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.

Description

Technical Field

[0001] This invention relates to diazonium alkylene Menin-MLL protein inhibitors, pharmaceutical compositions containing the same, and their use in the preparation of medicaments for the prevention, relief, or treatment of diseases associated with the interaction of Menin-MLL protein. Background Technology

[0002] MLL (Mixed Lineage Leukemia) protein is a histone methyltransferase, also known as KMT2A (Histone-lysine N-methyltransferase 2A). MLL rearrangement (MLL-r) leukemia is caused by a translocation at the 11q23 chromosomal locus containing the gene encoding KMT2A. This chromosomal translocation is known to produce more than 60 oncogenic fusion proteins—formed by the fusion of the N-terminus of MLL with various different proteins, among which the MLL-AF4 / 9 fusion mutation is the most malignant. MLL-r leukemia accounts for 5%–10% of adult acute leukemia and 70% of infant acute leukemia. Current treatment options are limited, mostly involving chemotherapy drugs, with a poor prognosis and a high relapse rate.

[0003] Menin is a nucleus-based protein encoded by the multiple endocrine neoplasia type 1 (MEN1) gene. It is a key cofactor of the oncogenic MLL-r fusion protein and exhibits a high affinity for MLL-r. Upon binding to MLL-r, Menin recruits chromatin-modifying enzymes such as the Dot1L or pTEFb complex, leading to enhanced transcription of genes including HOXA and MEIS1. Abnormal expression of these genes inhibits hematopoietic cell differentiation and promotes their proliferation. In vitro and in vivo experiments have shown 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 found that Menin inhibitors are effective against leukemia with NPM1 gene mutations, which account for approximately 20-30% of acute myeloid leukemia patients (Science 367, 586–590).

[0004] Several Menin inhibitors are currently undergoing phase 1 / 2 clinical trials for patients with relapsed / refractory acute leukemia harboring MLL-r or NPM1 mutations, such as Syndax's SNDX-5613, Kura Oncology's KO-539, and Daichi 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 (Nature 615, 913–919). Further research revealed that these resistant patients had MEN1 gene mutations, preventing Menin inhibitors from binding effectively to the Menin protein. Therefore, developing inhibitors that can bind to the mutated Menin protein is crucial and could offer hope for a cure to these resistant patients.

[0005] Furthermore, excessive Menin expression can inhibit β-cell proliferation, leading to relative insulin insufficiency. A Menin-MLL inhibitor has been shown to enhance β-cell proliferation, thus offering potential applications in the field of diabetes. Currently, one compound, BMF-219, is undergoing phase 2 clinical trials for type 2 diabetes.

[0006] In conclusion, Menin-MLL interaction inhibitors have promising applications in drug development, and there is a strong clinical need for their development. Summary of the Invention

[0007] This invention provides a compound of Formula I, a pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof:

[0008]

[0009] In the formula, X1, X2, X3, and X4 are all CRa; or any three of them are N and the rest are CRa; Ra at different positions can be the same or different, and Ra can be independently selected from hydrogen, halogen, or C1-C3 alkyl.

[0010] Y is either CH or N;

[0011] a is 0, 1, or 2; b is 0, 1, or 2;

[0012] R1 and R2 are each independently selected from hydrogen, C1-C3 alkyl, 3-6 membered cycloalkyl, or 3-6 membered heterocycloalkyl;

[0013] R3 is selected from hydrogen or C1-C3 alkyl, 3-6 membered cycloalkyl, or 3-6 membered heterocycloalkyl;

[0014] Ar is selected from substituted or unsubstituted 5-16 aryl groups, substituted or unsubstituted 5-16 heteroaryl groups, substituted or unsubstituted 5-16 cycloalkyl groups, and substituted or unsubstituted 5-16 heterocycloalkyl groups; Ar can be monocyclic or fused polycyclic; when Ar is monocyclic, it is an aromatic ring; when Ar is fused polycyclic, it is at least partially an aromatic ring, and the aromatic ring portion is connected to the host.

[0015] The skeletal atoms of the heterocyclic alkyl or heteroaryl groups may optionally contain 1-3 heteroatoms selected from N, O, and S.

[0016] In some implementations, one of X1, X2, X3, and X4 is N, and the rest are CRa. In some implementations, two of X1, X2, X3, and X4 are N, and the rest are CRa. In some implementations, X1 is N, and X2, X3, and X4 are CRa; or X2 is N, and X1, X3, and X4 are CRa; or X3 is N, and X1, X2, and X4 are CRa; or X4 is N, and X1, X2, and X3 are CRa. In some implementations, X1 and X3 are N, and X2 and X4 are CRa; or X1 and X4 are N, and X2 and X3 are CRa; or X2 and X4 are N, and X1 and X3 are CRa; or X1 and X2 are N, and X3 and X4 are CRa.

[0017] In some embodiments, Ra at all positions is independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, or isopropyl. In some embodiments, one of Ra is fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, or isopropyl, and the remainder is hydrogen. In some embodiments, two of Ra are independently selected from methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine, or iodine, and the remainder is hydrogen. In some embodiments, one of Ra is methyl, and the remainder is hydrogen. In some embodiments, one of Ra is fluorine, and the remainder is hydrogen. In some embodiments, Ra at all positions is hydrogen.

[0018] In some implementations, a is 1 and b is 1.

[0019] In some embodiments, R1 and R2 are independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxecyclopropyl, and azircyclopropane. In some embodiments, R1 is hydrogen, and R2 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxecyclopropyl, and azircyclopropane. In some embodiments, R1 is hydrogen, and R2 is isopropyl. In some embodiments, both R1 and R2 are hydrogen.

[0020] In some embodiments, R3 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxacyclopropane, and azircyclopropane. In some embodiments, R3 is hydrogen or isopropyl.

[0021] In some embodiments, Ar is selected from substituted or unsubstituted 5-14-membered aryl groups, substituted or unsubstituted 5-14-membered heteroaryl groups, substituted or unsubstituted 5-14-membered cycloalkyl groups, or substituted or unsubstituted 5-14-membered heterocycloalkyl groups. In some embodiments, Ar is selected from substituted or unsubstituted 5-10-membered aryl groups, substituted or unsubstituted 5-10-membered heteroaryl groups, substituted or unsubstituted 5-10-membered cycloalkyl groups, or substituted or unsubstituted 5-10-membered heterocycloalkyl groups.

[0022] In some implementations, Ar is a single ring. In some implementations, Ar is a fused bicyclic ring. In some implementations, Ar is a fused tricyclic ring.

[0023] In some embodiments, Ar may be selected from self-substituted or unsubstituted furanyl, thiophene, pyrrole, oxazolyl, thiazolyl, imidazole, pyrazolyl; phenyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl; indole, benzimidazolyl, benzofuranyl, benzothiazolyl, benzoisothiazolyl, benzooxazolyl, benzoisoxazole, benzothiaphenyl, indazole, indoleazinyl, imidazole[1,2-a]pyridinyl, imidazole[1, [5-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, imidazo[1,5-a]pyrazinyl, isoindoline-1-one, 3,4-dihydroisoquinoline-1(2H)-one; naphthyl, quinolinyl, isoquinoline, naphthidyl, pyridodiazinyl, benzotriazinyl, benzopyranyl, benzoγ-pyranone, tetrahydroisoquinoline, tetrahydronaphthyl, 3,4-dihydro-2H-1,4-benzoxazinyl, 2H-1,4-benzoxazin-3(4H)-one.

[0024] In some embodiments, Ar may be selected from the following substituted or unsubstituted fused bicyclic groups: indolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benziisothiazolyl, benzoxazolyl, benzoisoxazole, benzothiophene, indazole, indolazinyl, imidazo[1,2-a]pyridyl, imidazo[1,5-a]pyridyl, imidazo[1,5-a]pyrazinyl, pyrazo[1,5-a]pyridyl, 1,2,4-triazolo[4,3-A]pyr, naphthyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydronaphthyl.

[0025] In some implementations, Ar is a substituted or unsubstituted phenyl group.

[0026] In some embodiments, Ar is selected from the following groups, whether substituted or unsubstituted:

[0027]

[0028] In some embodiments, Ar may be substituted at any possible position with one or more of the following groups: halogen, cyano, C1-C6 alkyl, C1-C3 haloalkyl, C1-C3 alkylamino, C3-C8 cycloalkyl, 3-8 heterocyclic alkyl, 5-8 aryl or 5-8 heteroaryl.

[0029] In some embodiments, Ar may be substituted at any possible position with one or more independent groups selected from fluorine, chlorine, bromine, iodine, cyano, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, 2,2,2-trifluoroethyl, 2,2-difluoropropyl, 3-fluoropropyl, dimethylamino, diethylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolyl, 1-methylpyrrolyl, piperidinyl, tetrahydro-1H-pyrrolazinyl, pyrrolyl, phenyl, or selected from the following groups:

[0030]

[0031] In some embodiments, one or more hydrogen atoms at any possible position of the compound represented by Formula I, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof are replaced by deuterium.

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

[0033]

[0034]

[0035]

[0036] The present invention also provides a pharmaceutical composition comprising any of the compounds described above, a pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, and a pharmaceutically acceptable excipient and / or carrier.

[0037] The present invention also provides the use of any of the foregoing compounds, pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for the prevention, relief or treatment of diseases associated with the interaction of Menin-MLL protein.

[0038] In this invention, diseases associated with the interaction with the Menin-MLL protein include malignant tumors, diabetes, or complications related to said diseases. Malignant tumors include hematologic malignancies, lymphomas, and solid tumors.

[0039] Hematologic malignancies include leukemia and myeloma, including but not limited to acute lymphoblastic 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 neoplasm, plasmacytoma, and multiple myeloma.

[0040] Lymphomas include, but are not limited to, cutaneous T-cell lymphoma, lymphoid tumor, AIDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, or malignant lymphoma.

[0041] Solid tumors include, but are not limited to, pancreatic cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, glioblastoma, lung cancer, breast cancer, and prostate cancer.

[0042] Related complications include, but are not limited to, leukemic meningitis.

[0043] Experiments have shown that the Menin-MLL protein inhibitor of the present invention has excellent in vitro enzyme inhibitory activity and cell proliferation inhibitory activity, especially excellent activity in inhibiting the interaction between Menin mutant protein and MLL protein, thus showing good prospects for resisting drug resistance.

[0044] This invention was funded by the Sichuan Provincial Science and Technology Program. Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Definitions and General Descriptions

[0047] This indicates the connection position of the group with other structures.

[0048] Unless otherwise specified, "substitution" means that a hydrogen atom in a molecule is replaced by another different atom or group.

[0049] "Alkyl" refers to a saturated hydrocarbon group consisting only of carbon and hydrogen atoms, with single bonds connecting carbon atoms and hydrogen atoms. Alkyl groups can be straight-chain 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] "Heteroatoms" refer to non-carbon atoms in the carbon chain or framework. Typical heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur.

[0051] "Member" refers to the number of skeleton atoms that make up the ring. Typical 5-membered rings include, for example, cyclopentyl, pyrrole, tetrahydropyrrole, imidazole, thiazole, furan, tetrahydrofuran, and thiophene; typical 6-membered rings include, for example, cyclohexyl, piperidine, piperazine, pyridine, pyran, pyrazine, thiamphenicol, pyridazine, pyrimidine, and benzene.

[0052] "Cycloalkyl" refers to non-aromatic cyclic hydrocarbon groups whose skeleton atoms are all carbon atoms, including saturated or unsaturated monocyclic, bicyclic, or polycyclic systems (such as fused 2, 3, 4 rings, or spirocyclic rings). Specifically, it includes cyclic alkyl or alkenyl groups, examples of which include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, and cyclohexenyl. The definition of "cycloalkyl" also includes aromatic rings having one or more fused to a cycloalkyl ring, such as benzo[a] or pyrido[b] derivative groups of cyclopentane, cyclopentene, cyclohexane, etc., a specific example being tetrahydronaphthalene.

[0053] "Heterocyclic alkyl" refers to a non-aromatic cyclic hydrocarbon group whose skeleton contains one or more heteroatoms, including saturated or unsaturated monocyclic, bicyclic, or polycyclic systems (such as 2, 3, or 4 fused rings or spirocyclic rings). The heteroatoms are selected from nitrogen, oxygen, and sulfur, and the remaining ring atoms are carbon. Examples include, but are not limited to: nitrogen-heterocyclic butyl, oxocyclic butyl, pyrrolinyl, pyrrolylyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl, etc. The definition of "heterocyclic alkyl" also includes one or more aromatic rings fused to the aforementioned cycloalkyl or heterocyclic alkyl rings, wherein the aromatic ring may or may not contain the aforementioned heteroatoms. Specific examples include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, or benzoγ-pyranone.

[0054] "Aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group (e.g., having 2, 3, or 4 fused rings). Typical aryl groups include, but are not limited to, phenyl, naphthyl, anthracene, phenanthrene, etc.

[0055] "Heteroaryl" refers to a monocyclic or polycyclic aromatic heterocycle (e.g., having 2, 3, or 4 fused rings) having one or more heteroatom aromatic rings selected from N, S, and O. Typical heteroaryl groups include, but are not limited to, pyridyl, indolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothiopheneyl, benzofuranyl, benzothiopheneyl, benzopyranyl, benzothiapyranyl, furanyl, pyrroleyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thiopheneyl, oxadiazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, etc.

[0056] "Alkenyl" refers to an unsaturated hydrocarbon group having one or more -C=C- (carbon-carbon double bonds). Examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, etc.

[0057] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0058] “Cyano” refers to -CN.

[0059] "Halogenated alkyl" means that an alkyl group is replaced by one or more halogen atoms, wherein the alkyl group has the meaning described in this invention. Halogenated alkyl includes, but is not limited to, monohalogenated alkyl, dihalogenated alkyl, trihalogenated alkyl, perhalogenated alkyl, etc., such as chloromethyl, dichloromethyl, difluoromethyl, dibromomethyl, trifluoromethyl, 2,2,2-trifluoroethyl, perfluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, etc.

[0060] "Alkoxy" refers to -O-alkyl, where alkyl has the meaning as described in this invention.

[0061] The word "amino" alone or in combination with other terms refers to the -NH2 group.

[0062] "Aminoalkyl" indicates that an alkyl group is replaced by one or more amino groups, wherein the alkyl group has the meaning described in this invention. For example, C1-C6 aminoalkyl means a C1-C6 alkyl group replaced by one or more amino groups. Aminoalkyl includes, but is not limited to, aminomethyl and 2-aminoethyl.

[0063] "alkylamino" represents a -NH (alkyl) group 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 may be methylamino or ethylamino. The dialkylamino group is dimethylamino or diethylamino.

[0064] "Hydroxy" refers to -OH.

[0065] The compounds of the present invention may also include all isotopes of the atoms present in the intermediates or the final compound. Isotopes include atoms having the same number of atoms but different mass numbers. The isotopes of the component atoms of the compounds of the present invention can exist in natural or non-natural abundance. Examples of hydrogen isotopes include deuterium and tritium. In some embodiments, the compounds of the present invention are deuterated, i.e., at least one deuterium atom replaces 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 with deuterium in a molecule are known in the art.

[0066] In this document, unless otherwise stated, the term "Ca-Cb" as used refers to a portion having ab carbon atoms (b is greater than a, and both are integers). For example, C 1- C3 indicates that the modified part has 1 to 3 carbon atoms, such as 1 carbon atom, 2 carbon atoms, or 3 carbon atoms.

[0067] "pq-membered heterocyclic hydrocarbon group" refers to the portion modified by this term having pq carbon atoms (q is greater than p, and both are integers) and the number of heteroatoms involved in cyclization. For example, a 3-6 membered heterocyclic hydrocarbon group indicates that its modified cyclic structure has 3, 4, 5, or 6 atoms, including carbon atoms and at least one heteroatom.

[0068] "yz-membered (hetero)aryl" refers to the total number of carbon atoms (z is greater than y, and both are integers) and heteroatoms involved in ring formation in the modified part. For example, 5-7-membered aryl indicates that the modified aryl structure has 5, 6, or 7 carbon atoms; for example, 5-7-membered heteroaryl indicates that the modified heteroaryl structure has 5, 6, or 7 atoms, including carbon atoms and at least one heteroatom.

[0069] "Optional" means that the events or circumstances described below can be freely chosen or not chosen.

[0070] "Hydrate" refers to an aggregate of one or more water molecules contained in the compounds of this invention, including hemihydrates, monohydrates, dihydrates, trihydrates, etc.

[0071] "Isomer" refers to the fact that when the compounds of the present invention contain one or more asymmetric centers, they can exist as racemic mixtures and racemic mixtures, single enantiomers, diastereomer mixtures, and single diastereomers. The compounds of the present invention may have asymmetric centers, thereby resulting in two optical isomers. The scope of the present invention includes all possible optical isomers and mixtures thereof. If the compounds of the present invention contain an olefin double bond, the scope of the present invention includes cis and trans isomers unless otherwise specified. The compounds of the present invention can exist as tautomers (a type of functional group isomer) having different hydrogen connection points through one or more double bond shifts; for example, a ketone and its enol form are keto-enol tautomers. All tautomers and mixtures thereof are within the scope of the present invention. Enantiomers of all compounds. Diastereomers, racemates, mesomates, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof are all within the scope of the present invention.

[0072] "Prodrug" refers to a derivative compound that, upon administration to an individual, can directly or indirectly provide the compounds of the present invention. Particularly preferred derivative compounds or prodrugs are those that, upon administration to an individual, can improve the bioavailability of the compounds of the present invention (e.g., facilitate absorption into the bloodstream) or promote the delivery of the parent compound to its site of action (e.g., the lymphatic system). Unless otherwise stated, 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, see, for example, T. Higuchi, V. Stella, Prodrugs as Novel Drug Delivery Systems [J], American Chemical Society, Vol. 14, 1975. Furthermore, the present invention also covers compounds of the present invention containing a protecting group. In any process of preparing the compounds of the present invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the present invention. This can be achieved by conventional protecting groups, such as those described in T.W. Greene, P.G. M. W. Uts, Protective Groups in Organic Synthesis [M], John Wiley & Sons, 2006. These protective bases can be removed at appropriate subsequent stages using methods known in the art.

[0073] "Pharmaceutical composition" refers to a formulation of the compounds of the present invention with a medium generally accepted in the art for delivering a bioactive compound to a mammal (e.g., a human). This medium includes pharmaceutically acceptable carriers. The purpose of a pharmaceutical composition is to facilitate administration to the organism, thereby promoting the absorption of the active ingredient and the exertion of its bioactivity.

[0074] "Pharmaceutical acceptable" means a substance, such as a carrier, diluent, or excipient, that does not affect the biological activity or properties of the compounds of this invention and is relatively non-toxic, meaning that the substance can be administered to an individual without causing an adverse biological reaction or interacting adversely with any component contained in the composition. For example, "excipients" include, but are not limited to, any adjuvants, carriers, excipients, flow aids, sweeteners, diluents, preservatives, dyes / coloring agents, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are permitted by the relevant government regulatory authorities to be acceptable for human or animal use.

[0075] "Pharmaceutically acceptable salts" include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0076] The present invention also provides a method for synthesizing the above-mentioned compounds. The method of synthesis of the present invention mainly adopts the preparation methods reported in chemical literature or uses commercially available chemical reagents as starting materials for related synthesis.

[0077] Abbreviation Explanation

[0078] PhLi represents lithium phenylene.

[0079] s-BuLi represents sec-butyllithium

[0080] TMEDA represents N,N,N',N'-tetramethylethylenediamine.

[0081] CbzCl represents benzyl chloroformate.

[0082] MsCl represents methylsulfonyl chloride.

[0083] TEA represents triethylamine.

[0084] DMAP represents 4-dimethylaminopyridine

[0085] DCM represents dichloromethane.

[0086] THF represents tetrahydrofuran.

[0087] DMF stands for N,N-dimethylformamide

[0088] AcOH represents glacial acetic acid.

[0089] TsOH indicates p-toluenesulfonic acid

[0090] EtOH represents ethanol.

[0091] i-PrOH / IPA represents isopropanol.

[0092] mCPBA represents 3-chloroperoxybenzoic acid.

[0093] HATU represents O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0094] DIEA represents ethyl diisopropylamine.

[0095] NaBH4 represents sodium borohydride.

[0096] POCl3 represents phosphorus oxychloride

[0097] TMSCl represents trimethylchlorosilane.

[0098] Cs2CO3 represents cesium carbonate.

[0099] Pd(dppf)2Cl2 represents [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride.

[0100] Pd(OAc)2 represents palladium acetate.

[0101] Pd(PPh3)4 represents tetraphenylphosphine palladium.

[0102] Pd(Cy3P)2Cl2 represents bis(tricyclohexylphosphine)palladium dichloride.

[0103] Cy3P represents bis(tricyclohexylphosphine)

[0104] LiCl represents lithium chloride.

[0105] LiOH represents lithium hydroxide.

[0106] S-Phos represents 2-dicyclohexylphosphine-2′,6′-dimethoxy-biphenyl

[0107] TFA represents trifluoroacetic acid.

[0108] DCC represents N,N'-dicyclohexylcarbodiimide

[0109] NBS represents N-bromosuccinimide

[0110] AIBN represents azobisisobutyronitrile (AIBN).

[0111] HMTA represents Utopia.

[0112] TFAA represents trifluoroacetic anhydride.

[0113] CMBP represents cyanomethylenetri-n-butylphosphine.

[0114] Preparation of intermediates Example 1: Preparation of benzyl 1',4'-dihydro-2H,2'H-[3,3'-diazacyclobutanediyl]-1(4H)-carboxylic acid ester

[0115]

[0116] Step 1: Preparation of tert-butyl 3-(1-azabicyclo[1.1.0]but-3-yl)-3-hydroxyazacyclobutane-1-carboxylic acid

[0117]

[0118] 2,3-Dibromopropane-1-amine hydrobromide (4.8 g, 16.12 mmol) was dissolved in anhydrous tetrahydrofuran (60 mL), and the reaction system was cooled to -65 °C. Then, a solution of phenyllithium (2N, 48.35 mmol) in n-butyl ether was slowly added dropwise while maintaining the temperature below -60 °C. After the addition was complete, the reaction system was stirred at -65 °C for 2 hours. The cryogenic bath was then removed, and the temperature was rapidly raised to room temperature and stirred for 10 minutes. The reaction system was then cooled to -65 °C again, and 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 was added dropwise while maintaining the temperature below -60 °C. After the addition of the reactants was complete, the reaction was stirred at -65°C for 1 hour. Then, a tetrahydrofuran solution of 3-oxoazacyclobutane-1-carboxylic acid tert-butyl ester (3.59 g, 20.95 mmol) was slowly added dropwise. After the addition of the reactants was complete, the reaction system was stirred at -65°C for 1 hour. LCMS analysis showed that the reactants reacted completely. The reaction was quenched with water, extracted with ethyl acetate, and the organic phases were combined, dried, concentrated, and then used directly in the next step of the reaction.

[0119] Step 2: Preparation of 1-benzyl 1'-(tert-butyl)3'-hydroxy-3-iodo-[3,3'-diazacyclobutane]-1,1'-dicarboxylic acid

[0120]

[0121] 3-(1-azabicyclo[1.1.0]but-3-yl)-3-hydroxyazacyclobutane-1-carboxylic acid tert-butyl ester (2.41 g, 10.65 mmol) and sodium iodide (3.19 g, 21.30 mmol) were dissolved in acetonitrile (30 mL). The reaction system was cooled to 0 °C, and then benzyl chloroformate (2.18 g, 12.78 mmol) was slowly added dropwise. After the addition was complete, the reaction was stirred at 0 °C for 30 minutes. After the reaction was complete, the sample was purified by column chromatography to give 4.58 g of the title compound.

[0122] MS(ESI)m / z(M+H-100) + =389.1

[0123] Step 3: Preparation of 1-benzyl 1'-(tert-butyl)3-iodo-3'-((methanesulfonyl)oxy)-[3,3'-bis(azetane)-1,1'-dicarboxylic acid

[0124] 1-Benzyl-1'-(tert-butyl)3'-hydroxy-3-iodo-[3,3'-diazacyclobutane]-1,1'-dicarboxylic acid (2.12 g, 4.34 mmol) was dissolved in dichloromethane (50 mL). After cooling the reaction system to 0 °C, triethylamine (878 mg, 8.68 mmol) and 4-dimethylaminopyridine (1.06 g, 8.68 mmol) were added; subsequently, methanesulfonyl chloride (995 mg, 8.68 mmol) was slowly added dropwise. After the addition was complete, the reaction system was stirred at room temperature for 30 minutes. After the reaction was complete, the sample was purified by column chromatography to obtain 1.82 g of the title compound.

[0125] MS(ESI)m / z(M+H-100) + =467.1.

[0126] Step 4: Preparation of 1-benzyl 1'-(tert-butyl)2H,2'H-[3,3'-diazahexacyclic butanediyl]-1,1'(4H,4'H)-dicarboxylic acid

[0127]

[0128] 1-Benzyl1'-(tert-butyl)3-iodo-3'-((methanesulfonyl)oxy)-[3,3'-bis(azetane)-1,1'-dicarboxylic acid (1.8 g, 3.18 mmol) was dissolved in acetic acid (20 mL). Zinc powder (1.04 g, 15.89 mmol) was then added. After the addition was complete, the reaction mixture was stirred at 60 °C for 2 hours. After the reaction was complete, the mixture was purified to obtain 980 mg of the title compound.

[0129] MS(ESI)m / z(M+H-100) + =245.2.

[0130] 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).

[0131] Step 5: Preparation of benzyl 1',4'-dihydro-2H,2'H-[3,3'-diazacyclobutanediyl]-1(4H)-carboxylic acid ester

[0132]

[0133] 1-Benzyl 1'-(tert-butyl)2H,2'H-[3,3'-diazacyclobutanediyl]-1,1'(4H,4'H)-dicarboxylic acid ester (980 mg, 2.80 mmol) was dissolved in ethanol (32 mL), and then p-toluenesulfonic acid (1.6 g, 9.29 mmol) was added. The reaction mixture was stirred at 50 °C for 6 hours. After the reaction was complete, the product was purified by column chromatography to give 1.16 g of the title compound, p-toluenesulfonate.

[0134] MS(ESI)m / z(M+H) + =245.1.

[0135] Preparation Example 2: Preparation of benzyl 1'-(6-chloro-3-hydrazino-1,2,4-triazin-5-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid ester

[0136]

[0137] Step 1: Preparation of 1'-(3,6-dichloro-1,2,4-triazin-5-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester

[0138]

[0139] Under ice-water bath conditions, triethylamine (13.72 g, 135.57 mmol) and 3-(azacyclobutane-3-yl)azacyclobutane-1-carboxylic acid benzyl ester p-toluenesulfonate (14.57 g, 59.65 mmol) were added to a solution of trichloro-1,2,4-triazine (10 g, 54.23 mmol) in dichloromethane (200 mL). The reaction system was reacted at this temperature for 1 hour. The reaction was confirmed to be complete by LC-MS. The solvent was evaporated, and the crude product was subjected to silica gel column chromatography to give 15.14 g of the title compound.

[0140] MS(ESI)m / z(M+H) + =392.2.

[0141] Step 2: Preparation of 1'-(6-chloro-3-hydrazino-1,2,4-triazin-5-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester

[0142]

[0143] At room temperature, hydrazine hydrate (8.17 g, 163.2 mmol) was added to an ethanol (200 mL) solution of 1'-(3,6-dichloro-1,2,4-triazin-5-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester (8 g, 20.40 mmol). The mixture was stirred and heated to 70 °C for 4 hours. After the reaction was complete, the reaction system was cooled to room temperature. The mixture was filtered, and the filter cake was washed with water and ethanol and dried to give 6.51 g of the title compound.

[0144] MS(ESI)m / z(M+H) + =388.1.

[0145] Preparation Example 3: Preparation of 2-(2-cyclopropylphenyl)-4-fluorophenol

[0146]

[0147] At room temperature, (2-cyclopropylphenyl)boric acid (2.0 g, 12.1 mmol), 4-fluoro-2-iodophenol (3.2 g, 13.3 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (442.7 mg, 0.6 mmol), potassium carbonate (5.0 g, 36.3 mmol), 1,4-dioxane (20 mL), and water (5 mL) were added to a reaction flask. After purging with nitrogen, the mixture was stirred until homogeneous, and the temperature was slowly raised to 100 °C. The reaction was carried out for 2 hours, and the reaction was monitored by LCMS until complete. The product 2-(2-cyclopropylphenyl)-4-fluorophenol was obtained by rotary evaporation and purification by column chromatography.

[0148] MS(ESI)m / z(M+H) + =229.1.

[0149] Preparation Example 4: Preparation of 5-chloro-4-cyclopropyl-2-methylpyridine

[0150]

[0151] Step 1: Preparation of 5-chloro-2-methylpyridine-4-amine

[0152]

[0153] At room temperature, 2,5-dichloropyridin-4-amine (2.5 g, 15.3 mmol), trimethyl-1,3,5,2,4,6-trioxazolinone (4.2 g, 16.9 mmol), [1,1'-bis(diphenylphosphine)ferrocene]dichloropalladium(II) (1.1 g, 1.5 mmol), potassium carbonate (6.4 g, 46.0 mmol), and 1,4-dioxane (100 mL) were added to a reaction flask, purged with nitrogen, and heated to 120 °C. After 2 hours of reaction, the reaction was monitored by LCMS to indicate completion. The product, 5-chloro-2-methylpyridin-4-amine, was obtained by rotary evaporation and purified by column chromatography (2.1 g).

[0154] MS(ESI)m / z(M+H) + =143.1.

[0155] Step 2: Preparation of 4-bromo-5-chloro-2-methylpyridine

[0156]

[0157] Under ice-water bath conditions, 2.0 g (13.7 mmol) of 5-chloro-2-methylpyridin-4-amine and 40 mL of 48% hydrobromic acid were added to a reaction flask and stirred until homogeneous. Bromine (7.0 g, 43.8 mmol) was then slowly added dropwise. After the addition was complete, the mixture was cooled using an ice-salt bath. Sodium nitrite (2.6 g, 38.3 mmol) was dissolved in 30 mL of water and slowly added dropwise to the system. After the addition was complete, the system was heated to room temperature and reacted overnight. The reaction was monitored by LC-MS to ensure completeness. The reaction was quenched with sodium sulfite solution, the pH was adjusted to alkaline with sodium hydroxide solution, and the mixture was extracted with methyl tert-butyl ether. The organic phase was collected, evaporated to dryness, and purified by column chromatography to obtain 2.3 g of the product 4-bromo-5-chloro-2-methylpyridinium.

[0158] MS(ESI)m / z(M+H) + =206.1.

[0159] Step 3: Preparation of 5-chloro-4-cyclopropyl-2-methylpyridine

[0160]

[0161] At room temperature, 1.0 g (4.8 mmol) of 4-bromo-5-chloro-2-methylpyridine, 280 mg (0.2 mmol) of tetraphenylphosphine palladium, and 30 mL of tetrahydrofuran were added to a reaction flask. The mixture was purged with nitrogen, and 7.3 mL (7.3 mmol) of 1 M cyclopropylmagnesium bromide was slowly added. After the addition was complete, the mixture was heated to 65 °C and reacted for 2.5 hours. After the reaction was monitored by LCMS until complete, the reaction was quenched with saturated ammonium chloride solution, extracted with ethyl acetate, and washed with sodium chloride solution. The solution was evaporated to dryness and purified by column chromatography to obtain 603 mg of the product 5-chloro-4-cyclopropyl-2-methylpyridine.

[0162] MS(ESI)m / z(M+H) + =168.1.

[0163] Preparation Example 5: Preparation of 3-bromo-2-cyclopropyl-5-fluoropyridine

[0164]

[0165] Step 1: Preparation of 2-cyclopropyl-5-fluoropyridine-3-amine

[0166]

[0167] At room temperature, 2-bromo-5-fluoropyridine-3-amine (5.0 g, 26.2 mmol), cyclopropylboronic acid (2.2 g, 130.9 mmol), [1,1'-bis(diphenylphosphine)ferrocene]dichloropalladium(II) (1.9 g, 2.6 mmol), potassium carbonate (1.1 g, 78.5 mmol), and 1,4-dioxane (20 mL) were added to a reaction flask. After purging with nitrogen, the mixture was heated to 120 °C and reacted for 1 hour. After the reaction was complete as monitored by LCMS, the mixture was filtered, concentrated, and purified by column chromatography to obtain 3.7 g of the product 2-cyclopropyl-5-fluoropyridine-3-amine.

[0168] MS(ESI)m / z(M+H) + =153.1.

[0169] Step 2: Preparation of 3-bromo-2-cyclopropyl-5-fluoropyridine

[0170]

[0171] Under ice-water bath conditions, 2-cyclopropyl-5-fluoropyridin-3-amine (2.5 g, 16.4 mmol) and 20 mL of 48% hydrobromic acid were added to a reaction flask. Sodium nitrite (1.1 g, 16.4 mmol) was slowly added. After the addition was complete, stirring was continued for 1 hour. Cuprous bromide (2.4 g, 16.4 mmol) and 10 mL of 48% hydrobromic acid were mixed thoroughly and added dropwise to the system. The reaction was continued at this temperature for 2 hours, and the reaction was monitored by LCMS to ensure completeness. The pH was adjusted to weakly alkaline by adding sodium hydroxide solution. The mixture was extracted with dichloromethane, rotary evaporated, and the crude product was purified by column chromatography to obtain 259 mg of 3-bromo-2-cyclopropyl-5-fluoropyridinium.

[0172] MS(ESI)m / z(M+H) + =216.1.

[0173] Preparation Example 6: Preparation of 3-chloro-4-cyclopropyl-6-methylpyridazine

[0174]

[0175] Step 1: Preparation of 4-cyclopropyl-6-methylpyridazine-3-ol

[0176]

[0177] At room temperature, 2.5 g (13.23 mmol) of 4-bromo-6-methylpyridazin-3-ol, 3.41 g (39.69 mmol) of cyclopropylboronic acid, 0.15 g (0.66 mmol) of palladium acetate, 0.37 g (1.32 mmol) of tricyclohexylphosphine, 5.49 g (39.69 mmol) of potassium carbonate, 50 mL of 1,4-dioxane, and 10 mL of water were added to a 100 mL single-necked flask. After purging with nitrogen, the mixture was heated to 110 °C and reacted overnight. The reaction was confirmed to be complete by LC-MS. After cooling to room temperature, the mixture was filtered through diatomaceous earth, concentrated under reduced pressure, dissolved in acetonitrile, subjected to reversed-phase column chromatography, and freeze-dried to give 0.897 g of the title compound.

[0178] MS(ESI)m / z(M+H) + =151.2.

[0179] Step 2: Preparation of 3-chloro-4-cyclopropyl-6-methylpyridazine

[0180]

[0181] At room temperature, 780 mg (5.19 mmol) of 4-cyclopropyl-6-methylpyridazin-3-ol was added to phosphorus oxychloride (42 g, 273.92 mmol), and the mixture was heated to 90 °C and reacted for 3 hours. The reaction was confirmed to be complete by LC-MS. After concentration under reduced pressure, the mixture was dissolved in a small amount of dichloromethane, and then silica gel column chromatography was performed to give 0.41 g of the title compound.

[0182] MS(ESI)m / z(M+H) + =169.1.

[0183] Preparation Example 7: Preparation of 2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenol

[0184]

[0185] At room temperature, (5-fluoro-2-hydroxyphenyl)boronic acid (1 g, 6.41 mmol), 5-bromo-4-cyclopropylpyrimidine (1.28 g, 6.41 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (0.1 g, 0.14 mmol), sodium carbonate (2.38 g, 19.23 mmol), 1,4-dioxane (60 mL), and water (15 mL) were added to a 150 mL single-necked flask. After purging with nitrogen, the reaction system was heated to 100 °C and reacted for 5 hours. The reaction was confirmed to be complete by LC-MS. After concentration under reduced pressure, the sample was stirred with silica gel and purified by column chromatography to obtain 1.35 g of the title compound.

[0186] MS(ESI)m / z(M+H) + =231.2.

[0187] Following the synthetic method of Preparation Example 7, using commercial reagents or self-made intermediates as raw materials, the following intermediate compounds can be prepared, as shown in Table 1.

[0188] Table 1 Information on intermediate compounds obtained from the preparation examples.

[0189]

[0190]

[0191] Preparation Example 14: Preparation of 1-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,1',4,4'-tetrahydro-2H,2'H-3,3'-diazamethylene hydrocarbon

[0192]

[0193] Step 1: Preparation of benzyl 1'-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid ester

[0194]

[0195] At room temperature, 2-(4-cyclopropylpyrimidin-5-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester (0.726 g, 1.87 mmol) was added to a solution of 1'-(6-chloro-3-hydrazino-1,2,4-triazin-5-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester (15 mL) in N,N-dimethylformamide (0.43 g, 1.87 mmol) and cesium carbonate (1.37 g, 4.21 mmol). The mixture was then heated to 100 °C and reacted for 3 hours. LC-MS showed that the reaction was complete. After quenching with water, the mixture was extracted with ethyl acetate. The organic phase was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the title compound, which was used directly in subsequent reactions.

[0196] MS(ESI)m / z(M+H) + =552.2.

[0197] Step 2: Preparation of 1-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,1',4,4'-tetrahydro-2H,2'H-3,3'-diazamethylene hydrocarbon

[0198]

[0199] At room temperature, trifluoroacetic acid (20 mL) was added to the benzyl 1'-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid ester obtained in the previous step. The reaction was then heated to 60 °C and reacted for 1 hour. The reaction was confirmed to be complete by LCMS, concentrated under reduced pressure, purified by alkaline column chromatography, and lyophilized to give 234 mg of the title compound.

[0200] MS(ESI)m / z(M+H) + =418.2.

[0201] Following the synthetic method of Preparation Example 14, using commercial reagents or self-made intermediates as raw materials, the following intermediate compounds can be prepared, as shown in Table 2.

[0202] Table 2 Information on intermediate compounds obtained from the preparation examples.

[0203]

[0204] Preparation Example 22: Preparation of benzyl 1'-(5-(2-bromo-4-fluorophenoxy)pyrimidin-4-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid ester

[0205]

[0206] Step 1: Preparation of 5-(2-bromo-4-fluorophenoxy)pyrimidine

[0207]

[0208] 2-Bromo-4-fluorophenol (7.5 g, 39.27 mmol), 5-bromopyrimidine (12.49 g, 78.54 mmol), cesium carbonate (31.99 g, 98.18 mmol), and N,N-dimethylformamide were added to a 250 mL single-necked flask. After purging with nitrogen, the mixture was reacted overnight at 135 °C. LC-MS showed that some of the starting material remained. After dilution with water, the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to give 13.16 g of the title compound.

[0209] MS(ESI)m / z(M+H) + =269.0.

[0210] Step 2: Preparation of 5-(2-bromo-4-fluorophenoxy)pyrimidine 1-oxide

[0211]

[0212] Under ice-water bath conditions, m-chloroperoxybenzoic acid (29.80 g, 146.76 mmol) was added in portions to a solution of 5-(2-bromo-4-fluorophenoxy)pyrimidine (13.16 g, 48.92 mmol) in dichloromethane, and the reaction was allowed to proceed overnight at a naturally heated temperature. The reaction was confirmed to be complete by LC-MS. After quenching with sodium bisulfite aqueous solution and extraction with dichloromethane, the organic phase was concentrated and purified directly by column chromatography to give 18.29 g of the title compound.

[0213] MS(ESI)m / z(M+H) + =285.1.

[0214] Step 3: Preparation of 5-(2-bromo-4-fluorophenoxy)-4-chloropyrimidine

[0215]

[0216] In an ice-water bath, triethylamine (0.21 g, 2.1 mmol) and phosphorus oxychloride (0.40 g, 2.63 mmol) were added to a chloroform solution of 5-(2-bromo-4-fluorophenoxy)pyrimidine 1-oxide (0.24 g, 0.84 mmol). After nitrogen purging, the mixture was heated to 65 °C and reacted for 1 hour. The reaction was confirmed to be complete by LC-MS, quenched with aqueous sodium bicarbonate solution, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate before column chromatography to give 75 mg of the title compound.

[0217] MS(ESI)m / z(M+H) + =303.0.

[0218] Step 4: Preparation of 1'-(5-(2-bromo-4-fluorophenoxy)pyrimidin-4-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester

[0219]

[0220] At room temperature, triethylamine (76 mg, 0.30 mmol) and p-toluenesulfonate of 1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester (120 mg, 0.30 mmol) were added to an isopropanol solution of 5-(2-bromo-4-fluorophenoxy)-4-chloropyrimidine (75 mg, 0.25 mmol). The reaction was carried out overnight at 80 °C under a nitrogen atmosphere. After confirming the completion of the reaction by LCMS, the mixture was concentrated under reduced pressure and purified by column chromatography to give 100 mg of the title compound.

[0221] MS(ESI)m / z(M+H) + =511.1.

[0222] Preparation Example 23: Preparation of 1-(5-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)pyrimidin-4-yl)-1,1',4,4'-tetrahydro2H,2'H-3,3'-diazamethylene hydrocarbon

[0223]

[0224] Step 1: Preparation of 1'-(5-(4-fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)phenoxy)pyrimidin-4-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester

[0225]

[0226] At room temperature, 1'-(5-(2-bromo-4-fluorophenoxy)pyrimidin-4-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester (230 mg, 0.45 mmol), bis(pinacolyl)diboron (450.24 mg, 1.77 mmol), bis(tricyclohexylphosphine)palladium dichloride (33.22 mg, 0.045 mmol), and 1,4-dioxane (20 mL) were added to a reaction flask. After purging with nitrogen, the mixture was heated to 110 °C and reacted overnight. LC-MS analysis showed a good reaction. After cooling to room temperature, the mixture was filtered through diatomaceous earth and used directly in subsequent reactions.

[0227] MS(ESI)m / z(M+H) + =559.2.

[0228] Step 2: Preparation of benzyl 1'-(5-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)pyrimidin-4-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid ester

[0229]

[0230] At room temperature, 1'-(5-(4-fluoro-2-(4,4,5,5-tetramethyl-1,3,2-dioxoborazecyclopentan-2-yl)phenoxy)pyrimidin-4-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester (251.28 mg, 0.45 mmol), 5-bromo-4-cyclopropylpyrimidine (36 mg, 0.045 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane complex (33.22 mg, 0.045 mmol), potassium carbonate (311 mg, 2.25 mmol), and 1,4-dioxane (20 mL) were added to a reaction flask. After purging with nitrogen, the mixture was heated to 100 °C and reacted for 1 hour. The reaction was confirmed to be good by LCMS, and after concentration under reduced pressure, the title compound was purified by column chromatography to obtain 169 mg.

[0231] MS(ESI)m / z(M+H) + =551.2.

[0232] Step 3: Preparation of 1-(5-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)pyrimidin-4-yl)-1,1',4,4'-tetrahydro2H,2'H-3,3'-diazamethylene hydrocarbon

[0233]

[0234] At room temperature, 169 mg (0.29 mmol) of 1'-(5-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)pyrimidin-4-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-carboxylic acid benzyl ester was added to a flask. After adding 5 mL of trifluoroacetic acid, the mixture was heated to 65 °C and reacted for 1 hour. The reaction was confirmed to be complete by LC-MS. The mixture was concentrated under reduced pressure, diluted with methanol-dichloromethane (1:10), and 0.5 mL of pure water was added. Sodium bicarbonate solid was then added until no gas was produced. After drying with anhydrous sodium sulfate and concentrating the organic phase, 348 mg of the title compound was obtained and used directly in the next reaction.

[0235] MS(ESI)m / z(M+H) + =417.2.

[0236] Preparation Example 24: Preparation of (S)-3-((tert-Butoxycarbonyl)amino)-4-iodobutyrate benzyl ester

[0237]

[0238] Step 1: Preparation of 4-benzyl-1-(2,5-dioxopyrrolidone-1-yl)(tert-butoxycarbonyl)-L-aspartic acid ester

[0239]

[0240] Under ice-water bath conditions, 1-hydroxypyrrolidine-2,5-dione (19.04 g, 165.46 mmol) was added to a solution of (S)-4-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (50 g, 154.64 mmol) in ethyl acetate (250 mL), followed by the dropwise addition of a solution of 1,3-dicyclohexylcarbodiimide (32.39 g, 156.96 mmol). After allowing the reaction to proceed naturally overnight, the reaction was confirmed to be complete by LC-MS. The white insoluble matter was removed by filtration and sieving. The filtrate was washed with aqueous sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by silica gel column chromatography to give 61.21 g of the title compound.

[0241] MS(ESI)m / z(M+H) + =421.2.

[0242] Step 2: Preparation of (S)-3-((tert-Butoxycarbonyl)amino)-4-hydroxybutyrate benzyl ester

[0243]

[0244] Sodium borohydride (8.98 g, 237.33 mmol) was added to a mixture of tetrahydrofuran (150 mL) and water (20 mL) under ice-water bath conditions. After stirring until homogeneous, a solution of 4-benzyl-1-(2,5-dioxopyrrolidone-1-yl)(tert-butoxycarbonyl)-L-aspartic acid ester (61.21 g, 123.76 mmol) in tetrahydrofuran (100 mL) was added dropwise. The reaction mixture was then in an ice-water bath for 2 hours. The reaction was confirmed to be complete by LC-MS, quenched with water, and then 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 give 29.2 g of the title compound.

[0245] MS(ESI)m / z(M+H) + =310.2.

[0246] Step 3: Preparation of (3S)-3-{[(tert-butoxy)carbonyl]amino}-4-iodobutyrate benzyl ester

[0247]

[0248] In an ice-water bath, 1H-imidazolium (9.64 g, 141.59 mmol) and elemental iodine (35.94 g, 141.59 mmol) were added to a tetrahydrofuran (300 mL) solution of triphenylphosphine (37.14 g, 141.59 mmol). After stirring at this temperature for 10 min, a tetrahydrofuran (15 mL) solution of (S)-3-((tert-butoxycarbonyl)amino)-4-hydroxybutyrate benzyl ester (29.2 g, 94.39 mmol) was added. The ice-water bath was then removed, and the reaction was allowed to proceed at room temperature for 1 h. After confirming the completeness of the reaction by LCMS, the reaction was quenched with water, followed by extraction with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by column chromatography to give 24.49 g of the title compound.

[0249] MS(ESI)m / z(M+H) + =420.1.

[0250] Preparation Example 25: Preparation of 7-bromo-1-(2,2,2-trifluoroethyl)-1H-indazole

[0251]

[0252] 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 homogeneous. Sodium hydroxide (121.9 mg, 3.1 mmol) was then slowly added, and the mixture was stirred for another 5 minutes at room temperature. 2,2,2-trifluoroethyltrifluoromethanesulfonate (1.2 g, 5.1 mmol) was then added, and the reaction was continued at room temperature for two hours. The reaction was monitored by LC-MS until complete. The reaction was quenched with ammonium chloride solution, filtered, and the solvent was removed by rotary evaporation. The purified product was then purified by column chromatography to obtain 253 mg of the title compound, 7-bromo-1-(2,2,2-trifluoroethyl)-1H-indazole.

[0253] MS(ESI)m / z(M+H) + =279.1.

[0254] 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).

[0255] Preparation Example 26: Preparation of 8-bromo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline

[0256]

[0257] Preparation Example 26 was prepared using commercially available reagents as raw materials, following the same method as Preparation Example 25.

[0258] MS(ESI)m / z(M+H) + =294.1.

[0259] Preparation Example 27: Preparation of 7-bromo-1-(2,2,2-trifluoroethyl)-1H-indole-2-carboxynitrile

[0260]

[0261] Preparation Example 27 was prepared using commercially available reagents as raw materials, following the same method as Preparation Example 25.

[0262] MS(ESI)m / z(M+H) + =303.1.

[0263] Preparation Example 28: Preparation of (S)-7-bromo-1-(1-methylpyrrolidone-3-yl)-1H-indazole

[0264]

[0265] At room temperature, 7-bromo-1H-indazole (1 g, 5.1 mmol), (R)-(-)-1-methyl-3-hydroxypyrrolidine (566.4 mg, 5.6 mmol), and toluene (30 mL) were added sequentially to a reaction flask. After stirring until homogeneous, nitrogen gas was introduced, followed by the addition of cyanomethylenetri-n-butylphosphine (2.4 g, 10.2 mmol), and the temperature was raised to 120 °C. After reacting for half an hour, the reaction was monitored by LC-MS to ensure complete reaction. A small amount of water was added to quench the reaction, and the organic phase was extracted and collected. The crude product was purified by column chromatography to obtain 429 mg of the target product by rotary evaporation.

[0266] MS(ESI)m / z(M+H) + =280.1.

[0267] 1 H NMR(400MHz,Chloroform-d)δ8.04(s,1H),7.66(dd,J=8.0,0.9Hz,1H),7.54(dd,J=7.4,0.9Hz,1H),6.98(t,J=7.7Hz,1H),6. 31–6.19(m,1H),3.17–3.08(m,1H),3.00–2.93(m,1H),2.92–2.87(m,1H),2.83–2.72(m,1H),2.54–2.44(m,2H),2.45(s,3H).

[0268] Preparation Example 29: Preparation of 7-bromo-6-fluoro-1-(2,2,2-trifluoroethyl)-1H-indazole

[0269]

[0270] Step 1: Preparation of 7-bromo-6-fluoro-1H-indazole

[0271]

[0272] At room temperature, 2 g of 3-bromo-2,4-difluorobenzaldehyde (9.05 mmol), 4.53 g of hydrazine hydrate (90.5 mmol), and 50 mL of 1,2-dimethoxyethane were added to a 150 mL single-necked flask and heated to 100 °C overnight. The reaction was confirmed to be good by LC-MS. After concentration under reduced pressure, the sample was stirred with silica gel and purified by normal-phase column chromatography to obtain 1.444 g of the title compound.

[0273] MS(ESI)m / z(M+H) + =215.1.

[0274] Step 2: Preparation of 7-bromo-6-fluoro-1-(2,2,2-trifluoroethyl)-1H-indazole

[0275]

[0276] Sodium hydride (0.47 g, 11.66 mmol) was added to a solution of 7-bromo-6-fluoro-1H-indazole (1.444 g, 6.73 mmol) in N,N-dimethylformamide (20 mL) in an ice-water bath. The reaction was carried out in ice water for 20 min. Then, 2,2,2-trifluoroethyltrifluoromethanesulfonate (2.71 g, 11.69 mmol) was added. After 30 min, LC-MS showed that the reaction was complete. The reaction was quenched by adding sodium bicarbonate aqueous solution. Subsequently, water and ethyl acetate were added to separate the layers. The organic phase was concentrated and subjected to reversed-phase column chromatography and freeze-drying to give 41 mg of the title compound.

[0277] MS(ESI)m / z(M+H) + =297.1.

[0278] 1 H NMR (400MHz, Methanol-d4) δ8.19 (s, 1H), 7.82 (dd, J = 8.7, 4.9Hz, 1H), 7.15 (t, J = 8.9Hz, 1H), 5.56 (q, J = 8.3Hz, 2H).

[0279] Referring to the synthesis method in step 1 of Preparation Examples 28 and 29, the following intermediate compounds can be prepared using commercial reagents or self-made intermediates as raw materials, as shown in Table 3.

[0280] Table 3 Information on the intermediate compounds obtained.

[0281]

[0282]

[0283] Preparation Example 41: Preparation of (R)-3-((tert-butoxycarbonyl)amino)-4-(2-chlorophenyl)butyric acid

[0284]

[0285] Step 1: Preparation of (R)-3-((tert-Butoxycarbonyl)amino)-4-(2-chlorophenyl)butyrate benzyl ester

[0286]

[0287] 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. Under nitrogen protection, trimethylchlorosilane (0.042 g, 0.38 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, 1,2-dibromoethane (0.29 g, 1.53 mmol) was added, and the oil bath temperature was gradually increased to 80 °C. ℃ Continue the reaction for 30 minutes; after the reaction is complete, remove the system from the oil bath and cool to room temperature. Add trimethylchlorosilane (0.12 g, 1.15 mmol), elemental iodine (0.29 g, 1.15 mmol), and (S)-3-((tert-butoxycarbonyl)amino)-4-iodobutyrate benzyl ester (1.6 g, 3.82 mmol) to the above solution and react at room temperature for 30 minutes; after the reaction is complete, add the prepared solution 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 is carried out under nitrogen protection at 40 °C for 2 hours. After confirming the completeness of the reaction by LCMS, a small amount of ammonium chloride aqueous solution was added to quench the reaction. After filtration, the mixture was directly purified by reversed-phase column chromatography and freeze-dried to obtain 0.713 g of the title compound.

[0288] MS(ESI)m / z(M+H) + =404.2.

[0289] Step 2: Preparation of (R)-3-((tert-butoxycarbonyl)amino)-4-(2-chlorophenyl)butyric acid

[0290]

[0291] Lithium hydroxide (84.78 mg, 3.54 mmol) was added to a solution of (R)-3-((tert-butoxycarbonyl)amino)-4-(2-chlorophenyl)butyrate (713 mg, 1.77 mmol) in water (6 mL) and tetrahydrofuran (20 mL) under an ice-water bath. After the addition was complete, the reaction system was allowed to rise to room temperature. The reaction was monitored by LCMS until complete. A small amount of acetic acid was added under an ice-water bath, followed by the removal of the organic phase at low temperature. After dissolving in methanol, the mixture was subjected to reversed-phase column chromatography and freeze-dried to obtain 0.423 g of the title compound.

[0292] MS(ESI)m / z(M+H) + =314.2.

[0293] Preparation Example 42: Preparation of N-((6-bromopyridin-2-yl)methyl)-3,3,3-trifluoropropionamide

[0294]

[0295] Step 1: Preparation of 2-bromo-6-(bromomethyl)pyridine

[0296]

[0297] Under ice bath conditions, 2-bromo-6-methylpyridine (5.0 g, 29.1 mmol), carbon tetrachloride (80 mL), N-bromosuccinimide (6.2 g, 34.9 mmol), and azobisisobutyronitrile (480 mg, 2.9 mmol) were added sequentially to a reaction flask, stirred until homogeneous, and heated to reflux. After 4 hours of reaction, the reaction was monitored by LCMS to ensure complete reaction. The mixture was cooled to room temperature, and the organic phase was washed with water. The organic phase was collected and purified to obtain 5.4 g of the product 2-bromo-6-(bromomethyl)pyridine.

[0298] MS(ESI)m / z(M+H) + =250.1.

[0299] Step 2: Preparation of (6-bromopyridin-2-yl)methylamine hydrochloride

[0300]

[0301] At room temperature, 5.1 g (20.3 mmol) of 2-bromo-6-(bromomethyl)pyridine, 4.5 g (32.5 mmol) of hexamethylenetetramine, and 30 mL of dichloromethane were added to a reaction flask and stirred. After reacting for 4 hours, the mixture was filtered, and the solid was collected. Ethanol and concentrated hydrochloric acid were then added to the solid, and the mixture was reacted at 50 °C for 2 hours. The reaction was monitored by LCMS until complete. The mixture was then filtered, and the filtrate was collected and evaporated to dryness to obtain 4.5 g of the product (6-bromopyridin-2-yl)methylamine hydrochloride.

[0302] MS(ESI)m / z(M+H) + =187.1.

[0303] Step 3: Preparation of N-((6-bromopyridin-2-yl)methyl)-3,3,3-trifluoropropionamide

[0304]

[0305] At room temperature, dichloromethane (50 mL), 3,3,3-trifluoropropionic acid (2.3 g, 17.6 mmol), diisopropylethylamine (6.2 g, 48.1 mmol), and 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (7.3 g, 19.3 mmol) were added sequentially to a reaction flask, stirred thoroughly, and reacted at room temperature for 10 minutes. Subsequently, (6-bromopyridin-2-yl)methylamine hydrochloride (3.0 g, 16.0 mmol) was added, and the reaction was continued at room temperature for another half hour. The reaction was monitored by LCMS until complete, quenched with water, extracted with dichloromethane, and the organic phase was collected, evaporated to dryness, and purified by column chromatography to obtain 2.8 g of the product N-[(6-bromopyridin-2-yl)methyl]-3,3,3-trifluoropropionamide.

[0306] MS(ESI)m / z(M+H) + =297.1.

[0307] Preparation Example 43: Preparation of N-((6-bromo-5-fluoropyridin-2-yl)methyl)-3,3,3-trifluoropropionamide

[0308]

[0309] Using commercially available reagents as raw materials, Preparation Example 43 was obtained by referring to a similar method to Preparation Example 42.

[0310] MS(ESI)m / z(M+H) + =315.1.

[0311] Preparation Example 44: Preparation of ((R)-3-((tert-butoxycarbonyl)amino)-4-(3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)butyric acid

[0312]

[0313] Step 1: Preparation of (R)-3-((tert-Butoxycarbonyl)amino)-4-(6-((3,3,3-trifluoropropamido)methyl)pyridin-2-yl)benzyl butyrate

[0314] At room temperature, zinc powder (1.2 g, 19.0 mmol) and N,N-dimethylformamide (20 mL) were added to a reaction flask, purged with nitrogen, and stirred until homogeneous. Trimethylchlorosilane (20.6 mg, 0.19 mmol) was added, and the mixture was stirred for half an hour. Then, 1,2-dibromoethane (286 mg, 1.5 mmol) was added, and the mixture was stirred at 80°C for half an hour. After cooling to room temperature, trimethylchlorosilane (124 mg, 1.1 mmol) and iodine (289 mg, 1.1 mmol) were added, and the mixture was stirred for another half hour under nitrogen protection. (S)-3-((tert-Butoxycarbonyl)amino)-4-iodobutyrate benzyl ester (1.2 g, 2.9 mmol) was added to the above reaction solution, and the mixture was stirred for half an hour.

[0315] N-[(6-bromopyridin-2-yl)methyl]-3,3,3-trifluoropropamide (565 mg, 1.9 mmol), N,N-dimethylformamide (20 mL), tris(dibenzylacetone)dipalladium (174 mg, 0.2 mmol), 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (156 mg, 0.4 mmol), and lithium chloride (4.0 mg, 0.1 mmol) were added to a reaction flask, purged with nitrogen, and the supernatant of the zinc reagent was slowly added to the system. The mixture was stirred at 90 °C for 1 hour, and the reaction was monitored for completeness by LC-MS. The reaction was quenched with ammonium chloride solution, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography to obtain 632 mg of the target compound.

[0316] MS(ESI)m / z(M+H) + =510.2.

[0317] Step 2: Preparation of (R)-3-(2,2,2-trifluoroacetamido)-4-(6-((3,3,3-trifluoropropamido)methyl)pyridin-2-yl)benzyl butyrate

[0318]

[0319] At room temperature, (R)-3-((tert-butoxycarbonyl)amino)-4-(6-((3,3,3-trifluoropropamido)methyl)pyridin-2-yl)benzyl butyrate (300 mg, 0.6 mmol) was added to a reaction flask, followed by 10 mL of dichloromethane and 3 mL of trifluoroacetic acid. The mixture was heated to 40 °C and reacted for half an hour. The reaction was monitored by LCMS until complete. The mixture was then evaporated to dryness, and under ice bath conditions, 20 mL of dichloromethane and trifluoroacetic anhydride (186 mg, 0.9 mmol) were added. Triethylamine was then slowly added dropwise, and the mixture was allowed to react at room temperature for half an hour after the addition was complete. The reaction was monitored by LCMS until complete. Saturated sodium bicarbonate solution was added, and the mixture was extracted and the phases separated. The organic phase was collected, evaporated to dryness, and the crude product was purified by column chromatography to obtain 297 mg of the target compound.

[0320] MS(ESI)m / z(M+H) + =506.2.

[0321] Step 3: Preparation of (R)-3-(2,2,2-trifluoroacetamido)-4-(3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)benzyl butyrate

[0322]

[0323] At room temperature, (R)-3-(2,2,2-trifluoroacetamido)-4-(6-(((3,3,3-trifluoropropamido)methyl)pyridin-2-yl)benzyl butyrate (297 mg, 0.6 mmol) and phosphorus oxychloride (10 mL) were added to a reaction flask, stirred thoroughly, and heated to 90 °C overnight. The reaction was monitored by LCMS until complete. The solvent was removed by evaporation, and the organic phase was washed with ethyl acetate and sodium bicarbonate solution. The organic phase was evaporated to dryness, and the crude product was purified by column chromatography to obtain 100 mg of (3R)-3-(trifluoroacetamido)-4-(3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)benzyl butyrate.

[0324] MS(ESI)m / z(M+H) + =488.2

[0325] Step 4: Preparation of (R)-3-amino-4-(3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)butyric acid

[0326]

[0327] At room temperature, (R)-3-(2,2,2-trifluoroacetamido)-4-(3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)benzyl butyrate (100 mg, 0.2 mmol), lithium hydroxide (20 mg, 0.8 mmol), water (20 mL), and tetrahydrofuran (20 mL) were added to a reaction flask, stirred until homogeneous, and reacted overnight at 50 °C. After the reaction was monitored by LCMS until complete, the mixture was evaporated to dryness, and the crude product was purified by column chromatography to obtain 62 mg of (3R)-3-amino-4-(3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)butyric acid.

[0328] MS(ESI)m / z(M+H) + =302.2.

[0329] Step 5: Preparation of (R)-3-((tert-butoxycarbonyl)amino)-4-(3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)butyric acid

[0330] Under ice-water bath conditions, (R)-3-amino-4-(3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)butyric acid (62 mg, 0.2 mmol), dichloromethane (20 mL), and triethylamine (42 mg, 0.4 mmol) were sequentially added to a reaction flask and stirred until homogeneous. Di-tert-butyl dicarbonate (69 mg, 0.3 mmol) was then slowly added. After the addition was complete, the mixture was transferred to room temperature and reacted. The reaction was monitored by LC-MS for 2 hours until complete. Water was added, the mixture was extracted, the phases were separated, and the organic phase was collected. The organic phase was evaporated to dryness, and the crude product was purified by column chromatography to give 39 mg of the title compound.

[0331] MS(ESI)m / z(M+H) + =402.2.

[0332] Preparation Example 45: Preparation of (R)-3-((tert-butoxycarbonyl)amino)-4-(6-fluoro-3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)butyric acid

[0333]

[0334] Step 1: Preparation of (R)-3-((tert-butoxycarbonyl)amino)-4-(3-fluoro-6-((3,3,3-trifluoropropamido)methyl)pyridin-2-yl)benzyl butyrate

[0335]

[0336] At room temperature, zinc powder (0.93 g, 10.77 mmol) and N,N-dimethylformamide (10 mL) were added to a 100 mL three-necked flask. Trimethylchlorosilane (0.023 g, 0.21 mmol) was added under nitrogen protection, and the mixture was stirred at room temperature for 30 minutes. Then, 1,2-dibromoethane (0.22 g, 1.15 mmol) was added, and the mixture was heated to 80 °C in an oil bath for 30 minutes. The oil bath was then removed and the mixture was cooled to room temperature. At room temperature, trimethylchlorosilane (0.14 g, 1.28 mmol), elemental iodine (0.222 g, 0.87 mmol), and (3S)-3-{[(tert-butoxy)carbonyl]amino}-4-iodobutyrate benzyl ester (1.2 g, 2.86 mmol) were added to the above solution, and the mixture was reacted at room temperature for 30 minutes. The reaction solution was then set aside.

[0337] N-((6-bromo-5-fluoropyridin-2-yl)methyl)-3,3,3-trifluoropropionamide (417 mg, 1.32 mmol), tris[dibenzylacetone]palladium(0) (0.060 g, 0.066 mmol), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (0.11 g, 0.26 mmol), lithium chloride (0.0081 g, 0.19 mmol), and N,N-dimethylformamide (10 mL) were added to a 100 mL three-necked flask. The reaction mixture was then rapidly added under nitrogen protection, and the mixture was heated to 90 °C and reacted for 1 hour. The reaction was confirmed to be complete by LCMS. A small amount of ammonium chloride aqueous solution was added to quench the reaction, and the mixture was purified by direct reversed-phase column chromatography. The target fraction was concentrated under reduced pressure and freeze-dried to obtain 0.747 g of the title compound.

[0338] MS(ESI)m / z(M+H) + =428.2.

[0339] Step 2: Preparation of (R)-3-amino-4-(6-fluoro-3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)benzyl butyrate

[0340]

[0341] Under nitrogen protection, a solution of (R)-3-((tert-butoxycarbonyl)amino)-4-(3-fluoro-6-((3,3,3-trifluoropropamido)methyl)pyridin-2-yl)benzyl butyrate (636 mg, 1.21 mmol) in phosphorus oxychloride (33.6 g, 219.14 mmol) was heated to 90 °C and reacted for 7 hours. The reaction was confirmed to be complete by LC-MS. After concentration under reduced pressure, adjustment to a weakly alkaline state with sodium bicarbonate aqueous solution, and reversed-phase column chromatography, 0.281 g of the title compound was obtained.

[0342] MS(ESI)m / z(M+H) + =410.2.

[0343] Step 3: Preparation of (R)-3-((tert-butoxycarbonyl)amino)-4-(6-fluoro-3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)benzyl butyrate

[0344]

[0345] At room temperature, triethylamine (200.36 mg, 1.98 mmol), di-tert-butyl dicarbonate (720.23 mg, 3.30 mmol), and 4-dimethylaminopyridine (32.25 mg, 0.26 mmol) were added to a solution of (R)-3-amino-4-(6-fluoro-3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)butyrate (271 mg, 0.66 mmol) in dichloromethane (50 mL). The reaction was allowed to proceed for 2 hours, and LC-MS confirmed complete reaction. After concentration under reduced pressure and dissolution in methanol, reversed-phase column chromatography yielded 0.156 g of the title compound.

[0346] MS(ESI)m / z(M+H) + =510.2.

[0347] Step 4: Preparation of (R)-3-((tert-butoxycarbonyl)amino)-4-(6-fluoro-3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)butyric acid

[0348]

[0349] Lithium hydroxide monohydrate (50.45 mg, 1.2 mmol) was added to a mixed solution of (R)-3-((tert-butoxycarbonyl)amino)-4-(6-fluoro-3-(2,2,2-trifluoroethyl)imidazo[1,5-a]pyridin-5-yl)benzyl butyrate (150 mg, 0.24 mmol) in water (10 mL) and tetrahydrofuran (20 mL). The reaction was allowed to proceed naturally at room temperature for 4 hours, and the reaction was confirmed to be complete by LC-MS. The solution was then cooled in an ice-water bath, adjusted to a weakly acidic state with acetic acid, concentrated under reduced pressure, dissolved in methanol, subjected to reversed-phase column chromatography, and freeze-dried to give 95 mg of the title compound.

[0350] MS(ESI)m / z(M+H) + =420.2.

[0351] Following the synthetic methods of Preparation Examples 41-45, using commercial reagents or self-made intermediates as raw materials, the following intermediate compounds can be prepared, as shown in Table 4.

[0352] Table 4 Information on the intermediate compounds obtained.

[0353]

[0354]

[0355]

[0356] Preparation Example 65: Preparation of (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(6-fluoro-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl-1-d)butyric acid

[0357]

[0358] Step 1: Preparation of N-[(6-bromo-5-fluoropyridin-2-yl)methyl]-3-methylbutyramide

[0359]

[0360] At room temperature, pyridine (20 mL), 3-methylbutyric acid (634.2 mg, 6.2 mmol), (6-bromo-5-fluoropyridin-2-yl)methylamine hydrochloride (1.0 g, 4.1 mmol), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.2 g, 6.2 mmol) were added to a reaction flask, and the reaction was carried out at 50 °C for one hour. The reaction was monitored by LCMS until complete, and the mixture was evaporated to dryness. The crude product was purified by column chromatography to give 1.2 g of the title compound.

[0361] MS(ESI)m / z(M+H) + =289.2.

[0362] Step 2: Preparation of (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(3-fluoro-6-[(3-methylbutamido)methyl]pyridin-2-yl)benzyl butyrate

[0363]

[0364] At room temperature, zinc (830.7 mg, 12.7 mmol) and N,N-dimethylformamide (25 mL) were added to a reaction flask, purged with nitrogen, and stirred until homogeneous. Trimethylchlorosilane (13.8 mg, 0.1 mmol) was added, and the mixture was stirred for half an hour. Then, 1,2-dibromoethane (190.9 mg, 1.0 mmol) was added, and the mixture was stirred at 90°C for half an hour. After cooling to room temperature, trimethylchlorosilane (82.8 mg, 0.8 mmol) and iodine (193.4 mg, 0.8 mmol) were added, and the mixture was stirred for another half hour under nitrogen protection. Benzyl (3S)-3-{[(tert-butoxy)carbonyl]amino}-4-iodobutyrate (798.7 mg, 1.9 mmol) was added to the above reaction solution, and the mixture was stirred for half an hour.

[0365] N-[(6-bromo-5-fluoropyridin-2-yl)methyl]-3-methylbutyramide (367 mg, 1.3 mmol), N,N-dimethylformamide (25 mL), tris(dibenzylacetone)dipalladium (116.3 mg, 0.1 mmol), 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (104.3 mg, 0.3 mmol), and lithium chloride (2.7 mg, 0.06 mmol) were added to a reaction flask, purged with nitrogen, and the supernatant of the zinc reagent was slowly added to the system. The mixture was stirred for 1 hour, and the reaction was monitored by LC-MS to ensure complete reaction. The reaction was quenched with ammonium chloride solution, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography to obtain 636 mg of the title compound.

[0366] MS(ESI)m / z(M+H) + =502.2.

[0367] Step 3: Preparation of (3R)-4-(6-fluoro-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl)-3-(trifluoroacetamido)benzyl butyrate

[0368] At room temperature, (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(3-fluoro-6-[(3-methylbutamido)methyl]pyridin-2-yl)benzyl butyrate (636 mg, 1.27 mmol) was added to a reaction flask, followed by 10 mL of dichloromethane and 3 mL of trifluoroacetic acid. The mixture was heated to 40 °C and reacted for half an hour. The reaction was monitored by LCMS until complete. The mixture was then evaporated to dryness. Under ice bath conditions, 20 mL of dichloromethane and trifluoroacetic anhydride (666.8 mg, 3.2 mmol) were added, followed by the slow addition of triethylamine (642.6 mg, 6.4 mmol). After the addition was complete, the mixture was allowed to react at room temperature for half an hour. The reaction was monitored by LCMS until complete. Saturated sodium bicarbonate solution was added, and the mixture was extracted and separated. The organic phase was collected, evaporated to dryness, and the crude product was purified by column chromatography to give 271 mg of the title compound.

[0369] MS(ESI)m / z(M+H) + =480.2.

[0370] Step 4: Preparation of (3R)-4-(6-fluoro-1-iodo-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl)-3-(trifluoroacetamido)benzyl butyrate

[0371]

[0372] Under ice bath conditions, (3R)-4-(6-fluoro-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl)-3-(trifluoroacetamido)benzyl butyrate (118 mg, 0.3 mmol) and dichloromethane (30 mL) were added to a reaction flask and stirred until homogeneous. N-iodosuccinimide (84.4 mg, 0.4 mmol) was added, and the reaction was continued at 0 °C for 10 minutes. The reaction was monitored by LCMS until completion. Sodium bicarbonate solution was added, the mixture was extracted and separated, evaporated to dryness, and purified by column chromatography to obtain 113 mg of the title compound.

[0373] MS(ESI)m / z(M+H) + =606.2.

[0374] Step 5: Preparation of (3R)-4-(6-fluoro-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl-1-d)-3-(trifluoroacetamide)benzyl butyrate

[0375]

[0376] At room temperature, (3R)-4-(6-fluoro-1-iodo-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl)-3-(trifluoroacetamido)benzyl butyrate (100 mg, 0.2 mmol) and zinc (55.6 mg, 0.9 mmol) were added to a dry reaction flask, followed by the addition of deuterated acetic acid (2 mL). The reaction was carried out at 50 °C for 30 minutes. The reaction was confirmed to be complete by LCMS. The mixture was then evaporated to dryness, and sodium bicarbonate solution was added. The mixture was extracted and separated, evaporated to dryness, and purified by column chromatography to obtain 79 mg of the title compound.

[0377] MS(ESI)m / z(M+H) + =481.2.

[0378] Step 6: Preparation of (3R)-3-amino-4-(6-fluoro-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl-1-d)butyric acid

[0379]

[0380] At room temperature, (3R)-4-(6-fluoro-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl-1-d)-3-(trifluoroacetamide)benzyl butyrate (79 mg, 0.2 mmol), lithium hydroxide (15.3 mg, 0.6 mmol), water (10 mL), and tetrahydrofuran (10 mL) were added to a reaction flask, stirred until homogeneous, and reacted at 50 °C for 2 hours. After the reaction was monitored by LCMS until complete, the solution was evaporated to dryness and purified by column chromatography to obtain 48 mg of the title compound.

[0381] MS(ESI)m / z(M+H) +=295.2.

[0382] Step 7: Preparation of (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(6-fluoro-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl-1-d)butyric acid

[0383]

[0384] Under ice-water bath conditions, (3R)-3-amino-4-(6-fluoro-3-(2-methylpropyl)imidazo[1,5-a]pyridin-5-yl-1-d)butyric acid (48 mg, 0.2 mmol), methanol (20 mL), and triethylamine (32.4 mg, 0.3 mmol) were sequentially added to a reaction flask and stirred until homogeneous. Di-tert-butyl dicarbonate (69.8 mg, 0.3 mmol) was then slowly added. After the addition was complete, the mixture was transferred to room temperature and reacted. The reaction was monitored by LC-MS for 1 hour until complete. Water was added, the mixture was extracted, the phases were separated, and the organic phase was collected, evaporated to dryness, and purified by column chromatography to obtain 64 mg of the title compound.

[0385] MS(ESI)m / z(M+H) + =395.2.

[0386] Example 1: Preparation of (R)-3-amino-4-(2-chlorophenyl)-1-(1′-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1′,4′-dihydro-2H,2′H-[3,3′-diazamethylene]-1(4H)-yl)but-1-one

[0387]

[0388] Step 1: Preparation of tert-butyl(R)-(1-(2-chlorophenyl)-4-(1'-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-4-oxobutane-2-yl)carbamate

[0389]

[0390] At room temperature, N,N-diisopropylethylamine (0.037 g, 0.29 mmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.055 g, 0.14 mmol) were added to a solution of 1-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-1,1',4,4'-tetrahydro-2H,2'H-3,3'-diazamethylene hydrocarbon (0.040 g, 0.096 mmol) and (R)-3-((tert-butoxycarbonyl)amino)-4-(2-chlorophenyl)butyric acid (0.050 g, 0.16 mmol) in N,N-dimethylformamide (5 mL). The reaction was allowed to proceed for 30 minutes at room temperature, after which LC-MS confirmed a good reaction. The target product was obtained by direct reversed-phase column chromatography after quenching with water, and then freeze-dried to obtain 29 mg of the title compound.

[0391] MS(ESI)m / z(M+H) + =713.2.

[0392] Step 2: Preparation of (R)-3-amino-4-(2-chlorophenyl)-1-(1′-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1′,4′-dihydro-2H,2′H-[3,3′-diazamethylene]-1(4H)-yl)but-1-one

[0393]

[0394] At room temperature, trifluoroacetic acid (1.53 g, 13.42 mmol) was added to a solution of tert-butyl(R)-(1-(2-chlorophenyl)-4-(1'-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-4-oxobutane-2-yl)carbamate (29 mg, 0.041 mmol) in dichloromethane (15 mL). After reacting at room temperature for 2 hours, the reaction was confirmed to be complete by LC-MS. The solution was concentrated under reduced pressure and adjusted to a weakly basic pH to prepare and purify 24.55 mg of the title compound.

[0395] MS(ESI)m / z(M+H) + =613.2.

[0396] 1H NMR(400MHz, Methanol-d4)δ8.87(s,1H),8.40(s,1H),8.36(s,1H),7.49(dd,J= 9.0,4.8Hz,1H),7.40(dd,J=7.5,1.8Hz,1H),7.37–7.20(m,5H),4.90(s,2H),4. 68(s,4H),4.49(s,2H),3.65–3.47(m,1H),3.02–2.81(m,2H),2.41–2.16(m,2H) ,1.94–1.76(m,1H),1.11(t,J=4.3Hz,2H),1.08–1.00(m,1H),0.90–0.79(m,1H).

[0397] Using the intermediate compound obtained in the preparation example as a starting material, the following Examples 2-22 were prepared according to the synthesis method of Example 1. Specific information is shown in Table 5.

[0398] Table 5 Compound Information from Examples

[0399]

[0400]

[0401]

[0402]

[0403]

[0404] Example 23: Preparation of (R)-3-amino-1-(1'-(5-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)pyrimidin-4-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-3-isopropylamino-4-(1-(2,2,2-trifluoroethyl)-1H-indazol-7-yl)but-1-one

[0405]

[0406] At room temperature, acetone (49.95 mg, 0.86 mmol), sodium cyanoborohydride (10.81 mg, 0.17 mmol), and a catalytic amount of acetic acid were added to a methanol (20 mL) solution of (R)-3-amino-1-(1'-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-4-(1-(2,2,2-trifluoroethyl)-1H-indazol-7-yl)but-1-one (30 mg, 0.043 mmol). After the addition was complete, the reaction was allowed to proceed overnight at room temperature. The reaction was confirmed to be complete by LCMS. The reaction was quenched with water and concentrated under reduced pressure. The solution was dissolved in methanol and filtered to obtain 23.18 mg of the title compound after purification.

[0407] MS(ESI)m / z(M+H) + =743.2.

[0408] Example 24: Preparation of (R)-3-amino-1-(1'-(6-((2'-cyclopropyl-5-fluoro-[1,1'-biphenyl]-2-yl)oxy)-1,2,4-triazin-5-yl)-1',4'-dihydro-2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)-3-isopropylamino-4-(1-(2,2,2-trifluoroethyl)-1H-indazol-7-yl)but-1-one

[0409]

[0410] Using Example 2 as the raw material, Example 24 was synthesized by referring to the synthesis method of Example 23.

[0411] MS(ESI)m / z(M+H) + =741.2.

[0412] Similarly, using the intermediate compound obtained in the preparation example as a starting material, the following Examples 25-43 were prepared according to the synthesis method of Example 1. Specific information is shown in Table 6.

[0413] Table 6 Compound Information for Examples

[0414]

[0415]

[0416]

[0417]

[0418]

[0419] The analytical data described above, including NMR and LC-MS data, are shown in Table 7.

[0420] Table 7. NMR and LC-MS data of compounds in Examples 2-43

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427] Example 44: Synthesis of (S)-1-(5-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)pyrimidin-4-yl)-1'-prolyl-1,1',4,4'-tetrahydro2H,2'H-3,3'-diazaethylene

[0428]

[0429] Step 1: Preparation of (S)-2-(1'-(5-(2-(4-cyclopropylpyrimidin-5-yl)4-fluorophenoxy)pyrimidin-4-yl)-1,1',4,4'-tetrahydro2H,2'H-[3,3'-diazamethylene]-1-carbonyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0430]

[0431] At room temperature, to a solution of 1-(5-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)pyrimidin-4-yl)-1,1',4,4'-tetrahydro2H,2'H-3,3'-diazamethylene hydrocarbon (30 mg, 0.072 mmol) in N,N-dimethylformamide (3 mL), (tert-butoxycarbonyl)-L-proline (15 mg, 0.072 mmol), N,N-diisopropylethylamine (27.92 mg, 0.22 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (27.92 mg, 0.22 mmol) were added. The reaction was allowed to proceed for 10 minutes at room temperature, and LC-MS confirmed complete reaction. The reaction was quenched with water, followed by reversed-phase column chromatography to give 44 mg of the title compound.

[0432] MS(ESI)m / z(M+H) + =614.2.

[0433] Step 2: Preparation of (S)-1-(5-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)pyrimidin-4-yl)-1'-prolyl-1,1',4,4'-tetrahydro2H,2'H-3,3'-diazaethylene)

[0434]

[0435] At room temperature, trifluoroacetic acid (3.06 g, 26.84 mmol) was added to a solution of (S)-2-(1'-(5-(2-(4-cyclopropylpyrimidin-5-yl)4-fluorophenoxy)pyrimidin-4-yl)-1,1',4,4'-tetrahydro2H,2'H-[3,3'-diazamethylene]-1-carbonyl)pyrrolidine-1-carboxylic acid tert-butyl ester (44 mg, 0.072 mmol) in dichloromethane (6 mL). The reaction was heated to 65 °C and reacted for 30 minutes. The reaction was confirmed to be complete by LC-MS. After concentration under reduced pressure, the solution was adjusted to alkalinity with sodium bicarbonate aqueous solution, concentrated under reduced pressure, and acetonitrile (20 mL) was added. Inorganic salts were filtered off, and the filtrate was concentrated and purified to obtain 3 mg of the target compound.

[0436] MS(ESI)m / z(M+H) + =514.2.

[0437] 1 H NMR(400MHz, Methanol-d4)δ8.90(s,1H),8.47(s,1H),8.20(s,1H),7.70(s,1H),7.35–7.23(m,2H),7.22–7.16(m,1H ),4.83–4.51(m,8H),4.36–4.22(m,1H),3.42–3.22(m,2H),2.48–2.32(m,1H),2.12–1.83(m,4H),1.20–0.96(m,4H).

[0438] Example 45: Preparation of (1'-(5-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)pyrimidin-4-yl)-1',4'-dihydro2H,2'H-[3,3'-diazamethylene]-1(4H)-yl)((1S,3aR,6aS)-octahydrocyclopentan[c]pyrrole-1-yl) ketone

[0439]

[0440] Using commercially available reagents as raw materials, Example 45 was obtained by referring to the method of Example 44.

[0441] MS(ESI)m / z(M+H) +=554.2.

[0442] 1 H NMR(400MHz, Methanol-d4)δ8.90(s,1H),8.48(s,1H),8.20(s,1H),7.69(s,1H),7.35–7.25(m,2H),7.23–7.14(m,1H),4.84–4.45(m,9H) ),2.77–2.55(m,2H),2.50–2.41(m,1H),1.98–1.86(m,1H),1.88–1.74(m,1H),1.75–1.52(m,5H),1.49–1.39(m,1H),1.22–0.96(m,4H).

[0443] Experimental Example 1: Menin-MLL Protein Interaction Inhibition Activity Assay

[0444] (1) Inhibitory activity test of the interaction between Menin and MLL protein

[0445] The IC50 of the test compound inhibiting Menin-MLL protein interaction was detected using the Fluorescence Polarization method. 50 value.

[0446] The specific steps are as follows: The compound stock solution (10 mM, prepared in DMSO) was 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 an ECHO665 Series Acoustic Liquid Handler (BECKMAN Inc.), 50 nL of each concentration of the test compound (10 concentration gradients) and a DMSO solution without the compound (negative control well) were transferred to a 384-well plate and centrifuged at 1000 rpm. Using an I.DOT (DISPENDIX Inc.) syringe, 5 μL of Menin (ICE Inc., Cat No. E2208F-H15H) was added to each well and incubated at 25°C for 10 minutes. Using I.DOT, 5 μL of FITC-MLL4-43 (Genscrip Inc.) was added to each well, centrifuged at 1000 rpm, and incubated for 60 minutes. FP signal was measured using a Pherastar FSX multi-plate reader (BMGLabtech Inc.), and the data were processed.

[0447] The IC50 of each compound was fitted using a nonlinear regression equation: Inhibition% = (Signal from negative control wells - Signal from compound wells) / (Signal from negative control wells - Background signal) * 100% (Background signal is the signal value detected in wells containing only 10 μM SNDX-5613). The logarithmic value of the compound concentration was used as the X-axis, and the percentage inhibition rate (Inhibition%) as the Y-axis. A dose-response curve was fitted to derive the IC50 of each compound in inhibiting Menin-MLL protein interaction. 50 Value. The experimental results are shown in Table 8. Note: SNDX-5613 refers to N-ethyl-2-((4-(7-((trans-4-(ethylsulfonylamino)cyclohexyl)methyl)-2,7-diazaspiro[3.5]non-2-yl)pyrimidin-5-yl)oxy)-5-fluoro-N-isopropylbenzamide, purchased from Shanghai Loulan Biotechnology Co., Ltd., with the following structural formula:

[0448]

[0449] (2) Inhibition activity test of interaction between mutant Menin-M327I and Menin-T349M and MLL protein

[0450] The IC50 of the test compounds inhibiting the interaction between Menin-M327I, Menin-T349M and MLL protein was detected using fluorescence polarization. 50 value.

[0451] The specific steps are as follows: The compound stock solution (10 mM, prepared in DMSO) was 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 an ECHO665 Series Acoustic Liquid Handler (BECKMAN Inc.), 50 nL of each concentration of the test compound (10 concentration gradients) and a DMSO solution without the compound (negative control well) were transferred to a 384-well plate and centrifuged at 1000 rpm for later use. Using I.DOT (DISPENDIX Inc.), 5 μL of Menin-M327I (ICE Inc. Cat No. A130412011) and Menin-T349M (ICE Inc. Cat No. A130413011) were added to each well, and incubated at 25°C for 10 minutes. Using I.DOT, 5 μL of FITC-MLL4-43 (Genscrip Inc.) was added to each well, centrifuged at 1000 rpm, and incubated for 60 minutes. FP signals were measured using a Pherastar FSX multi-plate reader (BMG Labtech Inc.), and the data were processed.

[0452] Fitting compound IC with a nonlinear regression equation 50 Inhibition% = (Negative control well signal - Compound well signal) / (Negative control well signal - Background signal) * 100% (Background signal is the signal value detected in wells containing only 100 μM SNDX-5613). Using the log value of compound concentration as the X-axis and the percentage inhibition rate (Inhibition%) as the Y-axis, a dose-response curve was fitted to obtain the IC50 of each compound inhibiting the interaction between Menin-M327I, Menin-T349M, and MLL protein. 50 Values. The experimental results are shown in Table 8.

[0453] Experiment Example 2: Cell Proliferation Inhibition Experiment

[0454] (1) Cell plating:

[0455] Remove the cells from the incubator and place them on the worktable. Gently pipette them together and count them using CounterStar.

[0456] Dilute the cells to the required density using fresh complete culture medium. MV-4-11 cells (source: Nanjing Kebai, catalog number: CBP60522) were cultured in RPMI 1640 (containing HEPES) (BOSTER, catalog number: PYG0122) + 10% FBS (GIBCO, catalog number: 10099-141C) + 1% P / S (HyClone, catalog number: SV30010) at a density of 1×10^4 cells / well, 100 μL / well.

[0457] Use a power pipette to aspirate 100 μL of each of the above cell suspensions into a 96-well plate.

[0458] (2) Preparation of compounds:

[0459] The mother liquor of the compound was diluted from 10 mM to 4000, 1200, 400, 120, 40, 12, 4, 1.2 and 0 μM with DMSO, with 0 μM serving as the control well.

[0460] After thorough mixing, 1.3 μL was taken out with an electric pipette and added to 258.7 μL of culture medium. At this point, it was diluted 100 times, and the DMSO content was 0.5%.

[0461] After thoroughly mixing using a multi-channel pipette, remove the cells that have been plated above. Set up two replicates for each compound concentration. Add 100 μL of the compound diluted in step 2 to each replicate well. The final compound concentrations are: 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0 μM. At this point, there is a total of 200 μL of culture medium in each well of the cell culture plate, with a DMSO content of 0.25%. The well containing only 0.25% DMSO (compound concentration of 0) is the control well.

[0462] The cells were returned to a 37°C, 5% CO2 incubator for further culture, and then tested after 3 days of treatment with the added compound.

[0463] (3) CTG detection:

[0464] After the culture time is up, remove the cells and aspirate some of the culture medium, leaving 50 μL of culture medium in each well. Add 50 μL of CTG reagent (cellcounting-Lite 2.0, Vazyme, DD1101-02) per well using a multipipe.

[0465] Incubate in a shaker at room temperature for 15 minutes, then allow to stand at room temperature for 15 minutes to equilibrate.

[0466] Detection was performed using a multi-functional microplate reader.

[0467] (4) Data Analysis:

[0468] Calculate cell viability%, Cell viability% = As / Ac × 100%. As: Test wells (containing cell culture medium, CTG, and test compound), Ac: Control wells (containing cell culture medium, CTG, and no test compound).

[0469] Using the logarithm of compound concentration as the X-axis and cell viability (%) as the Y-axis, a dose-response curve was fitted to derive the IC50 of each compound's inhibitory activity on cell proliferation. 50 Values. Specific results are shown in Table 8.

[0470] Table 8 Activity data of the compounds of the present invention

[0471]

[0472] Note: In Table 8, "-" indicates that the compound was not detected. "Compound number" indicates the compound in the corresponding example.

[0473] The results in Table 8 show that the compounds of this invention possess excellent activity in inhibiting the interaction between Menin and MLL proteins, and also exhibit excellent activity in inhibiting the interaction between Menin mutant proteins and MLL proteins, thus demonstrating good potential for overcoming drug resistance. Simultaneously, the compounds of this invention can also effectively inhibit the proliferation of MV-4-11 cells. Therefore, the compounds of this invention have promising prospects for clinical development.

Claims

1. A compound represented by chemical formula I, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof: In the formula, X1, X2, X3, and X4 are all CRa; or any three of them are N and the rest are CRa; Ra at different positions may be the same or different, and Ra can be independently selected from H, halogens, and C1-C3 alkyl groups; Y is either CH or N; a is 0, 1, or 2; b is 0, 1, or 2; R1 and R2 are each independently selected from hydrogen, C1-C3 alkyl, 3-6 membered cycloalkyl, or 3-6 membered heterocycloalkyl; R3 is selected from hydrogen or C1-C3 alkyl, 3-6 membered cycloalkyl, or 3-6 membered heterocycloalkyl; Ar is selected from substituted or unsubstituted 5-16 aryl groups, substituted or unsubstituted 5-16 heteroaryl groups, substituted or unsubstituted 5-16 cycloalkyl groups, and substituted or unsubstituted 5-16 heterocycloalkyl groups; Ar can be monocyclic or fused polycyclic; when Ar is monocyclic, it is an aromatic ring; when Ar is fused polycyclic, it is at least partially an aromatic ring, and the aromatic ring portion is connected to the host. The skeletal atoms of the heterocyclic alkyl or heteroaryl groups may optionally contain 1-3 heteroatoms selected from N, O, and S.

2. The compound according to claim 1, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, X1 is N, and X2, X3, and X4 are CRa; or X2 is N, and X1, X3, and X4 are CRa; or X3 is N, and X1, X2, and X4 are CRa; or X4 is N, and X1, X2, and X3 are CRa; or X1 and X3 are N, and X2 and X4 are CRa; or X1 and X4 are N, and X2 and X3 are CRa; or X2 and X4 are N, and X1 and X3 are CRa; or X1 and X2 are N, and X3 and X4 are CRa.

3. The compound according to claim 1 or 2, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, Ra at different positions is independently selected from H, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl or isopropyl.

4. The compound according to any one of claims 1-3, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, R1 and R2 are independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxacyclopropyl, and azircyclopropyl, respectively.

5. The compound according to claim 4, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, R3 is selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxacyclopropane, and azircyclopropane.

6. The pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof of the compound according to any one of claims 1-5, characterized in that, Ar is selected from substituted or unsubstituted 5-10 aryl groups, substituted or unsubstituted 5-10 heteroaryl groups, substituted or unsubstituted 5-10 cycloalkyl groups, or substituted or unsubstituted 5-10 heterocycloalkyl groups; and Ar can be monocyclic, fused bicyclic, or fused tricyclic.

7. The pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof of the compound according to claim 6, characterized in that, Ar can be selected from substituted or unsubstituted furanyl, thiophene, pyrrolyl, oxazolyl, thiazolyl, imidazole, pyrazolyl; phenyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl; indole, benzimidazolyl, benzofuranyl, benzothiazolyl, benzoisothiazolyl, benzooxazolyl, benzoisoxazole, benzothiaphenyl, indazole, indoleazinyl, imidazole[1,2-a]pyridinyl, imidazole[1,5-a]pyridinyl Pyridyl, pyrazolo[1,5-a]pyridyl, imidazo[1,5-a]pyrazinyl, isoindoline-1-one, 3,4-dihydroisoquinoline-1(2H)-one; naphthyl, quinoline, isoquinoline, naphthidyl, pyridodiazinyl, benzotriazinyl, benzopyranyl, benzoγ-pyranone, tetrahydroisoquinoline, tetrahydronaphthyl, 3,4-dihydro-2H-1,4-benzoxazinyl, 2H-1,4-benzoxazin-3(4H)-one.

8. The pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof of the compound according to claim 7, characterized in that, Ar can be selected from the following groups, either substituted or unsubstituted:

9. The pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof of the compound according to claim 7 or 8, characterized in that, Ar can be substituted at any possible position by one or more groups selected from halogen, cyano, C1-C6 alkyl, C1-C3 haloalkyl, C1-C3 alkylamino, C3-C8 cycloalkyl, 3-8 heterocyclic alkyl, 5-8 aryl or 5-8 heteroaryl.

10. The pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof of the compound according to claim 9, characterized in that, Ar can be substituted at any possible position by one or more independent groups selected from fluorine, chlorine, bromine, iodine, cyano, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, 2,2,2-trifluoroethyl, 2,2-difluoropropyl, 3-fluoropropyl, dimethylamino, diethylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolyl, 1-methylpyrrolyl, piperidinyl, tetrahydro-1H-pyrrolazinyl, pyrrolyl, phenyl, or selected from the following groups:

11. The pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof of the compound according to any one of claims 1-10, characterized in that, One or more hydrogen atoms at any possible position are replaced by deuterium.

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

13. A pharmaceutical composition, characterized in that, Includes the compound of any one of claims 1-12, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, pharmaceutically acceptable excipients and / or carriers.

14. Use of the compound of any one of claims 1-12, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, or the pharmaceutical composition of claim 13, in the preparation of a medicament for the prevention, relief, or treatment of diseases related to the interaction of the Menin-MLL protein.

15. The use according to claim 14, characterized in that, Diseases associated with the interaction of the Menin-MLL protein include malignancies, diabetes, or complications related to these diseases; among which malignancies include hematologic malignancies, lymphomas, and solid tumors.

16. The use according to claim 15, characterized in that, Hematologic malignancies include leukemia and myeloma, including but not limited to acute lymphoblastic 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 neoplasm, plasmacytoma, and multiple myeloma; Lymphomas include, but are not limited to, cutaneous T-cell lymphoma, lymphoid tumors, AIDS-related lymphomas, Hodgkin lymphomas, non-Hodgkin lymphomas, or malignant lymphomas. Solid tumors include, but are not limited to, pancreatic cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, glioblastoma, lung cancer, breast cancer, and prostate cancer; Related complications include, but are not limited to, leukemic meningitis.

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