PYRIMIDINE COMPOUND OR SALT THEREOF IN APPLICATION FOR MANUFACTURING PHARMACEUTICAL PRODUCT FOR PREVENTING AND/OR TREATING FLT3-RELATED DISEASE OR DISORDER

Abstract

The present invention relates to a pyrimidine compound or salt thereof in an application for manufacturing a pharmaceutical product for preventing and /or treating an FLT3-related disease or disorder. The pyrimidine compound has a chemical structure as represented by formula (I).

##STR00001##

Claims

1. Use of a compound shown in Formula (I) or pharmaceutically acceptable salts thereof in preparing drugs for the prevention and/or treatment of FLT3-related diseases or disorders: ##STR00006##

2. The use according to claim 1, wherein the pharmaceutically acceptable salts are hydrochloride salt, hydrobromide, maleate, phosphate, succinate, sulphate, citrate, benzoate, mesylate, lactate, acetate, tosylate, palmitate, fumarate, tartrate, ascorbate, nitrate, formate, propionate, n-butyrate, isobutyrate, salicylate, oxalate, succinate, malate, glutamate, aspartate or gluconate; preferably, the pharmaceutically acceptable salts are hydrochloride salt, phosphate, mesylate, lactate, acetate, sulphate or hydrobromide; more preferably, the pharmaceutically acceptable salt is hydrochloride salt.

3. The use according to claim 1, wherein the pharmaceutically acceptable salts are present in a crystalline form; preferably, when the pharmaceutically acceptable salt is hydrochloride salt, the X-ray powder diffraction pattern of the crystalline form of the hydrochloride salt includes the diffraction peaks at 2 of 6.80.2, 9.50.2, 11.40.2, 15.00.2, 17.00.2, 19.90.2, 20.30.2, 20.60.2, 22.90.2, 23.60.2, 24.90.2, 26.10.2, 26.60.2; more preferably, the X-ray powder diffraction pattern of the crystalline form of the hydrochloride salt further includes the diffraction peaks at 2 of 12.00.2, 28.80.2, 29.10.2, 32.50.2, 34.70.2; further preferably, the X-ray powder diffraction pattern of the crystalline form of the hydrochloride salt is shown as FIG. 3.

4. The use according to claim 1, wherein the drugs are solid preparations or liquid preparations; preferably, the solid preparations are selected from capsule, tablet, pill, powder, granule, sugar pill, and lyophilized preparation; preferably, the liquid preparations are selected from solution, injection, suspension, oral liquid, syrup, tincture, and emulsion.

5. The use according to claim 1, wherein the FLT3-related diseases or disorders are myelodysplastic disorders, such as thrombocytosis, essential thrombocytosis, idiopathic extramedullary metaplasia, myelofibrosis, myelofibrosis with myeloid metaplasia, chronic idiopathic myelofibrosis, polycythemia vera, hematopenia and malignant anterior spinal cord dysplasia syndrome.

6. The use according to claim 1, wherein the FLT3-related diseases or disorders are selected from glioma, lung cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, esophageal cancer, colon cancer, pancreatic cancer, ovarian cancer, kidney cancer, thyroid cancer, neuron cancer and uterine cancer.

7. The use according to claim 1, wherein the FLT3-related diseases or disorders are selected from leukemia, lymphoma and myeloma; preferably, the leukemia is selected from acute myeloid leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, chronic neutrophilic leukemia, acute undifferentiated cell leukemia, prolymphocytic leukemia, juvenile myelomonocytic leukemia, adult T-cell acute lymphocytic leukemia, acute myeloid leukemia with trilineage myelodysplasia, mixed lineage leukemia, and acute mononuclear leukemia; preferably, the lymphoma is non-Hodgkin lymphoma or Hodgkin lymphoma, for example degenerative large cell lymphoma; preferably, the plasma cell diseases include multiple myeloma, macroglobulinemia or heavy chain disease; preferably, the myeloma is selected from myelodysplastic syndrome, myelodysplastic disorder, multiple myeloma and spinal cord sarcoma.

8. A method for preventing and/or treating FLT3-related diseases or disorders comprising administering a therapeutically effective amount of the compound shown in Formula (I) or the pharmaceutically acceptable salts thereof to a subject in need; preferably, the subject is a mammal.

9. The method according to claim 8, wherein, the pharmaceutically acceptable salts are hydrochloride salt, hydrobromide, maleate, phosphate, succinate, sulphate, citrate, benzoate, mesylate, lactate, acetate, tosylate, palmitate, fumarate, tartrate, ascorbate, nitrate, formate, propionate, butyrate, isobutyrate, salicylate, oxalate, succinate, malate, glutamate, aspartate or gluconate; preferably, the pharmaceutically acceptable salts are hydrochloride salt, phosphate, mesylate, lactate, acetate, sulphate or hydrobromide; preferably, the pharmaceutically acceptable salt is hydrochloride salt; preferably, the pharmaceutically acceptable salts are present in a crystalline form; preferably, when the pharmaceutically acceptable salt is hydrochloride salt, the X-ray powder diffraction pattern of the crystalline form of the hydrochloride salt includes the diffraction peaks at 2 of 6.80.2, 9.50.2, 11.40.2, 15.00.2, 17.00.2, 19.90.2, 20.30.2, 20.60.2, 22.90.2, 23.60.2, 24.90.2, 26.10.2, 26.60.2; more preferably, the X-ray powder diffraction pattern of the crystalline form of the hydrochloride salt further includes the diffraction peaks at 2 of 12.00.2, 28.80.2, 29.10.2, 32.50.2, 34.70.2; further preferably, the X-ray powder diffraction pattern of the crystalline form of the hydrochloride salt is shown as FIG. 3.

10. The method according to claim 8, wherein the FLT3-related diseases or disorders are myelodysplastic disorders, such as thrombocytosis, essential thrombocytosis, idiopathic extramedullary metaplasia, myelofibrosis, myelofibrosis with myeloid metaplasia, chronic idiopathic myelofibrosis, polycythemia vera, hematopenia and malignant anterior spinal cord dysplasia syndrome; preferably, the FLT3-related diseases or disorders are selected from glioma, lung cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, esophageal cancer, colon cancer, pancreatic cancer, ovarian cancer, kidney cancer, thyroid cancer, neuron cancer and uterine cancer; preferably, the FLT3-related diseases or disorders are selected from leukemia, lymphoma and myeloma; preferably, the leukemia is selected from acute myeloid leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, chronic neutrophilic leukemia, acute undifferentiated cell leukemia, prolymphocytic leukemia, juvenile myelomonocytic leukemia, adult T-cell acute lymphocytic leukemia, acute myeloid leukemia with trilineage myelodysplasia, mixed lineage leukemia, and acute mononuclear leukemia; preferably, the lymphoma is non-Hodgkin lymphoma or Hodgkin lymphoma, for example degenerative large cell lymphoma; preferably, the plasma cell diseases comprise multiple myeloma, macroglobulinemia or heavy chain disease; preferably, the myeloma is selected from myelodysplastic syndrome, myelodysplastic disorder, multiple myeloma and spinal cord sarcoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

[0044] FIG. 1 shows the .sup.1HNMR spectrum of the hydrochloride salt of the compound shown in formula (I) prepared in Example 2;

[0045] FIG. 2 shows the mass spectrum of the hydrochloride salt of the compound shown in formula (I) prepared in Example 2; and

[0046] FIG. 3 shows the X-ray powder diffraction pattern of the crystalline form of the hydrochloride salt of the compound shown in formula (I) prepared in Example 2.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] Hereinafter, the present invention will be further illustrated in conjunction with the specific examples. It should be understood that the examples of the present invention are only used for explaining the present invention, rather than limiting the scope of the present invention. The experimental methods without specific conditions in the following examples are usually carried out according to the conventional conditions or the conditions of suggested by manufacturers.

[0048] Unless otherwise defined, all professional and scientific terms in the description have the same meaning that is familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the method of the present invention. The preferred implementation method and materials herein are only used for demonstration.

EXAMPLE 1

Preparation of the Compound Shown in Formula (I)

1) Preparation of tert-butyl 4-((5-amino-pyrimidinyl-4-oxy) methyl)piperidine-1-carboxylate (SM-3)

[0049] ##STR00003##

[0050] At room temperature (25 C.), NaH (71 mg, 2.94 mmol) was added to tert-butyl 4-hydroxymethyl-piperidine-1-carboxylate (SM-2) (574 mg, 2.67 mmol) in THF (tetrahydrofuran) (10 mL) and stirred for 1 hour, and then 4-bromopyrimidine-5-amine (SM-1) (348 mg, 2.67 mmol) was added thereinto.

[0051] The reactant was heated to 100 C. under nitrogen protection, stirred for 4 hours, and then concentrated in a vacuum rotatory evaporator at room temperature (20-30 C). The residue after concentration was purified by silica gel chromatography (the used eluent was: 10-30% ethyl acetate/petrol) to give tert-butyl 4-((5-amino-pyrimidinyl-4-oxy) methyl) piperidine-1-carboxylate (SM-3) (370 mg, 1.2 mmol).

2) Preparation of tert-butyl 4-((5-(5-amino-2-(2,6-difluorophenyl)thiazole-4-carboxamide)pyrimidine-4-oxy)methyl)piperidine-1-carboxylate (SM-5)

[0052] ##STR00004##

[0053] A mixture of compound (SM-3) (52 mg, 0.169 mmol), compound 5-amino-2-(2,6-difluorophenyl) thiazole-4-carboxylic acid (1E) (SM-4) (40 mg, 0.169 mmol), HATU (77 mg, 0.203 mmol) and DIEA (93 L, 0.507 mmol) in DMF (5 mL) was stirred for 1 hour at 50 C., diluted with ethyl acetate (50 mL) after cooling and then washed with saturated salt water. The organic phase was concentrated in a vacuum rotatory evaporator at room temperature (20-30 C.) after drying with Na.sub.2SO.sub.4. The residue after concentration was purified by silica gel chromatography (the used eluent was: 10-30% ethyl acetate/petrol) to give tert-butyl 4-((5-(5-amino-2-(2,6-difluorophenyl) thiazole-4-carboxamide) pyrimidine-4-oxy) methyl) piperidine-1-carboxylate (SM-5) (32 mg, 0.0585 mmol).

3) Preparation of 5-amino-2-(2,6-difluorophenyl)-N-(4-(piperidine-4-methoxy)pyrimidine-5-) thiazole-4-formamide (Formula I)

[0054] ##STR00005##

[0055] At room temperature (25 C.), TFA (trifluoracetic acid) (0.5 mL) was added to compound (SM-5) (21 mg, 0.0384 mmol) in CH.sub.2Cl.sub.2 (1 mL), stirred for 10 minutes, and then concentrated in a vacuum rotatory evaporator at room temperature (25 C.) , the residue was dissolved in CH.sub.2Cl.sub.2 (10 mL), washed respectively with 1 equivalent NaOH (5 mL) and saturated salt water (5 mL), and the organic phase was dried with Na.sub.2SO.sub.4, and then concentrated in a vacuum rotatory evaporator at room temperature (25 C). to give the product of 5-amino-2-(2,6-difluorophenyl)-N-(4-(piperidine-4-methoxy) pyrimidine-5-) thiazole-4-formamide (the compound shown in formula (I)) (11 mg, 0.0246 mmol).

[0056] 1H NMR (400 MHz, CD3OD): ppm 1.26-1.29 (m, 2H), 1.91-1.94 (m, 2H), 2.04-2.12 (m, 1H), 2.66-2.69 (m, 2H), 3.10-3.13 (m, 2H), 3.90-3.98 (m, 2H), 6.99-7.02 (m, 2H), 7.31-7.41 (m, 1H), 8.33 (s, 1H), 9.40 (s, 1H). MS (ESI) 447m/z (M+H).sup.+.

EXAMPLE 2

Synthesis of Hydrochloride Salt of the Compound Shown in Formula (I)

[0057] 50 mg of the compound shown in Formula (I) prepared in EXAMPLE 1 (0.11 mmol), 2 ml methanol and 8 ml dichloromethane were added into a reaction bottle, stirred until the solution became clear at 2030 C., 165 l of 1N methanol solution of hydrochloric acid (0.165 mmol) was added in one portion, the solution became muddy slowly, stirred for half an hour at 2030 C., filtered,and then the filter cake was dried under vacuum at 50 C. to obtain 30 mg of almost white solid with a yield of 56.5%, mp: 236.1239.4 C.

[0058] The .sup.1HNMR (400 MHz, DMSO-d.sub.6) spectrum of the obtained almost white solid was shown as FIG. 1, the mass spectrum was shown as FIG. 2, wherein m/z: 446.9 [(M-HCl)+H]+.

[0059] The obtained almost white solid is a hydrochloride salt of the compound shown in Formula (I), which is in a crystalline form, and the X-ray powder diffraction pattern of the crystal was shown as FIG. 3, wherein the detecting condition is shown as below and the detecting results are listed in Table 1:

[0060] Detecting apparatus: Bruker D8AdvanceX-ray diffractometer

[0061] Detecting conditions: target material was Cu, 2 scan started at 3.000, 2 scan ended at 40.000, the voltage was 40 KV, the current was 40 mA, Ka1=1.54060, Ka2=1.54439, Ka2/Ka1=0.5, Ka=1.54186.

TABLE-US-00001 TABLE 1 X-ray Diffraction Pattern Datum of the Crystalline Form of Hydrochloride Salt of Compound Shown in Formula (I) No. Angle 2 Counts Intensity (%) 1 6.8 1590 100 2 9.5 747 47 3 11.4 760 47.8 4 12.0 391 24.6 5 15.0 957 60.2 6 17.0 692 43.5 7 19.9 659 41.4 8 20.3 547 34.4 9 20.6 846 53.2 10 22.9 491 30.9 11 23.6 642 40.4 12 24.9 635 39.9 13 26.1 870 54.7 14 26.6 861 54.2 15 28.8 475 29.9 16 29.1 597 37.5 17 32.5 365 23 18 34.7 345 21.7

EXAMPLE3

Synthesis of Phosphate of the Compound Shown in Formula (I)

[0062] 50 mg of the compound shown in Formula (I) prepared in EXAMPLE 1 (0.11 mmol), 2 ml methanol and 8 ml dichloromethane were added into a reaction bottle, stirred until the solution became clear at 2030 C., a mixed solution of 5 mg phosphoric acid (0.051 mmol) and 0.5 ml methanol was added in one portion, the solution became muddy slowly, stirred for half an hour at 2030 C., filtered, and then the filter cake was dried under vacuum at 50 C. to obtain 50 mg of almost white solid with a yield of 94.9%, mp: 222.3224.8 C. , m/z: 446.9[MH.sub.3PO.sub.4)+H]+.

EXAMPLE 4

Synthesis of Mesylate of the Compound Shown in Formula (I)

[0063] 50 mg of the compound shown in Formula (I) prepared in EXAMPLE 1 (0.11 mmol), 2 ml methanol and 8 ml dichloromethane were added into a reaction bottle, stirred until the solution became clear at 2030 C., a mixed solution of 16 mg methanesulfonic acid (0.166 mmol) and 0.5 ml methanol was added in one portion, the solution became muddy slowly, stirred for half an hour at 2030 C., the solvent was evaporated under reduced pressure at 50 C., 10 ml dichloromethane was added and stirred for half an hour at 2030 C., filtered, and then the filter cake was dried under vacuum at 50 C. to obtain 37 mg of almost white solid with a yield of 62%, mp: 242.1246.7 C., m/z: 446.9[(MCH.sub.4O.sub.3S)+H]+.

EXAMPLE 5

Synthesis of Lactate of the Compound Shown in Formula (I)

[0064] 50 mg of the compound shown in Formula (I) prepared in EXAMPLE 1 (0.11 mmol), 2 ml methanol and 8 ml dichloromethane were added into a reaction bottle, stirred until the solution became clear at 2030 C., a mixed solution of 15 mg lactic acid (0.167 mmol) and 0.5 ml methanol was added in one portion, the solution was clear, stirred for half an hour at 2030 C., the solvent was evaporated under reduced pressure at 50 C., 10 ml dichloromethane was added and stirred for half an hour at 2030 C., filtered, and then the filter cake was dried under vacuum at 50 C. to obtain 36 mg of almost white solid with a yield of 61%, mp: 210.1213.6 C., m/z: 446.9[(MC.sub.3H.sub.6O.sub.3)+H]+.

EXAMPLE 6

Synthesis of Acetate of the Compound Shown in Formula (I)

[0065] 50 mg of the compound shown in Formula (I) prepared in EXAMPLE 1 (0.11 mmol), 2 ml methanol and 8 ml dichloromethane were added into a reaction bottle, stirred until the solution became clear at 2030 C., a mixed solution of 10 mg acetic acid (0.167 mmol) and 0.5 ml methanol was added in one portion, the solution was clear, stirred for half an hour at 2030 C., the solvent was evaporated under reduced pressure at 50 C., 10 ml dichloromethane was added and stirred for half an hour at 2030 C., filtered, and then the filter cake was dried under vacuum at 50 C. to obtain 31 mg of almost white solid with a yield of 55.6%, mp: 232.2233.7 C., m/z: 446.9 R[(MCH.sub.3COOH)+H]+.

EXAMPLE 7

Synthesis of Sulphate of the Compound Shown in Formula (I)

[0066] 50 mg of the compound shown in Formula (I) prepared in EXAMPLE 1 (0.11 mmol), 2 ml methanol and 8 ml dichloromethane were added into a reaction bottle, stirred until the solution became clear at 20-30 C., a mixed solution of 8 mg sulphuric acid (0.081 mmol) and 0.5 ml methanol was added in one portion, the solution became muddy slowly, stirred for half an hour at 20-30 C., filtered, and then the filter cake was dried under vacuum at 50 C. to obtain 53 mg of almost white solid with a yield of 88.5%, mp: 231.5236.9 C. , m/z: 446.9 [(MH.sub.2SO.sub.4)+H]+.

EXAMPLE 8

Synthesis of hydrobromide of the Compound Shown in Formula (I)

[0067] 50 mg of the compound shown in Formula (I) prepared in EXAMPLE 1 (0.11 mmol), 2 ml methanol and 8 ml dichloromethane were added into a reaction bottle, stirred until the solution became clear at 2030 C., a mixed solution of 30% 44 mg hydrobromic acid (0.163 mmol) and 0.5 ml methanol was added in one portion, the solution became muddy slowly, stirred for half an hour at 2030 C., filtered, then the filter cake was dried under vacuum at 50 C. to obtain 24 mg of almost white solid with a yield of 41.3%, mp:225.1227.7 C. , m/z: 446.9[(MHBr)+H]+.

EXAMPLE 9

Activity of Inhibiting FLT3 Kinase

[0068] The used abbreviations in the following experiments represent the following meanings:

[0069] HEPES: 4-( 2-hydroxyethyl)-1-piperazineethanesulfonic acid;

[0070] Brij-35: polyethylene glycol dodecyl ether;

[0071] DTT: dithiothreitol;

[0072] EDTA: ethylene diamine tetraacetic acid;

[0073] EGFR: epidermal growth factor receptor;

[0074] HER2: human epidermal growth factor receptor 2;

[0075] EGFRT790M: epidermal growth factor receptor T790M mutant;

[0076] Peptide FAM-P22: fluorescein labeled peptide 22;

[0077] ATP: adenosine triphosphate;

[0078] DMSO: dimethyl sulfoxide;

[0079] Staurosporine: staurosporine;

[0080] Coating Reagent #3: coating reagent #3.

[0081] 1. Preparation of 1 Kinase Buffer and Termination Buffer:

[0082] (1) MnCl.sub.2-free 1 kinase buffer: 50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl.sub.2, 2 mM DTT;

[0083] (2) Termination buffer: 100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA.

[0084] 2. Preparation of compounds for testing kinase: the compounds are diluted continuously.

[0085] (1) the compound was diluted to 50 times the maximum final concentration with 100% DMSO. 100 mL of the solution containing the compound at this concentration was transferred to one hole of 96-well plate;

[0086] (2)10 concentrations of compounds were prepared by diluting 20 mL original solution with 60 mL DMSO;

[0087] (3) 100 mL solution of 100% DMSO was added into two empty holes as compound-free control and enzyme-free control; (4) a transitional plate was prepared, 10 mL compounds at different concentrations were transferred from the original plate to the transitional plate respectively, 90 mL 1 kinase buffer was added, and then mixed under oscillation for 10 minutes;

[0088] (5) preparation of an experimental plate: 5 mL solution containing compounds was transferred from the hole of the transitional plate of the 96-well plate to the corresponding hole of 384-well plate.

[0089] 3. Kinase reaction

[0090] (1) Preparation of 2.5 enzyme solution: enzyme was added to the 1 kinase buffer;

[0091] (2) Preparation of 2.5 peptide solution: a fluorescein labeled peptide and ATP were added to the 1 kinase buffer;

[0092] (3) 10 mL 2.5 enzyme solution was added to an experimental 384-well plate containing the compound solution with a concentration of 10% containing 5 mL DMSO and incubated at room temperature for 10 minutes;

[0093] (4) 10 mL 2.5 peptide solution was added to an experimental 384-well plate;

[0094] (5) kinase reaction and termination: Incubation was carried out at 28 C. for certain time, 25 mL termination buffer was added to terminate the reaction.

[0095] 4. Detection of data

[0096] Data was read and collected.

[0097] 5. Fitting of curve

[0098] (1) detected data was copied and converted

[0099] (2) data was converted to inhibitory rate

Inhibitory rate=(Maximum ValueSample Value)/(Maximum ValueMinimum Value)*100;

wherein Maximum Value is the value of DMSO control; Minimum Value is the value of kinase-free control hole.

[0100] (3) the data was input an analysis software to obtain IC.sub.50 value.

[0101] The experimental results were shown as Table 2:

TABLE-US-00002 TABLE 2 Inhibiting Activity of Compounds of the Present Invention Against FLT3 Kinase Samples FLT3(IC.sub.50, nM) Compound of Formula (I) 40 (prepared in EXAMPLE 1) Hydrochloride salt of 15 compound of Formula (I) (prepared in EXAMPLE 2) Phosphate of compound of 17 Formula (I) (prepared in EXAMPLE 3) Mesylate of compound of 28 Formula (I) (prepared in EXAMPLE 4) Lactate of compound of 25 Formula (I) (prepared in EXAMPLE 5) Acetate of compound of 27 Formula (I) (prepared in EXAMPLE 6) Sulphate of compound of 28 Formula (I) (prepared in EXAMPLE 7) Hydrobromide of compound of 39 Formula (I) (prepared in EXAMPLE 8)

[0102] The results show that the compounds of the present invention had quite strong activities of inhibiting FLT3 in vitro.

EXAMPLE 10

Effects on Proliferation of Tumor Cells

[0103] This example tested the effects of the compounds of the present invention (the compound shown in Formula (I), hydrochloride salt, phosphate, mesylate, lactate, acetate, sulphate, and hydrobromide thereof prepared in EXAMPLES 1-8 respectively) on proliferation of tumor cells, and further evaluated the inhibitory effects of the compounds on proliferation of tumor cells and selectivity of the compounds in inhibiting proliferation of tumor cells.

[0104] The present invention selected the following cells:

[0105] (1) human acute monocytic leukemia cell strain MV4-11 (expressing FLT3/ITD mutant gene);

[0106] (2) human acute myeloid leukemia cell strain MOLM-13 (expressing FLT3/ITD mutant gene and wild-type FLT3 gene);

[0107] (3) human acute myeloid leukemia cell strain MOLM-14 (expressing FLT3/ITD mutant gene and wild-type FLT3 gene);

[0108] (4) human acute myeloid leukemia cell strain OCI-AML-3 (expressing FLT3A680V mutant gene);

[0109] (5) human acute myeloid leukemia cell strain U937 (expressing wild-type FLT3 gene);

[0110] (6) mouse TEL-BaF3-FLT3/ITD (stably expressing FLT3/ITD mutant activated kinase);

[0111] (7) mouse TEL-BaF3-FLT3-D835Y (stably expressing FLT3D835Y mutant activated kinase);

[0112] (8) mouse TEL-FLT3-BaF3 (stably expressing FLT3 kinase);

[0113] (9) mouse BaF3-FLT3-ITD-D835Y (stably expressing FLT3/ITD D835Y mutant activated kinase);

[0114] (10) mouse BaF3-FLT3-ITD-F691L (stably expressing FLT3/ITD F691L mutant activated kinase).

[0115] The compounds at different concentration (0.000508 M, 0.00152 M, 0.00457 M, 0.0137 M, 0.0411 M, 0.123 M, 0.370 M, 1.11 M, 3.33 M, 10 M in DMSO) were added to the above-mentioned cells respectively, incubated for 72 hours, Cell Titer-Glo (Promega, United States of America) Chemiluminescence Kit was used to quantify ATP in living cells to detect the number of living cells, the results were shown as Table 3

TABLE-US-00003 TABLE 3 Effects of Compounds on Proliferation of Cells IC50 (M) Hydro- Hydro- chloride Phosphate Mesylate bromide salt of of of Lactate of Acetate of Sulphate of of Compound compound compound compound compound compound compound compound of of of of of of of of Formula Formula Formula Formula Formula Formula Formula Formula Cell (I) (I) (I) (I) (I) (I) (I) (I) MV4-11 0.51 0.13 0.33 0.43 0.34 0.41 0.23 0.42 MOLM-13 0.52 0.12 0.31 0.45 0.28 0.43 0.23 0.51 MOLM-14 0.27 0.08 0.12 0.13 0.31 0.12 0.22 0.13 OCI-AML-3 0.34 0.05 0.12 0.06 0.08 0.32 0.21 0.11 U937 13.41 6.21 6.43 7.42 9.05 2.09 3.56 4.66 TEL-BaF3- 0.11 0.02 0.03 0.02 0.04 0.02 0.04 0.02 FLT3/ITD TEL-BaF3- 0.32 0.21 0.22 0.31 0.13 0.32 0.22 0.31 FLT3-D835Y TEL-FLT3-BaF3 0.12 0.03 0.02 0.05 0.06 0.03 0.05 0.16 BaF3-FLT3- 0.61 0.32 0.23 0.55 0.34 0.53 0.23 0.66 ITD-D835Y BaF3-FLT3- 0.93 0.45 0.76 0.77 0.64 0.94 0.56 0.76 ITD-F691L

EXAMPLE 11

Therapeutic Effects on Subcutaneous Xenograft Tumor of Human MV-4-11 in Nude Mice

[0116] Preparation of test samples: all samples were prepared with 5% anhydrous ethanol, 5% Cremophor EL and 90% physiological saline.

[0117] Animals: BALB/cA-nude mice, 6-7 weeks, female purchased from Shanghai Lingchang Biotechnology Co., Ltd. Production license number was SCK(Hu)2013-0018; animal certification number was 2013001810589.

[0118] Feeding environment: SPF Level.

[0119] Experiment steps: human acute myeloid leukemia cells MV-4-11 were inoculated subcutaneously in nude mice, after the tumor grows to 80-150 mm.sup.3, animals were randomly divided into groups with 6 mice each group. The dosage was 15 mg/kg, the drugs were administrated by intraperitoneal injection, 3 times a day for 21 days. The volume of tumor was measured, the weight of mouse was weighed and the data were recorded.

[0120] The formula for calculating the volume of tumor (V) was:

V=1/2ab.sup.2

[0121] Wherein a and b represented length and width respectively.

T/C (%)=(TT.sub.0)/(CC.sub.0)100

[0122] Wherein T and C were the volume of tumor when the experiment was completed; T.sub.0 and C.sub.0 were the volume of tumor when the experiment was started.

[0123] The inhibited rate of tumor was calculated, the results were shown as Table 4.

TABLE-US-00004 TABLE 4 Therapeutic Effects on MV-4-11 Cell of Subcutaneous Xenograft Tumor in Nude Mice Samples Inhibited Rate of Tumor (%) Compound of Formula (I) 66 (prepared in EXAMPLE 1) Hydrochloride salt of 86 compound of Formula (I) (prepared in EXAMPLE 2) Phosphate of compound of 69 Formula (I) (prepared in EXAMPLE 3) Mesylate of compound of 74 Formula (I) (prepared in EXAMPLE 4) Lactate of compound of 71 Formula (I) (prepared in EXAMPLE 5) Acetate of compound of 77 Formula (I) (prepared in EXAMPLE 6) Sulphate of compound of 68 Formula (I) (prepared in EXAMPLE 7) Hydrobromide of compound of 66 Formula (I) (prepared in EXAMPLE 8)

EXAMPLE 12

Preparation of Tablet

[0124] 10 g of hydrochloride salt of the compound of Formula (I) prepared in EXAMPLE 2, 40 g of microcrystalline cellulose, 100 g of lactose and 1 g of polyvinylpolypyrrolidone were collected and screened through 80 mesh respectively, mixed evenly, 2% HPMC solution was used as a binder, screened through 18 mesh, granulated, dried, screened through 20 mesh and then broken, 1 g of polyvinylpolypyrrolidone, 1 g of micro powder silica gel and 1 g of magnesium stearate were added, mixed for 10 min, tableted (0.16 g/tablet) to obtain a tablet.

EXAMPLE 13

Preparation of Capsule

[0125] 10 g of the compound of Formula (I) prepared in EXAMPLE 1, 40 g of microcrystalline cellulose, 100 g of lactose were collected and screened through 80 mesh respectively, mixed evenly, 1 g of sodium carboxyl methyl starch and 1 g of magnesium stearate were added, mixed for 10 min, and then filled into a 3.sup.# capsule to obtain a capsule.

EXAMPLE 14

Preparation of Injection

[0126] 30 g of acetate of the compound of Formula (I) prepared in EXAMPLE 6 and 50 g of glucose were added into 900 ml of water for injection, stirred to dissolve, water for injection was added to 1000 ml, stirred evenly, filtered through 0.22 m filter membrane, and then filled to ampoules at 1 ml/branch, sealed, sterilized under 121 C. for 20 minutes to obtain an injection.

EXAMPLE 15

Preparation of Lyophilized Preparation

[0127] 20 g of hydrochloride salt of the compound of Formula (I) prepared in EXAMPLE 2 and 50 g of mannitol were added into 900 ml of water for injection, stirred to dissolve, water for injection was added to 1000 ml, stirred evenly, filtered through 0.22 m filter membrane, and then filled to vials at 1 ml/branch, freeze-dried, sealed to obtain a lyophilized preparation.

EXAMPLE 16

Preparation of Oral Liquid

[0128] 20 g of mesylate of the compound of Formula (I) prepared in EXAMPLE 4, 200 g of sucrose and 1 g of flavor were added into 900 ml of water for injection, stirred to dissolve, water for injection was filled to 1000 ml, stirred evenly, filtered through 0.22 m filter membrane, and then filled to oral liquid bottle at 1 ml/branch to obtain an oral liquid preparation.