Azacycloalkane Menin-MLL protein inhibitor as well as pharmaceutical composition and application thereof

Abstract

The invention provides an azacycloalkane compound as shown in a formula I, pharmaceutically acceptable salt, hydrate, isomer, prodrug or a mixture thereof, a pharmaceutical composition containing the azacycloalkane compound, and application of the azacycloalkane compound and the pharmaceutically acceptable salt, hydrate, isomer, prodrug or the mixture thereof in preparation of drugs for preventing, relieving or treating related diseases caused by interaction with Menin-MLL protein. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to azacycloalkane Menin-MLL protein inhibitors, pharmaceutical compositions containing the same, and their use in preparing drugs for preventing, alleviating or treating diseases related to Menin-MLL protein interaction. Background Art

[0002] Mixed Lineage Leukemia (MLL) is a histone methyltransferase, also known as KMT2A (Histone-lysine N-methyltransferase 2A). MLL-rearranged (MLL-r) leukemia is caused by a translocation at chromosomal locus 11q23, encompassing the gene encoding KMT2A. This chromosomal translocation is known to produce over 60 oncogenic fusion proteins—formed by the amino terminus of MLL fused to various proteins. The MLL-AF4 / 9 fusion mutation is the most malignant. MLL-r leukemia accounts for 5% to 10% of acute leukemia in adults and 70% of acute leukemia in infants. Current treatment options are limited, primarily chemotherapy, with a poor prognosis and a high risk of relapse.

[0003] Menin, a protein primarily localized in the nucleus and encoded by the Multiple Endocrine Neoplasia Type 1 (MEN1) gene, is a crucial cofactor of the oncogenic MLL-r fusion protein, binding to the MLL-r protein with high affinity. Upon binding to the MLL-r protein, menin recruits chromatin-modifying enzymes such as Dot1L or the pTEFb complex, leading to enhanced transcription of genes including HOXA and MEIS1. Aberrant expression of these genes impedes hematopoietic differentiation and promotes proliferation. In vitro and in vivo studies have demonstrated that menin inhibitors disrupt the interaction between menin and MLL-r and specifically induce growth inhibition and apoptosis in leukemia cells carrying MLL-r mutations (Cancer Cell 36, 660–673). Studies have also shown that menin inhibitors are effective against leukemias harboring NPM1 gene mutations, which occur in approximately 20–30% of acute myeloid leukemia patients (Science 367, 586–590).

[0004] Currently, several Menin inhibitors are undergoing Phase 1 / 2 clinical trials for patients with relapsed / refractory acute leukemia who carry MLL-r or NPM1 mutations, including Syndax's SNDX-5613, Kura Oncology's KO-539, and Daiichi Sankyo Group's DS-1594b. In a clinical trial called AUGMENT-101, 53% of the 60 evaluable patients responded to the drug. However, after the second treatment cycle, some patients developed resistance to SNDX-5613. The study found that these resistant patients had MEN1 gene mutations that resulted in amino acid changes in the Menin protein: M3271, M327V, G331R, G331D, T349M, and S160C. These amino acid point mutations within the drug-binding pocket of Menin interfere with drug binding to the target protein, thereby reducing drug affinity. Importantly, the affinity of these mutant Menin proteins for KMT2A peptides is minimally affected by structural changes. Studies have shown that proteins with the M327I and T349M point mutations significantly reduce sensitivity to reported inhibitors, thereby conferring a significant selective advantage (Nature 615, 913–919). Therefore, the development of inhibitors that bind to wild-type and mutant Menin proteins, particularly the M327I and T349M point mutations, is crucial and would offer hope for a cure for these drug-resistant patients.

[0005] Furthermore, excessive Menin expression can inhibit β-cell proliferation, leading to insufficient insulin secretion. A Menin-MLL inhibitor has been shown to enhance β-cell proliferation, offering potential applications in diabetes. Currently, one compound, BMF-219, is in Phase 2 clinical trials for type 2 diabetes.

[0006] In summary, Menin-MLL interaction inhibitors have great application prospects as drug research and development, and there is a good clinical demand for the development of Menin-MLL interaction inhibitors. Summary of the Invention

[0007] The present invention provides a compound represented by Formula I, a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof:

[0008]

[0009] Wherein, X is N or CH;

[0010] m=0, 1 or 2; n=0, 1 or 2.

[0011] R1 and R2 are independently selected from C1-C6 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl; or R1, R2 form a ring together with the nitrogen to which they are connected.

[0012] R3 and R4 are independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, -(CH2)pOR5, RaCO-, sulfonyl or phosphonyl, wherein p=1-4, R5 is alkyl; Ra is alkyl or alkenyl.

[0013] Ar is a substituted or unsubstituted 5-16 membered aryl or heteroaryl group, which may be a monocyclic or polycyclic ring, at least part of which has aromaticity.

[0014] In some embodiments, m = 1 or 2; n = 1 or 2. In some specific examples, m = 1; n = 1.

[0015] In some embodiments, R1 and R2 are independently C1-C4 alkyl. In some embodiments, R1 and R2 are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. In some specific implementations, R1 is isopropyl and R2 is methyl or ethyl.

[0016] In some embodiments, R1 or R2 may be substituted with one or more deuterium atoms.

[0017] In some embodiments, R1, R2, together with the nitrogen to which they are attached, form a 3-10 membered alicyclic ring. In some embodiments, R1, R2, together with the nitrogen to which they are attached, form a monocyclic or polycyclic ring, which may be a fused ring, a spirocyclic ring, or a bridged ring. In some embodiments, R1, R2, together with the nitrogen to which they are attached, form a 3-8 membered alicyclic ring. In some embodiments, R1, R2, together with the nitrogen to which they are attached, form a 3-6 membered alicyclic ring. In addition to the existing nitrogen, the alicyclic ring may optionally contain 0-3 heteroatoms selected from nitrogen, oxygen, and sulfur. In some specific embodiments, R1, R2, together with the nitrogen to which they are attached, form the following structure:

[0018]

[0019] In some embodiments, the heterocyclic ring formed by the R1, R2 and the nitrogen to which they are connected is substituted at any possible position. In some embodiments, the heterocyclic ring formed by the R1, R2 and the nitrogen to which they are connected is optionally substituted by one or more selected from oxygen, hydroxyl, amino, carboxyl, halogen, cyano, C1-C6 alkyl, C1-C3 alkylamino, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl. In some specific embodiments, the heterocycloalkyl formed by the R1, R2 and the nitrogen to which they are connected is optionally substituted by one or more selected from oxygen, hydroxyl, amino, carboxyl, fluorine, chlorine, bromine, iodine, cyano, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, dimethylamino, diethylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl, piperidinyl, tetrahydro-1H-pyrrolizinyl groups.

[0020] In some embodiments, R3 and R4 are independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, -(CH2)pOR5, RaCO-, sulfonyl or phosphonyl, wherein p=1-3, R5 is C1-C3 alkyl; Ra is C1-C3 alkyl or C2-C4 alkenyl.

[0021] In some embodiments, p is 1 or 2, and R5 is methyl, ethyl, n-propyl, or isopropyl.

[0022] In some embodiments, Ra is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, Ra is vinyl, 1-propenyl, or 2-propenyl.

[0023] In some embodiments, R3 and R4 are independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, -CH2CH2OCH3 or -CH2OCH2CH3.

[0024] In some embodiments, R3 is hydrogen. In some embodiments, R4 is hydrogen. In some embodiments, R3 is hydrogen, and R4 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, -CH2CH2OCH3, or -CH2OCH2CH3.

[0025] In some embodiments, Ar is a substituted or unsubstituted 5-7 membered aryl, 5-7 membered heteroaryl, 8-16 membered fused aryl, or 8-16 membered fused heteroaryl, wherein the backbone atoms of the heteroaryl optionally contain 1-3 heteroatoms selected from nitrogen, oxygen, and sulfur.

[0026] In some embodiments, Ar is a monocyclic ring. In some embodiments, Ar is a fused bicyclic ring. In some embodiments, Ar is a 5-7 membered aryl, a 5-7 membered heteroaryl, an 8-12 membered fused aryl, or an 8-12 membered fused heteroaryl.

[0027] In some specific embodiments, Ar can be selected from the following substituted or unsubstituted groups: furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl; phenyl, pyridyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl; indolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benzisothiazole, benzoxazolyl, benzisoxazole, benzothienyl, indazolyl, benzo[d][1,2,3]triazolyl, pyrrolo[1,2-a]pyridine 1-A, 1-B, 2-A, 3-H-Dihydro-1H-Benzoxazin-1-one, 1-A, 2-B, 3-H-Dihydro-2H-Benzoxazin-1-one, 1-A, 2-B, 3-H-Dihydro-3H-Benzoxazin-3-one, 1-A, 2-B, 3-H-Dihydro-3H-Benzoxazin-1-one, 1-A, 2-B, 3-H-Dihydro-2H-Benzoxazin-3-one, 1-A, 2-B, 3-H-Dihydro-3H-Benzoxazin-1-one, 1-A, 2-B, 3-H-Dihydro-3 ...1-one, 1-A, 2-B,

[0028] In some embodiments, Ar is optionally substituted at any possible position by one or more Ry, wherein one or more Ry can be independently selected from oxygen, hydroxyl, amino, carboxyl, cyano, halogen, phosphono, sulfonyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 aminoalkyl, C1-C3 alkylamino, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl, RbCO-, C2-C4 alkenyl, 5-8 membered aryl or 5-8 membered heteroaryl, wherein Rb represents alkyl or alkenyl. The backbone atoms of the heterocycloalkyl and heteroaryl groups optionally contain 1-3 heteroatoms selected from nitrogen, oxygen and sulfur.

[0029] In some embodiments, Rb is C1-C5 alkyl. In some specific embodiments, Rb is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, or neopentyl. In some embodiments, Rb is C2-C4 alkenyl. In some specific embodiments, Rb is vinyl, 1-propenyl, or 2-propenyl.

[0030] In some specific embodiments, Ry is one or more independently selected from oxygen, hydroxyl, amino, carboxyl, fluorine, chlorine, bromine, iodine, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, methoxy, ethoxy, chloromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, perfluoroethyl, 2,2,2-trifluoroethoxy yl, aminomethyl, 2-aminoethyl, dimethylaminoethyl, formyl, acetyl, acryloyl, methylphosphonyl, dimethylphosphonyl, methylsulfonyl, ethylsulfonyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl, piperidinyl, tetrahydro-1H-pyrrolazin-7a(5H)-yl)methyl, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazole, tetrazole, phenyl, pyridyl group.

[0031] In some embodiments, Ry can be substituted at any possible position by one or more independently selected from oxygen, hydroxyl, amino, carboxyl, halogen, cyano, C1-C6 alkyl, C1-C3 alkylamino, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl. In some embodiments, Ry can be substituted at any possible position by one or more independently selected from oxygen, hydroxyl, amino, carboxyl, fluorine, chlorine, bromine, iodine, cyano, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, dimethylamino, diethylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl, piperidinyl, tetrahydro-1H-pyrrolazinyl.

[0032] The present invention provides the following compounds, their pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof:

[0033]

[0034]

[0035]

[0036]

[0037] The present invention also provides a pharmaceutical composition comprising any of the aforementioned compounds, pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof, and pharmaceutically acceptable excipients and / or carriers.

[0038] The present invention also provides a use of any of the aforementioned compounds, their pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof, or pharmaceutical compositions, in the preparation of drugs for preventing, alleviating or treating diseases associated with the Menin-MLL protein interaction.

[0039] In the present invention, diseases related to the interaction with Menin-MLL protein include malignant tumors, diabetes or complications related to the diseases, wherein malignant tumors include hematological tumors, lymphomas, and solid tumors.

[0040] Hematological neoplasms include leukemias and myelomas, including but not limited to acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute monocytic leukemia, chronic monocytic leukemia, childhood leukemia, acute myeloid leukemia, chronic myeloid leukemia, mixed lineage leukemia, hairy cell leukemia, precursor T-cell lymphocytic leukemia, large granular lymphocytic leukemia, meningeal leukemia, myelodysplastic syndrome, myeloproliferative disorders, myeloproliferative neoplasia, plasmacytoma, and multiple myeloma.

[0041] Lymphomas include, but are not limited to, cutaneous T-cell lymphoma, lymphoma, AIDS-related lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or malignant lymphoma.

[0042] Solid tumors include, but are not limited to, pancreatic cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, glioblastoma, lung cancer, breast cancer, and prostate cancer.

[0043] Related complications include, but are not limited to, leukemic meningitis.

[0044] Experiments have shown that the Menin-MLL protein inhibitor of the present invention has excellent in vitro enzyme inhibition activity and cell proliferation inhibition activity. In particular, compared with existing compounds, it has excellent activity in inhibiting the interaction between Menin mutant protein and MLL protein, and thus has good prospects for resisting drug resistance. DETAILED DESCRIPTION

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Definitions and general descriptions:

[0046] Indicates the connection position of this group to other structures.

[0047] Unless otherwise specified, "substituted" means that a hydrogen atom in a molecule is replaced by another different atom or group.

[0048] "Oxygen substitution" means that an oxygen atom forms a "C=O" structure with a directly adjacent carbon atom.

[0049] "Alkyl" refers to a saturated hydrocarbon radical consisting solely of carbon and hydrogen atoms, with single bonds connecting the carbon atoms and the carbon atoms and hydrogen atoms. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two, or three branches. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl.

[0050] "Heteroatom" refers to a non-carbon atom in a carbon chain or backbone. Typical heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur.

[0051] "Membered" refers to the number of atoms that make up the ring. Typical five-membered rings include cyclopentyl, pyrrole, tetrahydropyrrole, imidazole, thiazole, furan, tetrahydrofuran, and thiophene. Typical six-membered rings include cyclohexyl, piperidine, piperazine, pyridine, pyran, pyrazine, thiopyran, pyridazine, pyrimidine, and benzene. Rings containing heteroatoms among their backbone atoms are heterocycles; aromatic groups containing heteroatoms are heteroaryls; and non-aromatic groups containing heteroatoms are heterocycloalkyls.

[0052] "Cycloalkyl" refers to a saturated or unsaturated monocyclic, bicyclic, or polycyclic ring system (e.g., fused 2, 3, or 4 rings or spirocyclic rings), wherein all backbone atoms are carbon atoms, including cyclized alkyl and alkenyl groups. Typical examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, and cyclohexenyl.

[0053] "Aliphatic heterocycle" refers to a saturated or unsaturated monocyclic, bicyclic, or polycyclic ring system (e.g., two, three, or four fused or spirocyclic rings) containing one or more heteroatoms in the backbone atoms. Where the heteroatoms are selected from nitrogen, oxygen, and sulfur, and the remaining ring atoms are carbon, the corresponding group is a heterocycloalkyl group, also known as a heterocyclyl group. Examples of heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, piperazinyl, tetrahydropyranyl, or morpholinyl.

[0054] "Aryl" refers to a monocyclic or polycyclic (eg, having 2, 3, or 4 fused rings) aromatic hydrocarbon group. Typical aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like.

[0055] "Heteroaryl" refers to a monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic heterocyclic moiety having one or more heteroatom ring members selected from N, S, and O. Typical heteroaryl groups include, but are not limited to, pyridyl, indolyl, quinoxalinyl, quinolyl, isoquinolyl, benzothiophenyl, benzofuranyl, benzothiophenyl, benzopyranyl, benzothiapyranyl, furanyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, oxadiazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, and the like. The definition of "heteroaryl" also includes aromatic rings (which can be aromatic or heteroaromatic) fused to saturated carbocyclic or saturated heterocyclic rings. In particular, the position where the group is connected to the other structure is located on the aromatic ring. Typical examples include but are not limited to 1,2,3,4-tetrahydroisoquinolin-5-yl and 1,2,3,4-tetrahydroisoquinolin-8-yl. "Alkenyl" refers to an unsaturated hydrocarbon group having one or more -C=C- (carbon-carbon double bonds). Example alkenyls include but are not limited to ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1propenyl, etc.

[0056] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0057] "Cyano" refers to -CN.

[0058] "Haloalkyl" means an alkyl group substituted with one or more halogen atoms, wherein alkyl has the meaning herein. Haloalkyl includes, but is not limited to, monohaloalkyl, dihaloalkyl, trihaloalkyl, and perhaloalkyl, such as chloromethyl, dichloromethyl, difluoromethyl, dibromomethyl, trifluoromethyl, 2,2,2-trifluoroethyl, perfluoroethyl, and 2,2,2-trifluoro-1,1-dichloroethyl.

[0059] "Alkoxy" refers to an -O-alkyl group, wherein alkyl has the meaning set forth herein.

[0060] "Amino," used alone or in combination with other terms, refers to a radical of the formula -NH2.

[0061] "Aminoalkyl" means an alkyl group substituted with one or more amino groups, wherein alkyl has the meaning herein. For example, C1-C6 aminoalkyl means a C1-C6 alkyl group substituted with one or more amino groups. Aminoalkyl groups include, but are not limited to, aminomethyl and 2-aminoethyl.

[0062] "Alkylamino" means a group of the formula -NH(alkyl) or a di-N(alkyl) group, wherein the alkyl group has 1-6 carbon atoms. In some embodiments, the alkyl group has 1-3 carbon atoms. In some specific embodiments, the alkylamino group can be methylamino or ethylamino. Dialkylamino is dimethylamino or diethylamino.

[0063] "Hydroxy" refers to -OH.

[0064] "Sulfonyl" refers to wherein Rd represents an alkyl or aryl group as defined in the present invention. Exemplary sulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, phenylsulfonyl, and p-toluenesulfonyl.

[0065] "Phosphono" refers to a group of the formula wherein R e represents a hydroxyl group or an alkyl or aryl group as described in the definition of the invention, and R f represents an alkyl or aryl group as described in the definition of the invention. Exemplary phosphoryl groups include, but are not limited to, methylphosphonyl, dimethylphosphonyl, and diethylphosphonyl.

[0066] The compounds of the present invention may also include all isotopes of atoms present in the intermediates or final compounds. Isotopes include atoms with the same atomic number but different mass numbers. Isotopes of the constituent atoms of the compounds of the present invention can exist in natural or unnatural abundance. Examples of hydrogen isotopes include deuterium and tritium. In certain embodiments, the compounds of the present invention are deuterated, i.e., at least one deuterium atom exists in place of a hydrogen atom. In some specific embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms in the compounds of the present invention are replaced by deuterium. Methods for replacing hydrogen atoms with deuterium in molecules are known in the art.

[0067] In this document, unless otherwise specified, the term "Ca-Cb" refers to a moiety having ab carbon atoms (b is greater than a, and both are integers). 1- C3 indicates that the modified moiety has 1 to 3 carbon atoms, such as 1 carbon atom, 2 carbon atoms, or 3 carbon atoms.

[0068] "pq-membered (hetero)cycloalkyl" means that the moiety modified by the term has pq (q is greater than p, and both are integers) carbon atoms and the number of heteroatoms participating in the ring. For example, a 3-6-membered heterocycloalkyl means that the modified ring structure has 3, 4, 5, or 6 atoms, including carbon atoms and at least one heteroatom.

[0069] "yz-membered (hetero)aryl" refers to a moiety modified by the term having a total of yz (z is greater than y, and both are integers) carbon atoms and heteroatoms participating in the ring formation. For example, a 5-7-membered aryl group indicates that the modified aryl structure has 5, 6, or 7 carbon atoms; for example, a 5-7-membered heteroaryl group indicates that the modified heteroaryl structure has 5, 6, or 7 atoms, including carbon atoms and at least one heteroatom.

[0070] "Optionally" means that the subsequently described event or circumstance can be freely chosen or not.

[0071] "Hydrate" refers to an aggregate of the compound of the present invention containing one or more water molecules, including hemihydrate, monohydrate, dihydrate, trihydrate and the like.

[0072] "Isomers" means that when the compounds of the present invention contain one or more asymmetric centers, they can exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and single diastereomers. The compounds of the present invention may have asymmetric centers, which may result in the existence of two optical isomers. The scope of the present invention includes all possible optical isomers and their mixtures. If the compounds of the present invention contain olefinic double bonds, the scope of the present invention includes cis-isomers and trans-isomers unless otherwise specified. The compounds of the present invention may exist as tautomers (a type of functional group isomers) that have different points of attachment of hydrogen through one or more double bond displacements, for example, a ketone and its enol form are keto-enol tautomers. Each tautomer and its mixtures are within the scope of the present invention. All enantiomers of the compounds. Diastereomers, racemates, mesomorphs, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof are all within the scope of the present invention.

[0073] "Prodrug" refers to a derivative compound that can directly or indirectly provide a compound of the present invention after being administered to an individual. Particularly preferred derivative compounds or prodrugs are compounds that can improve the bioavailability of the compound of the present invention when administered to an individual (e.g., more easily absorbed into the blood), or compounds that promote the delivery of the parent compound to the site of action (e.g., the lymphatic system). Unless otherwise indicated, all prodrug forms of the compounds of the present invention are within the scope of the present invention, and various prodrug forms are known in the art, for example, see T. Higuchi, V. Stella, Pro-drugs as Novel Drug Delivery Systems [J], American Chemical Society, Vol. 14, 1975. In addition, the present invention also encompasses compounds of the present invention containing protecting groups. In any process for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive groups or reactive groups on any related molecules, thereby forming a chemically protected form of the compound of the present invention. This can be achieved by conventional protecting groups, such as those described in TW Greene, PGM Wuts, Protective Groups in Organic Synthesis [M], John Wiley & Sons, 2006. These protecting groups may be removed at an appropriate subsequent stage using methods known in the art.

[0074] A "pharmaceutical composition" refers to a formulation of a compound of the present invention and a medium generally accepted in the art for delivering a biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration to an organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0075] "Pharmaceutically acceptable" refers to a substance, such as a carrier, diluent, or excipient, that does not affect the biological activity or properties of the compounds of the invention and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological reaction or interacting in an adverse manner with any component contained in the composition. For example, "excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the relevant governmental regulatory authorities as acceptable for use by humans or livestock.

[0076] "Pharmaceutically acceptable salts" include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0077] The present invention also provides a method for synthesizing the above-mentioned compound. The method of the present invention is mainly based on the preparation method reported in chemical literature or uses commercially available chemical reagents as starting materials for related synthesis.

[0078] Abbreviation

[0079] PhLi stands for phenyllithium

[0080] s-BuLi represents sec-butyllithium

[0081] TMEDA stands for N,N,N',N'-tetramethylethylenediamine

[0082] CbzCl represents benzyl chloroformate

[0083] MsCl stands for methanesulfonyl chloride

[0084] TEA stands for triethylamine

[0085] DMAP stands for 4-dimethylaminopyridine

[0086] DCM stands for dichloromethane

[0087] THF stands for tetrahydrofuran

[0088] ACN stands for acetonitrile

[0089] DMF stands for N,N-dimethylformamide

[0090] AcOH represents glacial acetic acid

[0091] TsOH represents p-toluenesulfonic acid

[0092] EtOH stands for ethanol

[0093] i-PrOH represents isopropyl alcohol

[0094] mCPBA stands for 3-chloroperoxybenzoic acid

[0095] HATU represents O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIEA represents ethyldiisopropylamine.

[0096] NaBH4 represents sodium borohydride

[0097] POCl3 represents phosphorus oxychloride

[0098] EA stands for ethyl acetate

[0099] TMSCl stands for trimethylsilyl chloride

[0100] Cs2CO3 represents cesium carbonate

[0101] BBr3 represents boron tribromide

[0102] Pd(OAc)2 represents palladium acetate

[0103] LiCl stands for lithium chloride

[0104] LiOH represents lithium hydroxide

[0105] S-Phos represents 2-dicyclohexylphosphino-2′,6′-dimethoxy-biphenyl

[0106] EDCI stands for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0107] HOBT stands for 1-hydroxybenzotriazole

[0108] TFA stands for trifluoroacetic acid

[0109] CMPB stands for cyanomethylenetri-n-butylphosphine

[0110] Preparation of intermediates Preparation Example 1: Preparation of 1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester

[0111]

[0112] Step 1: Preparation of tert-butyl 3-(1-azabicyclo[1.1.0]butan-3-yl)-3-hydroxyazetidine-1-carboxylate

[0113]

[0114] Dissolve 2,3-dibromopropane-1-amine hydrobromide (4.8 g, 16.12 mmol) in anhydrous tetrahydrofuran (60 mL) and cool the reaction system to -65°C. Then, slowly add a solution of phenyllithium (2N, 48.35 mmol) in n-butyl ether dropwise, maintaining the temperature below -60°C. After the addition is complete, stir the reaction system at -65°C for 2 hours. Remove the cryotherapy bath, rapidly warm to room temperature, and stir at room temperature for 10 minutes. Cool the reaction system again to -65°C, and add a mixed solution of N,N,N',N'-tetramethylethylenediamine (2.25 g, 19.34 mmol) and sec-butyllithium (1.3N, 19.34 mmol) in cyclohexane and n-hexane dropwise, maintaining the temperature below -60°C. After the addition was completed, the reaction was stirred at -65°C for 1 hour, and then a solution of tert-butyl 3-oxoazetidine-1-carboxylate (3.59 g, 20.95 mmol) in tetrahydrofuran was slowly added dropwise; after the addition was completed, the reaction system was stirred at -65°C for 1 hour. LCMS detection showed that the raw material reaction was complete. Water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried, concentrated, and used directly in the next reaction.

[0115] Step 2: Preparation of 1-benzyl 1'-(tert-butyl) 3'-hydroxy-3-iodo-[3,3'-diazetidine]-1,1'-dicarboxylate

[0116]

[0117] Dissolve tert-butyl 3-(1-azabicyclo[1.1.0]butan-3-yl)-3-hydroxyazetidine-1-carboxylate (2.41 g, 10.65 mmol) and sodium iodide (3.19 g, 21.30 mmol) in acetonitrile (30 mL). Cool the reaction system to 0°C, then slowly add benzyl chloroformate (2.18 g, 12.78 mmol) dropwise. After addition, stir the reaction at 0°C for 30 minutes. After completion of the reaction, purify by column chromatography to obtain 4.58 g of the title compound.

[0118] MS (ESI) m / z (M+H-100) + =389.1

[0119] Step 3: Preparation of 1-benzyl 1'-(tert-butyl) 3-iodo-3'-((methylsulfonyl)oxy)-[3,3'-diazetidine]-1,1'-dicarboxylate

[0120]

[0121] Dissolve 1-benzyl 1'-(tert-butyl) 3'-hydroxy-3-iodo-[3,3'-diazetidine]-1,1'-dicarboxylate (2.12 g, 4.34 mmol) in dichloromethane (50 mL). Cool the reaction system to 0°C, then add triethylamine (878 mg, 8.68 mmol) and 4-dimethylaminopyridine (1.06 g, 8.68 mmol). Methanesulfonyl chloride (995 mg, 8.68 mmol) was then slowly added dropwise. After addition, the reaction system was stirred at 0°C for 30 minutes. After completion of the reaction, the title compound (1.82 g) was purified by column chromatography.

[0122] MS (ESI) m / z (M+H-100) + =467.1.

[0123] Step 4: Preparation of 1-benzyl-1'-(tert-butyl)-2H,2'H-[3,3'-diazamethylene]-1,1'(4H,4'H)-dicarboxylate

[0124]

[0125] Dissolve 1-benzyl 1'-(tert-butyl) 3-iodo-3'-((methylsulfonyl)oxy)-[3,3'-diazetidine]-1,1'-dicarboxylate (1.8 g, 3.18 mmol) in acetic acid (20 mL). Then add zinc powder (1.04 g, 15.89 mmol). After addition, stir the reaction system at room temperature for 2 hours. After completion of the reaction, 980 mg of the title compound is obtained by purification.

[0126] MS (ESI) m / z (M+H-100) + =245.2.

[0127] 1 H NMR (400MHz, Chloroform-d) δ7.35 (d, J = 3.8Hz, 5H), 5.11 (s, 2H), 4.49 (m, 4H), 4.43–4.38 (m, 4H), 1.44 (s, 9H).

[0128] Step 5: Preparation of 1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester

[0129]

[0130] Dissolve 1-benzyl-1'-(tert-butyl)-2H,2'H-[3,3'-diazamethylene]-1,1'(4H,4'H)-dicarboxylate (980 mg, 2.80 mmol) in ethanol (32 mL), then add p-toluenesulfonic acid (1.6 g, 9.29 mmol). The reaction system is stirred at 50°C for 6 hours. After the reaction is complete, the title compound is purified by column chromatography to obtain 1.16 g of the p-toluenesulfonate salt.

[0131] MS (ESI) m / z (M+H) + =245.1.

[0132] Preparation Example 2: Preparation of 2-((5-(1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-1,2,4-triazin-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0133]

[0134] Step 1: Preparation of 1'-(3,6-dichloro-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester

[0135]

[0136] To a solution of trichloro-1,2,4-triazine (10 g, 54.23 mmol) in dichloromethane (200 mL) was added triethylamine (13.72 g, 135.57 mmol) and benzyl 3-(azetidin-3-ylidene)azetidine-1-carboxylate p-toluenesulfonate (14.57 g, 59.65 mmol) under an ice-water bath. The reaction was incubated at this temperature for 1 hour. LCMS confirmed the reaction was complete. The solvent was then evaporated, and the crude product was purified by silica gel column chromatography to yield 15.14 g of the title compound.

[0137] MS (ESI) m / z (M+H) + =392.2.

[0138] Step 2: Preparation of 1'-(6-chloro-3-hydrazino-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester

[0139]

[0140] To a solution of benzyl 1'-(3,6-dichloro-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylate (8 g, 20.40 mmol) in ethanol (200 mL) was added hydrazine hydrate (8.17 g, 163.2 mmol) at room temperature. The mixture was heated to 60°C with mechanical stirring for 4 hours. After completion of the reaction, the reaction system was cooled to room temperature. The filter cake was filtered, rinsed with water and ethanol, and dried to yield 6.51 g of the title compound.

[0141] MS (ESI) m / z (M+H) + =388.1.

[0142] Step 3: Preparation of 1'-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester

[0143]

[0144] To a solution of benzyl 1'-(6-chloro-3-hydrazino-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylate (6.511 g, 16.79 mmol) in N,N-dimethylformamide (40 mL) was added N-ethyl-5-fluoro-2-hydroxy-N-(propan-2-yl)benzamide (5.67 g, 25.18 mmol) and cesium carbonate (16.41 g, 50.37 mmol) at room temperature. After the addition, the reaction system was heated to 80°C for 6 hours. After completion of the reaction, the target compound was purified by reverse-phase column chromatography and freeze-dried to afford 4.1 g of the title compound.

[0145] MS (ESI) m / z (M+H) + =547.2.

[0146] Step 4: Preparation of 2-((5-(1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-1,2,4-triazin-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0147]

[0148] To benzyl 1'-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylate (4.1 g, 7.50 mmol) was added trifluoroacetic acid (15 mL) at room temperature, followed by heating to 60°C for 3 hours. After completion of the reaction as monitored by LCMS, the solvent was evaporated, and the crude product was purified by column chromatography to yield 2.41 g of the title compound.

[0149] MS (ESI) m / z (M+H) + =413.2.

[0150] Preparation Example 3: Preparation of 5-fluoro-2-hydroxy-N-isopropyl-N-(methyl-d3)benzamide

[0151]

[0152] Step 1: Preparation of 5-fluoro-2-methoxy-N-(propan-2-yl)benzamide

[0153]

[0154] To a solution of 5-fluoro-2-methoxybenzoic acid (5 g, 29.39 mmol) in dichloromethane (100 mL) was added ethyldiisopropylamine (5.70 g, 44.09 mmol), propan-2-amine (2.08 g, 35.27 mmol), and 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (13.41 g, 35.27 mmol) under an ice-water bath. The reaction was allowed to warm naturally for approximately 4 hours. After LCMS confirmed the reaction was complete, water was added to quench the reaction. The organic phase was washed with water and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to yield the crude product, which was purified by column chromatography to afford 6.537 g (90% purity) of the title compound.

[0155] MS (ESI) m / z (M+H) + =212.2.

[0156] Step 2: Preparation of 5-fluoro-2-methoxy-N-(methyl-d3)-N-(propan-2-yl)benzamide

[0157]

[0158] Under nitrogen in an ice-water bath, sodium hydride (1.34 g, 60%, 33.42 mmol) was added to a solution of 5-fluoro-2-methoxy-N-(propan-2-yl)benzamide (6.537 g, 27.85 mmol) in tetrahydrofuran (100 mL). The mixture was allowed to react at this temperature for 1 hour. Subsequently, deuterated iodomethane (12.11 g, 83.55 mmol) was added and the temperature was naturally increased to react overnight. LCMS indicated the reaction was complete. The product was quenched with water and extracted with ethyl acetate. The organic phase was washed with water and brine, concentrated under reduced pressure, and purified by column chromatography to yield 7.822 g of the title compound (80% purity).

[0159] MS (ESI) m / z (M+H) + =229.2.

[0160] Step 3: Preparation of 5-fluoro-2-hydroxy-N-isopropyl-N-(methyl-d3)benzamide

[0161]

[0162] To a solution of 5-fluoro-2-methoxy-N-(methyl-d3)-N-(propan-2-yl)benzamide (7.822 g, 80%, 27.41 mmol) in dichloromethane (50 mL) was added dropwise boron tribromide (13.73 g, 54.82 mmol) under an ice-water bath. The reaction was allowed to warm naturally overnight. The boron tribromide and solvent were removed under reduced pressure at 40°C. The remaining solution was poured into ice water and extracted with dichloromethane. The organic phase was dried, concentrated, and then purified by column chromatography to yield 4.145 g of the title compound.

[0163] MS (ESI) m / z (M+H) + =215.2.

[0164] Preparation Example 4: Preparation of 2-((5-(1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-1,2,4-triazin-6-yl)oxy)-5-fluoro-N-isopropyl-N-(methyl-d3)benzamide

[0165]

[0166] The title compound was prepared using the above synthetic or commercial reagents as raw materials according to the synthetic method of Preparation Example 2.

[0167] MS (ESI) m / z (M+H) + =402.2.

[0168] Preparation Example 5: Preparation of 2-((4-chloropyrimidin-5-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0169]

[0170] Step 1: Preparation of N-ethyl-5-fluoro-N-(propan-2-yl)-2-(pyrimidin-5-yloxy)benzamide

[0171]

[0172] At room temperature, N-ethyl-5-fluoro-2-hydroxy-N-(propan-2-yl)benzamide (25 g, 110.98 mmol), 5-bromopyrimidine (23 g, 144.27 mmol), cesium carbonate (108 g, 332.94 mmol), and N,N-dimethylformamide (250 mL) were added sequentially to a 500 mL single-necked reaction flask. After stirring, the temperature was slowly raised to 135°C and the reaction was allowed to proceed overnight. After completion of the reaction, water was added, the mixture was extracted with ethyl acetate, and the organic phases were combined and concentrated under reduced pressure to obtain a crude product. The crude product was separated by column chromatography to obtain 30 g of N-ethyl-5-fluoro-N-(propan-2-yl)-2-(pyrimidin-5-yloxy)benzamide.

[0173] MS (ESI) m / z (M+H) + =304.2.

[0174] Step 2: Preparation of 5-(2-(ethyl(propan-2-yl)carbamoyl)-4-fluorophenoxy)pyrimidin-1-ium-1-oate

[0175]

[0176] Under an ice bath, 3-chloroperoxybenzoic acid (17 g, 98.90 mmol) was slowly added to a mixture of N-ethyl-5-fluoro-N-(propan-2-yl)-2-(pyrimidin-5-yloxy)benzamide (30 g, 98.90 mmol) and dichloromethane (300 mL). After the addition, the mixture was allowed to warm to room temperature under nitrogen and allowed to react overnight. After completion, the reaction was quenched by the addition of saturated sodium bisulfite solution, and the solvent was removed by rotary evaporation to obtain 30 g of 5-(2-(ethyl(propan-2-yl)carbamoyl)-4-fluorophenoxy)pyrimidin-1-ium-1-oate. The crude product was used directly in the next reaction without purification.

[0177] MS (ESI) m / z (M+H) + =320.2.

[0178] Step 3: Preparation of 2-((4-chloropyrimidin-5-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0179]

[0180] 5-(2-bromo-4-fluorophenoxy)pyrimidin-1-ium-1-oate (39 g, 122.13 mmol) was dissolved in chloroform (400 mL) under ice-cooling conditions, followed by the addition of triethylamine (31 g, 305.32 mmol). After stirring, phosphorus oxychloride (2.58 g, 168.55 mmol) was slowly added. Stirring was continued under ice-cooling for 5 minutes, and the temperature was slowly raised to 65°C for 2 hours. Upon completion of the reaction, the solvent was removed by rotary evaporation. The reaction was quenched by the addition of 100 mL of aqueous sodium bicarbonate. The reaction was extracted three times with ethyl acetate, and the organic phases were combined, backwashed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by column chromatography afforded 15 g of 2-((4-chloropyrimidin-5-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide.

[0181] MS (ESI) m / z (M+H) + =338.2.

[0182] Preparation Example 6: 2-((4-(1',4'-dihydro-2H,2'H-[3,3'-diazomethylene]-1(4H)-yl)pyrimidin-5-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0183]

[0184] Step 1: Preparation of benzyl 3-(1-(5-(2-(ethyl(propan-2-yl)carbamoyl)phenoxy)pyrimidin-4-yl)azetidin-3-ylidene)azetidine-1-carboxylate

[0185]

[0186] At room temperature, 2-[(4-chloropyrimidin-5-yl)oxy]-N-ethyl-N-(propan-2-yl)benzamide (2.5 g, 7.82 mmol), isopropanol (80 mL), and benzyl 3-(azetidin-3-ylidene)azetidine-1-carboxylate p-toluenesulfonate (3.91 g, 9.38 mmol) were added to a reaction flask; the mixture was stirred thoroughly, and triethylamine (2.37 g, 23.46 mmol) was slowly added. The temperature was gradually raised to 85°C. After 2 hours of reaction, the reaction was complete as monitored by LCMS. Water was added, the mixture was extracted with ethyl acetate, and the organic phases were combined and concentrated under reduced pressure to obtain a crude product. The crude product was separated by column chromatography to obtain 3.72 g of benzyl 3-(1-(5-(2-(ethyl(propan-2-yl)carbamoyl)phenoxy)pyrimidin-4-yl)azetidin-3-ylidene)azetidine-1-carboxylate.

[0187] MS (ESI) m / z (M+H) + =546.2.

[0188] Step 2: Preparation of 2-((4-(1',4'-dihydro-2H,2'H-[3,3'-diazomethylene]-1(4H)-yl)pyrimidin-5-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0189]

[0190] Trifluoroacetic acid (20 mL) was added to the starting material, benzyl 3-(1-(5-(2-(propan-2-yl)carbamoylethyl)-4-fluorophenoxy)pyrimidin-4-yl)azetidin-3-ylidene)azetidine-1-carboxylate (3.7 g, 6.78 mmol) at room temperature. The temperature was slowly raised to 65°C and the reaction was allowed to react for 1 hour. LCMS confirmed the reaction was complete, and the trifluoroacetic acid was removed by concentration under reduced pressure. The resulting crude product was dissolved in a mixture of methanol and dichloromethane, and sodium bicarbonate was added and stirred for 0.5 hour. The sodium bicarbonate was removed by filtration, and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography to obtain 2.79 g of 2-((4-(1',4'-dihydro-2H,2'H-[3,3'-diazomethylene]-1(4H)-yl)pyrimidin-5-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide.

[0191] MS (ESI) m / z (M+H) + =412.2.

[0192] Preparation Example 7: Preparation of (R)-3-((tert-Butoxycarbonyl)amino)-4-(2-chlorophenyl)butanoic acid

[0193]

[0194] Step 1: Preparation of 1-benzyl 2,5-dioxopyrrolidin-1-yl (3S)-3-{[(tert-butoxy)carbonyl]amino}succinate

[0195]

[0196] To a solution of (2S)-4-(benzyloxy)-2-{[(tert-butoxy)carbonyl]amino}-4-oxobutanoic acid (50 g, 154.64 mmol) in ethyl acetate (250 mL) was added 1-hydroxypyrrolidine-2,5-dione (19.04 g, 165.46 mmol), followed by the dropwise addition of a solution of 1,3-dicyclohexylcarbodiimide (32.39 g, 156.96 mmol) in ethyl acetate under an ice-water bath. After allowing to react overnight at naturally elevated temperature, LCMS confirmed the reaction was complete. The white insoluble material was filtered off, and the filtrate was washed with aqueous sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to yield 61.21 g of the title compound.

[0197] MS (ESI) m / z (M+H) + =421.2.

[0198] Step 2: Preparation of benzyl (3S)-3-{[(tert-butoxy)carbonyl]amino}-4-hydroxybutyrate

[0199]

[0200] Under ice-water bath, sodium borohydride (8.98 g, 237.33 mmol) was added to a mixture of tetrahydrofuran (150 mL) and water (20 mL). After stirring, a solution of 1-benzyl 2,5-dioxopyrrolidin-1-yl (3S)-3-{[(tert-butoxy)carbonyl]amino}succinate (61.21 g, 123.76 mmol) in tetrahydrofuran (100 mL) was added dropwise. The reaction system was allowed to react in an ice-water bath for 2 hours. LCMS confirmed the reaction was complete, and the mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to yield 29.2 g of the title compound.

[0201] MS (ESI) m / z (M+H) + =310.2.

[0202] Step 3: Preparation of benzyl (3S)-3-{[(tert-butoxy)carbonyl]amino}-4-iodobutyrate

[0203]

[0204] To a solution of triphenylphosphine (37.14 g, 141.59 mmol) in tetrahydrofuran (300 mL) in an ice-water bath, 1H-imidazole (9.64 g, 141.59 mmol) and iodine (35.94 g, 141.59 mmol) were added. The system was stirred at this temperature for 10 minutes, followed by the addition of a solution of (3S)-3-{[(tert-butoxy)carbonyl]amino}-4-hydroxybutyric acid benzyl ester (29.2 g, 94.39 mmol) in tetrahydrofuran (15 mL). The ice-water bath was then removed and the mixture was allowed to react at room temperature for 1 hour. After LCMS confirmed the reaction was complete, the mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by column chromatography to yield 24.49 g of the title compound.

[0205] MS (ESI) m / z (M+H) + =420.1.

[0206] Step 4: Preparation of benzyl (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(2-chlorophenyl)butanoate

[0207]

[0208] At room temperature, zinc powder (1.25 g, 19.10 mmol) and N,N-dimethylformamide (10 mL) were added to a 100 mL three-necked flask. Trimethylsilyl chloride (0.042 g, 0.38 mmol) was added under nitrogen protection and stirred at room temperature for 30 minutes. Subsequently, 1,2-dibromoethane (0.29 g, 1.53 mmol) was added and the oil bath was gradually heated to 80°C and the reaction was continued for 30 minutes. After the reaction was completed, the oil bath was removed and the temperature was lowered to room temperature. To the above solution were added trimethylsilyl chloride (0.12 g, 1.15 mmol), iodine (0.29 g, 1.15 mmol) and benzyl (3S)-3-{[(tert-butoxy)carbonyl]amino}-4-iodobutyrate (1.6 g, 3.82 mmol), and the mixture was reacted at room temperature for 30 min. After completion of the reaction, the prepared solution was added to a dry reaction flask containing 1-chloro-2-iodobenzene (455.44 mg, 1.91 mmol), palladium acetate (0.086 g, 0.38 mmol), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (0.31 g, 0.76 mmol), lithium chloride (0.0081 g, 0.19 mmol) and N,N-dimethylformamide (10 mL). The reaction system was reacted at 40°C under nitrogen protection for 2 h. After LCMS confirmed the completion of the reaction, a small amount of aqueous ammonium chloride solution was added to quench the reaction. After filtration, the product was directly purified by reverse phase column chromatography and freeze-dried to obtain 0.713 g of the title compound.

[0209] MS (ESI) m / z (M+H) + =404.2.

[0210] Step 5: Preparation of (R)-3-((tert-Butoxycarbonyl)amino)-4-(2-chlorophenyl)butanoic acid

[0211]

[0212] To a solution of benzyl (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(2-chlorophenyl)butanoate (713 mg, 1.77 mmol) in water (6 mL) and tetrahydrofuran (20 mL) was added lithium hydroxide (84.78 mg, 3.54 mmol) under ice-water bath. After the addition was complete, the reaction system was naturally warmed to room temperature. LCMS monitored the reaction until complete. A small amount of acetic acid was added under ice-water bath, and the organic phase was removed by rotary evaporation at low temperature. The clear phase was dissolved in methanol and purified by reverse-phase column chromatography and freeze-dried to obtain 0.423 g of the title compound.

[0213] MS (ESI) m / z (M+H) + =314.2.

[0214] Preparation Example 8: Preparation of 7-bromo-1-(2,2,2-trifluoroethyl)-1H-indazole

[0215]

[0216] At room temperature, 7-bromo-1H-indazole (500 mg, 2.5 mmol) and N,N-dimethylformamide (10 mL) were added to a reaction flask and stirred until uniform. Sodium hydroxide (121.9 mg, 3.1 mmol) was then slowly added and stirred at room temperature for 5 minutes. 2,2,2-Trifluoroethyl trifluoromethanesulfonate (1.2 g, 5.1 mmol) was then added and the reaction continued at room temperature for two hours. LCMS confirmed the reaction was complete and the reaction was quenched by adding ammonium chloride solution. The mixture was filtered, and the solvent was removed by rotary evaporation. The mixture was then purified by column chromatography to yield 253 mg of the title compound, 7-bromo-1-(2,2,2-trifluoroethyl)-1H-indazole.

[0217] MS (ESI) m / z (M+H) + =279.1.

[0218] 1 H NMR (400MHz, DMSO-d6) δ8.37(s,1H),7.89(dd,J=8.0,1.0Hz,1H),7.75(dd,J=7.5,0.9Hz,1H),7.17(dd,J=8.0,7.5Hz,1H),5.67(q,J=8.7Hz,2H).

[0219] Preparation Example 9: Preparation of 3-iodo-2-(2,2,2-trifluoroethoxy)benzonitrile

[0220]

[0221] Step 1: Preparation of 2-hydroxy-3-iodobenzaldehyde

[0222]

[0223] At room temperature, 2-iodophenol (2 g, 9.1 mmol), formaldehyde (818.92 mg, 27.3 mmol), magnesium chloride (1.7 g, 18.2 mmol), triethylamine (1.8 g, 18.2 mmol), and tetrahydrofuran (10 mL) were added to a reaction flask in that order. After nitrogen displacement, the temperature was slowly raised to reflux. After two hours of reaction, LCMS monitored the reaction for completion. Water was added, and the mixture was extracted with ethyl acetate. The organic phase was collected, the solvent was evaporated, and the title compound, 2-hydroxy-3-iodobenzaldehyde, was obtained by column chromatography (1.9 g).

[0224] MS (ESI) m / z (M+H) + =249.1.

[0225] Step 2: Preparation of 3-iodo-2-(2,2,2-trifluoroethoxy)benzaldehyde

[0226]

[0227] At room temperature, 2-hydroxy-3-iodobenzaldehyde (1.9 g, 7.7 mmol), potassium carbonate (1.3 g, 9.2 mmol), and N,N-dimethylformamide (30 mL) were added to a reaction flask in sequence. After stirring, 2,2,2-trifluoroethyl trifluoromethanesulfonate (2.0 g, 8.4 mmol) was slowly added and the reaction continued at room temperature for two hours. LCMS monitored the reaction completion. Saturated sodium bicarbonate was added, and the mixture was extracted with ethyl acetate. The organic phase was collected and the solvent was evaporated. The crude product was purified by column chromatography to obtain 1.7 g of the title compound, 3-iodo-2-(2,2,2-trifluoroethoxy)benzaldehyde.

[0228] MS (ESI) m / z (M+H) + =331.1.

[0229] Step 3: Preparation of 3-iodo-2-(2,2,2-trifluoroethoxy)benzaldehyde oxime

[0230]

[0231] At room temperature, 3-iodo-2-(2,2,2-trifluoroethoxy)benzaldehyde (800 mg, 2.4 mmol), hydroxylamine hydrochloride (252.3 mg, 3.6 mmol), methanol (15 mL), and water (5 mL) were added to a reaction flask in that order. After stirring, sodium acetate (297.8 mg, 3.6 mmol) was slowly added and the reaction continued at room temperature for two hours. LCMS monitored the reaction completion, and water and dichloromethane were added for extraction. The organic phase was collected, dried, and rotary evaporated to obtain 836 mg of crude 3-iodo-2-(2,2,2-trifluoroethoxy)benzaldehyde oxime, which was used directly in the next reaction.

[0232] MS (ESI) m / z (M+H) + =346.1.

[0233] Step 4: Preparation of 3-iodo-2-(2,2,2-trifluoroethoxy)benzonitrile

[0234]

[0235] At room temperature, N-[(3-iodo-2-(2,2,2-trifluoroethoxy)phenyl)methylene]hydroxylamine (450 mg, 1.3 mmol), dimethyl sulfoxide (15 mL), potassium carbonate (270 mg, 2.0 mmol), and acetic anhydride (200 mg, 2.0 mmol) were added to a reaction flask in sequence. After stirring, the reaction system was heated to 50°C for 1 hour. After completion of the reaction as monitored by LCMS, water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phase was collected and dried, and the crude product was purified by column chromatography to yield 303 mg of the title compound, 3-iodo-2-(2,2,2-trifluoroethoxy)benzonitrile.

[0236] MS (ESI) m / z (M+H) + =328.1.

[0237] Preparation Example 10: Preparation of 7-bromo-1-(1-methylpiperidin-4-yl)-1H-indazole

[0238]

[0239] At room temperature, 7-bromo-1H-indazole (1 g, 5.1 mmol), 1-methylpiperidin-4-ol (643.6 mg, 5.6 mmol), and toluene (30 mL) were added to a reaction flask in sequence. After stirring, the atmosphere was replaced with nitrogen, and cyanomethylenetri-n-butylphosphine (2.4 g, 10.2 mmol) was added. The temperature was then raised to 120°C. After half an hour of reaction, the reaction was complete as monitored by LCMS. The mixture was quenched with a small amount of water, extracted, and the organic phase was collected. The crude product was rotary evaporated and purified by column chromatography to yield 429 mg of 7-bromo-1-(1-methylpiperidin-4-yl)-1H-indazole.

[0240] MS (ESI) m / z (M+H) + =294.2.

[0241] 1 H NMR (400MHz, DMSO-d6) δ8.19(s,1H),7.81(dd,J=8.0,0.9Hz,1H),7.63(dd,J=7.4,0.9Hz,1H),7.06(t, J=7.7Hz,1H),5.40–5.25(m,1H),3.03–2.88(m,2H),2.24(s,3H),2.20–2.03(m,4H),2.01–1.90(m,2H).

[0242] Referring to the synthetic method of Preparation Example 10, the following intermediate compounds can be prepared using commercial reagents as raw materials, as shown in Table 1.

[0243] Table 1 Information on intermediate compounds obtained in the preparation examples

[0244]

[0245]

[0246] Preparation Example 19: Preparation of 7-bromo-1-[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]-1H-indazole

[0247]

[0248] Step 1: Preparation of tert-butyl (3R,4R)-4-(7-bromo-1H-indazol-1-yl)-3-fluoropiperidine-1-carboxylate

[0249]

[0250] At room temperature, 7-bromo-1H-indazole (1 g, 5.1 mmol), tert-butyl (3S,4R)-3-fluoro-4-hydroxypiperidine-1-carboxylate (1.1 g, 5.1 mmol), and toluene (30 mL) were added to a reaction flask in sequence. After stirring, the atmosphere was replaced with nitrogen, and cyanomethylenetri-n-butylphosphine (2.0 g, 10.2 mmol) was added. The reaction system was then heated to 120°C. After half an hour of reaction, the reaction was complete as monitored by LCMS. A small amount of water was added to quench the reaction, and the organic phase was extracted and collected. The solvent was removed by rotary evaporation, and the title compound, tert-butyl (3R,4R)-4-(7-bromo-1H-indazol-1-yl)-3-fluoropiperidine-1-carboxylate, was obtained by purification by column chromatography. 820 mg of the title compound was obtained.

[0251] MS (ESI) m / z (M+H) + =398.2.

[0252] Step 2: Preparation of 7-bromo-1-(1-methylpiperidin-4-yl)-1H-indazole

[0253]

[0254] At room temperature, tert-butyl (3R,4R)-4-(7-bromo-1H-indazol-1-yl)-3-fluoropiperidine-1-carboxylate was added to a reaction flask, followed by dichloromethane (10 mL) and trifluoroacetic acid (3 mL). The reaction system was heated to 40°C for half an hour. LCMS monitored the reaction completion, and the solvent was removed by rotary evaporation. A small amount of methanol was added, and the pH was adjusted to a weak base with sodium bicarbonate solution. The solvent was removed by rotary evaporation, and the residue was purified to obtain 613 mg of 7-bromo-1-[(3R,4R)-3-fluoropiperidin-4-yl]-1H-indazole.

[0255] MS (ESI) m / z (M+H) + =298.1.

[0256] Step 3: Preparation of 7-bromo-1-[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]-1H-indazole

[0257]

[0258] Under ice-cooling, 7-bromo-1-[(3R,4R)-3-fluoropiperidin-4-yl]-1H-indazole (613 mg, 2.1 mmol), potassium carbonate (433.3 mg, 3.1 mmol), and N,N-dimethylformamide (30 mL) were added to a reaction flask in sequence. After stirring, iodomethane (296.7 mg, 2.1 mmol) was slowly added dropwise. The reaction was continued under ice-cooling for 1 hour. LCMS monitored the reaction completion, and ammonium chloride solution was added to quench the reaction. The solvent was evaporated, and the crude product was purified by column chromatography to obtain 392 mg of the title compound, 7-bromo-1-[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]-1H-indazole.

[0259] MS (ESI) m / z (M+H) + =312.2.

[0260] 1 H NMR(400MHz,Chloroform-d)δ8.07(s,1H),7.69(dd,J=8.0,0.9Hz,1H),7.57(dd,J=7.4,0.9Hz,1H),7.02(t,J=7.7Hz,1H),5. 89–5.71(m,1H),5.29–4.99(m,1H),4.70–4.44(m,1H),4.36–4.11(m,1H),3.13–2.84(m,2H),2.33–2.07(m,2H),1.65(s,3H).

[0261] Preparation Example 20: Preparation of 2-(7-bromo-1H-indazol-1-yl)-N,N-dimethylethan-1-amine

[0262]

[0263] Step 1: Preparation of 7-bromo-1-(2,2-dimethoxyethyl)-1H-indazole

[0264]

[0265] To a solution of 7-bromo-1H-indazole (0.5 g, 2.54 mmol) in acetonitrile (50 mL) at room temperature were added potassium carbonate (1.76 g, 12.7 mmol) and 2-bromo-1,1-dimethoxyethane (2.15 g, 12.7 mmol). The temperature was then raised to 100°C and the reaction was allowed to proceed for 3 days. After completion of the reaction, the mixture was cooled to room temperature, and the inorganic salts were filtered off. The filtrate was concentrated under reduced pressure and purified by reverse-phase column chromatography to obtain isomers, which were then freeze-dried to afford 122 mg of the title compound.

[0266] MS (ESI) m / z (M+H) + =285.1.

[0267] 1 H NMR (400MHz, Methanol-d4) δ8.08 (s, 1H), 7.76 (d, J = 8.0Hz, 1H), 7.62 (d, J = 7.4Hz, 1H), 7.04 (t, J = 7.7Hz, 1H), 4.92 (d, J = 5.6Hz, 2H), 4.85–4.81 (m, 1H), 3.31 (s, 6H).

[0268] Step 2: Preparation of 2-(7-bromo-1H-indazol-1-yl)acetaldehyde

[0269]

[0270] To a solution of 7-bromo-1-(2,2-dimethoxyethyl)-1H-indazole (122 mg, 0.43 mmol) in dichloromethane (30 mL) was added concentrated hydrochloric acid (1 mL) at room temperature. The mixture was reacted at room temperature for 0.5 h. LCMS confirmed the completion of the reaction. The mixture was concentrated under reduced pressure and purified by reverse phase column chromatography to obtain 90 mg of the title compound.

[0271] MS (ESI) m / z (M+H) + =240.1.

[0272] Step 3: Preparation of 2-(7-bromo-1H-indazol-1-yl)-N,N-dimethylethan-1-amine

[0273]

[0274] To a solution of 2-(7-bromo-1H-indazol-1-yl)acetaldehyde (90 mg, 0.38 mmol) in ethanol (25 mL) was added dimethylamine hydrochloride (61.97 mg, 0.76 mmol) and ethyldiisopropylamine (98.22 mg, 0.76 mmol) at room temperature, followed by sodium cyanoborohydride (47.76 mg, 0.76 mmol). The mixture was allowed to react at room temperature for 30 minutes. LCMS confirmed the reaction was complete. After concentration under reduced pressure, the mixture was dissolved in water and methanol, and purified by reverse-phase column chromatography to afford 36 mg of the title compound.

[0275] MS (ESI) m / z (M+H) + =268.1.

[0276] The target product of step 3 in Preparation Example 7 was used as the reaction substrate, and the commercial or homemade reagents shown in Table 2 were used as raw materials. The following intermediate compounds were prepared according to the synthetic method of Preparation Example 7. The specific information is shown in Table 2.

[0277] Table 2 Information on intermediate compounds obtained in the preparation examples

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284] Example 1: Preparation of (R)-2-((5-(1'-(3-amino-4-(2-chlorophenyl)butyryl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-1,2,4-triazin-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0285]

[0286] Step 1: Preparation of tert-butyl (R)-(1-(2-chlorophenyl)-4-(1'-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-4-oxobutan-2-yl)carbamate

[0287]

[0288] To a solution of (R)-3-((tert-butoxycarbonyl)amino)-4-(2-chlorophenyl)butanoic acid (0.11 g, 0.35 mmol) in N,N-dimethylformamide (4 mL) were added ethyldiisopropylamine (0.075 g, 0.58 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.083 g, 0.43 mmol) and 1-hydroxybenzotriazole (0.059 g, 0.43 mmol) at room temperature. After reacting at room temperature for 0.5 hour, 2-((5-(1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-1,2,4-triazin-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide (0.12 g, 0.29 mmol) was added. After the addition was complete, the reaction system was allowed to react at room temperature for 1 hour. After the reaction was complete as monitored by LCMS, the crude product was purified by reverse phase column chromatography to obtain 88 mg of the title compound.

[0289] MS (ESI) m / z (M+H) + =708.3.

[0290] Step 2: Preparation of (R)-2-((5-(1'-(3-amino-4-(2-chlorophenyl)butanoyl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-1,2,4-triazin-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0291]

[0292] To a solution of (tert-butyl (R)-(1-(2-chlorophenyl)-4-(1'-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-4-oxobutan-2-yl)carbamate (88 mg, 0.12 mmol) in dichloromethane (21 mL) was added trifluoroacetic acid (2 mL) and the mixture was reacted at room temperature for 1 hour. After completion of the reaction as monitored by LCMS, the crude product was preparatively purified and freeze-dried to give 32 mg of the title compound.

[0293] MS (ESI) m / z (M+H) + =608.2.

[0294] 1H NMR(400MHz,Methanol-d4)δ8.44(s,1H),7.46–7.36(m,2H),7.36–7.17(m,5H),5.17(s,2H),4.77(s,2H),4.71(s,2H),4.51(s,2H), 3.87–3.76(m,1H),3.58–3.43(m,2H),3.28–3.09(m,1H),2.99–2.82(m,2H),2.37–2.16(m,2H),1.26–1.02(m,7H),0.88–0.74(m,2H).

[0295] Using the target compound of Preparation Example 2, Preparation Example 4, or Preparation Example 6 as the reaction substrate and the intermediate compound prepared in the Preparation Example as the raw material, Examples 2-43, 45-49 were prepared by referring to the synthetic method of Example 1. Specific information is shown in Tables 3-4.

[0296] Table 3 Information on compounds of Examples 2-43, 45-49

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307] The analytical data of the above described embodiments, including NMR and HPLC data, are shown in Table 4.

[0308] Table 4 NMR and LC-MS data of compounds in Examples 2-43, 45-49

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316] Example 44: Preparation of (R)-2-((5-(1′-(2-chlorophenyl)-3-((2-methoxyethyl)amino)-butyryl)-1′,4′-dihydro-2H,2′H-[3,3′-diazamethylene]-1(4H)-yl)-1,2,4-triazine-6-oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0317]

[0318] To a solution of (R)-2-((5-(1'-(3-amino-4-(2-chlorophenyl)butanoyl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-1,2,4-triazin-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide (75 mg, 0.12 mmol) in acetonitrile (30 mL) was added potassium carbonate (0.083 g, 0.60 mmol) and 1-bromo-2-methoxyethane (25.02 mg, 0.18 mmol) at room temperature. After the addition was complete, the reaction system was heated to 100°C and reacted overnight. 1-Bromo-2-methoxyethane (100 mg, 0.72 mmol) was added, and the reaction was continued at 100°C for 3 hours before being stopped. The insoluble matter was removed by filtration, and the filtrate was concentrated, purified by preparative method, and freeze-dried to obtain 15 mg of the title compound.

[0319] 1 H NMR(400MHz,Methanol-d4)δ8.44(s,1H),7.44–7.37(m,2H),7.35–7.18(m,5H) ,5.16(s,2H),4.77(s,2H),4.65(s,2H),4.48(s,2H),3.85–3.77(m,1H),3.52–3 .42(m,3H),3.43–3.31(m,1H),3.29(s,3H),3.26–3.20(m,1H),3.16–3.04(m,1 H),2.92–2.73(m,3H),2.31–2.21(m,2H),1.27–1.03(m,7H),0.85–0.77(m,2H).

[0320] MS (ESI) m / z (M+H) + =666.3.

[0321] Example 50: Preparation of (S)-N-ethyl-5-fluoro-N-isopropyl-2-((4-(1'-prolyl-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)pyrimidin-5-yl)oxy)benzamide

[0322]

[0323] Step 1: Preparation of tert-butyl (S)-2-(1'-(5-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)pyrimidin-4-yl)-1,1',4,4'-tetrahydro-2H,2'H-[3,3'-diazamethylene]-1-carbonyl)pyrrolidine-1-carboxylate

[0324]

[0325] At room temperature, (2S)-1-[(tert-butoxy)carbonyl]pyrrolidine-2-carboxylic acid (21.92 mg, 0.10 mmol), diisopropylethylamine (50 mg, 0.39 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38.73 mg, 0.10 mmol) and N,N-dimethylformamide (5 mL) were added sequentially into the reaction flask and stirred evenly. Subsequently, 2-((4-(1',4'-dihydro-2H,2'H-[3,3'-diazomethylene]-1(4H)-yl)pyrimidin-5-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide (40 mg, 0.097 mmol) was added and stirring was continued for 10 minutes. LCMS monitored the complete reaction of the starting material, and water was added to quench the reaction. The reaction was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to give 59 mg of crude tert-butyl (2S)-2-(3-(1-(5-(2-(ethyl(propan-2-yl)carbamoyl)-4-fluorophenoxy)pyrimidin-4-yl)azetidine-3-ylidene)azetidine-1-carbonyl)pyrrolidine-1-carboxylate, which was used directly in the next reaction without purification.

[0326] MS (ESI) m / z (M+H) + =609.3.

[0327] Step 2: Preparation of (S)-N-ethyl-5-fluoro-N-isopropyl-2-((4-(1'-prolyl-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)pyrimidin-5-yl)oxy)benzamide

[0328]

[0329] At room temperature, tert-butyl (S)-2-(1'-(5-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)pyrimidin-4-yl)-1,1',4,4'-tetrahydro-2H,2'H-[3,3'-diazamethylene]-1-carbonyl)pyrrolidine-1-carboxylate (59 mg, 0.097 mmol), trifluoroacetic acid (3 mL) and dichloromethane (10 mL) obtained in the previous step were added to the reaction flask in sequence and stirred evenly. The reaction system was then heated to 40°C for 20 minutes. LCMS monitoring showed that the reaction of the raw material was complete, and the solvent was removed by rotary evaporation. The residue was dissolved in a mixed solvent of dichloromethane and methanol (10:1), solid sodium bicarbonate was added, and a small amount of water was added dropwise to adjust the pH to alkaline. The product was filtered through celite, and the filtrate was collected and rotary evaporated to obtain the residue. The title compound (S)-N-ethyl-5-fluoro-N-isopropyl-2-((4-(1'-prolyl-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)pyrimidin-5-yl)oxy)benzamide was obtained by preparative purification (8 mg).

[0330] MS (ESI) m / z (M+H) + =509.3.

[0331] 1 H NMR(400MHz,Methanol-d4)δ8.30(s,1H),7.83(s,1H),7.24–7.13(m,2H),7 .04–6.94(m,1H),4.84–4.71(m,6H),4.62–4.38(m,2H),3.97–3.80(m,1H),3 .70–3.62(m,1H),3.58–3.44(m,1H),3.43–3.21(m,1H),3.16–3.01(m,1H), 2.89–2.74(m,1H),2.21–2.01(m,1H),1.90–1.64(m,3H),1.38–1.00(m,9H).

[0332] Experimental Example 1: Menin-MLL protein interaction inhibitory activity test

[0333] (1) Menin and MLL protein interaction inhibitory activity test

[0334] Fluorescence Polarization method was used to detect the IC of the test compound to inhibit Menin-MLL protein interaction. 50 value.

[0335] The specific steps were as follows: Compound stock solutions (10 mM, prepared in DMSO) were serially diluted three-fold with DMSO to ten concentrations: 10000.00, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52, and 0.51 nM. Using ECHO 665 Series Acoustic Liquid Handlers (BECKMAN Inc.), 50 nL of each of the test compound concentrations (10 gradients) and a DMSO solution without compound (negative control well) were transferred to a 384-well plate and centrifuged at 1000 rpm. Using I.DOT (DISPENDIX Inc.), 5 μL of Menin (ICE Inc., Cat. No. E2208F-H15H) was added to each assay well and incubated at 25°C for 10 minutes. 5 μL of FITC-MLL4-43 (Genscrip Inc.) was pipetted into each assay well using I.DOT, centrifuged at 1000 rpm, and incubated for 60 minutes. FP signals were measured using a PHERAstar FSX multifunctional microplate reader (BMG Labtech Inc.) and data were processed.

[0336] Compound IC50 values were calculated using a nonlinear regression equation: Inhibition % = (Signal in negative control well - Signal in compound well) / (Signal in negative control well - Background signal) * 100% (background signal is the signal detected in wells containing only 10 μM SNDX-5613). Dose-response curves were fitted using the logarithmic value of compound concentration as the X-axis and the percentage inhibition (Inhibition %) as the Y-axis to determine the IC50 value for each compound's inhibition of the Menin-MLL protein interaction. The experimental results are shown in Table 4.

[0337] (2) Inhibitory activity test of the interaction between mutant Menin-M327I and Menin-T349 M and MLL protein

[0338] Fluorescence Polarization method was used to detect the IC of the test compounds that inhibited the interaction between Menin-M327I, Menin-T349 M and MLL protein. 50 value.

[0339] The specific steps were as follows: the compound stock solution (10 mM, prepared in DMSO) was diluted three-fold with DMSO to a concentration of 10,000.00, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52, and 0.51 nM. Using ECHO665 Series Acoustic Liquid Handlers (BECKMAN Inc.), 50 nL of each of the test compound (10 concentrations) and a DMSO solution without compound (negative control well) were transferred to a 384-well plate and centrifuged at 1000 rpm for later use. 5 μL of Menin-M327I (ICE Inc., Cat. No. A130412011) and Menin-T349M (ICE Inc., Cat. No. A130413011) were pipetted into each assay well using an I.DOT (DISPENDIX Inc.) and incubated at 25°C for 10 minutes. 5 μL of FITC-MLL4-43 (Genscrip Inc.) was pipetted into each assay well using an I.DOT, centrifuged at 1000 rpm, and incubated for 60 minutes. FP signals were measured and data processed using a PHERAstar FSX multi-function microplate reader (BMG Labtech Inc.).

[0340] Fitting compound IC with nonlinear regression equation 50 Inhibition % = (Signal of negative control well - Signal of compound well) / (Signal of negative control well - Background signal) * 100% (Background signal is the signal value detected in the well containing only 100 μM SNDX-5613). The log value of compound concentration is used as the X-axis and the percentage inhibition rate (Inhibition %) is used as the Y-axis. Dose-effect curves were fitted to obtain the IC value of each compound for inhibiting the interaction between Menin-M327I, Menin-T349 M and MLL protein. 50 The experimental results are shown in Table 4.

[0341] Experimental Example 2: Cell proliferation inhibition experiment

[0342] (1) Cell plating:

[0343] Remove the cells from the incubator and place them on the operating table. Mix them by gently pipetting and count them using a CounterStar.

[0344] Dilute cells to the desired density with fresh complete medium. MV-4-11 cells (Nanjing Kebai, Catalog No. CBP60522) were plated in RPMI1640 (containing HEPES) (BOSTER, Catalog No. PYG0122) + 10% FBS (GIBCO, Catalog No. 10099-141C) + 1% P / S (HyClone, Catalog No. SV30010). The cell density was 1 × 10^4 cells / well, and 100 μL / well was used.

[0345] Use an electric dispenser to pipette 100 μL of each of the above cell suspensions into a 96-well plate.

[0346] (2) Preparation of compounds:

[0347] The stock solution of the compound was diluted with DMSO from 10 mM to 4000, 1200, 400, 120, 40, 12, 4, 1.2, and 0 μM, with 0 μM being used as a control well.

[0348] After thorough mixing, 1.3 μL was taken out with an electric dispenser and added to 258.7 μL of culture medium, which was diluted 100 times and the DMSO content was 0.5%.

[0349] After thorough mixing using a spray gun, the cells were removed and two replicate wells were set for each compound concentration. 100 μL of the compound diluted in step 2 was added to each replicate well. The final compound concentrations were: 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0 μM. At this time, a total of 200 μL of culture medium was placed in each well of the cell culture plate, and the DMSO content was 0.25%. The wells containing only 0.25% DMSO (compound concentration of 0) served as control wells.

[0350] The cells were returned to the 37°C, 5% CO2 incubator for continued culture and tested 3 days after compound treatment.

[0351] (3) CTG test:

[0352] After the culture time is up, the cells are removed and a portion of the culture medium is aspirated to leave 50 μL of culture medium in each well. 50 μL / well of CTG reagent (cellcounting-Lite 2.0, Vazyme, DD1101-02) is added using a dispenser.

[0353] Incubate on a shaker at room temperature for 15 min and allow to equilibrate at room temperature for 15 min.

[0354] Detect using a multi-function microplate reader.

[0355] (4) Data analysis:

[0356] Calculate cell viability: Cell viability % = As / Ac × 100%. As: test wells (culture medium containing cells, CTG, test compound), Ac: control wells (culture medium containing cells, CTG, no test compound).

[0357] The log value of compound concentration was used as X-axis and cell viability (Cell viability%) was used as Y-axis to fit the dose-effect curve to obtain the IC value of each compound on cell proliferation inhibition. 50 The specific results are shown in Table 5.

[0358] Table 5 Activity data of compounds of the present invention

[0359]

[0360]

[0361] Note: In Table 5, “-” indicates that the compound has not yet been tested. “Compound No.” indicates the compound in the corresponding example. SNDX5613 refers to N-ethyl-2-((4-(7-((trans-4-(ethylsulfonylamino)cyclohexyl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)oxy)-5-fluoro-N-isopropylbenzamide, purchased from Shanghai Loulan Biotechnology Co., Ltd., with the following structural formula:

[0362]

[0363] The results in Table 5 demonstrate that the compounds of the present invention exhibit excellent activity in inhibiting the interaction between Menin and MLL proteins, as well as the interaction between mutant Menin proteins and MLL proteins, thus showing promising prospects for combating drug resistance. Furthermore, the compounds of the present invention also effectively inhibit the proliferation of MV-4-11 cells. Therefore, the compounds of the present invention have promising prospects for clinical development.

Claims

1. A compound of formula I, a pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof: in, X is N or CH; m=0, 1 or 2; n=0, 1 or 2; R1 and R2 are independently selected from C1-C6 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl; or R1, R2 and the nitrogen to which they are attached form a ring; R3 and R4 are independently selected from hydrogen, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, -(CH2)pOR5, RaCO-, sulfonyl or phosphonyl, wherein p=1-4, R5 is alkyl; Ra is alkyl or alkenyl; Ar is a substituted or unsubstituted 5-16 membered aryl or heteroaryl group, which may be a monocyclic or polycyclic ring, at least part of which has aromaticity.

2. The compound according to claim 1, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: m=1 or 2, n=1 or 2; or m=1, n=1.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: R1 and R2 are independently C1-C4 alkyl.

4. The compound according to claim 3, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: R1 is isopropyl, and R2 is methyl or ethyl.

5. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: R1, R2 and the nitrogen to which they are connected together form a 3-8 membered aliphatic heterocycle; or R1, R2 and the nitrogen to which they are connected together form a 3-6 membered aliphatic heterocycle; in addition to the existing nitrogen, the aliphatic heterocycle optionally contains 0-3 heteroatoms selected from nitrogen, oxygen, and sulfur; the aliphatic heterocycle formed by R1, R2 and the nitrogen to which they are connected may optionally be substituted by one or more groups selected from oxygen, hydroxyl, amino, carboxyl, halogen, cyano, C1-C6 alkyl, C1-C3 alkylamino, C3-C8 cycloalkyl, and 3-8 membered heterocycloalkyl.

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: R3 and R4 are independently selected from hydrogen, hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, -(CH2)pOR5, RaCO-, sulfonyl or phosphonyl, wherein p=1-3, R5 is C1-C3 alkyl; Ra is C1-C3 alkyl or C2-C4 alkenyl.

7. The compound according to claim 6, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: R3 is hydrogen and / or R4 is hydrogen; or R3 is hydrogen, R4 is selected from hydroxy, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, -CH2CH2OCH3 or -CH2OCH2CH3.

8. The compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: Ar is a substituted or unsubstituted 5-7 membered aryl, 5-7 membered heteroaryl, 8-12 membered fused aryl or 8-12 membered fused heteroaryl, wherein the backbone atoms of the heteroaryl optionally contain 1-3 heteroatoms selected from nitrogen, oxygen and sulfur.

9. The compound according to claim 8, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: Ar can be selected from the following substituted or unsubstituted groups: furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl; phenyl, pyridyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl; indolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benzisothiazole, benzoxazolyl, benzisoxazole, benzothienyl, indazolyl, benzo[d][1,2,3]triazolyl, pyrrolo[1,2-a]pyridinyl, imidazolyl [1,2-a]pyridinyl, imidazo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, isoindolin-1-one, 3,4-dihydroisoquinolin-1(2H)-one; naphthyl, quinolinyl, isoquinolinyl, naphthyridinyl, pyridadiazinyl, benzotriazinyl, benzotriazinyl, benzopyranyl, benzoγ-pyrone, tetrahydroisoquinolinyl, tetrahydronaphthyl, 3,4-dihydro-2H-1,4-benzoxazinyl, 2H-1,4-benzoxazin-3(4H)-one.

10. The compound according to claim 9, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: Ar is optionally substituted by one or more Ry at any possible position, and one or more Ry may be independently selected from oxygen, hydroxyl, amino, carboxyl, cyano, halogen, phosphonyl, sulfonyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 aminoalkyl, C1-C3 alkylamino, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl, RbCO-, C2-C4 alkenyl, 5-8 membered aryl or 5-8 membered heteroaryl, wherein Rb represents C1-C5 alkyl or C2-C4 alkenyl; the skeleton atoms of the heterocycloalkyl and the heteroaryl optionally contain 1-3 heteroatoms selected from nitrogen, oxygen and sulfur.

11. The compound according to claim 10, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that: Ry is selected from oxygen, hydroxy, amino, carboxyl, fluorine, chlorine, bromine, iodine, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, methoxy, ethoxy, chloromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, perfluoroethyl, 2,2,2-trifluoroethoxy, aminomethyl, 2-aminoethyl, dimethylaminoethyl, formyl, acetyl, acryloyl, methylphosphonyl, dimethylphosphonyl, methylsulfonyl, ethylsulfonyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl, piperidinyl, tetrahydro-1H-pyrrolazin-7a(5H)-yl)methyl, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazole, tetrazole, phenyl or pyridyl.

12. The following compounds, their pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof:

13. A pharmaceutical composition, characterized in that The invention comprises the compound according to any one of claims 1 to 12, a pharmaceutically acceptable salt, hydrate, isomer, prodrug or a mixture thereof, and a pharmaceutically acceptable excipient and / or carrier.

14. Use of the compound according to any one of claims 1 to 12, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, or the pharmaceutical composition according to claim 13, in the preparation of a medicament for preventing, alleviating or treating diseases associated with the interaction of Menin-MLL protein.

15. The use according to claim 14, characterized in that Diseases related to the interaction with Menin-MLL protein include malignant tumors, diabetes or complications related to the diseases; wherein malignant tumors include hematological tumors, lymphomas, and solid tumors.

16. The use according to claim 15, characterized in that Hematological neoplasms include leukemias and myelomas, including but not limited to acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute monocytic leukemia, chronic monocytic leukemia, childhood leukemia, acute myeloid leukemia, chronic myeloid leukemia, mixed lineage leukemia, hairy cell leukemia, precursor T-cell lymphocytic leukemia, large granular lymphocytic leukemia, meningeal leukemia, myelodysplastic syndrome, myeloproliferative disorders, myeloproliferative neoplasia, plasma cell neoplasm, multiple myeloma; Lymphoma includes, but is not limited to, cutaneous T-cell lymphoma, lymphoma, AIDS-related lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or malignant lymphoma; Solid tumors include but are not limited to pancreatic cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, glioblastoma, lung cancer, breast cancer, and prostate cancer; Related complications include, but are not limited to, leukemic meningitis.

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