Heteroaromatic ring compounds, pharmaceutical compositions thereof and uses thereof

Abstract

The application relates to a heteroaromatic ring compound with a structure shown in formula (I) or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule thereof, and a pharmaceutical composition and application thereof. The compound can be used as an FLT3 kinase inhibitor, has excellent anti-proliferation activity on acute myeloid leukemia cells and Ba / F3-FLT3-ITD-TKD stable strain cells containing drug-resistant point mutations, and can overcome the acquired drug resistance of FLT3-F691L of existing clinical drugs. The heteroaromatic ring compound provided by the application can be used for preparing a drug for preventing or treating diseases (such as acute myeloid leukemia) mediated by FLT3 tyrosine kinase, and can be used for treating various malignant blood diseases of human beings and other mammals. (I).

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and relates to antitumor drugs, specifically heterocyclic aromatic compounds, their pharmaceutical compositions, and applications. Background Technology

[0002] FLT3 (Fms-like tyrosine kinase 3) is a transmembrane ligand-activated receptor tyrosine kinase, typically expressed by hematopoietic stem / progenitor cells, playing a crucial role in the early stages of bone marrow and lymphoid development. Generally, FLT3 ligands bind to the FLT3 extracellular domain receptor, activating FLT3, inducing receptor dimerization, and causing autophosphorylation of tyrosine residues within the kinase domain. This further activates downstream signaling pathways, primarily including RAS / RAF / MEK, PI3K / AKT, and JAK / STAT5, participating in the regulation of cell proliferation, differentiation, and cell survival. Mutations in the FLT3 gene lead to its abnormal activation, causing dimerization even without ligand binding, activating downstream signaling pathways, and resulting in abnormal proliferation of hematopoietic cells and lymphocytes, thereby triggering various malignant hematological diseases such as acute myeloid leukemia (AML).

[0003] In newly diagnosed AML patients, approximately 30% have FLT3 mutations, primarily classified into two types: internal tandem repeat mutations in the juxtamembrane domain (FLT3-ITD) and point mutations in the tyrosine kinase domain (FLT3-TKD). FLT3 mutations are highly associated with poor prognosis in AML patients, leading to reduced survival. First-generation FLT3 inhibitors exhibit low selectivity and lack specificity for FLT3, often causing off-target toxicity and adverse reactions, limiting clinical efficacy. Second-generation selective FLT3 inhibitors, giglitinib and quezatinib, were approved by the FDA in 2018 and 2023, respectively, and can overcome FLT3-ITD mutations. However, remission rates in patients with FLT3-TKD remain low, particularly with poor inhibition of the gate residue F691L mutation. Acquired resistance to second-generation FLT3 inhibitors has significantly reduced the clinical efficacy of existing drugs. Currently, there are no marketed inhibitors targeting the F691L mutation. Therefore, developing novel FLT3 inhibitors that effectively overcome acquired drug resistance has significant practical and scientific value. Summary of the Invention

[0004] Based on this, the purpose of this invention is to provide an FLT3 inhibitor that can overcome the F691L mutation.

[0005] The technical solutions for achieving the above objectives include the following.

[0006] In a first aspect, the present invention provides a heterocyclic compound having the structure shown in Formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof.

[0007]

[0008] (I)

[0009] Among them, each of W, Y, and Z is independently selected from CH and N;

[0010] R1 is selected from: H, C1~C6 alkyl, C1~C6 alkoxy, -NR2R3;

[0011] R2 is selected from: hydrogen, C1~C6 alkyl, C3~C8 alkyl, -S(=O)2R4;

[0012] R3 and R4 are each independently selected from: H, C1~C6 alkyl groups;

[0013] Ring A is selected from: one or more R5-substituted or unsubstituted C6~C rings. 10 A aryl group, or one or more R5-substituted or unsubstituted 5- to 10-membered heteroaryl groups;

[0014] Each R5 group is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, C6~C. 10 Aryl, (C1~C6 alkyl)2amino, (C1~C6 alkyl)amino, C1~C6 alkyl, C1~C6 alkoxy;

[0015] B is selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, C6~C 10 Aryl, 5-10 heteroaryl, C1-C6 alkyl, C1-C6 alkoxy, (C1-C6 alkyl)2amino, (C1-C6 alkyl)amino, halogen-substituted C1-C6 alkyl, cyano-substituted C1-C6 alkyl, carbamoyl, C3-C 12 Cycloalkyl, -X1-C(=O)-X2-R6, -X1-C(=NH)-X2-R6;

[0016] X1 and X2 are independently selected from: -N(R7)-, -CH(R8)-;

[0017] R6 is selected from: H, C1-C8 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, carbamoyl, cyano-substituted C1-C6 alkyl, cyano, one or more R'9 substituted or unsubstituted 3-14 membered heterocyclic groups, one or more R'9 substituted or unsubstituted C3-C6 alkyl groups. 14 Cycloalkyl, one or more R9-substituted or unsubstituted C6~C 10Aryl, one or more R9-substituted or unsubstituted 5- to 10-membered heteroaryl groups;

[0018] R7 and R8 are each independently selected from: H, C1~C6 alkyl groups;

[0019] Each R'9 is independently selected from: H, halogen, C1-C6 alkyl, C1-C6 alkoxy, (C1-C6 alkyl)2amino, (C1-C6 alkyl)amino, 3-14 membered heterocyclic group, C3-C 14 cycloalkyl;

[0020] Each R9 group is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, C1~C 10 Alkyl, C1~C 10 Alkoxy groups, (C1~C 10 alkyl)2amino, (C1~C 10 Alkyl)amine group, C3~C 12 cycloalkyl groups, 3-12 membered heterocyclic groups;

[0021] L1 is selected from: ethynyl group, one or more R groups 10 Substituted or unsubstituted C6~C 10 Aromatic, one or more R 10 Substituted or unsubstituted 5-10 heteroaryl groups;

[0022] Each R 10 Each group is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, (C1~C6 alkyl)2amino, (C1~C6 alkyl)amino, C1~C6 alkyl, C1~C6 alkoxy;

[0023] L2 is selected from: C1~C 12 Alkylene, 2-12 atom oxaalkylene, 2-12 atom azeaalkylene, C2-C 12 Unsaturated chain hydrocarbon groups;

[0024] L3 is selected from: One or more R 10 Substituted or unsubstituted 3- to 14-membered heterocyclic groups;

[0025] R 11 Selected from: H, C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl; n is selected from: 1, 2, 3, 4, 5, 6;

[0026] Q is selected from: -C(=O)R 12 -S(=O)R 12 -S(=O)2R 12 ;

[0027] R 12Selected from: one or more R 13 Substituted or unsubstituted C1-C6 alkyl groups ;

[0028] R 13 Selected from: hydrogen, deuterium, C1-C6 alkyl, halogen, cyano, amino, hydroxyl, hydroxyl-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, (C1-C6 alkyl)NH-substituted C1-C6 alkyl, (C1-C6 alkyl)2N-substituted C1-C6 alkyl;

[0029] R 14 R 15 R 16 Each of the following is independently selected from: hydrogen, deuterium, C1-C6 alkyl, halogen, cyano, hydroxy-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, (C1-C6 alkyl)NH-substituted C1-C6 alkyl, and (C1-C6 alkyl)2N-substituted C1-C6 alkyl.

[0030] In some embodiments, ring A is selected from one or more R5-substituted or unsubstituted phenyl groups.

[0031] In some embodiments, each R5 is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, phenyl, (C1~C3 alkyl)2amino, (C1~C3 alkyl)amino, C1~C3 alkyl, C1~C3 alkoxy.

[0032] In some implementations, Q is selected from: -C(=O)R 12 -S(=O)R 12 -S(=O)2R 12 ;R 12 Selected from: one or more R 13 Substituted or unsubstituted C1-C3 alkyl groups .

[0033] In some implementations, R 13 Selected from: hydrogen, deuterium, C1-C6 alkyl, halogen, cyano, amino, hydroxyl, hydroxyl-substituted C1-C3 alkyl, amino-substituted C1-C3 alkyl, (C1-C3 alkyl)NH-substituted C1-C3 alkyl, (C1-C3 alkyl)2N-substituted C1-C3 alkyl.

[0034] In some implementations, R 14 R 15 R 16Each is independently selected from: hydrogen, deuterium, C1-C3 alkyl, halogen, cyano, hydroxyl-substituted C1-C3 alkyl, amino-substituted C1-C3 alkyl, (C1-C3 alkyl)NH-substituted C1-C3 alkyl, (C1-C3 alkyl)2N-substituted C1-C3 alkyl, or R 14 With R 15 They connect to form C4-C6 cycloalkenyl groups.

[0035] In some implementations, Q is selected from:

[0036] , , , .

[0037] In some embodiments, the heterocyclic compound has the structure shown in formula (II) or formula (III):

[0038] .

[0039] In some embodiments, the heterocyclic compound has the structure shown in formula (IV) or formula (V):

[0040] .

[0041] In some implementations, R 14 R 15 R 16 Each is independently selected from: hydrogen, deuterium, methyl, ethyl, chlorine, fluorine, cyano, (C1-C3 alkyl)NH-substituted C1-C3 alkyl, (C1-C3 alkyl)2N-substituted C1-C3 alkyl, or R 14 With R 15 They connect to form C5-C6 cycloalkenyl groups.

[0042] In some implementations, R 14 R 16 Both are hydrogen, R 15 Selected from: hydrogen, cyano, .

[0043] In some embodiments, R1 is selected from: H, -NHR2, C1~C3 alkyl, C1~C3 alkoxy.

[0044] In some embodiments, R2 is selected from: hydrogen, C1~C4 alkyl, C3~C6 alkylalkyl, -S(=O)2R4, and R4 is selected from C1~C4 alkyl.

[0045] In some embodiments, each R2 is independently selected from: H, methyl, ethyl, propyl, butyl, cyclopropyl, propylsulfonyl.

[0046] In some embodiments, R1 is selected from: H, propylamino, ethylamino, methylamino, propylsulfonamide, methoxy, ethoxy, propoxy.

[0047] In some embodiments, B is selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, phenyl, 5-6 heteroaryl, C1-C3 alkyl, C1-C3 alkoxy, (C1-C3 alkyl)2amino, (C1-C3 alkyl)amino, halogen-substituted C1-C3 alkyl, cyano-substituted C1-C3 alkyl, carbamoyl, C5-C8 cycloalkyl, -X1-C(=O)-X2-R6, -X1-C(=NH)-X2-R6;

[0048] X1 and X2 are independently selected from: -N(R7)-, -CH(R8)-.

[0049] In some embodiments, R7 and R8 are each independently selected from H and C1-C3 alkyl groups.

[0050] In some embodiments, B is selected from: cyano, cyanomethyl, carbamoyl, -NH-C(=O)-NH-R6, -CH2-C(=O)-NH-R6, -NH-C(=NH)-NH-R6.

[0051] In some embodiments, R6 is selected from: H, C1-C4 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl, carbamoyl, cyano-substituted C1-C3 alkyl, cyano, one or more R'9 substituted or unsubstituted 3- to 8-membered heterocyclic groups, one or more R'9 substituted or unsubstituted C3- to C8 cycloalkyl, one or more R9 substituted or unsubstituted phenyl groups, and one or more R9 substituted or unsubstituted 5- to 6-membered heteroaryl groups.

[0052] In some embodiments, each R9 is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, C1-C6 alkyl, C1-C6 alkoxy, (C1-C6 alkyl)2amino, (C1-C6 alkyl)amino, C3-C8 cycloalkyl, 3-8 heterocyclic group.

[0053] In some embodiments, each R'9 is independently selected from: H, halogen, C1-C3 alkyl, C1-C3 alkoxy, (C1-C3 alkyl)2amino, (C1-C3 alkyl)amino, 3-8 membered heterocyclic group, C3-C8 cycloalkyl.

[0054] In some embodiments, R6 is selected from: H, methyl, ethyl, tert-butyl, methoxy, ethoxy, cyanomethyl, cyano, one or more R'9 substituted or unsubstituted cyclopropyl, one or more R'9 substituted or unsubstituted cyclobutyl, one or more R'9 substituted or unsubstituted cyclopentyl, one or more R'9 substituted or unsubstituted cyclohexyl, one or more R'9 substituted or unsubstituted tetrahydrofuranyl, one or more R'9 substituted or unsubstituted oxacyclobutyl, one or more R9 substituted or unsubstituted phenyl, one or more R9 substituted or unsubstituted 5-membered heteroaryl;

[0055] Preferably, each R9 is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, methyl, ethyl, propyl, butyl, C1~C3 alkoxy, (C1~C3 alkyl)2amino, (C1~C3 alkyl)amino, C5~C6 cycloalkyl, 5~6 heterocyclic group.

[0056] In some embodiments, B is selected from: cyano, , , , , , , , , , , , , , .

[0057] In some embodiments, L1 is selected from: acetylenic, one or more R groups. 10 Substituted or unsubstituted phenylene, one or more R 10 Substituted or unsubstituted 5-6 heteroaryl groups.

[0058] In some of these implementations, each R 10 Each is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, (C1~C3 alkyl)2amino, (C1~C3 alkyl)amino, C1~C3 alkyl, C1~C3 alkoxy.

[0059] In some embodiments, L2 is selected from: C1-C8 alkylene groups, 2-8 atoms of oxaalkylene groups, 2-8 atoms of aziralkylene groups, and C2-C8 unsaturated chain hydrocarbon groups.

[0060] In some implementations, L3 is selected from: One or more R 10 Substituted or unsubstituted 4- to 12-membered heterocyclic groups.

[0061] In some of these implementations, each R 10 Each is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, (C1~C3 alkyl)2amino, (C1~C3 alkyl)amino, C1~C3 alkyl, C1~C3 alkoxy.

[0062] In some implementations, R 11 Selected from: H, C1~C3 alkyl, hydroxyl-substituted C1-C3 alkyl; n is selected from: 1, 2, 3.

[0063] In some embodiments, L2 is selected from: C1-C4 alkylene groups, and 2-6 azaalkylene groups.

[0064] In some implementations, L3 is selected from: One or more R 10 Substituted or unsubstituted 5- to 10-membered heterocyclic groups.

[0065] In some implementations, L3 is selected from: One or more R 10 Substituted or unsubstituted 5- to 10-membered heterocyclic groups containing 1 to 3 nitrogen atoms.

[0066] In some of these embodiments, L2 is selected from methylene, ethylene, and propylene.

[0067] In some embodiments, L3-L2, which consists of L2 and L3, is selected from the following structures:

[0068] , 5- to 10-membered heterocyclic -C1- to C4 alkylene groups containing 1-3 nitrogen atoms;

[0069] m is selected from: 1, 2, 3, 4.

[0070] In some embodiments, L3-L2, which consists of L2 and L3, is selected from the following structures:

[0071] , , , .

[0072] Secondly, the present invention provides the use of the aforementioned heterocyclic compound, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, in the preparation of an FLT3 inhibitor; preferably, the FLT3 is a genetically mutated FLT3, preferably an FLT3-ITD mutation, an FLT3-TKD mutation; more preferably an FLT3-F691L mutation, an FLT3-D835Y mutation, an FLT3-D835Y / F691L mutation, an FLT3-ITD-D835Y mutation, or an FLT3-ITD-F691L mutation.

[0073] Thirdly, the present invention provides the use of the aforementioned heterocyclic aromatic compounds, or their pharmaceutically acceptable salts, or their stereoisomers, or their prodrug molecules, in the preparation of medicaments for treating and / or preventing FLT3-mediated diseases;

[0074] Preferably, the FLT3-mediated disease is a malignant hematological disease; the malignant hematological disease is preferably acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, or myeloproliferative disorder.

[0075] Fourthly, the present invention provides a pharmaceutical composition for the prevention or treatment of FLT3-mediated diseases, prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the heterocyclic aromatic compound of the present invention or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof.

[0076] The present invention has the following beneficial effects:

[0077] This invention provides a novel heterocyclic aromatic compound or its pharmaceutically acceptable salt or stereoisomer, which can act as a protein kinase inhibitor, exhibiting strong inhibitory activity against FLT3 mutant kinases. Furthermore, it demonstrates strong proliferative inhibitory activity against Ba / F3-FLT3-ITD cells containing resistance point mutations, specifically Ba / F3-FLT3-ITD-D835Y and Ba / F3-FLT3-ITD-F691L cells. This inhibitor can suppress the proliferation, migration, and invasion of various tumor cells, overcoming drug resistance in existing clinical drugs, particularly acquired resistance to FLT3-F691L. The aromatic heterocyclic compounds provided by this invention can be used to prepare drugs for the prevention or treatment of FLT3 kinase-mediated diseases, and can be used to treat various malignant hematological diseases in humans and other mammals, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and myeloproliferative disorders (MPD). Attached Figure Description

[0078] Figure 1The results show the in vivo anti-leukemia activity of compound KJB-C2 on the BaF3-FLT3-ITD-F691L cell transplantation model. In the figure, A is the weight of the mouse spleen, B is the body weight of the mouse, C, D, and E are the proportions of leukemia cells in peripheral blood, bone marrow, and spleen, respectively, and F is the survival time of the mouse. Detailed Implementation

[0079] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0080] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0081] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0082] In this invention, when any variable (e.g., R2, etc.) appears more than once in any component, the definition of each occurrence is independent of the definitions of other occurrences. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. Lines drawn from substituents into the ring system indicate that the bond referred to can be attached to any substituted ring atom. If the ring system is polycyclic, it means that such a bond is attached only to any suitable carbon atom of a neighboring ring. It will be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent itself is substituted by more than one group, it should be understood that these groups can be on the same carbon atom or different carbon atoms, as long as structural stability is achieved.

[0083] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including both branched and straight-chain groups with a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups with 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0084] The term "alkylene" refers to a group that is one hydrogen atom less than the corresponding group. For example, the term "alkylene" refers to a group that is one hydrogen atom less than "alkyl", such as -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc.

[0085] The term "unsaturated chain hydrocarbon group" refers to a branched or straight-chain unsaturated aliphatic hydrocarbon group with a specific number of carbon atoms, i.e., a non-cyclic chain hydrocarbon group, and the carbon chain contains one or more carbon-carbon double bonds or carbon-carbon triple bonds, such as: -CH=CH-, -CH2CH=CH-, -CH2CH=CHCH2-, -CH=CHCH2CH2-, -(CH2)2(CH=CH)(CH2)2CH2-, -CH2CH=CHCH2CH=CHCH2-, etc.

[0086] The term "alicyclic hydrocarbon group" refers to cycloalkyl or cycloalkenyl groups. Specifically, cycloalkyl refers to a saturated monocyclic, bicyclic, or polycyclic cyclic hydrocarbon group whose ring atoms are composed of carbon atoms. Bicyclic or polycyclic groups include spirocyclic, fused, and bridged rings. For example, "cycloalkyl" includes, but is not limited to, the following groups: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. , , , , , etc. Cycloalkenyl groups refer to partially unsaturated monocyclic, bicyclic, or polycyclic cyclic hydrocarbon groups whose ring atoms are composed of carbon atoms. Bicyclic or polycyclic groups include spirocyclic, fused, and bridged rings. For example, "cycloalkenyl" includes, but is not limited to, the following groups: , wait.

[0087] The term "alkoxy" refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0088] The term "heterocyclic alkyl" or "heterocyclic group" refers to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic cyclic substituent (including spirocyclic, bridged, fused, and fused rings), wherein one or more ring atoms are selected from heteroatoms of N, O, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Examples include: morpholinyl, piperidinyl, tetrahydropyrrolyl, pyrrolylalkyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazoleyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiopheneyl, dihydrotriazolyl, dihydroazacyclobutane, tetrahydrofuranyl, tetrahydrothiopheneyl, etc. , , , , , And so on, and their N-oxides. The connection of heterocyclic substituents can be achieved through carbon atoms or through heteroatoms.

[0089] The term "heteroaryl" or "heteroary ring" refers to an aromatic ring containing one or more heteroatoms selected from O, N, or S. Heteroaryl groups within the scope of this invention include, but are not limited to: quinolinyl, pyrazolyl, pyrroloyl, thiophene, furanyl, pyridinyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, benzimidazolyl, benzoxazolyl, pyridinimidazolyl, benzopyrimidinyl, etc. "Heteroaryl" is also understood to include any N-oxide derivative of a nitrogen-containing heteroaryl group. The linkage of heterocyclic substituents can be achieved through carbon atoms or through heteroatoms.

[0090] The term "halogen" or "halogen" refers to chlorine, fluorine, bromine, and iodine.

[0091] The pharmaceutical compositions of the present invention are prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the heterocyclic aromatic compounds of the present invention or their pharmaceutically acceptable salts or stereoisomers or their prodrug molecules.

[0092] This invention includes the free forms of compounds of formulas I-V, as well as their pharmaceutically acceptable salts and stereoisomers. Some specific exemplary compounds in this invention are amine compounds. The term "free form" refers to an amine compound in its non-salt form. Pharmaceutically acceptable salts include not only exemplary salts of the specific compounds described herein, but also typical pharmaceutically acceptable salts of the free forms of all compounds of formulas I-V. The free forms of specific salts of said compounds can be separated using techniques known in the art. For example, the free form can be regenerated by treating the salt with a suitable dilute aqueous solution of an alkali, such as dilute aqueous solution of NaOH, potassium carbonate, dilute ammonia, or sodium bicarbonate. The free form may differ somewhat from its respective salt form in certain physical properties, such as solubility in polar solvents, but for the purposes of this invention, such acid salts and alkali salts are otherwise pharmaceutically equivalent to their respective free forms.

[0093] Pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention containing either a basic or acidic moiety using conventional chemical methods. Typically, salts of basic compounds are prepared by ion-exchange chromatography or by reacting a free base with a stoichiometric or excess amount of the desired salt form of an inorganic or organic acid in a suitable solvent or a combination of solvents. Similarly, salts of acidic compounds are formed by reacting with a suitable inorganic or organic base.

[0094] Therefore, pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting an alkaline compound of the present invention with an inorganic or organic acid. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc., and also include salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pyric acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethylsulfonic acid, trifluoroacetic acid, etc.

[0095] If the compounds of this invention are acidic, then a suitable "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic alkali, including inorganic and organic bases. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganese salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases, including salts of primary, secondary, and tertiary amines, wherein substituted amines include naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxycobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, guanidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, aminobutanetriol, etc.

[0096] Since the deprotonated acidic portion of the compound, such as the carboxyl group, can be anionic under physiological conditions, and this charge can then be balanced by the protonated or alkylated basic portion, such as the tetravalent nitrogen atom, which carries a cation, it should be noted that the compounds of the present invention are potential internal salts or zwitterions.

[0097] The present invention provides a pharmaceutical composition or method for the prevention and / or treatment of FLT3-mediated diseases, comprising (administered to a patient or subject) an active ingredient (i.e., the heterocyclic compound described in this invention, or its pharmaceutically acceptable salt, stereoisomer, or prodrug molecule), and pharmaceutically acceptable excipients within a safe and effective range, and pharmaceutically acceptable amounts of the active ingredient. When administering the drug, a safe and effective amount of the active ingredient is given to the mammal (e.g., a human) requiring treatment, wherein the dose administered is a pharmaceutically considered effective dose. Of course, the specific dose should also consider factors such as the route of administration and the patient's health condition, which are within the scope of a skilled physician's expertise.

[0098] "Safe and effective dose" means that the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects.

[0099] "Pharmaceutical acceptable excipients" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0100] "Compatibility" here refers to the ability of the components in the composition to interact with and blend with the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients.

[0101] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0102] There are no particular limitations on the administration of the active ingredients or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0103] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0104] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components:

[0105] (a) Fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silica;

[0106] (b) Adhesives, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0107] (c) Moisturizers, such as glycerin;

[0108] (d) Disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginate, certain complex silicates, and sodium carbonate;

[0109] (e) Slow solvents, such as paraffin;

[0110] (f) Absorption accelerators, for example, quaternary ammonium compounds;

[0111] (g) Wetting agents, such as cetyl alcohol and glyceryl monostearate;

[0112] (h) Adsorbents, such as kaolin; and

[0113] (i) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0114] The solid dosage form can also be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient from this composition can be delayed in a portion of the digestive tract. Examples of suitable encapsulating components are polymers and waxes.

[0115] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0116] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0117] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0118] The present invention will be further described in detail below with reference to specific embodiments.

[0119] Example 1: Preparation of (S,E)-N-(1-((5-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-4-(dimethylamino)-N-methylbut-2-enamide (compound KJB-C1)

[0120]

[0121] Step 1: Preparation of ethyl 2-(4-((4-bromo-2-nitrophenyl)amino)phenyl)ethyl acetate (compound M1)

[0122]

[0123] 4-Bromo-1-fluoro-2-nitrobenzene (3.3 g, 15 mmol) and ethyl 4-aminophenylacetate (3.5 g, 20 mmol) were dissolved in 20 mL of N,N-dimethylacetamide in a 200 mL round-bottom flask. The mixture was microwaved to 160 °C and reacted for 30 minutes. The reaction solution was then added to water (200 mL), and the mixture was extracted three times with ethyl acetate. The solvent was removed under reduced pressure, and the mixture was stirred with silica gel and subjected to column chromatography to obtain 6.1 g of a red oily substance (yield: 87%). 1 H NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.21 (d, J= 2.4 Hz, 1H), 7.61 (dd, J = 9.2, 2.4 Hz, 1H), 7.34 – 7.25 (m, 4H), 7.09 (d,J = 9.2 Hz, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.68 (s, 2H), 1.21 (d, J = 7.1 Hz, 3H). LC-MS (ESI) m / z 380.0 [M+H] + .

[0124] Step 2: Preparation of ethyl 2-(4-((2-amino-4-bromophenyl)amino)phenyl)ethyl acetate (compound M2)

[0125]

[0126] Compound M1 (3.1 g, 8 mmol) was dissolved in 40 mL of anhydrous ethanol, and reduced iron powder (4.4 g, 80 mmol) and 2 mL of glacial acetic acid were added. The mixture was stirred at 80 °C for 1 hour, extracted 2-3 times with ethyl acetate and water, and the organic layer was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively. After drying with anhydrous Na2SO4, the solvent was removed under reduced pressure, and column chromatography was performed to give 2.2 g of white solid (yield: 77%). 1 H NMR (400 MHz, DMSO-d6) δ 7.11 (s, 1H), 7.04 (d, J = 8.4 Hz, 2H), 6.94 – 6.87 (m, 2H), 6.70 (d, J = 8.4 Hz, 2H), 6.65 (dd, J = 8.3, 2.3 Hz, 1H), 5.06 (s, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.50 (s,2H), 1.18 (t, J = 7.1 Hz, 3H). LC-MS (ESI) m / z 350.0 [M+H]+ .

[0127] Step 3: Preparation of ethyl 2-(4-(5-bromo-1H-benzo[d]imidazol-1-yl)phenyl)ethyl acetate (compound M3)

[0128]

[0129] Compound M2 (2.1 g, 6 mmol) was dissolved in 30 mL of trimethyl orthoformate, and p-toluenesulfonic acid (51.6 mg, 0.3 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, an aqueous sodium bicarbonate solution was added, followed by extraction with ethyl acetate three times. The solvent was removed under reduced pressure, and the mixture was dried and used directly in the next reaction step. 1 H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H),8.00 (d, J = 1.9 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.6 Hz, 1H),7.53 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 1.9 Hz, 1H), 4.12 (d, J = 7.1 Hz, 2H), 3.81 (s, 2H), 1.22 (t, J = 7.1 Hz, 3H). LC-MS (ESI) m / z 360.2 [M+H] + .

[0130] Step 4: Preparation of 2-(4-(5-bromo-1H-benzo[d]imidazol-1-yl)phenyl)acetic acid (compound M4)

[0131]

[0132] The crude product from the previous step was dissolved in a 1:1 mixture of tetrahydrofuran and water (50 mL), and lithium hydroxide (4.3 g, 10 mmol) was added. The mixture was stirred at 100 °C for 1 hour. After the reaction was complete, the pH of the system was adjusted to 2 with 4 M hydrochloric acid solution, and 1.6 g of a white solid precipitated out (total yield of two steps: 80%). 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s,1H), 7.99 (d, J = 1.9 Hz, 1H), 7.59 (dd, J = 13.2, 8.3 Hz, 3H), 7.51 (d, J =8.1 Hz, 2H), 7.46 (dd, J = 8.6, 1.9 Hz, 1H), 3.67 (s, 2H). LC-MS (ESI) m / z229.0 [MH] + .

[0133] Step 5: Preparation of 2-(4-(5-bromo-1H-benzo[d]imidazol-1-yl)phenyl)-N-(5-(tert-butyl)isoxazol-3-yl)acetamide (compound M5)

[0134]

[0135] Compound M4 (250 mg, 0.6 mmol) was dissolved in 10 mL of anhydrous N,N-dimethylformamide. 5-(tert-butyl)isoxazole-3-amine (100.9 mg, 0.72 mmol), HATU (456 mg, 1.2 mmol), and N,N-diisopropylethylamine (232 mg, 1.8 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The solution was purified by column chromatography to give 210 mg of a white solid (yield: 61%). 1 H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 8.61 (s, 1H), 7.99 (d, J = 1.9 Hz, 1H), 7.64 (d, J= 8.2 Hz, 2H), 7.61 – 7.54 (m, 3H), 7.46 (dd, J = 8.6, 1.9 Hz, 1H), 6.59 (s,1H), 3.80 (s, 2H), 1.28 (s, 9H). LC-MS (ESI) m / z 453.1 [M+H] + .

[0136] Step 6: Preparation of (S)-(1-oxo-1-(pent-4-yn-1-ylamino)prop-2-yl)carbamate tert-butyl ester (compound S1-1)

[0137]

[0138] N-(tert-Butoxycarbonyl)-N-methyl-L-alanine (304.5 mg, 1.5 mmol) and pentyl-4-yn-1-amine hydrochloride (119 mg, 1.0 mmol) were dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF), and HATU (760 mg, 2 mmol) and N,N-diisopropylethylamine (388 mg, 3 mmol) were added. The mixture was reacted at room temperature for 12 hours. After the reaction was completed, the reaction solution was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. Column chromatography yielded 316 mg of a colorless oil (yield: 79%). 1 H NMR (400 MHz, DMSO-d6) δ 7.79 (t, J = 5.7 Hz, 1H), 4.37 (m, 1H), 3.10 (m, 2H), 2.77 (t, J = 2.7 Hz, 1H), 2.74 (s, 3H), 2.15 (m, 2H), 1.58 (m,2H), 1.39 (s, 9H), 1.22 (d, J = 6.9 Hz, 3H).

[0139] Step 7: Preparation of (S)-(1-((5-(1-(4-(2-((5-(tert-butyl)isoxazo-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)(methyl)carbamate (compound M6)

[0140]

[0141] Compound M5 (113 mg, 0.25 mmol) and compound S1-1 (84 mg, 0.5 mmol) were dissolved in DMF, and cuprous iodide (5 mg, 10% mmol), Pd(PPh3)4 (17.3 mg, 5% mmol) and diisopropylamine (1 mL) were added. The reaction was carried out under an argon atmosphere at 100 °C for 2 h. After the reaction was completed, the reaction solution was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous Na2SO4, the solvent was removed under reduced pressure, and the solution was purified by column chromatography to give 94 mg of white solid (yield: 44%). 1H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 7.86 (t, J = 5.7 Hz, 1H), 7.77 – 7.53 (m, 5H), 7.43(d, J = 8.3 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 6.59 (s, 1H), 4.40 (m, 1H), 3.80 (s, 2H), 3.22 (m, 2H), 2.75 (s, 3H), 2.44 (t, J = 7.1 Hz, 2H), 1.81 –1.55 (m, 2H), 1.39 (s, 9H), 1.28 (s, 9H), 1.24 (d, J = 8.3 Hz, 3H). LC-MS(ESI) m / z 641.1 [M+H] + .

[0142] Step 8: Preparation of (S,E)-N-(1-((5-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-4-(dimethylamino)-N-methylbut-2-enamide (compound KJB-C1)

[0143]

[0144] Compound M6 (64 mg, 0.1 mmol) was dissolved in 4 M dioxane hydrochloride solution (2 mL) and stirred at room temperature for 1 hour. After the reaction was complete, the solvent was removed under reduced pressure and used directly in the next step. The compound prepared in the previous step was dissolved in 10 mL of anhydrous DMF, and (E)-4-(dimethylamino)but-2-enoic acid (24.8 mg, 0.15 mmol), EDCI (38 mg, 0.2 mmol), and triethylamine (303 mg, 0.3 mmol) were added. The reaction was carried out at 50 °C for 12 hours. After the reaction was complete, the reaction solution was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. Column chromatography yielded 31 mg of a colorless oil (yield: 47%). 1H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 8.60 (s, 1H), 7.89 (t, J =6.3 Hz, 1H), 7.78 (d, J = 1.5 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.60 – 7.53(m, 3H), 7.34 (dd, J = 8.4, 1.5 Hz, 1H), 6.67 – 6.52 (m, 3H), 5.00 (m, 1H), 3.79 (s, 2H), 3.20 (m, 2H), 3.04 (m , 2H), 2.43 (t, J = 7.1 Hz, 2H), 2.16 (d,J = 6.4 Hz, 6H), 1.75 – 1.67 (m, 2H), 1.28 (s, 9H), 1.23 (d, J = 7.2 Hz, 3H).HRMS (ESI) for C 37 H 46 N7O4 [M+H] + : calcd, 652.3606, found. 652.3597.

[0145] Example 2: Preparation of (S)-N-(1-((5-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yne-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C2)

[0146]

[0147] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 8.60 (s, 1H), 7.78 (s, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.57 (t, J = 7.4 Hz, 3H), 7.34 (d, J = 8.5 Hz, 1H), 6.76 (dd, J = 16.6, 10.5 Hz, 1H), 6.59 (s, 1H), 6.18 – 6.07 (m, 1H), 5.68 (m, 1H), 5.00 (q, J = 7.2 Hz, 1H), 3.79 (s, 2H),3.22 (m, 2H), 2.95 (s, 3H), 2.44 (t, J = 7.2 Hz, 2H), 1.71 (m, 2H), 1.28 (s,9H), 1.25 (d, J = 7.5 Hz, 3H). HRMS (ESI) for C 34 H 39 N6O4 [M+H] +: calcd,595.3027, found. 595.3021.

[0148] Example 3: Preparation of (S)-N-(1-((5-(1-(4-cyanophenyl)-1H-benzis[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C3)

[0149]

[0150] Step 1: Preparation of 4-((4-bromo-2-nitrophenyl)amino)benzonitrile (compound M1-1)

[0151]

[0152] The synthesis method is the same as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.26 (d,J = 2.4 Hz, 1H), 7.85 – 7.72 (m, 3H), 7.46 (d, J = 9.0 Hz, 1H), 7.37 – 7.29(m, 2H). LC-MS (ESI) m / z 317.5 [M+H] + .

[0153] Step 2: Preparation of 4-((2-amino-4-bromophenyl)amino)benzonitrile (compound M1-2)

[0154]

[0155] The synthesis method is the same as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ 8.05 (s, 1H), 7.53 –7.47 (m, 2H), 6.97 – 6.90 (m, 2H), 6.72 – 6.64 (m, 3H), 5.19 (s, 2H). LC-MS(ESI) m / z 288.3 [M+H] + .

[0156] Step 3: Preparation of 4-(5-bromo-1H-benzo[d]imidazol-1-yl)benzonitrile (compound M1-3)

[0157]

[0158] The synthesis method is the same as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.17 –8.11 (m, 2H), 8.03 (d, J = 1.9 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.71 (d, J= 8.7 Hz, 2H), 7.53 (dd, J = 8.7, 1.9 Hz, 1H). LC-MS (ESI) m / z 298.0 [M+H] + .

[0159] Step 4: Preparation of (S)-(1-((5-(1-(4-cyanophenyl)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)(methyl)carbamate-4-(5-bromo-1H-benzo[d]imidazol-1-yl)benzonitrile (compound M1-4)

[0160]

[0161] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.12 (d,J = 8.2 Hz, 2H), 7.95 (d, J = 8.2 Hz, 2H), 7.86 (t, J = 5.8 Hz, 1H), 7.74 (d,J = 8.6 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 4.39 (m, 1H), 3.21 (m, 2H), 2.75(s, 3H), 2.44 (t, J = 7.2 Hz, 2H), 1.72 (t, J = 7.0 Hz, 2H), 1.39 (s, 9H),1.25 (d, J = 7.1 Hz, 3H). LC-MS (ESI) m / z 486.1 [M+H] + .

[0162] Step 5: Preparation of (S)-N-(1-((5-(1-(4-cyanophenyl)-1H-benzis[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C3)

[0163]

[0164] The synthesis method is the same as in Example 1. 1 H NMR (600 MHz, DMSO-d6) δ 8.74 (s, 1H), 8.12 (d,J = 8.5 Hz, 2H), 7.95 (d, J = 8.5 Hz, 2H), 7.92 (t, J = 6.7 Hz, 1H), 7.81 (d,J = 1.5 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.41 – 7.35 (m, 1H), 6.75 (dd, J =11.9, 4.8 Hz, 1H), 6.14 (dd, J = 16.6, 2.6 Hz, 1H), 5.71 (dd, J = 10.5, 2.4Hz, 1H), 5.00 (q, J = 7.1 Hz, 1H), 3.22 (td, J = 11.5, 5.8 Hz, 2H), 2.95 (s,2H), 2.78 (s, 1H), 2.47 – 2.41 (m, 2H), 1.75 – 1.67 (m, 2H), 1.25 (d, J = 7.2Hz, 3H). HRMS (ESI) for C 28 H 32 N5O3 [M+H] +: calcd, 440.2081, found. 440.2077.

[0165] Example 4: Preparation of (S)-N-(1-((5-(1-(4-cyanophenyl)-6-(propylamino)-1H-benzi[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C4)

[0166]

[0167] Step 1: Preparation of (2-bromo-5-fluoro-4-nitrophenyl)carbamate tert-butyl ester (compound M2-1)

[0168]

[0169] 2-Bromo-5-fluoro-4-nitroaniline (660 mg, 2 mmol) was dissolved in dichloromethane, and Boc₂O anhydride (654 mg, 1.5 mmol) and DMAP (24 mg, 0.2 mmol) were added. The reaction was carried out at room temperature for 1 hour. After the 2-bromo-5-fluoro-4-nitroaniline starting material disappeared, the solvent was removed under reduced pressure, the residue was dissolved in acetonitrile, lithium bromide (172 mg, 2 mmol) was added, and the reaction was carried out at 50 °C for 4 hours. After the reaction was completed, the reaction solution was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous Na₂SO₄, the solvent was removed under reduced pressure, and the solution was purified by column chromatography to give 628 mg of white solid (yield: 94%). 1 H NMR (400 MHz, DMSO-d6) δ8.89 (s, 1H), 8.40 (d, J = 7.9 Hz, 1H), 7.95 (d, J = 14.0 Hz, 1H), 1.50 (s,9H). LC-MS (ESI) m / z 335.3 [M+H] + .

[0170] Step 2: Preparation of (2-bromo-5-fluoro-4-nitrophenyl)(propyl)carbamate tert-butyl ester (compound M2-2-1)

[0171]

[0172] Compound 2-1 (334 mg, 1 mmol) was dissolved in DMF at 0 °C. NaH (60 mg, 1.5 mmol, 60%) was added under argon protection, and the mixture was stirred for 15 minutes. Iodopropane (225 mg, 1.5 mmol) was then added dropwise to the reaction mixture, and the temperature was raised to 50 °C. The reaction was allowed to proceed for 4 hours. After the reaction was complete, water was added to quench the reaction mixture. The reaction mixture was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The purified solid was obtained by column chromatography, yielding 318 mg of a white solid (yield: 84%). 1 H NMR (400 MHz, DMSO-d6) δ 8.23 ​​(d, J = 8.1 Hz, 1H), 7.87 (d, J = 14.0 Hz, 1H), 4.05 (m, 2H), 1.72 (m, 2H), 1.42 (s, 9H), 1.05 (t, J = 7.1 Hz, 3H). LC-MS (ESI) m / z 377.8[M+H] + .

[0173] Step 3: Preparation of (2-bromo-5-((4-cyanophenyl)amino)-4-nitrophenyl)(propyl)carbamate tert-butyl ester (compound M2-3-1)

[0174]

[0175] The synthesis method is the same as in Example 1. 1 H NMR (400 MHz, CDCl3) δ 9.40 (s, 1H), 8.51 (s,1H), 7.71 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H), 3.50 (m, 2H), 1.37(m, 9H), 1.29 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H). LC-MS (ESI) m / z 746.0 [M+H] + .

[0176] Step 4: Preparation of (4-amino-2-bromo-5-((4-cyanophenyl)amino)phenyl)(propyl)carbamate tert-butyl ester (compound M2-4-1)

[0177]

[0178] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 7.52 (d,J = 8.5 Hz, 2H), 7.04 (s, 1H), 6.86 (s, 1H), 6.68 (d, J = 8.6 Hz, 2H), 5.21(s, 2H), 3.58 (m, 2H), 1.41 (m, 2H), 1.29 (s, 9H), 0.82 (t, J = 7.4 Hz, 3H).LC-MS (ESI) m / z 445.3 [M+H] + .

[0179] Step 5: Preparation of (4-amino-2-bromo-5-((4-cyanophenyl)amino)phenyl)(propyl)carbamate tert-butyl ester (compound M2-5-1)

[0180]

[0181] The synthesis method is the same as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.11 (m,3H), 7.93 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 12.6 Hz, 1H), 3.72 – 3.42 (m,2H), 1.64 – 1.40 (m, 2H), 1.25 (s, 9H), 0.83 (q, J = 8.3 Hz, 3H). LC-MS (ESI)m / z 455.1 [M+H] + .

[0182] Step 5: Preparation of (S)-(1-((5-(6-((tert-butoxycarbonyl)(propyl)amino)-1-(4-cyanophenyl)-1H-benzimidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)(methyl)carbamate (compound M2-6-1)

[0183]

[0184] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, DMSO-d6) δ 8.75 (s, 1H), 8.13 (d,J = 8.5 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 7.83 (m, 2H), 7.55 (s, 1H), 3.54 –4.24 (m, 1H), 3.54 – 3.51 (m, 2H), 3.21 – 3.17 (m, 2H), 2.75 (s, 3H), 2.40(m, 2H), 1.70 – 1.68 (m, 2H), 1.40 (s, 9H), 1.55 – 1.45 (m, 2H), 1.25 (d,12H), 0.81 (t, J = 7.6 Hz, 3H). LC-MS (ESI) m / z [M+H] + = 643.3.

[0185] Step 6: Preparation of (S)-N-(1-((5-(1-(4-cyanophenyl)-6-(propylamino)-1H-benzi[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C4)

[0186]

[0187] The synthesis method is the same as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ 8.37 (s, 1H), 8.11 (d,J = 8.7 Hz, 2H), 7.91 (d, J = 8.7 Hz, 2H), 7.58 (s, 1H), 6.76 (m, 1H), 6.14(m, 1H), 5.73 – 5.58 (m, 1H), 5.23 (t, J = 5.7 Hz, 1H), 5.01 (m, 1H), 3.26(m, 2H), 3.13 (m, 2H), 2.87 (m, 3H), 1.77 – 1.67 (m, 2H), 1.63 (m, 2H), 1.35– 1.22 (m, 5H), 0.94 (t, J = 7.4 Hz, 3H). HRMS (ESI) for C 29 H 32 N6O2 [M+H] +:calcd, 497.2660, found. 497.2655.

[0188] Example 5: Preparation of (S)-N-(1-((5-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-6-(propylamino)-1H-benzi[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methacrylamide (compound KJB-C5)

[0189]

[0190] The synthesis method is the same as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.59(s, 1H), 8.01 (s, 1H), 7.63 (m, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.30 (d, J =8.1 Hz, 2H), 6.74 (m, 1H), 6.60 (s, 1H), 6.14 (dd, J = 16.8, 2.4 Hz, 1H), 5.71 (dd, J = 10.4, 2.5 Hz, 1H), 5.01 (m, 1H), 3.80 (m, 2H), 3.32 (s, 2H),2.76 (m, 5H), 1.73 (m, 2H), 1.28 (s, 9H), 1.23 (m, 4H), 1.06 (t, J = 7.4 Hz, 3H). LC-MS (ESI) m / z 653.3[M+H] + .

[0191] Example 6: Preparation of (S)-N-(1-((4-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)phenethyl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C6)

[0192]

[0193] Step 1: Preparation of (S)-(1-((4-bromophenylethyl)amino)-1-oxopropane-2-yl)(methyl)carbamate tert-butyl ester (compound S2-1)

[0194]

[0195] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, DMSO-d6) δ 7.45 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 8.3 Hz, 2H), 4.15 (m, 1H), 2.89 (m, 3H), 2.73 (d, J = 7.4Hz, 2H), 2.62 (d, J = 7.4 Hz, 2H), 1.05-0.91 (m, 12H). LC-MS (ESI) m / z 433.2[M+H] + .

[0196] Step 2: Preparation of (S)-(1-oxo-1-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenethyl)amino)propyl-2-yl)carbamate tert-butyl ester (compound S2-2)

[0197]

[0198] Bis(pinacol)diboron (631 mg, 2.5 mmol) and compound S2-1 (478 mg, 1.25 mmol) were dissolved in 1,4-dioxane solvent. 2-Bicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (120 mg, 0.25 mmol), Pd2(dba)3 (119 mg, 0.13 mmol), and potassium acetate (244 mg, 2.5 mmol) were added. The reaction mixture was carried out overnight at 90°C under an argon atmosphere. After the reaction was complete, the reaction solution was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The solution was purified by column chromatography to give 381 mg of a white solid (yield: 71%). 1 H NMR (400 MHz, DMSO-d6) δ7.78 (s, 1H), 7.59 (d, J = 7.5Hz, 2H), 7.21 (d, J = 7.6 Hz, 2H), 4.62 – 4.23(m, 1H), 3.32 – 3.19 (m, 2H), 2.71 (m, 5H), 1.37 (s, 9H), 1.28 (s, 12H), 1.16(m, 3H). LC-MS (ESI) m / z 433.6[M+H] + .

[0199] Step 3: Preparation of (S)-(1-((4-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)phenethyl)amino)-1-oxopropane-2-yl)(methyl)carbamate (compound M3-1)

[0200]

[0201] Compound M5 (245 mg, 0.54 mmol) and compound S2-2 (181 mg, 0.65 mmol) were dissolved in 50% DMF aqueous solution. Sodium carbonate (229 mg, 2.16 mmol) and Pd(dppf)Cl2 (40 mg, 10% mmol) were added, and the mixture was reacted overnight at 130 °C under an argon atmosphere. After the reaction was complete, the reaction solution was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous Na2SO4, the solvent was removed under reduced pressure, and the solution was purified by column chromatography to give 109 mg of white solid (yield: 30%). 1 H NMR (400MHz, DMSO-d6) δ 9.01 (s, 1H), 8.79 (s, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.41 –7.32 (m, 4H), 7.24 (d, J = 8.2 Hz, 2H), 6.94 (m, 3H), 6.53 (s, 1H), 4.06 (m,1H), 3.07 (s, 2H), 2.96 (m, 2H), 2.89 – 2.81 (m, 5H), 1.46 (s, 9H), 1.04 (s,9H), 0.89 (m, 3H). LC-MS (ESI) m / z 679.9[M+H] + .

[0202] Step 4: Preparation of (S)-N-(1-((4-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)phenethyl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C6)

[0203]

[0204] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.55 (s,1H), 7.56 – 7.43 (m, 6H), 7.22 (d, J = 8.1 Hz, 2H), 6.94 – 6.72 (m, 4H), 6.50(s, 1H), 6.29 – 6.43 (m, 1H), 6.85 (m, 1H), 6.70 – 6.53 (m, 1H), 4.36 (m,1H), 3.07 (s, 2H), 2.96 (m, 2H), 2.68 (m, 3H), 1.50 (s, 9H), 1.21 – 0.90 (d,12H). LC-MS (ESI) m / z 634.3 [M+H] + .

[0205] Example 7: Preparation of (S)-N-(1-((5-(1-(4-cyanophenyl)-6-(propylsulfonamido)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C7)

[0206]

[0207] The synthesis method is the same as in Example 4. 1 H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 8.20 –8.16 (m, 3H), 8.02 – 7.97 (m, 2H), 7.93 (s, 1H), 7.88 (m, 1H), 6.76 (m, 1H),6.15 (m, 1H), 5.65 (m, 1H), 3.47 – 3.41 (m, 2H), 3.18 (m, 2H), 2.94 (m, 2H), 2.75 (s, 3H), 1.89 (m, 2H), 1.53 – 1.44 (m, 2H), 1.20 (m, 6H), 0.82 (d, J =7.5 Hz, 3H). LC-MS (ESI) m / z 560.9 [M+H] + .

[0208] Example 8: Preparation of (S)-N-(1-((5-(1-(4-cyanophenyl)-6-(propylsulfonamido)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C8)

[0209]

[0210] Step 1: Preparation of N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(5-(5-chloro-1-pentyn-1-yl)-1H-benzo[d]imidazol-1-yl)phenyl)acetamide (compound M5-1)

[0211]

[0212] Compound M5 (113 mg, 0.25 mmol) and 5-chloro-1-pentyne (51 mg, 0.5 mmol) were dissolved in DMF, and cuprous iodide (5 mg, 10% mmol), Pd(PPh3)4 (17.3 mg, 5% mmol) and diisopropylamine (1 mL) were added. The reaction was carried out under an argon atmosphere at 100 °C for 2 h. After the reaction was completed, the reaction solution was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous Na2SO4, the solvent was removed under reduced pressure, and the solution was purified by column chromatography to give 59 mg of yellow solid (yield: 49%). 1 H NMR (400MHz, CDCl3) δ 7.98 (s, 1H), 7.77 (s, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.35 (d, J= 8.1 Hz, 2H), 7.30 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 6.39 (s,1H), 3.83 (s, 2H), 3.2 (m, 2H), 2.53 (m, 2H), 1.65 (m, 2H), 1.26 (s, 9H). LC-MS (ESI) m / z 474.1 [M+H] + .

[0213] Step 2: Preparation of tert-butyl 4-(5-(1-(4-(2-((5-tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)piperazine-1-carboxylate (compound M5-2-1)

[0214]

[0215] Compound M5-1 (237 mg, 0.5 mmol) was dissolved in DMF, and 1-Boc-piperazine (186 mg, 1 mmol) and potassium carbonate (192 mg, 1.5 mmol) were added. The mixture was reacted at 80 °C for 12 h. After the reaction was complete, 100 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The solution was purified by column chromatography to give 201 mg of a yellow oil (yield: 64%). 1 H NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.75 (m, 3H), 7.40 (d,J = 8.4 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.33 (d, J = 8.1 Hz, 2H), 6.64 (s,1H), 3.85 (s, 2H), 3.30 (m, 4H), 2.75 (m, 4H), 2.46 (t, J = 7.2 Hz, 2H), 2.30(m, 2H),1.70 (m, 2H), 1.41 (s, 9H), 1.30 (s, 9H). LC-MS (ESI) m / z 625.9 [M+H] + .

[0216] Step 3: Preparation of 2-(4-(5-(5-(4-acryloylpiperazin-1-yl)pent-1-yn-1-yl)-1H-benzo[d]imidazol-1-yl)phenyl)-N-(5-(tert-butyl)isoxazol-3-yl)acetamide (compound KJB-C8)

[0217]

[0218] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.80 (s,1H), 7.46 – 7.44 (m, 2H), 7.44 – 7.31 (d, J = 8.3 Hz, 1H), 7.28 (m, 2H), 6.75(m, 1H), 6.50 (m, 1H), 6.40 (s, 1H), 5.65(m, 1H), 5.11 (m, 1H), 3.79 (s, 2H), 3.45 (m, 4H), 2.75 (m, 4H), 2.61 (m, 2H), 2.44 (t, J = 7.1 Hz, 2H), 1.71 (m,2H), 1.28 (s, 9H). LC-MS (ESI) m / z 578.3 [M+H] + .

[0219] Example 9: Preparation of 2-(4-(5-(5-(2-acryloyl-2,7-diazaspiro[3.5]nonane-7-yl)pent-1-yn-1-yl)-1H-benzo[d]imidazol-1-yl)phenyl)-N-(5-(tert-butyl)isoxazol-3-yl)acetamide (compound KJB-C9)

[0220]

[0221] The synthesis method is as described in Example 8. 1 H NMR (400 MHz, CDCl3) δ 7.93 (s, 1H), 7.85 (s,1H), 7.51 (m, 1H), 7.40 – 7.33 (m, 4H), 6.33 – 6.29 (m, 1H), 6.88 (s, 1H), 6.31 (m, 1H), 5.79 (m, 1H), 3.68 (d, 6H), 2.63 – 2.55 (m, 6H), 2.47 (m, 2H),1.73 (m, 2H), 1.65 – 1.60 (m, 4H), 1.30 (s, 9H).LC-MS (ESI) m / z 618.8 [M+H] + .

[0222] Example 10: Preparation of 2-(4-(5-(5-(4-acryloylpiperazin-1-yl)pent-1-yn-1-yl)-1H-benzi[d]imidazol-1-yl)phenyl)-N-(5-(tert-butyl)isoxazol-3-yl)acetamide (compound KJB-C10)

[0223]

[0224] The synthesis method is as described in Example 8. 1 H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 8.10 (d, J= 8.4 Hz, 2H), 7.97(d, J = 8.5 Hz, 2H), 7.76 (s, 1H), 7.69 (d, J = 8.5 Hz,1H), 7.39 (m, 1H), 6.56 (m, 1H), 6.27 (m, 1H), 5.70 (m, 1H), 5.08 (m, 1H), 3.48 (m, 4H), 2.71 (m, 4H), 2.47 (t, J = 7.2 Hz, 2H), 1.74 (m, 2H).

[0225] Example 11: Preparation of 4-(5-(5-(2-acryloyl-2,7-diazaspiro[3.5]nonane-7-yl)pent-1-yn-1-yl)-1H-benzo[d]imidazol-1-yl)benzonitrile (compound KJB-C11)

[0226]

[0227] The synthesis method is as described in Example 8. 1 H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.82 (d, J= 8.0 Hz, 1H), 7.75 – 7.68 (m, 3H), 7.65 (m, 2H), 7.50 (m, 1H), 6.36 (m, 1H),5.78 (m, 1H), 5.68 (m, 1H), 3.56 (s, 4H), 2.55 – 2.47 (m, 6H), 2.31 (t, J =7.1 Hz, 2H), 1.73 (m, 2H), 1.64 – 1.56 (m, 4H)..

[0228] Example 12: Preparation of (S)-N-(1-((5-(1-(4-(2-amino-2-oxoethyl)phenyl)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C12)

[0229]

[0230] Step 1: Preparation of 2-(4-(5-bromo-1H-benzo[d]imidazol-1-yl)phenyl)acetamide (compound M6-1)

[0231]

[0232] Under an argon atmosphere, ammonium chloride (80 mg, 1.5 mmol) was dissolved in 10 mL of anhydrous toluene. A toluene solution of trimethylaluminum (0.75 mL, 2 M toluene solution) was added dropwise at 0 °C. After the addition was complete, the mixture was transferred to room temperature and stirred for 2 h. Compound M3 (180 mg, 0.5 mmol) was dissolved in 2 mL of anhydrous toluene and added dropwise to the above reaction system. The reaction was carried out at 50 °C for 12 h. After the reaction was complete, a solid precipitated. The reaction was quenched by slowly adding 50 mL of 5% hydrochloric acid aqueous solution to the reaction system at 0 °C. The mixture was filtered, the filter cake was washed three times with water, and dried under vacuum to obtain compound M6-1 (yield: 78%). 1 H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 7.99 (d, J = 1.9 Hz, 1H), 7.68 – 7.58 (m, 3H), 7.57 –7.41 (m, 4H), 6.96 (s, 1H), 3.50 (s, 2H). LC-MS (ESI) m / z 329.8[M+H] + .

[0233] Step 2: Preparation of tert-butyl(S)-(1-((5-(1-(4-(2-amino-2-oxoethyl)phenyl)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)(methyl)carbamate (compound M6-2)

[0234]

[0235] The synthesis method is the same as in Example 1. 1 H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.87 (s,1H), 7.65 – 7.58 (m, 3H), 7.43 – 7.05 (m, 4H), 5.14 (m, 1H), 3.66 (s, 2H), 3.32 (m, 2H), 2.95 (s, 3H), 2.83 (m, 2H), 2.30 (m, 2H), 1.45 (s, 12H), 1.23(m, 3H). LC-MS (ESI) m / z 517.1 [M+H] + .

[0236] Step 3: Preparation of (S)-N-(1-((5-(1-(4-(2-amino-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C12)

[0237]

[0238] The synthesis method is the same as in Example 1. 1 H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.86 (s,1H), 7.50 – 7.28 (m, 4H), 6.75 (m, 3H), 6.38 (dd, J = 16.7, 1.8 Hz, 1H), 5.76(dd, J = 10.4, 1.7 Hz, 1H), 5.18 (m, 1H), 3.65 (s, 2H), 3.38 (m, 2H), 2.99(s, 3H), 2.91 (m, 2H), 2.43 (t, J = 7.1 Hz, 2H), 1.35 (s, 3H), 1.23 (d, J =2.8 Hz, 3H). LC-MS (ESI) m / z 472.2 [M+H] + .

[0239] Example 13: Preparation of (S)-N-methyl-N-(1-((5-(1-(4-(2-(methylamino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)acrylamide (compound KJB-C13)

[0240]

[0241] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 7.83 (s,1H), 7.48 – 7.44 (m, 2H), 7.43 – 7.29 (m, 4H), 6.77 (t, J = 6.2 Hz, 1H), 6.54(m, 1H), 6.41 – 6.22 (m, 2H), 5.72 (m, 1H), 5.18 (q, J = 7.1 Hz, 1H), 3.61(s, 2H), 3.38 (q, J = 6.7 Hz, 2H), 2.98 (s, 3H), 2.80 (d, J = 4.8 Hz, 3H),2.43 (t, J = 7.0 Hz, 2H), 1.80 (p, J = 7.0 Hz, 2H), 1.34 (d, J = 7.2 Hz, 3H).LC-MS (ESI) m / z 486.2[M+H] + .

[0242] Example 14: Preparation of (S)-N-(1-((5-(1-(4-(2-(ethylamino)-2-oxoethyl)phenyl)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C14)

[0243]

[0244] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 8.02 (s, 1H), 7.82 (s,1H), 7.40 (q, J = 8.3 Hz, 4H), 7.35 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.6 Hz,1H), 6.61 – 6.45 (m, 2H), 6.35 – 6.26 (m, 1H), 5.72 – 5.58 (m, 2H), 5.12 (q,J = 7.2 Hz, 1H), 3.56 (s, 2H), 3.34 (d, J = 7.0 Hz, 2H), 3.28 – 3.20 (m, 2H),2.93 (s, 3H), 2.38 (t, LC-MS (ESI) m / z 500.3[M+H] + .

[0245] Example 15: Preparation of (S)-N-(1-((5-(1-(4-(2-(tert-butylamino)-2-oxoethyl)phenyl)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C15)

[0246]

[0247] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 8.08 (s, 1H), 7.87 (s,1H), 7.44 (d, J = 2.6 Hz, 4H), 7.40 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.5 Hz,1H), 6.61 (t, J = 6.0 Hz, 1H), 6.55 (dd, J = 16.8, 10.4 Hz, 1H), 6.35 (d, J =16.7 Hz, 1H), 5.74 (d, J = 10.4 Hz, 1H), 5.49 (s, 1H), 5.18 (q, J = 7.2 Hz,1H), 3.53 (s, 2H), 3.39 (d, J LC-MS (ESI) m / z528.5[M+H] + .

[0248] Example 16: Preparation of (S)-N-(1-((5-(1-(4-(2-(cyclopropylamino)-2-oxoethyl)phenyl)-1H-benzi[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C16)

[0249]

[0250] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 7.98 (s, 1H), 7.77 (s,1H), 7.38 (d, J = 8.0 Hz, 2H), 7.34 – 7.28 (m, 3H), 7.24 (d, J = 8.8 Hz, 1H), 6.78 (t, J = 5.9 Hz, 1H), 6.66 – 6.54 (m, 1H), 6.48 (dd, J = 16.8, 10.4 Hz,1H), 6.26 (d, J = 16.2 Hz, 1H), 5.66 (d, J = 10.4 Hz, 1H), 5.12 (d, J = 7.1Hz, 1H), 3.51 (s, 2H), 3.32 (d, J = 6.8 Hz, 2H), 2.92 (s, 3H), 2.66 (m, 1H), 2.36 (t, J = 7.1 Hz, 2H), 1.77 – 1.71 (m, 2H), 1.28 (d, J = 7.1 Hz, 3H), 0.72– 0.63 (m, 2H), 0.42 (m, 2H). LC-MS (ESI) m / z 512.3[M+H] + .

[0251] Example 17: Preparation of (S)-N-(1-((5-(1-(4-(2-(cyclobutylamino)-2-oxoethyl)phenyl)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C17)

[0252]

[0253] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 7.99 (s, 1H), 7.78 (s,1H), 7.39 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.4 Hz,1H), 7.25 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 5.8 Hz, 1H), 6.53 – 6.45 (m, 1H), 6.40 (s, 1H), 6.27 (d, J = 16.8 Hz, 1H), 5.66 (d, J = 10.5 Hz, 1H), 5.13 (q,J = 7.1 Hz, 1H), 4.32 (m, 1H), 3.51 (s, 2H), 3.32 (d, J = 6.7 Hz, 2H), 2.92(s, 3H), 2.37 (t, J = 7.1 Hz, 2H), 2.24 (m, 2H), 1.85 – 1.69 (m, 4H), 1.65 –1.57 (m, 2H), 1.28 (d, J = 7.3 Hz, 3H). LC-MS (ESI) m / z 525.9[M+H] + .

[0254] Example 18: Preparation of (S)-N-methyl-N-(1-((5-(1-(4-(2-(oxecyclobutane-3-ylamino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yne-1-yl)amino)-1-oxopropane-2-yl)acrylamide (compound KJB-C18)

[0255]

[0256] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 8.51 (d, J = 8.2 Hz, 2H), 8.13 (s, 1H), 7.84 (s, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.44 (d, J = 8.3Hz, 1H), 7.34 (m, 2H), 6.54 – 6.46 (m, 1H), 6.32 (m, 1H), 5.71 (d, J = 10.4Hz, 1H), 5.12 (d, J = 7.8 Hz, 1H), 3.95 – 3.93 (m, 1H), 3.34 (s, 2H), 3.19(t, J = 6.2 Hz, 1H), 3.10 (d, J = 6.7 Hz, 1H), 2.93 (s, 3H), 2.47 (m, 1H), 2.40 (m, 1H), 2.28 (s, 3H), 1.06 (d, J = 7.4 Hz, 3H). LC-MS (ESI) m / z 528.2[M+H] + .

[0257] Example 19: Preparation of (S)-N-(1-((5-(1-(4-(2-(cyclopentylamino)-2-oxoethyl)phenyl)-1H-benzi[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C19)

[0258]

[0259] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 7.99 (s, 1H), 7.78 (s,1H), 7.39 (d, J = 8.0 Hz, 2H), 7.36 – 7.28 (m, 3H), 7.27 – 7.23 (m, 1H), 6.77(t, J = 5.8 Hz, 1H), 6.48 (dd, J = 16.8, 10.4 Hz, 1H), 6.27 (d, J = 16.8 Hz,1H), 6.24 – 6.16 (m, 1H), 5.66 (d, J = 10.4 Hz, 1H), 5.12 (d, J = 7.2 Hz,1H), 4.13 (q, J = 7.1 Hz, 1H), 3.52 (s, 2H), 2.92 (s, 3H), 2.36 (t, J = 7.1Hz, 2H), 1.93 – 1.86 (m, 2H), 1.73 (t, J = 7.1 Hz, 2H), 1.56 (m, 2H), 1.52 –1.44 (m, 2H), 1.29 (m, 5H). LC-MS (ESI) m / z 540.1[M+H] + .

[0260] Example 20: Preparation of (S)-N-(1-((5-(1-(4-(2-((cyanomethyl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yne-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C20)

[0261]

[0262] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 7.96 (m, 1H), 7.76 (m,1H), 7.35 (d, J = 8.0 Hz, 2H), 7.30 – 7.21 (m, 3H), 7.11 (m, 1H), 6.76 (t, J= 5.8 Hz, 1H), 6.65 (s, 1H), 6.48 (dd, J = 16.8, 10.4 Hz, 1H), 6.26 (d, J =16.7 Hz, 1H), 6.03 (m, 1H), 5.66 (d, J = 10.6 Hz, 1H), 5.12 (q, J = 7.1 Hz,1H), 3.87 (d, J = 5.0 Hz, 2H), 3.57 (s, 2H), 3.31 (m, 2H), 2.93 (s, 3H), 2.36(t, J = 7.0 Hz, 2H), 1.73 (t, J = 7.1 Hz, 2H), 1.32 – 1.26 (m, 3H). LC-MS(ESI) m / z 511.0[M+H] + .

[0263] Example 21: Preparation of (S)-N-(5-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yn-1-yl)-2-(N-methylpropamido)propionamide (compound KJB-C21)

[0264]

[0265] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 10.54 (s, 1H), 8.03 (s,1H), 7.80 (s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.32(d, J = 8.4 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 6.72 (t, J = 5.8 Hz, 1H), 6.69(s, 1H), 5.15 (q, J = 7.1 Hz, 1H), 3.84 (s, 2H), 3.32 (d, J = 6.7 Hz, 2H),2.87 (s, 3H), 2.36 (t, J = 7.1 Hz, 2H), 2.30 (q, J = 7.4 Hz, 2H), 1.73 (t, J= 7.1 Hz, 2H), 1.26 (d, J = 4.1 Hz, 12H), 1.08 (t, J = 7.4 Hz, 3H). LC-MS(ESI) m / z 597.4[M+H] + .

[0266] Example 22: Preparation of (S)-N-(1-((5-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-6-methoxy-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methacrylamide (compound KJB-C22)

[0267]

[0268] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, CDCl3) δ 9.70 (s, 1H), 8.00 (s,1H), 7.86 (s, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 6.90(s, 1H), 6.77 (s, 1H), 6.71 (s, 1H), 6.55 (dd, J = 16.7, 10.3 Hz, 1H), 6.36 (dd, J = 16.8, 1.9 Hz, 1H), 5.73 (dd, J = 10.3, 1.9 Hz, 1H), 5.22 (m, 1H),3.89 (d, 5H), 3.46 (m, 2H), 3.02 (s, 3H), 2.53 (t, J = 7.0 Hz, 2H), 1.86 (t,J = 7.0 Hz, 3H), 1.36 (s, 9H), 1.29 (m, 3H). LC-MS (ESI) m / z 625.3 [M+H] + .

[0269] Example 23: Preparation of (S)-N-(1-((5-(1-(4-(2-cyanamido-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-N-methylacrylamide (compound KJB-C23)

[0270]

[0271] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 8.01 (s, 1H), 7.81 (s,1H), 7.41 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 7.8 Hz, 2H), 7.31 (d, J = 8.6 Hz,1H), 7.27 (d, J = 8.4 Hz, 1H), 6.50 (dd, J = 16.7, 10.4 Hz, 1H), 6.34 – 6.24(m, 1H), 5.72 – 5.66 (m, 1H), 5.12 (d, J = 7.2 Hz, 1H), 3.59 (d, J = 6.4 Hz,3H), 3.33 (d, J = 7.8 Hz, 2H), 2.92 (d, J = 10.1 Hz, 3H), 2.38 (t, J = 7.1Hz, 2H), 1.75 (t, J = 7.0 Hz, 2H), 1.20 (m, 3H). LC-MS (ESI) m / z 497.2 [M+H] + .

[0272] Example 24: Preparation of (S)-N-(1-((5-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzo[d]imidazol-5-yl)pent-4-yn-1-yl)amino)-3-hydroxy-1-oxopropane-2-yl)-N-methacrylamide (compound KJB-C24)

[0273]

[0274] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 9.46 (s, 1H), 8.06 (s,1H), 7.83 (s, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 8.7 Hz, 2H), 7.36 –7.26 (m, 2H), 6.67 (s, 1H), 6.33 – 6.27 (m, 1H), 6.01 (dd, J = 18.0, 11.3 Hz,1H), 5.82 – 5.67 (m, 2H), 5.39 – 5.30 (m, 1H), 4.53 (m, 2H), 3.81 (s, 2H),3.42 – 3.25 (m, 2H), 3.01 (s, 3H), 2.38 (m, 2H), 1.82 – 1.61 (m, 2H), 1.31 –1.21 (m, 9H), 1.18 (m, 3H). LC-MS (ESI) m / z 611.2 [M+H] + .

[0275] Example 25: Preparation of (S)-N-(1-((5-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-6-methoxy-1H-benzi[d]imidazol-5-yl)-but-4-yn-1-yl)amino)-3-hydroxy-1-oxopropane-2-yl)-N-methacrylamide (compound KJB-C25)

[0276]

[0277] The synthesis method is the same as in Example 1. 1H NMR (600 MHz, CDCl3) δ 10.12 (s, 1H), 7.92 (s,1H), 7.75 (s, 1H), 7.53 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.9 Hz, 2H), 6.81(s, 2H), 6.69 (s, 1H), 6.45 (dd, J = 16.8, 10.4 Hz, 1H), 6.34 – 6.17 (m, 2H), 5.99 (dd, J = 17.3, 10.4 Hz, 1H), 5.79 – 5.70 (m, 1H), 4.60 – 4.43 (m, 2H),3.84 (s, 2H), 3.78 (s, 3H), 3.51 – 3.31 (m, 2H), 3.01 (s, 3H), 2.45 (m, 2H), 1.78 (m, 2H), 1.27 (m, 12H). LC-MS (ESI) m / z 641.6 [M+H] + .

[0278] Example 26: Preparation of (S)-N-(1-((5-(1-(4-(2-((5-(tert-butyl)isoxazol-3-yl)amino)-2-oxoethyl)phenyl)-1H-benzimidazol-5-yl)-pent-4-yn-1-yl)amino)-1-oxopropane-2-yl)-2-cyano-N-methacrylamide (compound KJB-C26)

[0279]

[0280] The synthesis method is the same as in Example 1. 1H NMR (400 MHz, CDCl3) δ 9.64 (s, 1H), 8.12 (s,1H), 7.91 (d, J = 7.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.3 Hz,2H), 7.45 – 7.35 (m, 2H), 6.77 (s, 1H), 6.33 (m, 1H), 6.21 (m, 1H), 5.24 –5.02 (m, 1H), 3.91 (s, 3H), 3.59 (m, 1H), 3.52 – 3.36 (m, 2H), 3.08 (t, J =3.1 Hz, 2H) , 1.84 (m, 2H), 1.36 (m, 12H),. LC-MS (ESI) m / z 620.4 [M+H] + .

[0281] Example 27: IC50 of the compound against FLT3-ITD kinase 50 test

[0282] Kinase activity assay: LanthaScreen ® Eukinase binding assay is based on proprietary Alexa Fluor ® The binding and translocation of a 647-labeled ATP-competitive kinase inhibitor scaffold (kinase tracer) to the kinase was observed. Binding of the tracer to the kinase was detected using a europium (Eu)-labeled anti-tag antibody, which also binds to the kinase. Binding of both the tracer and antibody to the kinase resulted in a high fluorescence resonance energy transfer (FRET) pattern, with the Eu donor fluorophore transforming into the Alexa Fluor. ® 647 receptor fluorophore. Conversely, the inhibitor competes with the kinase for binding with the tracer, leading to the loss of FRET.

[0283] Enzymatic reaction: Add 5 μL of enzyme-antibody mixture to a 384-well plate, transfer 5 nL of the test compound (concentration gradient) using an Echo550 micro-volume liquid transfer system, vortex to mix and centrifuge, then add 5 μL of tracer, vortex to mix and centrifuge, and react at room temperature for 1 h.

[0284] Detection reaction: Add 2.5 µL of development solution (1:128 dilution) to each well and incubate at 37°C in the dark for 1 h, then add 5 µL of stop solution.

[0285] Plate reading: The Perkin Elmer EnVision Multimode Plate Reader was used to detect fluorescence signals (excitation wavelength 340 nm, emission wavelengths 665 nm and 615 nm).

[0286] Calculation: The inhibition rate of each well was calculated using the fully active wells and the control signal wells. The data analysis method is as follows:

[0287] Phosphorylation ratio = 1 – { (emission ratio × F100% – C100%) / [C0% – C100% + emission ratio × (F100% – F0%)]} × 100;

[0288] Inhibition rate = 100 × (1 – compound phosphorylation rate / negative control phosphorylation rate).

[0289] IC 50 The values ​​were calculated using medical graphing software (GraphPad Prism 5.0).

[0290] The results of the kinase activity test are shown in Table 1.

[0291] Example 28: IC50 of the compound against FLT3-ITD-F691L kinase 50 test

[0292] Kinase activity assay: using Z´-LYTE TM The technology (using fluorescence detection, enzyme-coupled formulation, based on the difference in sensitivity of phosphorylated and non-phosphorylated peptides to protein hydrolysis) employs the fluorescence resonance energy transfer (FRET) principle and uses Z'-LYTEK. TM FRET peptide substrates, secondary reaction detection of compounds for FLT3 D835Y / E69L Inhibitory activity of kinases.

[0293] Enzymatic reaction: 5 μL of enzyme-nonphosphorylated substrate system [50 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) pH 7.5, 0.01% BRIJ-35, 10 mM magnesium chloride (MgCl2), 1 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 2 µM Tyr O2 nonphosphorylated substrate] was added to a 384-well plate. 5 nL of the test compound (concentration gradient) was transferred using an Echo550 microfluidic pipette system. After shaking at room temperature for 15 min, 250 nL of ATP (final concentration 500 µM) was transferred to each well using the Echo550 microfluidic pipette system. After shaking and mixing, the mixture was centrifuged and reacted at 30°C in the dark for 1.5 h. Meanwhile, a separate 5 µL phosphorylated substrate system [50 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) pH 7.5, 0.01% BRIJ-35, 10 mM magnesium chloride (MgCl2), 1 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 2 µM Tyr O2 phosphorylated peptide substrate] was prepared as a control.

[0294] Detection of reaction: Add 2.5 µL of development solution (1:128 dilution) to each well and incubate at 37°C in the dark for 1 h, then add 5 µL of stop solution.

[0295] Plate reading: The Perkin Elmer EnWision Multimode Plate Reader was used to detect fluorescence signals (excitation wavelength 400 nm, emission wavelengths 460 nm and 535 nm).

[0296] Calculation: The inhibition rate of each well was calculated using the fully active wells and the control signal wells. The data analysis method is as follows:

[0297] Phosphorylation ratio = 1 – { (emission ratio × F100% – C100%) / [C0% – C100% + emission ratio × (F100% – F0%)]} × 100;

[0298] Inhibition rate = 100 × (1 – compound phosphorylation rate / negative control phosphorylation rate).

[0299] IC 50 The values ​​were calculated using medical graphing software (GraphPad Prism 5.0).

[0300] The results of the kinase activity test are shown in Table 1.

[0301] Table 1. Inhibition results of compounds on FLT3-ITD and FLT3-ITD-F691L kinases (IC50, 100%) 50 :nM)

[0302]

[0303] The data in Table 1 show that the novel benzimidazole compounds of the present invention have strong inhibitory activity against FLT3 mutant kinase.

[0304] Example 29: Study on the antiproliferative activity of the compound against AML cells expressing FLT3-ITD

[0305] MOLM-13 and MV4-11 cells were cultured in RPMI-1640 medium containing 10% FBS, penicillin (100 U / ml), and streptomycin (100 μg / ml) and incubated at 37°C in a 5% CO2 incubator. The compound was prepared to a concentration of 10 mM using DMSO and stored at -20°C. MOLM-13 and MV4-11 cells in good growth condition were seeded at appropriate cell densities in 96-well plates and treated with different concentrations (0-10 μM) of inhibitors for 48 hours. Then, 100 μl of CellTiter-Glo reagent was added to each well, incubated at room temperature for 30 minutes, and 50 μl was transferred to a 384-well plate. The corresponding fluorescence values ​​were detected using a microplate reader, and cell proliferation rate or cell viability was calculated based on the fluorescence values. The IC50 was calculated using a nonlinear fitting curve fitted with GraphPad Prism 8.0.1 software. 50 Values. Each measurement should be repeated at least three times.

[0306] The test results are shown in Table 2.

[0307] Table 2. Results of the antiproliferative activity test of the compounds against AML cells (IC50). 50 :nM)

[0308]

[0309] The data in Table 2 show that the compounds of the present invention have excellent antiproliferative activity against AML cell lines MV4-11 and MOLM-13.

[0310] Example 30: Study on the antiproliferative activity of the compound against Ba / F3-FLT3-ITD-TKD cells

[0311] FLT3-ITD, FLT3-ITD-D835Y, and FLT3-ITD-F691L plasmids were transfected into Ba / F3 cells to construct Ba / F3 cells dependent on FLT3-ITD-TKD growth. Well-grown Ba / F3-FLT3-ITD, Ba / F3-FLT3-ITD-D835Y, and Ba / F3-FLT3-ITD-F691L cells were seeded at appropriate cell densities in 96-well plates. After treatment with different concentrations of inhibitors (0-10 μM) for 48 hours, 100 μl of CellTiter-Glo reagent was added to each well, and the cells were incubated at room temperature for 30 minutes. After mixing, 50 μl of the incubated solution was transferred to 384-well plates, and the corresponding fluorescence values ​​were detected using a microplate reader. Cell proliferation rate or cell viability was calculated based on the fluorescence values. The IC50 was calculated using nonlinear fitting curves with GraphPad Prism 8.0.1 software. 50 Values. Each measurement should be repeated at least three times.

[0312] The test results are shown in Table 3.

[0313] Table 3. Results of the antiproliferative activity assay of the compounds against BaF3-FLT3-ITD-TKD cells (IC50). 50 :nM)

[0314]

[0315]

[0316] The data in Table 3 show that the compounds of the present invention have excellent anti-proliferative activity against Ba / F3-FLT3-ITD and Ba / F3-FLT3-ITD-TKD cells, while exhibiting low cytotoxicity against normal Ba / F3 cells.

[0317] Example 31: In vivo antileukemic activity of compound KJB-C2 against Ba / F3-FLT3-ITD-F691L cell transplantation model

[0318] Collect Ba / F3-FLT3-ITD-F691L cells in good growth condition at a quantity of 5 × 10⁻⁶. 5Each mouse was injected intravenously via tail vein into a Balbc mouse and then randomly divided into four groups: a methylcellulose solvent control group, a Quizartinib group (10 mg / kg / d), a Gilteritinib group (30 mg / kg / d), and two KJB-C2 groups (one group at 10 mg / kg / d and the other at 25 mg / kg / d). Administration began two days later. Ten days after treatment, peripheral blood was collected from the orbital sinus for flow cytometry analysis of the proportion of leukemia cells (BaF3-FLT3-ITD-F691L cells). Ten days after treatment, three mice from each group were randomly euthanized, and their spleens and bone marrow were harvested for flow cytometry analysis of the leukemia cell proportion. The spleens were also photographed and weighed. Mice in all four treatment groups continued to be administered the drug via gavage daily until the control group mice began to die. Administration was then discontinued to observe the survival and mortality of the mice and to record survival time.

[0319] The results are as follows Figure 1 As shown, Figure 1 Data shows that: Figure 1 As indicated by A, compound KJB-C2 can significantly reduce the infiltration of AML cells in spleen tissue; Figure 1 As indicated by B, compound KJB-C2 had no significant effect on mouse body weight during administration, suggesting low potential toxicity. Figure 1 As shown in C, D, and E, compound KJB-C2 can significantly eliminate leukemia cells in the peripheral blood, spleen, and bone marrow of mice, and its anti-leukemia effect is significantly superior to the marketed positive control drugs Gilteritinib and Quizartinib; Figure 1 From F, we can see that compound KJB-C2 can significantly prolong the survival time of mice.

[0320] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A heteroaromatic ring compound having the structure shown in Formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof. ； in, Each of W, Y, and Z is independently selected from: CH and N; R1 is selected from: H, C1~C6 alkyl, C1~C6 alkoxy, -NR2R3; R2 is selected from: hydrogen, C1~C6 alkyl, C3~C8 alkyl, -S(=O)2R4; R3 and R4 are each independently selected from: H, C1~C6 alkyl groups; Ring A is selected from: one or more R5-substituted or unsubstituted C6~C rings. 10 A aryl group, or one or more R5-substituted or unsubstituted 5- to 10-membered heteroaryl groups; Each R5 group is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, C6~C. 10 Aryl, (C1~C6 alkyl)2amino, (C1~C6 alkyl)amino, C1~C6 alkyl, C1~C6 alkoxy; B is selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, C6~C 10 Aryl, 5-10 heteroaryl, C1-C6 alkyl, C1-C6 alkoxy, (C1-C6 alkyl)2amino, (C1-C6 alkyl)amino, halogen-substituted C1-C6 alkyl, cyano-substituted C1-C6 alkyl, carbamoyl, C3-C 12 Cycloalkyl, -X1-C(=O)-X2-R6, -X1-C(=NH)-X2-R6; X1 and X2 are independently selected from: -N(R7)-, -CH(R8)-; R6 is selected from: H, C1-C8 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, carbamoyl, cyano-substituted C1-C6 alkyl, cyano, one or more R'9 substituted or unsubstituted 3-14 membered heterocyclic groups, one or more R'9 substituted or unsubstituted C3-C6 alkyl groups. 14 Cycloalkyl, one or more R9-substituted or unsubstituted C6~C 10 Aryl, one or more R9-substituted or unsubstituted 5- to 10-membered heteroaryl groups; R7 and R8 are each independently selected from: H, C1~C6 alkyl groups; Each R'9 is independently selected from: H, halogen, C1-C6 alkyl, C1-C6 alkoxy, (C1-C6 alkyl)2amino, (C1-C6 alkyl)amino, 3-14 membered heterocyclic group, C3-C 14 cycloalkyl; Each R9 group is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, C1~C 10 Alkyl, C1~C 10 Alkoxy groups, (C1~C 10 alkyl)2amino, (C1~C 10 Alkyl)amine group, C3~C 12 cycloalkyl groups, 3-12 membered heterocyclic groups; L1 is selected from: ethynyl group, one or more R groups 10 Substituted or unsubstituted C6~C 10 Aromatic, one or more R 10 Substituted or unsubstituted 5-10 heteroaryl groups; Each R 10 Each group is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, (C1~C6 alkyl)2amino, (C1~C6 alkyl)amino, C1~C6 alkyl, C1~C6 alkoxy; L2 is selected from: C1~C 12 Alkylene, 2-12 atom oxaalkylene, 2-12 atom azeaalkylene, C2-C 12 Unsaturated chain hydrocarbon groups; L3 is selected from: One or more R 10 Substituted or unsubstituted 3- to 14-membered heterocyclic groups; R 11 Selected from: H, C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl; n is selected from: 1, 2, 3, 4, 5, 6; Q selection: -C(=O)R 12 -S(=O)R 12 -S(=O)2R 12 ; R 12 Selected from: one or more R 13 Substituted or unsubstituted C1-C6 alkyl groups ; R 13 Selected from: hydrogen, deuterium, C1-C6 alkyl, halogen, cyano, amino, hydroxyl, hydroxyl-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, (C1-C6 alkyl)NH-substituted C1-C6 alkyl, (C1-C6 alkyl)2N-substituted C1-C6 alkyl; R 14 R 15 R 16 Each is independently selected from: hydrogen, deuterium, C1-C6 alkyl, halogen, cyano, hydroxy-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, (C1-C6 alkyl)NH-substituted C1-C6 alkyl, and (C1-C6 alkyl)2N-substituted C1-C6 alkyl.

2. The heterocyclic compound according to claim 1, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, Ring A is selected from one or more R5-substituted or unsubstituted phenyl groups; Preferably, each R5 is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, phenyl, (C1~C3 alkyl)2amino, (C1~C3 alkyl)amino, C1~C3 alkyl, C1~C3 alkoxy.

3. The heterocyclic compound according to claim 1, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, Q selection: -C(=O)R 12 -S(=O)R 12 -S(=O)2R 12 ; R 12 Selected from: one or more R 13 Substituted or unsubstituted C1-C3 alkyl groups ; Preferably, R 13 Selected from: hydrogen, deuterium, C1-C6 alkyl, halogen, cyano, amino, hydroxyl, hydroxyl-substituted C1-C3 alkyl, amino-substituted C1-C3 alkyl, (C1-C3 alkyl)NH-substituted C1-C3 alkyl, (C1-C3 alkyl)2N-substituted C1-C3 alkyl; Preferably, R 14 R 15 R 16 Each is independently selected from: hydrogen, deuterium, C1-C3 alkyl, halogen, cyano, hydroxyl-substituted C1-C3 alkyl, amino-substituted C1-C3 alkyl, (C1-C3 alkyl)NH-substituted C1-C3 alkyl, and (C1-C3 alkyl)2N-substituted C1-C3 alkyl. Preferably, Q is selected from: 、 、 、 。 4. The heterocyclic compound according to claim 3, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, The heterocyclic compound has the structure shown in formula (II) or formula (III): ； Preferably, the heterocyclic compound has the structure shown in formula (IV) or formula (V): ； Preferably, R 14 R 15 R 16 Each is independently selected from: hydrogen, deuterium, methyl, ethyl, chlorine, fluorine, cyano, (C1-C3 alkyl)NH-substituted C1-C3 alkyl, (C1-C3 alkyl)2N-substituted C1-C3 alkyl, or R 14 With R 15 They connect to form C5-C6 cycloalkenes; Preferably, R 14 R 16 Both are hydrogen, R 15 Selected from: hydrogen, cyano, .

5. The heterocyclic compound according to any one of claims 1-4, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, R1 is selected from: H, -NHR2, C1~C3 alkyl, C1~C3 alkoxy; Preferably, R2 is selected from: hydrogen, C1~C4 alkyl, C3~C6 alkylalkyl, -S(=O)2R4, and R4 is selected from C1~C4 alkyl; Preferably, each R2 is independently selected from: H, methyl, ethyl, propyl, butyl, cyclopropyl, propylsulfonyl; Preferably, R1 is selected from: H, propylamino, ethylamino, methylamino, propylsulfonamide, methoxy, ethoxy, propoxy.

6. The heterocyclic compound according to any one of claims 1-4, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, B is selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, phenyl, 5-6 membered heteroaryl, C1-C3 alkyl, C1-C3 alkoxy, (C1-C3 alkyl)2amino, (C1-C3 alkyl)amino, halogen-substituted C1-C3 alkyl, cyano-substituted C1-C3 alkyl, carbamoyl, C5-C8 cycloalkyl, -X1-C(=O)-X2-R6, -X1-C(=NH)-X2-R6; X1 and X2 are independently selected from: -N(R7)-, -CH(R8)-; Preferably, R7 and R8 are each independently selected from: H, C1~C3 alkyl groups; Preferably, B is selected from: cyano, cyanomethyl, carbamoyl, -NH-C(=O)-NH-R6, -CH2-C(=O)-NH-R6, -NH-C(=NH)-NH-R6.

7. The heterocyclic compound according to claim 6, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, R6 is selected from: H, methyl, ethyl, tert-butyl, methoxy, ethoxy, cyanomethyl, cyano, one or more R'9 substituted or unsubstituted cyclopropyl, one or more R'9 substituted or unsubstituted cyclobutyl, one or more R'9 substituted or unsubstituted cyclopentyl, one or more R'9 substituted or unsubstituted cyclohexyl, one or more R'9 substituted or unsubstituted tetrahydrofuranyl, one or more R'9 substituted or unsubstituted oxacyclobutyl, one or more R9 substituted or unsubstituted phenyl, one or more R9 substituted or unsubstituted 5-membered heteroaryl; Preferably, each R9 is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, methyl, ethyl, propyl, butyl, C1~C3 alkoxy, (C1~C3 alkyl)2amino, (C1~C3 alkyl)amino, C5~C6 cycloalkyl, 5~6 heterocyclic group.

8. The heterocyclic compound according to claim 7, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, B is selected from: cyano, , , , , , , , , , , , , , .

9. The heterocyclic compound according to any one of claims 1-4, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, L1 is selected from: ethynyl group, one or more R groups 10 Substituted or unsubstituted phenylene, one or more R 10 Substituted or unsubstituted 5-6 member heteroaryl groups; Preferably, each R 10 Each is independently selected from: H, halogen, nitro, hydroxyl, amino, cyano, alkynyl, (C1~C3 alkyl)2amino, (C1~C3 alkyl)amino, C1~C3 alkyl, C1~C3 alkoxy.

10. The heterocyclic compound according to any one of claims 1-4, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, L2 is selected from: C1~C4 alkylene groups, and 2~6 atom-azide alkylene groups; And / or, L3 is selected from: One or more R 10 Substituted or unsubstituted 5- to 10-membered heterocyclic groups; Preferably, L3 is selected from: One or more R 10 Substituted or unsubstituted 5- to 10-membered heterocyclic groups containing 1-3 nitrogen atoms; Preferably, L2 is selected from methylene, ethylene, and propylene.

11. The heterocyclic compound according to claim 10, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, L3-L2, composed of L2 and L3, is selected from the following structure: , 5- to 10-membered heterocyclic -C1- to C4 alkylene groups containing 1-3 nitrogen atoms; m is selected from: 1, 2, 3, 4; Preferably, L3-L2, composed of L2 and L3, is selected from the following structures: 、 、 、 。 12. The heterocyclic compound according to claim 1, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, characterized in that, The heterocyclic aromatic compounds are selected from the following compounds: ； ； ； 。 13. The use of the heterocyclic compound of any one of claims 1-12, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, in the preparation of an FLT3 inhibitor; preferably, the FLT3 is a genetically mutated FLT3, preferably an FLT3-ITD mutation, an FLT3-TKD mutation; more preferably an FLT3-F691L mutation, an FLT3-D835Y mutation, an FLT3-D835Y / F691L mutation, an FLT3-ITD-D835Y mutation, or an FLT3-ITD-F691L mutation.

14. The use of the heterocyclic compound of any one of claims 1-12, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, in the preparation of a medicament for treating and / or preventing FLT3-mediated diseases; Preferably, the FLT3-mediated disease is a malignant hematological disease; the malignant hematological disease is preferably acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, or myeloproliferative disorder.

15. A pharmaceutical composition for the prevention or treatment of FLT3-mediated diseases, characterized in that, It is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises a heterocyclic aromatic compound as described in any one of claims 1-12, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof.

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