutant lentivirus, take an appropriate amount of lentivirus to infect 32D and 293T cells.

[0055] 2) After 72 hours of infection, culture medium containing puromycin was added and the cells were cultured until all negative control cells died.

[0056] 3) Well-grown 293T / 32D cells were lysed, and Western Blot was used to detect whether HA-tag was expressed to identify whether the cells stably expressed FLT3 wild-type / mutant protein.

[0057] 3. CCK8 assay for cell viability

[0058] Cell viability was detected using a CCK-8 assay kit. The CCK-8 method is a reliable and sensitive method for detecting cell viability. The CCK-8 reagent contains... -8 reacts in the presence of the electron carrier 1-Methoxy PMS to generate water-soluble formazan dye. -8, the absorbance at 450 nm can reflect cell viability. Raw data were processed using GraphPad 8.0 software (USA) to obtain drug susceptibility curves and IC50 values.50 data.

[0059] Example 1: Functional Verification of F-50

[0060] 1. At the cellular level, F-50 is more sensitive to cell lines with ITD mutations than to cell lines without FLT3 mutations.

[0061] Based on the results of previous virtual screening in a small molecule library, dozens of small molecule drugs were constructed. According to the inhibitory effects of these small molecule drugs on the 32D parent strain and the 32D-ITD mutant strain (8#) at 100 μM, more than ten drugs that are more sensitive to the 32D-ITD mutant strain (8#) were initially screened. Further drug sensitivity testing at multiple concentration gradients confirmed that F-50 showed a more significant inhibitory effect on the 32D-ITD mutant strain (8#) at multiple concentration gradients, and this difference was statistically significant (see...). Figure 3 ).

[0062] Subsequently, the growth-inhibiting effect of F-50 on cell lines with and without the FLT3 mutation was examined. Specifically, normal 32D cells and the stable transgenic 32D-ITD mutant (8#) were treated with different concentrations of F-50 for 72 hours, and cell viability was detected using CCK8 reagent. The results showed that F-50 had a stronger inhibitory effect on the stable transgenic 32D-ITD mutant (8#) than on the normal 32D cells without the FLT3 mutation (IC50). 50 The corresponding speeds are 18.18µM and 6.824µM respectively (see...). Figure 4 In normal 32D cells without FLT3 mutations, stable 32D-ITD mutant cells (8#), FLT3 mutant leukemia cell lines (MV-4-11, Molm13), FLT3-free leukemia cell lines (THP-1, HL60, K562), and hematopoietic stem cells (HSCs), the half-maximal inhibitory concentration (IC50) of F-50 against FLT3-mutant myeloid leukemia cell lines (MV-4-11, Molm13) was [not specified]. 50 Overall, the levels were lower than those of myeloid leukemia cell lines without FLT3 mutations (THP-1, HL60, K562) (see...). Figure 5 ).

[0063] 2. Molecular docking results showed that F-50 can specifically bind to the FLT3 protein.

[0064] Targeted inhibitors affect the biological function of target proteins by specifically binding to them and altering their conformation. This invention utilizes virtual molecular docking, specifically:

[0065] The FLT3 protein structure was obtained from the PDB database (PDB: 1rjb), and the F-50 structure was obtained from the PubChem database (cid: 329305). Autodock vina (v1.1.2) was used for docking, and the docking result shows the configuration with the lowest binding energy (-8.1 kcal / mol). Protein-Ligand Interaction Profiler (PLIP) was used for protein-liquid interaction analysis, and PyMOL was used for image editing and output.

[0066] The results are as follows Figure 6 As shown, it demonstrates that F-50 can specifically bind to the FLT3 protein (forming hydrogen bonds with FLT3 protein Q575 and R849 residues, and forming hydrophobic interactions with I836, Y842, and R849). This binding may exert its targeted inhibitory effect by stabilizing the self-inhibitory conformation of FLT3.

[0067] 3. F-50 kills FLT3-mutant cells by inhibiting FLT3 phosphorylation and activation of its downstream pathways, thereby promoting apoptosis.

[0068] 3.1 F-50 can effectively reduce the phosphorylation level of FLT3.

[0069] FLT3 is a member of the tyrosine kinase family. It promotes cell proliferation and survival by phosphorylating downstream signaling proteins through its kinase activity. The phosphorylation capacity of FLT3 partially reflects its activation capacity of downstream signaling pathways. This invention used a constructed 293T-FLT3-ITD cell line to detect the inhibitory capacity of F-50 cells on FLT3 phosphorylation. Specifically:

[0070] Cells were collected after treatment with DMSO / 100μM F-50 / 100nMAC220 in the same state. Cells were then lysed, and proteins were purified using HA beads. Western blotting was used to detect the expression level of phosphorylated FLT3 protein. The results showed that after 6 hours of treatment with F-50 on the 293T cell line with the ITD mutation, the phosphorylation level of FLT3 was significantly reduced compared to the DMSO group (see...). Figure 7 (A) indicates that F-50 has an inhibitory effect on the phosphorylation of FLT3.

[0071] We also investigated the effects of F-50 on downstream FLT3 pathways. Specifically, after treating the 32D / 293T-FLT3-ITD stable cell line with DMSO, F-50 100 μM, and AC220 100 nM for 6 hours, cells were harvested, proteins were lysed, and Western blotting was used to detect the phosphorylation levels of FLT3 and its downstream pathways STAT5, AKT, and ERK. The results are as follows: Figure 7 As shown in Figure B, the results indicate that F-50 significantly inhibits the phosphorylation of FLT3 and downstream STAT5, AKT, and ERK in 293T-ITD and 32D-ITD cell lines.

[0072] 3.2 F-50 has a significant pro-apoptotic effect on FLT3 mutant cells.

[0073] To investigate the effect of F-50 on apoptosis in AML cells carrying the FLT3 mutation, this invention used the Annexin V-APC apoptosis detection kit for experiments. Specifically, 32D parental cells and 32D-ITD mutant cells in the same state were treated with 100 μM F-50 and 100 nMAC220 for 24 h. Cells were harvested and apoptosis was detected by flow cytometry. The changes in apoptosis between the drug-treated group and the untreated group were compared.

[0074] Flow cytometry results as follows Figure 8 As shown, the results indicate that F-50 has a similar effect to AC220, inducing a certain degree of apoptosis in both 32D-ITD stable cell lines and parental cells. Among them, the 32D-ITD stable cell line is more sensitive to the apoptosis induced by the lead small molecule than the parental cell line, and a high proportion of late apoptosis is induced.

[0075] 3.3 F-50 can overcome cell line resistance to inhibitors AC220 or sorafenib.

[0076] This invention utilizes a gradient escalation method to induce F-50 treatment in FLT3-ITD mutant AML cell lines MV-4-11AC200-R / MV-4-11Sor-R resistant to inhibitors AC220 or Sorafenib, and observes the drug sensitivity of F-50.

[0077] like Figure 9As shown, the drug susceptibility test results indicated that MV-4-11AC200-R / MV-4-11Sor-R had developed significant resistance to the inhibitors AC220 and Sorafenib, respectively. By comparing the drug susceptibility of the parental MV-4-11 strain, the resistant strain MV-4-11AC200-R, and the resistant strain MV-4-11Sor-R to F-50, it can be seen that F-50 still has the same killing effect on AC220 or Sorafenib-resistant cell lines as it does on the parental MV-4-11 strain. That is, F-50 can overcome the resistance mechanism of the parental MV-4-11 strain to the inhibitors AC220 and Sorafenib.

[0078] The inventors recognized that current FLT3 small molecule inhibitors in clinical trials or already approved suffer from various problems, such as specificity issues, toxic side effects, secondary resistance, and mutations. The primary cause is design flaws and the limited range of reported FLT3 structural state models. Therefore, based on homologous molecular models, the inventors constructed wild-type and mutant (FLT3-ITD, FLT3-ITD with D835Y double mutant, and FLT3-ITD with F691L double mutant) FLT3 activation state structural models to identify novel small molecule binding sites specific to FLT3. Through computer-aided virtual screening, optimization, and subsequent biochemical, cellular, and other activity verifications, they identified and obtained the highly sensitive and specific small molecule compound F-50, which can reduce resistance issues associated with secondary TKD region mutations, offering new hope for precise targeted therapy in FLT3 mutation-positive AML patients.

[0079] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the FLT3 kinase inhibitor F-50 in the preparation of drugs for treating acute myeloid leukemia with FLT3 mutations, characterized in that, The chemical structural formula of the FLT3 kinase inhibitor F-50 is shown below:

2. The application as described in claim 1, characterized in that, The acute myeloid leukemia with FLT3 mutations includes acute myeloid leukemia with FLT3-ITD mutation, FLT3-TKD mutation, FLT3-ITD-TKD mutation or FLT3-ITD-F691L mutation.

3. The application as described in claim 1, characterized in that, The FLT3 kinase inhibitor F-50 achieves therapeutic effects on acute myeloid leukemia with FLT3 mutations by inhibiting the growth of FLT3-mutant acute myeloid leukemia cells, inhibiting the phosphorylation level of FLT3 kinase in the cells, promoting apoptosis of the cells, or arresting the cell cycle of the cells.

4. The application of the FLT3 kinase inhibitor F-50 in the preparation of drugs for treating FLT3-ITD resistant mutant acute myeloid leukemia, characterized in that, The chemical structural formula of the FLT3 kinase inhibitor F-50 is shown below:

5. The application as described in claim 4, characterized in that, The FLT3 kinase inhibitor F-50 achieves therapeutic effects on FLT3-ITD resistant mutant acute myeloid leukemia by inhibiting the growth of FLT3-ITD resistant mutant acute myeloid leukemia cells, inhibiting the phosphorylation level of FLT3 kinase in the cells, promoting apoptosis of the cells, or arresting the cell cycle of the cells.

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