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

Abstract

The invention provides a compound as shown in a formula I, pharmaceutically acceptable salts, hydrates, isomers, prodrugs or a mixture of the salts, hydrates, isomers and prodrugs, a pharmaceutical composition containing the compound, and application of the compound and the pharmaceutically acceptable salts, hydrates, isomers and prodrugs 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 Menin-MLL protein inhibitors, pharmaceutical compositions containing the same, and their use in the preparation of medicaments for the prevention or treatment of diseases related to the interaction with 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 CH; or any three of them are N, and the rest are CH.

[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 independently selected from hydrogen, C1-C3 alkyl, 3-6 membered cycloalkyl, or 3-6 membered heterocycloalkyl.

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

[0014] R4 and R5 may be the same or different, and can be independently selected from hydrogen or fluorine.

[0015] R6 and R7 may be the same or different, and can be independently selected from hydrogen or fluorine.

[0016] 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, at least part of the ring is an aromatic ring, and the aromatic ring part is connected to the host.

[0017] The skeleton atoms of the heteroaryl and heterocycloalkyl groups may optionally contain 1-3 heteroatoms selected from N, O, and S.

[0018] In some implementations, Y is N. In some implementations, Y is CH.

[0019] In some implementations, one of X1, X2, X3, and X4 is N, and the rest are CH. In some implementations, two of X1, X2, X3, and X4 are N, and the rest are CH. In some implementations, X1 is N, and X2, X3, and X4 are CH. In some implementations, X2 is N, and X1, X3, and X4 are CH, or X3 is N, and X1, X2, and X4 are CH, or X4 is N, and X1, X2, and X3 are CH. In some implementations, X1 and X3 are N, and X2 and X4 are CH, or X1 and X4 are N, and X2 and X3 are CH, or X2 and X4 are N, and X1 and X3 are CH, or X1 and X2 are N, and X3 and X4 are CH.

[0020] In some implementations, a is 1 and b is 1.

[0021] 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 or methyl, and R2 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxecyclopropyl, and azircyclopropane. In some embodiments, R1 is methyl, and R2 is methyl. In some embodiments, both R1 and R2 are hydrogen.

[0022] In some embodiments, R3 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, oxecyclopropyl, and azircyclopropane. In some embodiments, R3 is methyl. In some embodiments, R3 is hydrogen.

[0023] In some embodiments, R4 and R5 are both hydrogen. In some embodiments, R4 is hydrogen and R5 is fluorine. In some embodiments, R4 and R5 are both fluorine.

[0024] In some embodiments, R6 and R7 are both hydrogen. In some embodiments, R6 is hydrogen and R7 is fluorine. In some embodiments, R6 and R7 are both fluorine.

[0025] In some embodiments, R4 and R5 are both hydrogen, and R6 and R7 are both fluorine. In some embodiments, R4 and R5 are both fluorine, and R6 and R7 are both hydrogen. In some embodiments, R4, R5, R6, and R7 are all hydrogen.

[0026] In some embodiments, Ar is selected from 5-14 aryl, 5-14 heteroaryl, 5-14 cycloalkyl, or 5-14 heterocycloalkyl. In some embodiments, Ar is selected from 5-10 aryl, 5-10 heteroaryl, 5-10 cycloalkyl, or 5-10 heterocycloalkyl.

[0027] 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.

[0028] 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, pyrazol[ 1,5-a]pyridyl, isoindoline-1-one, 3,4-dihydroisoquinoline-1(2H)-one; 2,3-dihydrobenzo[b][1,4]dioxane, benzo[d][1,3]dihydroxy, 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.

[0029] In some implementations, Ar is a substituted or unsubstituted phenyl group.

[0030] In some embodiments, Ar may be selected from the following substituted or unsubstituted fused bicyclic groups: indolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benzoisothiazolyl, benzooxazolyl, benzoisoxazole, benzothiophenyl, indazoleyl, indolazinyl, imidazo[1,2-a]pyridyl, imidazo[1,5-a]pyridyl, pyrazolo[1,5-a]pyridyl, 2,3-dihydrobenzo[b][1,4]dioxane, benzo[d][1,3]dihydroxy, naphthyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, tetrahydronaphthyl.

[0031] In some embodiments, Ar is selected from the following groups, substituted or unsubstituted:

[0032]

[0033] In some embodiments, Ar may be substituted at any possible position by one or more groups selected from cyano, halogen, phosphono, sulfonyl, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, RaCO-, C2-C4 alkenyl, -RpOH, N(RxRy)CO(CH2)-, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-6 heterocyclic alkyl, substituted or unsubstituted 5-7 aryl or substituted or unsubstituted 5-7 heteroaryl, wherein Ra is a C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl or 3-6 heterocyclic alkyl, and Rp, Rx, and Ry are all C1-C3 alkyl.

[0034] In some embodiments, Ar may be substituted at any possible position by one or more groups selected from cyano, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, methoxy, ethoxy, n-propoxy, isopropoxy, trifluoromethyl, 2,2,2-trifluoroethyl, cyclopropyl, oxecyclopropyl, azicyclopropyl, oxecyclobutyl, azicyclobutyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydropyranyl, methanesulfonyl, ethanesulfonyl, -CH2CO(CH3)2 or selected from the following groups.

[0035]

[0036] This invention provides the following compounds, pharmaceutically acceptable salts, hydrates, isomers, prodrugs, or mixtures thereof:

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Lymphomas include, but are not limited to, cutaneous T-cell lymphoma, lymphoid tumor, AIDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, or malignant lymphoma.

[0047] 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.

[0048] Related complications include, but are not limited to, leukemic meningitis.

[0049] 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.

[0050] This invention was funded by the Sichuan Provincial Science and Technology Program. Detailed Implementation

[0051] 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 the embodiments. 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. Definitions and general descriptions:

[0052] This indicates the connection position of the group with other structures.

[0053] Unless otherwise specified, "substitution" means that a hydrogen atom in a molecule is replaced by another different atom or group.

[0054] "Oxygen substitution" refers to the formation of a "C=O" structure between an oxygen atom and its immediate neighboring carbon atom.

[0055] "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.

[0056] "Heteroatoms" refer to non-carbon atoms in the carbon chain or framework. Typical heteroatoms include, but are not limited to, N, O, and S.

[0057] "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.

[0058] "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 cyclized alkyl and 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.

[0059] "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 N, O, and S, 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, or morpholinyl. 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.

[0060] "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.

[0061] "Heteroaryl" refers to a monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic heterocyclic moiety having one or more heteroatom ring members selected from N, S, and O. Typical heteroaryl groups include, but are not limited to, pyridyl, indolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothiopheneyl, benzofuranyl, benzothiopheneyl, benzopyranyl, benzothiapyranyl, furanyl, pyrroleyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thiopheneyl, oxadiazolyl, benzimidazolyl, benzothiazolyl, benzoxoxazolyl, etc.

[0062] "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.

[0063] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0064] “Cyano” refers to -CN.

[0065] "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.

[0066] "Alkoxy" refers to -O-alkyl, where alkyl has the meaning as described in this invention.

[0067] The word "amino" alone or in combination with other terms refers to the -NH2 group.

[0068] "Hydroxy" refers to -OH.

[0069] "Sulfonyl" formula The indicated group, wherein Rb represents an alkyl or aryl group as defined in this invention. Exemplary sulfonyl groups include, but are not limited to, methanesulfonyl, ethanesulfonyl, benzenesulfonyl, and p-toluenesulfonyl.

[0070] "phosphonoyl" formula The indicated group, wherein Rc represents a hydroxyl group or an alkyl or aryl group as defined in the invention, and Rd represents an alkyl or aryl group as defined in the invention. Exemplary phosphoryl groups include, but are not limited to, methylphosphonoyl, dimethylphosphonoyl, and diethylphosphonoyl.

[0071] 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, C1-C3 indicates that the portion it modifies has 1-3 carbon atoms, such as 1 carbon atom, 2 carbon atoms, or 3 carbon atoms.

[0072] "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.

[0073] "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.

[0074] "Optional" means that the events or circumstances described below can be freely chosen or not chosen.

[0075] "Hydrate" refers to an aggregate of one or more water molecules contained in the compounds of this invention, including hemihydrates, monohydrates, dihydrates, trihydrates, etc.

[0076] "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.

[0077] "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, Pro-drugs 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 TW Greene, PGM 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.

[0078] "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.

[0079] "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.

[0080] "Pharmaceutically acceptable salts" include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0081] 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.

[0082] Preparation of intermediates

[0083] Preparation Example 1: Preparation of 7-bromo-1-(2,2,2-trifluoroethyl)-1H-indole-2-nitrile

[0084]

[0085] Step 1: Preparation of 7-bromo-1H-indole-2-carboxamide

[0086] 7-Bromo-1H-indole-2-carboxylic acid (3 g) was dissolved in 40 mL of tetrahydrofuran, and N,N'-carbonyldiimidazole (3.04 g) was added. The reaction was carried out at room temperature for 1.5 h. Then, ammonia water (5.78 mL) was added, and the reaction was continued for another 1 h. The reaction was monitored by TLC and LCMS until complete. The mixture was concentrated under reduced pressure and purified by column chromatography to give the title compound (3.0 g).

[0087]

[0088] MS(ESI) m / z 239.0(M+H) +

[0089] Step 2: Preparation of 7-bromo-1H-indole-2-nitrile

[0090] 7-Bromo-1H-indole-2-carboxamide (3 g) was dissolved in 40 mL of toluene, and phosphorus oxychloride (5.86 mL) was added. The mixture was purged with nitrogen three times and reacted at 120 °C for 2 h. The reaction was monitored by TLC until complete, concentrated under reduced pressure, and purified by column chromatography to give the title compound (2.63 g).

[0091]

[0092] 1 H NMR (400MHz, DMSO-d6) δ12.62(s,1H),7.71(d,J=8.0Hz,1H),7.59(d,J=7.5Hz,1H),7.51(d,J=2.0Hz,1H),7.11(t,J=7.8Hz,1H).

[0093] Step 3: Preparation of 7-bromo-1-(2,2,2-trifluoroethyl)-1H-indole-2-nitrile

[0094] 7-Bromo-1H-indole-2-onitrile (600 mg) was dissolved in 15 mL of N,N-dimethylformamide, and potassium carbonate (1.13 g) and 2,2,2-trifluoroethyltrifluoromethanesulfonate (0.79 mL) were added. The reaction was carried out at 80 °C for 6 h. The reaction was monitored by TLC, quenched with water, extracted twice with ethyl acetate, washed with saturated brine, and purified by column chromatography to give the title compound (800 mg).

[0095]

[0096] 1 H NMR (400MHz, DMSO-d6) δ7.82(dd,J=8.0,1.0Hz,1H),7.78(s,1H),7.74(dd,J=7.6,1.1Hz,1H),7.21(t,J=7.8Hz,1H),5.65(q,J=8.6Hz,2H).

[0097] Preparation Example 2: Preparation of 7-bromo-1-(oxetane-3-ylmethyl)-1H-indole-2-nitrile

[0098]

[0099] Step 1: Preparation of 7-bromo-1-(oxetane-3-ylmethyl)-1H-indole-2-nitrile

[0100]

[0101] 7-Bromo-1H-indole-2-carbamate (0.5 g, 2.3 mmol) was dissolved in toluene, followed by the addition of 3-hydroxymethyl-1-oxetane (0.21 g, 2.3 mmol) and CMBP (1.11 g, 4.6 mmol). The reaction was carried out at 120 °C for 3 hours. The LCMS response was weak, and the reaction was confirmed by TLC and ¹H-NMR. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL * 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to give 0.38 g of the target compound.

[0102] MS(ESI)m / z(M+H) + =291.1

[0103] Using 7-bromo-1H-indole-2-nitrile (the title compound in step 2 of Preparation Example 1) as a substrate, different intermediates were obtained by reacting with different commercially available halogenated reagents, as shown in Table 1, following step 3 of Preparation Example 1.

[0104] Table 1 Intermediate Compounds

[0105]

[0106]

[0107]

[0108]

[0109] Preparation Example 18: Preparation of (S)-3-((tert-Butoxycarbonyl)amino)-4-iodobutyrate methyl ester

[0110]

[0111] Step 1: Preparation of 1-(2,5-dioxopyrrolidone-1-yl)4-methyl(tert-butoxycarbonyl)-L-aspartic acid

[0112] (S)-2-((tert-Butoxycarbonyl)amino)-4-methoxy-4-oxobutyric acid was dissolved in ethyl acetate (400 mL). 1-Hydroxypyrrolidine-2,5-dione (15.4 g) was added under ice-water bath, followed by dropwise addition of 100 mL of ethyl acetate solution of N,N'-dicyclohexylcarbodiimide (26.3 g). After the addition was complete, the mixture was allowed to stand at room temperature overnight, and the reaction was monitored by TLC until complete. The mixture was filtered, and the filtrate was washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the crude title compound (41.8 g).

[0113]

[0114] MS(ESI)m / z(M+H) + =345.1.

[0115] Step 2: Preparation of (S)-3-((tert-Butoxycarbonyl)amino)-4-hydroxybutyrate methyl ester.

[0116] Sodium borohydride (7.4 g) was added to a mixed solvent of tetrahydrofuran (300 mL) and water (40 mL) under ice-water bath conditions. At the same temperature, a tetrahydrofuran solution (100 mL) of 1-(2,5-dioxopyrrolidone-1-yl)4-methyl(tert-butoxycarbonyl)-L-aspartic acid (41.8 g) was added dropwise. After the addition was completed over 40 minutes, the reaction was continued for another 40 minutes under ice-water bath conditions. The reaction was monitored by TLC until the starting material disappeared. The reaction was quenched by adding saturated ammonium chloride solution. The mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the crude product of the title compound (17.3 g).

[0117]

[0118] MS(ESI)m / z(M+Na) + =256.1

[0119] Step 3: Preparation of (S)-3-((tert-Butoxycarbonyl)amino)-4-iodobutyrate methyl ester

[0120] Triphenylphosphine (19.5 g), imidazole (5.1 g), and iodine (18.9 g) were mixed in 300 mL of dry dichloromethane. Under nitrogen protection, a solution of (S)-3-((tert-butoxycarbonyl)amino)-4-hydroxybutyrate (17.3 g) in 100 mL of dichloromethane was added dropwise in an ice-water bath. The mixture was heated to room temperature and reacted for 3 h. The starting materials were monitored by TLC until they were completely consumed. Some triphenylphosphine oxide was removed by filtration. The filtrate was added to water and quenched with sodium thiosulfate solution until the system was colorless. The mixture was extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the title compound (15.9 g).

[0121]

[0122] MS(ESI)m / z(M+Na) + =366.1.

[0123] Preparation Example 19: Preparation of (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(3-methanesulfonylphenyl)butyric acid

[0124] Step 1: Preparation of methyl (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(3-methanesulfonylphenyl)butyrate

[0125] Under nitrogen protection, zinc (0.3 g) was added to N,N-dimethylformamide (15 mL), followed by iodine (0.24 g). The mixture was stirred until the solution was colorless, then trimethylchlorosilane (0.3 mL) was added and reacted at room temperature for half an hour. Then, methyl (3S)-3-{[(tert-butoxy)carbonyl]amino}-4-iodobutyrate (0.6 g) was added and reacted at room temperature for twenty minutes. Finally, 3-bromophenylmethyl sulfone (0.42 g), 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (0.07 g), and tris(dibenzylideneacetone)dipalladium (0.16 g) were added sequentially. The mixture was then heated to 70 °C and reacted for 2 hours. LC-MS analysis confirmed the reaction was complete. Sodium bicarbonate was added to the reaction system to quench the reaction. The mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the title compound (0.22 g).

[0126]

[0127] MS(ESI)[M+H] + =272.1.

[0128] Step 2: Preparation of (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(3-methanesulfonylphenyl)butyric acid

[0129] Methyl (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(3-methanesulfonylphenyl)butyrate (0.22 g) was added to a mixed solution of ethanol and water (10 mL, ethanol:water = 4:1), followed by the addition of lithium hydroxide (0.12 g). The reaction was carried out at 80 °C for one hour, and the reaction was monitored by LCMS until complete. The solution was adjusted to weakly acidic by adding glacial acetic acid, extracted three times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the title compound (0.1 g).

[0130]

[0131] MS(ESI)[M+H] + =258.0.

[0132] Preparation Example 20: Preparation of (R)-3-(tert-butylcarbonyl)amino-4-(2-cyano-1-(oxy-3-methyl)-1H-indole-7-yl)butyric acid

[0133] Step 1: Preparation of methyl (R)-3-((tert-butoxycarbonyl)amino)-4-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indole-7-yl)butyrate

[0134] Zinc powder (1.77 g) was weighed and dissolved in 35 mL of dry N,N-dimethylformamide. Iodine (689 mg) was added, and the mixture was purged with nitrogen three times. When the system turned colorless, a DMF solution of trimethylchlorosilane (0.34 mL) was added and stirred at room temperature for 30 min. Then, a DMF solution of (S)-3-((tert-butoxycarbonyl)amino)-4-iodobutyrate (3.1 g) was added and stirred at room temperature for another 30 min. Subsequently, 7-bromo-1-(2,2,2-trifluoroethyl)-1H-indole-2-nitrile (3.0 g), tris(dibenzylideneacetone)dipalladium (827 mg), and 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (371 mg) were added and the mixture was reacted at 90 °C for 12 h. The reaction was monitored by TLC and LCMS until complete. The reaction was quenched with water, diluted with ethyl acetate, filtered through diatomaceous earth, extracted twice with ethyl acetate, washed with saturated brine, and purified by column chromatography to obtain the title compound (1.3 g).

[0135]

[0136] MS(ESI)m / z(M-99) + =340.1

[0137] Step 2: Preparation of (R)-3-((tert-butoxycarbonyl)amino)-4-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indole-7-yl)butyric acid

[0138] Methyl (R)-3-((tert-butoxycarbonyl)amino)-4-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)butyrate (1.3 g) was dissolved in a mixed solvent of 12 mL tetrahydrofuran, 4 mL methanol, and 4 mL water. Lithium hydroxide monohydrate (311 mg) was added, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until complete. The reaction was quenched with water, and the pH was adjusted to acidic with 2 M hydrochloric acid. The mixture was extracted twice with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound (1.27 g).

[0139]

[0140] MS(ESI)m / z(M-55) + = 370.1 MS (ESI) m / z (M-99) + =326.1

[0141] Using self-made compounds or compounds available from different commercial sources as starting materials and reaction substrates, different intermediate compounds were prepared by referring to the steps of Preparation Examples 19 or 20, as shown in Table 2.

[0142] Table 2 Intermediate Compounds

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] Preparation Example 57: Preparation of (3-((tert-butylsulfinyl)amino)-2,2-difluoro-4-phenylbutyric acid

[0149]

[0150] Step 1: Preparation of 2-methyl-N-(2-phenylethylidene)propane-2-sulfinamide:

[0151] Phenylacetaldehyde (2.0 g, 16.6 mmol) was dissolved in tetrahydrofuran (20 mL), cooled to 0 °C, and then tert-butylsulfinamide (2.4 g, 19.9 mmol) and tetraethyl titanate (9.5 g, 33.3 mmol) were added sequentially. After the addition was complete, the reaction mixture was heated to 60 °C and stirred for 16 hours. TLC showed that the reaction was complete. The reaction solution was quenched and diluted with ice water, the aqueous phase was extracted with ethyl acetate, the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the solution was purified by column chromatography to give the title compound (1.8 g). MS (ESI) m / z 224.3 (M+H) +

[0152]

[0153] Step 2: Preparation of ethyl 3-((tert-butylsulfinyl)amino)-2,2-difluoro-4-phenylbutyrate:

[0154] Ethyl difluorobromoacetate (4.4 g, 21.5 mmol) and zinc powder (1.4 g, 21.5 mmol) were dissolved in tetrahydrofuran (20 mL) and stirred at 30 °C for 1 h under a nitrogen atmosphere. The reaction mixture was then added dropwise at 0 °C to a tetrahydrofuran (20 mL) solution of 2-methyl-N-(2-phenylethylidene)propane-2-sulfinamide (1.6 g, 7.16 mmol). After the addition was complete, the reaction mixture was stirred at 0 °C for 2 h. LC-MS showed that the reaction was complete. The reaction mixture was quenched with saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrate was then purified by column chromatography to give the title compound (1.5 g). MS (ESI) m / z 348.4 (M+H) +

[0155]

[0156] Step 3: Preparation of (3-((tert-butylsulfinyl)amino)-2,2-difluoro-4-phenylbutyric acid:

[0157] Ethyl 3-((tert-butylsulfinyl)amino)-2,2-difluoro-4-phenylbutyrate (1.5 g, 4.32 mmol) was dissolved in a mixed solvent of ethanol (10 mL) and water (5 mL), and sodium hydroxide (345 mg, 8.64 mmol) was added. After the addition was complete, the reaction system was stirred at 25 °C for 2 hours. LC-MS showed that the reaction was complete. Dilute hydrochloric acid (0.5 M) was slowly added dropwise to the reaction solution until the pH reached 5-6. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the title compound (910 mg) was obtained. MS (ESI) m / z 320.3 (M+H) +

[0158]

[0159] Preparation Example 58: Preparation of 3-((tert-butylthionyl<sulfinyl>)amino)-3-methyl-4-(naphth-1-yl)butyric acid

[0160] Step 1: Preparation of N-methoxy-N-methyl-2-(naphth-1-yl)acetamide:

[0161] 2-(naphthyl-1-yl)acetic acid (10.0 g, 53.70 mmol) was dissolved in dichloromethane (100 mL). Under nitrogen protection, the mixture was cooled to 0 °C, and N,N-carbonyldiimidazole (7.8 g, 54.03 mmol) and dimethylhydroxylamine hydrochloride (5.3 g, 53.70 mmol) were added. After the addition was complete, the reaction mixture was gradually heated to 25 °C and stirred for 2 hours. LC-MS showed that the starting material reacted completely. The reaction solution was quenched with saturated ammonium chloride aqueous solution, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the title compound (8.3 g). MS (ESI) m / z 230.1 (M+H) +

[0162]

[0163] Step 2: Preparation of 1-(naphth-1-yl)propane-2-one:

[0164] N-methoxy-N-methyl-2-(naphth-1-yl)acetamide (7.3 g, 35.20 mmol) was dissolved in anhydrous tetrahydrofuran (80 mL). Under nitrogen protection, the mixture was cooled to 0 °C, and methyl magnesium bromide (23.5 mL, 70.40 mmol, 3 M) was added. The reaction mixture was stirred at 0 °C for 2 hours. LC-MS showed that the starting material reacted completely. The reaction solution was quenched with saturated ammonium chloride, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give the title compound (4.5 g). MS (ESI) m / z 185.1 (M+H) +

[0165]

[0166] Step 3: Preparation of 2-methyl-N-(1-(naphth-1-yl)propane-2-ylidene)propane-2-sulfinamide:

[0167] 1-(naphthyl-1-yl)propane-2-one (4.5 g, 24.43 mmol) was dissolved in tetrahydrofuran (50 mL). Under a nitrogen atmosphere, the mixture was cooled to 0 °C, and tert-butylsulfinamide (3.0 g, 24.43 mmol) and tetraethyl titanate (8.4 g, 36.64 mmol) were added sequentially. After the addition was complete, the reaction mixture was heated to 60 °C and stirred for 3 hours. LC-MS showed that the starting material reacted completely. The reaction solution was cooled, quenched with saturated ammonium chloride, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the title compound (3.6 g). MS (ESI) m / z 288.1 (M+H) +

[0168]

[0169] Step 4: Preparation of methyl 3-((tert-butylthionyl<sulfinyl>)amino)-3-methyl-4-(naphthyl-1-yl)butyrate:

[0170] 2-Methyl-N-(1-(naphthyl-1-yl)propane-2-ethylene)propane-2-sulfinamide (3.3 g, 15.10 mmol) was dissolved in tetrahydrofuran (40 mL). Under a nitrogen atmosphere, the mixture was cooled to 0 °C, and (2-methoxy-2-carbonylethyl)zinc(II) bromide (30 mL, 30.20 mmol, 1 M) was added. After the addition was complete, the reaction mixture was heated to 25 °C and stirred for 16 hours. LC-MS showed that the starting material reacted completely. The reaction solution was quenched with saturated ammonium chloride, the aqueous phase was extracted with ethyl acetate, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give the title compound (2.5 g). MS (ESI) m / z 362.1 (M+H) +

[0171]

[0172] Step 5: Preparation of 3-((tert-butylthionylsulfinyl)amino)-3-methyl-4-(naphthyl-1-yl)butyric acid:

[0173] Methyl 3-((tert-butylthionylsulfinyl)amino)-3-methyl-4-(naphthyl-1-yl)butyrate (2.5 g, 6.9 mmol) was dissolved in methanol (25 mL) and water (5 mL). Sodium hydroxide (830 mg, 20.7 mmol) was added at 25 °C. After the addition was complete, the reaction mixture was stirred at 40 °C for 2 hours. LC-MS showed complete reaction of the starting material. The reaction solution was cooled to room temperature, and dilute hydrochloric acid (0.5 M) was slowly added dropwise until the pH reached 4–5. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrate yielded the title compound (1.5 g). MS (ESI) m / z 348.2 (M+H) + .

[0174]

[0175] Starting with phenylacetyl chloride, and following the synthetic steps of Preparation Example 58 described above, compound 3-((tert-butylthionylsulfinyl)amino)-3-methyl-4-phenylbutyric acid was prepared as described in Preparation Example 59. The structure and characterization data are shown below:

[0176]

[0177] MS(ESI) m / z 298.2(M+H) +

[0178] Preparation Example 60: Preparation of 3-((tert-butylthionyl(sulfinyl)amino)-4,4-difluoro-4-phenylbutyric acid Step 1: Preparation of 2,2-difluoro-2-phenylethane-1-ol:

[0179] 2,2-Difluoro-2-phenylacetic acid (9.0 g, 52.29 mmol) was dissolved in tetrahydrofuran (90 mL), and carbonyl diimidazole (9.3 g, 57.52 mmol) was added. The reaction mixture was stirred at 25 °C for 1 hour. Sodium borohydride (4.0 g, 104.57 mmol) was added slowly in portions. After the addition was complete, the reaction mixture was stirred at 25 °C for 6 hours. LC-MS showed that the starting material reacted completely. The reaction solution was diluted with saturated ammonium chloride aqueous solution, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the solution was purified by column chromatography to obtain the title compound (5.7 g).

[0180]

[0181] Step 2: Preparation of 2,2-difluoro-2-phenylacetaldehyde:

[0182] Dimethyl sulfoxide (5.9 g, 75.38 mmol) was dissolved in dichloromethane (30 mL). Under a nitrogen atmosphere, the mixture was cooled to -70 °C, and oxaloyl chloride (4.8 g, 37.80 mmol) was added. The reaction mixture was stirred at -70 °C for 0.5 h. 2,2-Difluoro-2-phenylethane-1-ol (3.0 g, 18.99 mmol) was added, and the reaction mixture was stirred at -70 °C for 0.5 h. Triethylamine (11.5 g, 114.06 mmol) was added, and the reaction mixture was stirred at -70 °C for 0.5 h. The mixture was then heated to 25 °C and stirred for 0.5 h. TLC showed that the reactants reacted completely. The reaction solution was quenched with saturated ammonium chloride, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrate yielded the title compound (2.2 g).

[0183]

[0184] Step 3: Preparation of (E)-N-(2,2-difluoro-2-phenylethylene)-2-methylpropane-2-sulfinamide:

[0185] 2,2-Difluoro-2-phenylacetaldehyde (2.0 g, 12.81 mmol) was dissolved in tetrahydrofuran (20 mL). Under a nitrogen atmosphere, the mixture was cooled to 0 °C, and tert-butylsulfinamide (1.6 g, 12.81 mmol) and tetraethyl titanate (8.8 g, 38.43 mmol) were added sequentially. After the addition was complete, the reaction mixture was heated to 60 °C and stirred for 16 hours. LC-MS showed that the starting material reacted completely. The reaction mixture was cooled, diluted with saturated ammonium chloride aqueous solution, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (1.5 g). MS (ESI) m / z 260.1 (M+H) +

[0186]

[0187] Step 4: Preparation of ethyl 3-((tert-butylthionyl(sulfinyl)amino)-4,4-difluoro-4-phenylbutyrate:

[0188] (E)-N-(2,2-difluoro-2-phenylethylene)-2-methylpropane-2-sulfinamide (1.5 g, 5.78 mmol) was dissolved in tetrahydrofuran (10 mL), and the mixture was cooled to 0 °C under a nitrogen atmosphere. Zinc(II) bromide (2-methoxy-2-carbonylethyl) (22 mL, 21.68 mmol, 1 M) was then added. After the addition was complete, the reaction mixture was stirred at 25 °C for 16 hours. LC-MS showed that the reactants reacted completely. The reaction solution was quenched with saturated ammonium chloride, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the solution was purified by column chromatography to obtain the title compound (1.0 g).

[0189]

[0190] Step 5: Preparation of 3-((tert-butylthionyl(sulfinyl)amino)-4,4-difluoro-4-phenylbutyric acid:

[0191] 3-((tert-butylthionylsulfinyl)amino)-4,4-difluoro-4-phenylbutyric acid (900 mg, 2.59 mmol) was dissolved in ethanol (10 mL) and water (2 mL), and sodium hydroxide (207 mg, 5.18 mmol) was added. The reaction mixture was stirred at 25 °C for 2 hours. LC-MS showed that the starting material reacted completely. Dilute hydrochloric acid (0.5 M) was slowly added dropwise to the reaction mixture until the pH reached 4–5. The aqueous phase was extracted with dichloromethane, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the title compound (570 mg) was obtained. MS (ESI) m / z 320.2 (M+H) +

[0192]

[0193] Preparation Example 61: Preparation of tert-butyl 5-(3,6-dichloro-1,2,4-triazine-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid

[0194]

[0195] Step 1: Preparation of tert-butyl 3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid p-toluenesulfonate

[0196] 1.7 g of 4,6-dihydropyrrolo[3,4-c]pyrrole-2,5(1H,3H)-dicarboxylic acid di-tert-butyl ester was dissolved in isopropanol (30 mL), and 1.04 g of p-toluenesulfonic acid was added. The reaction was carried out overnight at 55 °C. The reaction was monitored by LC-MS until complete. After cooling to room temperature, the mixture was filtered, the filter cake was washed with ethyl acetate, and dried to give the title compound (1.65 g).

[0197]

[0198] MS(ESI)m / z(M+H) + =211.1.

[0199] Step 2: Preparation of tert-butyl 5-(3,6-dichloro-1,2,4-triazine-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid.

[0200] 1.64 g of 3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid tert-butyl ester p-toluenesulfonate was added to dichloromethane (30 mL), followed by the dropwise addition of triethylamine (1.30 g), and then the addition of trichloro-1,2,4-triazine (0.95 g) in portions. The reaction was allowed to proceed at room temperature for 2 h. The reaction was monitored by LCMS until complete. The mixture was quenched with water, extracted three times with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give the title compound (1.5 g).

[0201]

[0202] MS(ESI)m / z(M+H) + =358.1

[0203] Preparation Example 62: Preparation of 2-(3-cyclopropylpyridin-2-yl)-4-fluorophenol

[0204]

[0205] Step 1: Preparation of 2-chloro-3-cyclopropylpyridine:

[0206]

[0207] 2-Chloro-3-iodopyridine (5.0 g, 20.96 mmol), cyclopropylboronic acid (1.8 g, 20.96 mmol), and cesium carbonate (20.5 g, 62.87 mmol) were dissolved in 1,4-dioxane (50 mL) and water (10 mL), and 1,1-bis(diphenylphosphine)ferrocene palladium dichloride (1.5 g, 2.10 mmol) was added. After the addition was complete, the reaction system was stirred at 95 °C for 2 hours under a nitrogen atmosphere. LC-MS showed that the starting material reacted completely. The reaction solution was concentrated and purified by column chromatography to obtain the title compound (2.30 g). MS (ESI) m / z 154.1 (M+H) +

[0208] Step 2: Preparation of 2-(3-cyclopropylpyridin-2-yl)-4-fluorophenol:

[0209]

[0210] 2-Chloro-3-cyclopropylpyridine (1.0 g, 6.57 mmol), (5-fluoro-2-hydroxyphenyl)boronic acid (1.2 g, 7.89 mmol), and cesium carbonate (6.4 g, 19.72 mmol) were dissolved in 1,4-dioxane (10 mL) and water (2.0 mL), and 1,1-bis(diphenylphosphine)ferrocene palladium dichloride (0.48 g, 0.66 mmol) was added. After the addition was complete, the reaction system was stirred at 95 °C for 2 hours under a nitrogen atmosphere. LC-MS showed that the starting material reacted completely. The reaction solution was concentrated and purified by column chromatography to obtain the title compound (1.10 g). MS (ESI) m / z 230.1 (M+H) +

[0211] Using cyclopropylboronic acid as a substrate, different intermediates were obtained by reacting with halogenated reagents available from different commercial sources, following the synthesis method described in the preparation example above. The specific results are shown in Table 3.

[0212] Table 3 Intermediate Compounds

[0213]

[0214]

[0215] Preparation Example 67: Preparation of 2-(4-cyclopropylpyridin-3-yl)-4-fluorophenol

[0216]

[0217] Step 1: Preparation of 3-chloro-4-cyclopropylpyridine:

[0218]

[0219] 3-Chloro-4-iodopyridine (4.5 g, 18.79 mmol) was dissolved in tetrahydrofuran (50 mL), and tetratetraphenylphosphine palladium (0.65 g, 0.56 mmol) and cyclopropyl zinc bromide (28 mL, 28.19 mmol, 1 M) were added under a nitrogen atmosphere. After the addition was complete, the reaction system was stirred at 65 °C for 16 hours under a nitrogen atmosphere. LC-MS showed that the starting material reacted completely. The reaction system was cooled to 25 °C, the reaction solution was quenched with water, the aqueous phase was extracted with ethyl acetate, the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the compound was purified by column chromatography to give the title compound (550 mg). MS (ESI) m / z 154.2 (M+H) + .

[0220] Step 2: Preparation of 2-(4-cyclopropylpyridin-3-yl)-4-fluorophenol:

[0221]

[0222] 3-Chloro-4-cyclopropylpyridine (500 mg, 3.26 mmol) and (5-fluoro-2-hydroxy-phenyl)boronic acid (660 mg, 4.23 mmol) were dissolved in 1,4-dioxane (6 mL) and water (2 mL). Potassium fluoride (946 mg, 16.28 mmol) and chloro(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (47 mg, 0.065 mmol) were added sequentially. After the addition was complete, the reaction system was stirred at 100 °C for 2 hours under a nitrogen atmosphere. LC-MS showed that the starting material reacted completely. The reaction solution was concentrated and purified by column chromatography to obtain the title compound (730 mg). MS (ESI) m / z 230.1 (M+H) +

[0223] Preparation Example 68: Preparation of 2-(2-cyclopropylpyridin-3-yl)-4-fluorophenol

[0224]

[0225] Step 1: Preparation of 3-chloro-2-cyclopropylpyridine:

[0226]

[0227] 2,3-Dichloropyridine (5.0 g, 33.79 mmol) was dissolved in a mixed solvent of tetrahydrofuran (80 mL) and N-methylpyrrolidone (20 mL). Under a nitrogen atmosphere, the mixture was cooled to 0 °C, and then ferric triacetylacetone (1.2 g, 3.38 mmol) and cyclopropylmagnesium bromide (50.7 mL, 50.68 mmol, 1 M tetrahydrofuran solution) were added. The reaction mixture was stirred at 25 °C for 3 hours. LC-MS showed complete reaction of the starting material. The reaction solution was quenched with saturated ammonium chloride, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (2.3 g, yield 44.3%). MS (ESI) m / z 154.1 (M+H) +

[0228] Step 2: Preparation of 2-(2-cyclopropylpyridin-3-yl)-4-fluorophenol:

[0229]

[0230] 3-Chloro-2-cyclopropylpyridine (1.0 g, 6.51 mmol) and 5-fluoro-2-hydroxyphenylboronic acid (1.55 g, 6.51 mmol) were dissolved in a mixed solvent of 1,4-dioxane (10 mL) and water (2 mL). Under nitrogen protection, dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium (439 mg, 0.65 mmol) and sodium carbonate (1.38 g, 13.02 mmol) were added sequentially. The reaction mixture was gradually heated to 90 °C and stirred for 2 hours. LC-MS showed that the starting material reacted completely. The reaction solution was cooled to room temperature, quenched with saturated ammonium chloride solution, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (550 mg). MS (ESI) m / z 230.1 (M+H) +

[0231] Preparation Example 69: Preparation of 2-(4-cyclopropyl-2-methylpyrimidin-5-yl)-4-fluorophenol

[0232]

[0233] Step 1: Preparation of 5-bromo-4-cyclopropyl-2-methylpyrimidine:

[0234]

[0235] 5-Bromo-2-methylpyrimidine (5.0 g, 28.90 mmol) was dissolved in tetrahydrofuran (50 mL). Under nitrogen protection, at 0 °C, cyclopropylmagnesium bromide (34.4 mL, 34.42 mmol, 1 M tetrahydrofuran solution) was added. The reaction mixture was gradually heated to 25 °C and stirred for 4 hours. The mixture was then cooled to 0 °C, and a tetrahydrofuran solution of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.78 g, 34.42 mmol) (50 mL) was added. The reaction mixture was gradually heated to 25 °C and stirred for 12 hours. LC-MS showed that the reactants reacted completely. The reaction solution was quenched with saturated ammonium chloride, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (2.1 g). MS (ESI) m / z 213.1 (M+H) +

[0236] Step 2: Preparation of 2-(4-cyclopropyl-2-methylpyrimidin-5-yl)-4-fluorophenol:

[0237]

[0238] 5-Bromo-4-cyclopropyl-2-methylpyrimidine (1.0 g, 4.69 mmol) and 5-fluoro-2-hydroxyphenylboronic acid (1.12 g, 4.69 mmol) were dissolved in a mixed solvent of 1,4-dioxane (15 mL) and water (3 mL). Under nitrogen protection, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (389 mg, 0.47 mmol) and sodium carbonate (994 mg, 9.39 mmol) were added sequentially. The reaction system was gradually heated to 100 °C and stirred for 4 hours. LC-MS showed that the starting material reacted completely. The reaction solution was cooled to room temperature, diluted with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (510 mg). MS (ESI) m / z 245.2 (M+H) +

[0239] Preparation Example 70: Preparation of 4-cyclopropyl-5-(5-fluoro-2-hydroxyphenyl)pyrimidine-2-carboxynitrile

[0240]

[0241] Step 1: Preparation of 5-bromo-2-chloro-4-cyclopropylpyrimidine:

[0242]

[0243] 5-Bromo-2-chloropyrimidine (6.0 g, 31 mmol) was dissolved in acetonitrile (50 mL) and water (50 mL), followed by the sequential addition of cyclopropionic acid (2.7 g, 31 mmol), ammonium persulfate (42.5 g, 187 mmol), and silver nitrate (527 mg, 3.1 mmol). After the addition was complete, the reaction mixture was stirred at 25 °C for 16 hours. LC-MS showed complete reaction of the starting material. The reaction solution was quenched with water, the aqueous phase was extracted with ethyl acetate, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the title compound (3.5 g). MS (ESI) m / z 233.1 (M+H) + .

[0244] Step 2: Preparation of 5-bromo-4-cyclopropylpyrimidine-2-carboxynitrile:

[0245]

[0246] 5-Bromo-2-chloro-4-cyclopropylpyrimidine (1.0 g, 4.3 mmol) was dissolved in acetonitrile (10 mL), and tetrabutylamine cyanide (1.7 g, 6.4 mmol) and triethylenediamine (721 mg, 6.4 mmol) were added. The reaction mixture was stirred at 25 °C for 2 hours under nitrogen protection. LC-MS showed complete reaction of the starting material. The reaction solution was quenched with water, extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrate was purified by column chromatography to give the title compound (800 mg). MS (ESI) m / z 224.1 (M+H) + .

[0247] Step 3: Preparation of 4-cyclopropyl-5-(5-fluoro-2-hydroxyphenyl)pyrimidine-2-carboxynitrile:

[0248]

[0249] 5-Bromo-4-cyclopropylpyrimidin-2-carboxynitrile (500 mg, 2.2 mmol) and (5-fluoro-2-hydroxy-phenyl)boronic acid (452 ​​mg, 2.9 mmol) were dissolved in 1,4-dioxane (6 mL) and water (2 mL). Under nitrogen protection, cesium carbonate (2.2 g, 6.7 mmol) and 1,1-bis(diphenylphosphine)diberberine palladium dichloride (163 mg, 0.22 mmol) were added sequentially. The reaction mixture was heated to 90 °C and stirred for 16 hours. LC-MS showed that the starting material reacted completely. The reaction solution was cooled to room temperature, diluted with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (540 mg). MS (ESI) m / z 256.1 (M+H) +

[0250] Preparation Example 71: Preparation of 2-(3-cyclopropylpyridazine-4-yl)-4-fluorophenol

[0251]

[0252] Step 1: Preparation of 4,5-dibromo-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one:

[0253]

[0254] 4,5-Dibromopyridazin-3-one (50.0 g, 196 mmol) was dissolved in tetrahydrofuran (500 mL), cooled to 0 °C, and then 3,4-dihydro-2H-pyran (82.8 g, 984 mmol) and p-toluenesulfonic acid hydrate (3.40 g, 19.7 mmol) were added sequentially. After the addition was complete, the reaction mixture was heated to 70 °C and stirred for 16 hours. LC-MS showed that the reaction was complete. The reaction solution was diluted with water, the aqueous phase was extracted with dichloromethane, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify and give the title compound 4 (55.2 g). MS (ESI) m / z 338.0 (M+H) +

[0255] Step 2: Preparation of 4-bromo-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one:

[0256]

[0257] 55 g (162 mmol) of 4,5-dibromo-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one was dissolved in ethylene glycol diethyl ether (550 mL), cooled to 0 °C, and sodium borohydride (15.39 g, 406 mmol) was added in portions. After the addition was complete, the reaction mixture was heated to 25 °C and stirred for 12 hours. LC-MS showed that the reaction was complete. The reaction solution was quenched with saturated ammonium chloride aqueous solution, the aqueous phase was extracted with dichloromethane, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (8.9 g). MS (ESI) m / z 259.1 (M+H) +

[0258] Step 3: Preparation of 4-(5-fluoro-2-hydroxyphenyl)-2-(tetrahydro-2H-pyran-2-yl)pyridazine-3(2H)-one:

[0259]

[0260] 4-Bromo-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one (8.9 g, 34.7 mmol) and 5-fluoro-2-hydroxyphenylboronic acid (5.9 g, 38.2 mmol) were dissolved in a mixed solvent of 1,4-dioxane (150 mL) and water (30 mL). Under nitrogen protection, tetrakis(triphenylphosphine)palladium (4.0 g, 3.47 mmol) and sodium carbonate (11.0 g, 104 mmol) were added sequentially. The reaction mixture was heated to 100 °C and stirred for 4 hours. LC-MS showed that the starting material reacted completely. After cooling to room temperature, the reaction solution was quenched with saturated ammonium chloride aqueous solution, extracted with ethyl acetate, and the combined organic phases were washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to give the title compound (4.5 g). LC-MS m / z (ESI): 290.3 [M+H] + .

[0261] Step 4: Preparation of 4-(2-(benzyloxy)-5-fluorophenyl)-2-(tetrahydro-2H-pyran-2-yl)pyridazine-3(2H)-one:

[0262]

[0263] 4-(5-fluoro-2-hydroxyphenyl)-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one (4.0 g, 13.78 mmol) was dissolved in acetone (40 mL), cooled to 0 °C, and potassium carbonate (9.6 g, 69.4 mmol) and benzyl bromide (3.58 g, 28.2 mmol) were added. After the addition was complete, the reaction mixture was heated to 60 °C and stirred for 12 hours. LC-MS showed that the reaction was complete. The reaction solution was diluted with aqueous solution, the aqueous phase was extracted with dichloromethane, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (3.0 g). MS (ESI) m / z 380.4 (M+H) +

[0264] Step 5: Preparation of 4-(2-(benzyloxy)-5-fluorophenyl)pyridazine-3-ol:

[0265]

[0266] 3.0 g (7.89 mmol) of 4-(2-(benzyloxy)-5-fluorophenyl)-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one was dissolved in a mixed solvent of methanol (30 mL) and water (10 mL), cooled to 0 °C, and then hydrochloric acid (10 mL, 7.89 mmol, 37 wt%) was added. After the addition was complete, the reaction mixture was heated to 50 °C and stirred for 1 hour. LC-MS showed that the reaction was complete. The mixture was cooled to room temperature, quenched with saturated sodium carbonate solution, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. Concentration and purification by column chromatography yielded the title compound (1.8 g). MS (ESI) m / z 296.3 (M+H) +

[0267] Step 6: Preparation of 4-(2-(benzyloxy)-5-fluorophenyl)-3-chloropyridazine:

[0268]

[0269] 4-(2-(benzyloxy)-5-fluorophenyl)pyridazin-3-ol (1.8 g, 6.08 mmol) was dissolved in phosphorus oxychloride (18 mL). Under nitrogen protection, the reaction mixture was stirred at 100 °C for 5 hours. LC-MS showed the reaction was complete. The mixture was cooled to room temperature, quenched slowly with water, and then saturated sodium carbonate solution was added. The aqueous phase was extracted with dichloromethane, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. Concentration and purification by column chromatography yielded the title compound (1.4 g). MS (ESI) m / z 316.7 (M+H) +

[0270] Step 7: Preparation of 4-(2-(benzyloxy)-5-fluorophenyl)-3-chloropyridazine:

[0271]

[0272] 4-(2-(benzyloxy)-5-fluorophenyl)pyridazin-3-ol (1.2 g, 3.81 mmol) and cyclopropylboronic acid (1.2 g, 13.3 mmol) were dissolved in a mixed solvent of toluene (10 mL) and water (1 mL). Under nitrogen protection, palladium acetate (171 mg, 0.76 mmol), 1,1'-bis(diphenylphosphine)ferrocene (844 mg, 1.53 mmol), and sodium carbonate (808 mg, 7.63 mmol) were added sequentially. The reaction mixture was heated to 100 °C and stirred for 2 hours. LC-MS showed that the starting material reacted completely. After cooling to room temperature, the reaction solution was diluted with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (610 mg). MS (ESI) m / z 321.3 (M+H)+

[0273] Step 8: Preparation of 2-(3-cyclopropylpyridazine-4-yl)-4-fluorophenol:

[0274]

[0275] 4-(2-(benzyloxy)-5-fluorophenyl)-3-chloropyridazine (550 mg, 1.72 mmol) was dissolved in methanol (15 mL). Under nitrogen protection, palladium / carbon (55 mg, 10%) was added, and the mixture was purged with hydrogen three times. The reaction mixture was heated to 30 °C and stirred for 2 hours. LC-MS showed that the starting material reacted completely. The mixture was filtered through diatomaceous earth, washed with methanol, and the filtrate was concentrated to give the title compound (310 mg). MS (ESI) m / z 231.2 (M+H) +

[0276] Preparation Example 72: Preparation of 2-(4-cyclopropylpyridazine-3-yl)-4-fluorophenol:

[0277]

[0278] Step 1: Preparation of 4-chloro-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one:

[0279]

[0280] 4-Chlorpyridazin-3-ol (11.0 g, 84 mmol) was dissolved in tetrahydrofuran (100 mL), followed by the addition of 3,4-dihydro-2H-pyran (28.4 g, 337 mmol) and p-toluenesulfonic acid (1.5 g, 8.4 mmol). After the addition was complete, the reaction mixture was stirred at 70 °C for 16 hours. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was concentrated and purified by column chromatography to obtain the title compound (16.0 g). MS (ESI) m / z 215.1 (M+H) + .

[0281] Step 2: Preparation of 4-cyclopropyl-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one:

[0282]

[0283] 15.0 g (70 mmol) of 4-chloro-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one was dissolved in 1,4-dioxane (150 mL) and water (30 mL). Under a nitrogen atmosphere, cesium carbonate (68.3 g, 210 mmol), cyclopropylboronic acid (7.8 g, 91 mmol), and 1,1-bis(diphenylphosphine)diberberine palladium dichloride (5.1 g, 7 mmol) were added sequentially. After the addition was complete, the reaction mixture was stirred at 90 °C for 16 hours. LC-MS showed that the starting material reacted completely. The reaction solution was cooled to room temperature, diluted with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the solution was purified by column chromatography to give the title compound (6.0 g). MS (ESI) m / z 221.1 (M+H) + .

[0284] Step 3: Preparation of 4-cyclopropylpyridazine-3(2H)-one:

[0285]

[0286] 5.5 g (70 mmol) of 4-cyclopropyl-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one was dissolved in methanol (45 mL), and concentrated hydrochloric acid (15 mL) was added. After the addition was complete, the reaction mixture was stirred at 50 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction solution was cooled to room temperature and quenched with ice-cold saturated sodium carbonate solution. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrate yielded the title compound (2.4 g). MS (ESI) m / z 137.1 (M+H) + .

[0287] Step 4: Preparation of 3-chloro-4-cyclopropylpyridazine:

[0288]

[0289] 2.4 g (18 mmol) of 4-cyclopropylpyridazine-3(2H)-one was dissolved in acetonitrile (30 mL), and phosphorus oxychloride (10 mL) was added. After the addition was complete, the reaction mixture was stirred at 80 °C for 12 hours. LC-MS showed that the starting material reacted completely. The reaction solution was cooled to room temperature, quenched slowly with water, and then saturated sodium carbonate aqueous solution was added. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 800 mg of the product. MS (ESI) m / z 155.1 (M+H) + .

[0290] Step 5: Preparation of 2-(4-cyclopropylpyridazine-3-yl)-4-fluorophenol:

[0291]

[0292] 3-Chloro-4-cyclopropylpyridine (500 mg, 3.2 mmol) and (5-fluoro-2-hydroxy-phenyl)boronic acid (656 mg, 4.2 mmol) were dissolved in 1,4-dioxane (6.0 mL) and water (2.0 mL). Under a nitrogen atmosphere, cesium carbonate (3.2 g, 9.4 mmol) and 1,1-bis(diphenylphosphine)diberberine palladium dichloride (236 mg, 0.32 mmol) were added sequentially. After the addition was complete, the reaction system was stirred at 90 °C for 16 hours. LC-MS showed that the starting material reacted completely. The reaction solution was cooled to room temperature, diluted with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify the title compound (290 mg). MS (ESI) m / z 231.1 (M+H) +

[0293] Preparation Example 73: Preparation of 2-(6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole:

[0294]

[0295] Step 1: Preparation of tert-butyl 5-(3-chloro-6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid ester:

[0296]

[0297] 2-(3-cyclopropylpyridin-2-yl)-4-fluorophenol (500.0 mg, 2.18 mmol) and tert-butyl-5-(3,6-dichloro-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid ester (781.3 mg, 2.18 mmol) were dissolved in anhydrous tetrahydrofuran (10 mL), followed by the addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (1.1 g, 4.36 mmol). After the addition was complete, the reaction mixture was stirred at 25 °C for 16 hours. LC-MS showed that the starting material reacted completely. The reaction mixture was diluted with water, the aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrate was then purified by column chromatography to obtain the title compound (500 mg). MS(ESI) m / z 551.3(M+H) +

[0298] Step 2: Preparation of tert-butyl 5-(6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid ester:

[0299]

[0300] tert-Butyl 5-(3-chloro-6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid ester (600.0 mg, 1.09 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), followed by the addition of N,N,N',N'-tetramethylethylenediamine (253.1 mg, 2.18 mmol), sodium borohydride (206.9 mg, 5.44 mmol), and 1,1-bis(diphenylphosphine)ferrocene palladium dichloride (79.7 mg, 108.89 mmol). After the addition was complete, the reaction system was stirred at 45 °C for 16 hours under a nitrogen atmosphere. LC-MS showed that the starting material reacted completely. The reaction solution was diluted with saturated ammonium chloride aqueous solution, the aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the solution was purified by column chromatography to obtain the title compound (480 mg). MS (ESI) m / z 517.2 (M+H) +

[0301] Step 3: Preparation of 2-(6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole:

[0302]

[0303] 100.0 mg (193.59 μmol) of tert-butyl-5-(6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid ester was dissolved in dichloromethane (2 mL), followed by the addition of trifluoroacetic acid (0.5 mL). The reaction mixture was stirred at 25 °C for 2 hours. LC-MS showed complete reaction of the starting material. The reaction solution was concentrated and adjusted to basicity with N,N-diisopropylethylamine. Further concentration yielded the title compound (72.0 mg). MS (ESI) m / z 417.2 (M+H) +

[0304] Using tert-butyl5-(3,6-dichloro-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid ester as a substrate, different intermediates were obtained by reacting with different intermediates prepared in the preparation example, following the synthetic method of preparation example 73 described above, as shown in Table 4.

[0305] Table 4 Intermediate Compounds

[0306]

[0307]

[0308]

[0309] Example 1: Preparation of (3R)-3-amino-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-(3-methanesulfonylphenyl)but-1-one

[0310]

[0311] Step 1: Preparation of 5-bromo-4-cyclopropylpyrimidine

[0312] 5-Bromopyrimidine (3.0 g) was dissolved in tetrahydrofuran (80 mL), and cyclopropylmagnesium bromide (2.74 g) was slowly added under nitrogen protection at 0 °C, followed by reaction at room temperature for 4 h. Then, 2,3-dichloro-5,6-dicyanobenzoquinone (4.28 g) was dissolved in THF and added dropwise to the reaction solution under nitrogen protection at 0 °C, and the reaction was allowed to proceed overnight at room temperature. The reaction was monitored by LCMS until completion. The reaction was quenched by adding an aqueous solution of ammonium chloride to the reaction system, followed by extraction three times with ethyl acetate and drying with anhydrous sodium sulfate to obtain the crude product. After concentration, column purification yielded the title compound (0.81 g).

[0313]

[0314] MS(ESI)m / z(M+H) + =199.0.

[0315] Step 2: Preparation of 2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenol.

[0316] Under nitrogen protection, 0.81 g of 5-bromo-4-cyclopropylpyrimidine and 0.95 g of 5-fluoro-2-hydroxyphenylboronic acid were dissolved in a mixed solution of dioxane and water (30 mL, dioxane:water = 4:1). Potassium carbonate (1.69 g) and 0.33 g of 1,1'-bis(diphenylphosphine)ferrocene palladium(II) dichloride were then added, and the reaction was carried out at 100 °C for 2 hours. The reaction was monitored by LC-MS until complete. The reaction was quenched with water, extracted three times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give the title compound (1.16 g).

[0317]

[0318] MS(ESI)m / z(M+H) + =231.2

[0319] Step 3: Preparation of tert-butyl 5-(3-chloro-6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid

[0320] 1.8 g of 5-(3,6-dichloro-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid tert-butyl ester was added to tetrahydrofuran (30 mL), followed by 2.29 g of DBU and 1.16 g of 2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenol. The reaction was allowed to proceed overnight at room temperature. The reaction was monitored by LCMS until complete. The reaction was then stopped, cooled to room temperature, quenched with water, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give the title compound (2.0 g).

[0321]

[0322] MS(ESI)m / z(M+H) + =552.2.

[0323] Step 4: Preparation of tert-butyl 5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid

[0324] 2.0 g of 5-(3-chloro-6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylic acid tert-butyl ester was added to tetrahydrofuran (30 mL), followed by the addition of sodium borohydride (0.68 g) in portions, then tetramethylethylenediamine (0.84 g) and 1,1'-bis(diphenylphosphine)ferrocene palladium(II) dichloride (0.29 g). The reaction was carried out overnight at room temperature under nitrogen protection. The reaction was monitored by LCMS until complete. The reaction was quenched with water, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give the title compound (1.8 g).

[0325]

[0326] MS(ESI)m / z(M+H) + =518.2.

[0327] Step 5: Preparation of 6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-5-(1H,2H,3H,4H,5H,6Hpyrrolo[3,4-c]pyrrolo-2-yl)-1,2,4-triazine

[0328] 1.8 g of 5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylic acid tert-butyl ester was added to trifluoroethanol (20 mL), followed by 0.88 mL of trimethylchlorosilane. The reaction was carried out at room temperature for 1 hour. The reaction was monitored by LCMS until complete. The solvent was evaporated under reduced pressure, and the residue was adjusted to alkalinity with saturated sodium bicarbonate solution. After lyophilization, column purification yielded the title compound (1.57 g).

[0329]

[0330] MS(ESI)[M+H] + =418.2.

[0331] Step 6: Preparation of N-[(2R)-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-1-(3-methanesulfonylphenyl)-4-oxobut-2-yl]tert-butyl carbamate

[0332] (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-(3-methanesulfonylphenyl)butyric acid (42 mg) was added to N,N-dimethylformamide (2 mL), followed by N,N-diisopropylethylamine (46 mg) and 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylbromohexafluorophosphate (91 mg). After reacting at room temperature for half an hour, 6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-5-(1H,2H,3H,4H,5H,6Hpyrrolo[3,4-c]pyrrolo-2-yl)-1,2,4-triazine (50 mg) was added, and the reaction was carried out at room temperature for 1 hour. The reaction was monitored by LCMS until complete, quenched with water, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give the title compound (73 mg).

[0333]

[0334] MS(ESI)[M+H] + =757.2.

[0335] Step 7: Preparation of (3R)-3-amino-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-(3-methanesulfonylphenyl)but-1-one

[0336] 73 mg of N-[(2R)-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-1-(3-methanesulfonylphenyl)-4-oxobut-2-yl]carbamate tert-butyl ester was added to trifluoroethanol (4 mL), followed by trimethylchlorosilane (0.1 mL). The reaction was carried out at room temperature for 1 hour. The reaction was monitored by LCMS until complete. The solvent was evaporated under reduced pressure. The residue was adjusted to weakly basic solution with saturated sodium bicarbonate solution, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by reverse-phase preparative column chromatography to obtain the title compound (13.05 mg).

[0337]

[0338] MS(ESI)[M+H] + =657.2.

[0339] 1H NMR (400MHz, DMSO-d6) δ8.92(d,J=2.3Hz,1H),8.48(s,1H),8.42(d,J=2.5Hz,1H),7.7 7(d,J=8.2Hz,2H),7.61-7.55(m,3H),7.46(t,J=7.3Hz,2H),4.51(s,2H),4.29(s,4H) ,4.11(d,J=17.0Hz,2H),3.20(s,4H),2.84(dd,J=13.7,5.2Hz,1H),2.69(dd,J=13.3, 7.7Hz,1H),2.34-2.25(m,2H),1.79(q,J=6.4Hz,1H),1.06-0.92(m,3H),0.78(s,1H).

[0340] Using the intermediate compound or commercially available compound prepared in the preparation examples as raw materials, and the title compound of step 5 in Example 1 as the reaction substrate, the following Examples 2-24 were prepared by referring to the synthesis steps 6-7 of Example 1. Specific information is shown in Tables 5-6.

[0341] Table 5 Compound Information from Examples

[0342]

[0343]

[0344]

[0345]

[0346]

[0347] The analytical data described above, including NMR and LC-MS data, are shown in Table 6.

[0348] Table 6. NMR and LC-MS data of compounds in Examples 2-21

[0349]

[0350]

[0351]

[0352]

[0353] Example 22: Preparation of (R)-7-(2-amino-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-oxobutyl)-1-(oxoethyl-3-methyl)-1H-indole-2-carbamate

[0354] Step 1: Preparation of tert-butyl(R)-(1-(2-cyano-1-(oxyethyl-3-methyl)-1H-indol-7-yl)-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-oxobut-2-yl)carbamate

[0355]

[0356] 2-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,2,3,4,5,6-hexahydropyrrole[3,4-c]pyrrole (30.47 mg, 0.073 mmol), (R)-3-(tert-butylcarbonyl)amino-4-(2-cyano-1-(oxy-3-methyl)-1H-indol-7-yl)butyric acid (30 mg, 0.073 mmol), HOBt (19.73 mg, 0.15 mmol), EDCI (27.99 mg, 0.15 mmol), and triethylamine (0.06 mL) were dissolved in DMF (8 mL) and reacted overnight at room temperature. After the raw material disappeared as monitored by LCMS, it was quenched with water, extracted three times with ethyl acetate (5 mL * 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 36 mg of the target compound.

[0357] MS(ESI)m / z(M+H) + =813.2

[0358] Step 2: Preparation of (R)-7-(2-amino-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-oxobutyl)-1-(oxoethyl-3-methyl)-1H-indole-2-carbamate

[0359]

[0360] 30 mg (0.037 mmol) of tert-butyl(R)-(1-(2-cyano-1-(oxyethyl-3-methyl)-1H-indol-7-yl)-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-oxobut-2-yl)carbamate was dissolved in DCM, and then TFA (21.09 mg (0.18 mmol) was added. The reaction was carried out at room temperature for 0.5 hours. After the starting material disappeared as monitored by LCMS, the mixture was quenched with water and saturated sodium bicarbonate was added to adjust the alkali. The mixture was extracted three times with ethyl acetate (5 mL * 3), and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography and then reverse-phase to obtain 6 mg of the target compound.

[0361] MS(ESI)m / z(M+H) + =713.2

[0362] 1 H NMR (400MHz, DMSO-d6) δ8.84(t,J=7.3Hz,1H),8.41(s,1H),8.37–8.32(m,1H),7.55–7.49(m,2H),7.44(s,1H),7.40(t,J=7.4 Hz,2H),7.16(dd,J=7.3,1.3Hz,1H),7.08(t,J=7.5Hz,1H),4.90–4.81(m,1H),4.75(dd,J=15.7,7.3Hz,1H),4.52(ddd,J=7.8, 6.2,4.3Hz,3H),4.41(d,J=17.3Hz,2H),4.40–4.28(m,3H),4.19(d,J=22.5Hz,4H),4.03(d,J=18.7Hz,2H),3.37(s,2H),3.07 (d,J=11.2Hz,1H),2.79(d,J=12.1Hz,1H),2.33(q,J=7.1,6.2Hz,2H),1.73(s,1H),0.99–0.87(m,3H),0.75(d,J=13.4Hz,1H).

[0363] Using the intermediate compound prepared in the preparation examples or a commercially available compound as a starting material and / or substrate, or using the title compound from step 5 of Example 1 as a reaction substrate, the following Examples 23-37 were prepared by referring to the synthesis steps of Example 22. Specific information is shown in Tables 7-8.

[0364] Table 7 Information on compounds in Examples 23-37

[0365]

[0366]

[0367]

[0368]

[0369]

[0370] The analytical data described above, including NMR and LC-MS data, are shown in Table 8.

[0371] Table 8. NMR and LC-MS data of compounds in Examples 23-37

[0372]

[0373]

[0374]

[0375] Example 38: Preparation of (R)-7-(2-amino-4-(5-(6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-5,6-dihydropyrrolo[3,4-c]pyrrolo-2(1H,3H,4H)-yl)-4-carbonylbutyl)-1-(2,2,2-trifluoroethyl)-1H-indole-2-carboxynitrile

[0376] Step 1: Preparation of tert-butyl(R)-(1-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)-4-(5-(6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-carbonylbutane-2-yl)carbamate:

[0377]

[0378] 2-(6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (72.0 mg, 171.6 μmol), (R)-3-((tert-butoxycarbonyl)amino)-4-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indole- 7-yl)butyric acid (73.0 mg, 171.6 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (84.8 g, 223.08 μmol), and N,N-diisopropylethylamine (66.4 mg, 514.81 mmol) were dissolved in N,N-dimethylformamide (3 mL). The reaction mixture was stirred at 0 °C for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was diluted with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the title compound (70.0 mg) was obtained. MS (ESI) m / z 824.5 (M+H) +

[0379] Step 2: Preparation of (R)-7-(2-amino-4-(5-(6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-5,6-dihydropyrrolo[3,4-c]pyrrolo-2(1H,3H,4H)-yl)-4-carbonylbutyl)-1-(2,2,2-trifluoroethyl)-1H-indole-2-carboxynitrile:

[0380]

[0381] 70.00 mg (84.97 μmol) of tert-butyl(R)-(1-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)-4-(5-(6-(2-(3-cyclopropylpyridin-2-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-carbonylbutane-2-yl)carbamate was dissolved in dichloromethane (2 mL), followed by the addition of trifluoroacetic acid (0.5 mL). The reaction mixture was stirred at 0 °C for 1 hour. LC-MS showed complete reaction of the starting material. The reaction solution was adjusted to alkaline with saturated sodium bicarbonate solution, extracted with dichloromethane, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by preparative separation to obtain the title compound (32.6 mg). MS(ESI) m / z 724.1(M+H) +

[0382] 1H-NMR(400MHz,DMSO-d6)δ8.45(s,1H),8.32(s,1H),7.70-7.63(m,2H),7.59-7.52(m,1H ),7.44-7.32(m,2H),7.32-7.28(m,1H),7.28-7.18(m,3H),5.79-5.57(m,1H),5.56-5.36 (m,1H),4.38-4.16(m,6H),4.14-3.95(m,2H),3.50-3.38(m,1H),3.29-3.27(m,1H),3.03 -2.87(m,1H),2.53-2.51(m,2H),1.66-1.53(m,1H),0.78-0.65(m,2H),0.65-0.49(m,2H)

[0383] Using (R)-3-((tert-butoxycarbonyl)amino)-4-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)butyric acid as a substrate and different intermediates prepared in the preparation examples as reactants, the compounds of Examples 39-48 were obtained by following the synthetic steps of Example 38. Specific details are shown in Table 9.

[0384] Table 9 Information on compounds 39-48 in Examples 9

[0385]

[0386]

[0387]

[0388] The analytical data described above for Examples 39-48 include NMR and LC-MS data, as shown in Table 10.

[0389] Table 10. NMR and LC-MS data of compounds 39-48 in Examples 10

[0390]

[0391]

[0392] Example 49: Preparation of (R)-7-(4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2-(methylamino)-4-oxobutyl)-1-(2,2,2-trifluoroethyl)-1H-indole-2-nitrile

[0393] Step 1: Preparation of tert-butyl((R)-(1-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)4-oxobut-2-yl)carbamate

[0394] Weigh (R)-3-((tert-butoxycarbonyl)amino)-4-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)butyric acid (306 mg) and dissolve it in 12 mL of N,N-dimethylformamide. Then, add N,N-diisopropylethylamine (0.36 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (206 mg), and 1-hydroxybenzotriazole (146 mg) in sequence. After stirring for 40 min, add 2-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,2,3,4,5,6-hexahydropyrrole[3,4-c]pyrrole (300 mg) and react at room temperature for 15 hours. The reaction was monitored by TLC and LCMS until complete. The reaction was quenched with water, extracted twice with ethyl acetate, washed with saturated brine, and purified by column chromatography to obtain the title compound (400 mg).

[0395]

[0396] MS(ESI) m / z 825.3(M+H) +

[0397] Step 2: Preparation of tert-butyl(R)-(1-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)4-oxobut-2-yl)(methyl)carbamate

[0398] 180 mg of tert-butyl(R)-(1-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)4-oxobut-2-yl)carbamate was dissolved in 2 mL of N,N-dimethylformamide and placed at 0 °C. Sodium hydride (13 mg) was added and stirred for 15 min, followed by the addition of 27 μL of iodomethane and stirring for 40 min. The mixture was monitored by TLC and LCMS, quenched with water, extracted twice with ethyl acetate, washed with saturated brine, and purified by Prep-TLC to obtain the title compound (142 mg).

[0399]

[0400] MS(ESI) m / z 839.3(M+H) +

[0401] Step 3: Preparation of (R)-7-(4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-3,4,5,6-tetrahydropyrrole[3,4-c]pyrrole-2(1H)-yl)-2-(methylamino)-4-oxobutyl)-1-(2,2,2-trifluoroethyl)-1H-indole-2-nitrile

[0402] 142 mg of tert-butyl(R)-(1-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)4-oxobut-2-yl)(methyl)carbamate was dissolved in 6 mL of trifluoroethanol. 107 μL of trimethylchlorosilane was added, and the reaction was carried out at room temperature for 40 min. The reaction was monitored by LCMS until complete, concentrated under vacuum, and separated by reverse-phase preparative column chromatography to obtain the title compound (13.29 mg).

[0403]

[0404] MS(ESI) m / z 739.3(M+H) + MS(ESI) m / z 370.2(M / 2+H) +

[0405] 1H NMR (400MHz, DMSO-d6) δ8.92(d,J=4.6Hz,1H),8.48(s,1H),8.42(d,J=2.6Hz,1H),7.67(s,1H),7.65–7.57(m,2H),7.5 0–7.43(m,2H),7.29(dd,J=7.4,1.2Hz,1H),7.20(t,J=7.6Hz,1H),5.80(d,J=8.9Hz,1H),5.43(dq,J=17.4,8.7Hz,1H) ,4.62–4.41(m,2H),4.30–4.26(m,4H),4.10(t,J=15.4Hz,2H),3.25(d,J=14.8Hz,2H),3.15–3.08(m,1H),2.96(t,J=3 .9Hz,1H),2.66–2.62(m,1H),2.37–2.25(m,1H),2.16(s,3H),1.83–1.77(m,1H),1.01–097(m,3H),0.87–0.73(m,1H).

[0406] Example 50: Preparation of (R)-7-(4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2-(dimethylamino)-4-oxobutyl)-1-(2,2,2-trifluoroethyl)-1H-indole-2-nitrile

[0407] Step 1: Preparation of (R)-7-(2-amino-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-oxobutyl)-1-(2,2,2-trifluoroethyl)-1H-indole-2-nitrile

[0408] 200 mg of tert-butyl(R)-(1-(2-cyano-1-(2,2,2-trifluoroethyl)-1H-indol-7-yl)-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)4-oxobut-2-yl)carbamate was dissolved in 8 mL of trifluoroethanol. 0.15 mL of trimethylchlorosilane was added, and the mixture was reacted at room temperature for 40 min. The reaction was monitored by LCMS until complete. The reaction was quenched with saturated sodium bicarbonate solution, extracted twice with dichloromethane / methanol (10 / 1), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the title compound (187 mg).

[0409]

[0410] MS(ESI) m / z 725.3(M+H) +

[0411] Step 2: Preparation of (R)-7-(4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-3,4,5,6-tetrahydropyrrole[3,4-c]pyrrole-2(1H)-yl)-2-(dimethylamino)-4-oxobutyl)-1-(2,2,2-trifluoroethyl)-1H-indole-2-nitrile

[0412] (R)-7-(2-amino-4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4-oxobutyl)-1-(2,2,2-trifluoroethyl)-1H-indole-2-nitrile (177 mg) was dissolved in 3 mL of acetonitrile. Formaldehyde (71 μL) was added, followed by a catalytic amount of acetic acid (14 μL). The reaction was carried out at room temperature for 20 min, and then sodium borohydride (18 mg) was added, with the reaction continuing at room temperature for another 20 min. The reaction was monitored by LCMS until complete, quenched with saturated sodium bicarbonate solution, dried twice with dichloromethane, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by reverse-phase preparative column chromatography to obtain the compound (17.80 mg).

[0413]

[0414] MS(ESI) m / z 753.3(M+H) + MS(ESI) m / z 377.2(M / 2+H) +

[0415] 1H NMR (400MHz, DMSO-d6) δ8.92(d,J=5.9Hz,1H),8.49(s,1H),8.43(d,J=6.0Hz,1H),7.66(s,1H),7.63–7.56(m,2H),7.4 7(t,J=7.3Hz,2H),7.33–7.26(m,1H),7.15(t,J=7.6Hz,1H),5.80(dd,J=18.8,9.6Hz,1H),5.35(dd,J=17.3,8.8Hz,1H ),4.60–4.26(m,6H),3.99(q,J=19.7,17.6Hz,2H),3.30(d,J=6.0Hz,1H),3.08(td,J=14.9,12.8,7.0Hz,2H),2.62(d, J=15.7Hz,1H),2.27(dd,J=15.6,8.6Hz,1H),2.20(s,6H),1.81(d,J=6.5Hz,1H),1.01–0.98(m,3H),0.85–0.77(m,1H).

[0416] Example 51: Preparation of 3-amino-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2,2-difluoro-4-phenylbut-1-one

[0417]

[0418] Step 1: Preparation of N-(4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-3,3-difluoro-4-oxo-1-phenylbut-2-yl)-2-methylpropane-2-thioamide:

[0419]

[0420] 3-((tert-butylsulfinyl)amino)-2,2-difluoro-4-phenylbutyric acid (170 mg, 0.41 mmol) was dissolved in dichloromethane (2 mL), followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (116 mg, 0.60 mmol), N,N-diisopropylethylamine (104 mg, 0.80 mmol), 1-hydroxybenzotriazole (81.5 mg, 0.60 mmol), and 2-(6-(2-(3-cyclopropylpyridazin-4-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (130 mg, 0.41 mmol). After the addition was complete, the reaction system was stirred at 45 °C for 2 hours. LC-MS showed that the starting material reacted completely. The reaction solution was diluted with ice water, the aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the solution was purified by column chromatography to give the title compound (72 mg). MS (ESI) m / z 719.8 (M+H) +

[0421] Step 2: Preparation of 3-amino-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2,2-difluoro-4-phenylbut-1-one:

[0422]

[0423] N-(4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-3,3-difluoro-4-oxo-1-phenylbut-2-yl)-2-methylpropane-2-thioamide (72 mg, 0.10 mmol) was dissolved in methanol (1 mL), followed by the addition of a methanol solution of hydrochloric acid (2 mL, 4 M). After the addition was complete, the reaction mixture was stirred at 25 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction mixture was quenched with ice-cold saturated sodium bicarbonate solution. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the mixture was purified by preparative separation to obtain the title compound (14.95 mg). MS (ESI) m / z 615.4 (M+H) +

[0424] 1H-NMR(400MHz,DMSO-d6)δ8.92(s,1H),8.48(s,1H),8.42(s,1H),7.61-7.42(m,3H),7.34-7.20(m,5H),4.67-4.44(m, 4H),4.35-4.21(m,4H),3.55-3.42(m,1H),3.00-2.92(m,1H),2.60-2.52(m,1H),1.84-1.76(m,1H),1.05-0.75(m,4H).

[0425] Example 52: Preparation of (3-amino-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-3-methyl-4-(naphth-1-yl)butane-1-one

[0426]

[0427] Step 1: Preparation of N-(4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2-methyl-1-(naphth-1-yl)-4-carbonylbutane-2-yl)-2-methylpropane-2-sulfinamide:

[0428]

[0429] 3-((tert-butylthionylsulfinyl)amino)-3-methyl-4-(naphthyl-1-yl)butyric acid (100 mg, 0.29 mmol) was dissolved in N,N-dimethylformamide (2 mL). Under a nitrogen atmosphere, the mixture was cooled to 0 °C, and then 2-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (120 mg, 0.29 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (142 mg, 0.37 mmol), and N,N-diisopropylethylamine (111 mg, 0.86 mmol) were added sequentially. After the addition of the additives, the reaction mixture was stirred at 0 °C for 2 hours. LC-MS showed the reaction was essentially complete. The reaction solution was quenched with ice water, the aqueous phase was extracted with ethyl acetate, the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate, and concentrated to give the title compound (95 mg). MS (ESI) m / z 747.3 (M+H) +

[0430] Step 2: Preparation of (3-amino-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-3-methyl-4-(naphth-1-yl)butane-1-one:

[0431]

[0432] N-(4-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2-methyl-1-(naphthyl-1-yl)-4-carbonylbutane-2-yl)-2-methylpropane-2-sulfinamide (85 mg, 0.11 mmol) was dissolved in a 4.0 M methanol-hydrochloric acid solution. After the addition was complete, the reaction mixture was stirred at 25 °C for 1 hour. LC-MS showed that the reaction was essentially complete. The reaction solution was quenched with saturated sodium bicarbonate, the aqueous phase was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the solution was purified by preparative high-performance liquid chromatography to give the title compound (17.60 mg). MS (ESI) m / z 643.5 (M+H) +

[0433] 1 H-NMR (400MHz, DMSO-d6) δ8.92(s,1H),8.48(s,1H),8.42(s,1H),8.30(d,J=8.0Hz,1H),7.91-7.88(m,1H),7.79(d,J=7.8Hz,1H) ,7.62-7.56(m,1H),7.52-7.40(m,6H),4.58-4.11(m,8H),3.38-3.34(m,2H),2.39(s,2H),1.84-1.76(m,1H),1.07-0.73(m,7H).

[0434] Using the different intermediates prepared in the preparation examples as reaction substrates, and referring to the synthesis steps of Example 52, the compounds of Examples 53 and 54 were obtained.

[0435] Example 53: 3-amino-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-3-methyl-4-phenylbutane-1-one

[0436]

[0437] MS(ESI) m / z 593.5(M+H) +

[0438] 1 H-NMR (400MHz, DMSO-d6) δ8.92(s,1H),8.48(s,1H),8.42(s,1H),7.61-755(m,1H),7.50-7.42(m,2H),7.31-7.25(m,2H),7. 23-7.16(m,3H),4.61-4.10(m,8H),2.83-2.72(m,2H),2.24(s,2H),2.20-2.00(m,2H),1.85-1.75(m,1H),1.10-0.70(m,7H).

[0439] Example 54: 3-amino-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-4,4-difluoro-4-phenylbutane-1-one

[0440]

[0441] MS(ESI) m / z 615.5(M+H) +

[0442] 1 H-NMR(400MHz,DMSO-d6)δ8.91(s,1H),8.44(d,J=25.0Hz,2H),7.61-7.57(m,1H),7.55-7.41(m,7 H),4.65-4.07(m,8H),3.79-3.64(m,1H),2.33-1.90(m,2H),1.84-1.75(m,1H),1.08-0.72(m,4H).

[0443] Example 55: Preparation of (S)-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2-(2-methylpyrrolidin-2-yl)ethane-1-one

[0444]

[0445] Step 1: Preparation of (S)-(S)-1-(tert-butoxycarbonyl)-2-methylpyrrolidine-2-carboxylic acid (isobutyl carbonate) anhydride:

[0446]

[0447] (S)-1-(tert-butoxycarbonyl)-2-methylpyrrolidine-2-carboxylic acid (4.0 g, 17.4 mmol) was dissolved in tetrahydrofuran (40 mL) under nitrogen protection. Triethylamine (2.8 g, 27.4 mmol) and isobutyl chloroformate (2.5 g, 18.3 mmol) were added sequentially at 0 °C. After the addition was complete, the reaction mixture was stirred at 0 °C for 2 hours. TLC showed that the starting material reacted completely. The reaction solution was diluted with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrate yielded the title compound (4.2 g).

[0448] Step 2: Preparation of (S)-2-(2-diazoacetyl)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester:

[0449]

[0450] (S)-(S)-1-(tert-butoxycarbonyl)-2-methylpyrrolidine-2-carboxylic acid (isobutylcarbonic) anhydride (4.2 g, 12.8 mmol) was dissolved in acetonitrile (42 mL) under nitrogen protection. Trimethylsilyldiazomethane (2 M, 19.1 mL) was added at 0 °C. After the addition was complete, the reaction mixture was gradually heated to room temperature and stirred for 12 hours. LC-MS showed that the reaction was complete. The reaction solution was diluted with saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. Concentration and column chromatography were used to purify the title compound (1.3 g). MS (ESI) m / z 254.3 (M+H) +

[0451] Step 3: Preparation of (S)-2-(2-methoxy-2-carbonylethyl)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester:

[0452]

[0453] (S)-2-(2-diazonylacetyl)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (1.3 g, 5.13 mmol) was dissolved in methanol (13 mL). Under nitrogen protection, triethylamine (1.03 g, 10.2 mmol) and silver benzoate (246 mg, 1.02 mmol) were added sequentially. The reaction mixture was gradually heated to 60 °C and stirred for 3 hours. LC-MS showed that the starting material reacted completely. After cooling to room temperature, the reaction solution was dissolved in saturated ammonium chloride water, extracted with ethyl acetate, and the combined organic phases were washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure and purified by column chromatography to give the title compound (260 mg). LC-MS m / z (ESI): 258.3 [M+H] +.

[0454] Step 4: Preparation of (S)-2-(1-(tert-butoxycarbonyl)-2-methylpyrrolidine-2-yl)acetic acid:

[0455]

[0456] (S)-2-(1-(tert-butoxycarbonyl)-2-methylpyrrolidine-2-yl)acetic acid (260 mg, 1.01 mmol) was dissolved in a mixed solvent of methanol (2 mL) and water (1 mL), and sodium hydroxide (80.8 mg, 2.02 mmol) was added. After the addition was complete, the reaction system was heated to 40 °C and stirred for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was added to a saturated ammonium chloride aqueous solution, the aqueous phase was extracted with dichloromethane, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, column chromatography was used to purify and give the title compound (210 mg). MS (ESI) m / z 244.4 (M+H) +

[0457] Step 5: Preparation of (S)-2-(2-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2-carbonylethyl)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester:

[0458]

[0459] (S)-2-(1-(tert-butoxycarbonyl)-2-methylpyrrolidine-2-yl)acetic acid (70 mg, 0.29 mmol) was dissolved in N,N-dimethylformamide (2 mL), followed by the addition of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (131 mg, 0.34 mmol), N,N-diisopropylethylamine (89 mg, 0.69 mmol), and 2-(6-(2-(3-cyclopropylpyridazin-4-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-1,2,3,4,5,6-hexahydropyrrolo[3,4-c]pyrrole (96 mg, 0.23 mmol). After the addition was complete, the reaction mixture was stirred at 0 °C for 2 hours. LC-MS showed that the reaction proceeded completely. The reaction solution was diluted with ice water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the solution was purified by column chromatography to give the title compound (90 mg). MS (ESI) m / z 643.7 (M+H) +

[0460] Step 6: Preparation of (S)-1-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2-(2-methylpyrrolidin-2-yl)ethane-1-one:

[0461]

[0462] (S)-2-(2-(5-(6-(2-(4-cyclopropylpyrimidin-5-yl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)-2-carbonylethyl)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (90 mg, 0.14 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. After the addition was complete, the reaction system was stirred at 25 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction solution was mixed with ice-cold saturated sodium bicarbonate solution, the aqueous phase was extracted with ethyl acetate, the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate, concentrated and purified by preparative separation to obtain the title compound (27.83 mg). MS (ESI) m / z 543.3 (M+H) +

[0463] 1 H-NMR(400MHz,DMSO-d6)δ8.91(s,1H),8.48(s,1H),8.41(s,1H),7.61-7.44(m,3H),4.60-4.42(m,2H),4.37-4.25(m,4H),4.10 (d,J=13.6Hz,2H),2.90-2.78(m,2H),2.38-2.32(m,2H),1.82-1.64(m,4H),1.54-1.46(m,1H),1.15(s,3H),1.02-0.74(m,4H).

[0464] Experimental Example 1: Menin-MLL Protein Interaction Inhibition Activity Assay

[0465] (1) Inhibitory activity test of the interaction between Menin and MLL protein

[0466] The IC50 of the test compound inhibiting Menin-MLL protein interaction was detected using the Fluorescence Polarization method. 50 Value. The specific steps are as follows:

[0467] 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 ECHO 665 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 the plates were 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 (BMG Labtech Inc.), and the data were processed.

[0468] 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 10 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 is fitted to obtain the IC50 of each compound inhibiting Menin-MLL protein interaction. 50 value.

[0469] Note: SNDX-5613 refers to N-ethyl-2-((4-(7-(((1r,4r)-4-(ethylsulfonamide)cyclohexyl)methyl)-2,7-diazaspiro[3.5]non-2-yl)pyrimidin-5-yl)oxy)-5-fluoro-N-isopropylbenzamide (hereinafter the same), purchased from Shanghai Loulan Biotechnology Co., Ltd., with the following structural formula:

[0470]

[0471] (2) Inhibition activity test of interaction between mutant Menin-M327I and Menin-T349M and MLL protein

[0472] The IC50 of the test compounds inhibiting the interaction between Menin-M327I, Menin-T349M and MLL protein was detected using fluorescence polarization. 50 Value. The specific steps are as follows:

[0473] 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 ECHO 665 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.

[0474] 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 value.

[0475] Experiment Example 2: Cell Proliferation Inhibition Experiment

[0476] (1) Cell plating:

[0477] Remove cells from the incubator and place them on the worktable. Gently pipette to mix and count using CounterStar. Dilute cells to the desired density with fresh complete culture medium. Use MV-4-11 cells (source: Nanjing Kebai, catalog number: CBP60522) in a medium of RPMI 1640 (containing HEPES) (BOSTER, catalog number: PYG0122) + 10% FBS (GIBCO, catalog number: 10099-141C) + 1% P / S (HyClone, catalog number: SV30010). The plating density is 1×10^4 cells / well, 100 μL / well. Use a multipipe pipette to transfer 100 μL of each cell suspension into a 96-well plate.

[0478] (2) Preparation of compounds:

[0479] The stock solution of the compound was diluted from 10 mM with DMSO to 4000, 1200, 400, 120, 40, 12, 4, 1.2, and 0 μM, with the 0 μM well serving as a control. After thorough mixing, 1.3 μL was taken out with a multi-channel pipette and added to 258.7 μL of culture medium, resulting in a 100-fold dilution and a DMSO concentration of 0.5%. After thorough mixing with the multi-channel pipette, the cells were taken out, and two replicates were set up for each compound concentration. 100 μL of the compound diluted in step 2 was added to each replicate well, resulting in the following final compound concentrations: 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0 μM. At this point, there were a total of 200 μL of culture medium per well in the cell culture plate, with a DMSO concentration of 0.25%. The well containing only 0.25% DMSO (compound concentration of 0) served as a control well. 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.

[0480] (3) CTG detection:

[0481] After the culture time is up, remove the cells and aspirate a portion of the culture medium, leaving 50 μL of medium in each well. Add 50 μL of CTG reagent (cellcounting-Lite 2.0, Vazyme, DD1101-02) per well using a multipipe pipette. Incubate with shaking on a shaker at room temperature for 15 min, then allow to equilibrate at room temperature for 15 min. Detect using a multi-mode microplate reader.

[0482] (4) Data Analysis:

[0483] Cell viability% was calculated as As / Ac × 100%. As: test wells (containing cell culture medium, CTG, and the test compound); Ac: control wells (containing cell culture medium, CTG, and no test compound). A dose-response curve was fitted using the logarithmic value of the compound concentration as the X-axis and cell viability% as the Y-axis to obtain the IC50 value of each compound's inhibitory activity on cell proliferation. 50 value.

[0484] The compounds of the present invention were tested using the above-described activity testing method, and the results are shown in Table 11.

[0485] Table 11 Activity data of the compounds of the present invention

[0486]

[0487]

[0488] Note: In Table 11, "-" indicates that the compound was not detected. "Compound number" indicates the compound in the corresponding example.

[0489] The results in Table 11 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. The compound represented by Formula I, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, In Formula I: X1, X2, X3, and X4 are all CH; or any three of them are N, and the rest are CH. Y is either CH or N; a is 0, 1, or 2; b is 0, 1, or 2; R1 and R2 are independently selected from hydrogen, C1-C3 alkyl, 3-6 membered cycloalkyl, or 3-6 membered heterocycloalkyl. R3 is selected from hydrogen, C1-C3 alkyl, 3-6 membered cycloalkyl, or 3-6 membered heterocycloalkyl; R4 and R5 may be the same or different, and can be independently selected from hydrogen or fluorine; R6 and R7 may be the same or different, and can be independently selected from hydrogen or fluorine; 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, or 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 skeleton atoms of the heteroaryl and heterocycloalkyl 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, X2, X3, X4 are CH; or X2 is N, X1, X3, X4 are CH; or X3 is N, X1, X2, X4 are CH; or X4 is N, X1, X2, X3 are CH; or X1 and X3 are N, X2 and X4 are CH; or X1 and X4 are N, X2 and X3 are CH; or X2 and X4 are N, X1 and X3 are CH; or X1 and X2 are N, X3 and X4 are CH.

3. The compound according to claim 1, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, R1 and R2 are independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxecyclopropyl, and azircyclopropyl.

4. The compound according to any one of claims 1-3, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, R3 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, oxecyclopropyl, and azircyclopropyl.

5. The compound according to any one of claims 1-4, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, Ar is selected from 5-10 aryl, or 5-10 heteroaryl, 5-10 cycloalkyl, or 5-10 heterocycloalkyl; and Ar is monocyclic, fused bicyclic, or fused tricyclic.

6. The compound according to claim 5, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, Ar is 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, pyrazol[1,5-a]pyridinyl ]Pyridyl, isoindoline-1-one, 3,4-dihydroisoquinoline-1(2H)-one; 2,3-dihydrobenzo[b][1,4]dioxane, benzo[d][1,3]dihydroxy, 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.

7. The compound according to claim 6, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, Ar is selected from the following substituted or unsubstituted groups:

8. The compound, pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof according to any one of claims 1-7, characterized in that, Ar can be substituted at any possible position by one or more groups selected from cyano, halogen, phosphono, sulfonyl, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, RaCO-, C2-C4 alkenyl, -RpOH, N(RxRy)CO(CH2)-, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-6 heterocyclic alkyl, substituted or unsubstituted 5-7 aryl or substituted or unsubstituted 5-7 heteroaryl, wherein Ra is C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl or 3-6 heterocyclic alkyl, and Rp, Rx and Ry are all C1-C3 alkyl.

9. The compound according to claim 8, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, characterized in that, Ar can be substituted at any possible position by one or more groups selected from cyano, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, methoxy, ethoxy, n-propoxy, isopropoxy, trifluoromethyl, 2,2,2-trifluoroethyl, cyclopropyl, oxecyclopropyl, azicyclopropyl, oxecyclobutyl, azicyclobutyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydropyranyl, methanesulfonyl, ethanesulfonyl, -CH2CO(CH3)2, or groups selected from the following groups:

10. The following compounds, their pharmaceutically acceptable salts, hydrates, isomers, prodrugs, or mixtures thereof, 11. A pharmaceutical composition, characterized in that, It includes the compound of any one of claims 1-10, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, and pharmaceutically acceptable excipients and / or carriers.

12. The use of any compound of claims 1-10, its pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, or the pharmaceutical composition of claim 11, in the preparation of a medicament for the prevention, relief, or treatment of diseases related to the interaction with the Menin-MLL protein.

13. The use according to claim 12, characterized in that, Diseases associated with the interaction of the Menin-MLL protein include malignancies, diabetes, or complications associated with those diseases, including hematologic malignancies, lymphomas, and solid tumors.

14. The use according to claim 13, 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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