Aromatic amide derivative as well as preparation method and application thereof

Abstract

The invention relates to an aromatic amide derivative, a preparation method of the aromatic amide derivative and application of a pharmaceutical composition containing the aromatic amide derivative in medicine. Specifically, the present invention relates to an aromatic amide derivative represented by general formula (I), a preparation method and a pharmaceutically acceptable salt thereof, and uses of the aromatic amide derivative and the pharmaceutically acceptable salt thereof as therapeutic agents, especially KIF18A inhibitors, in which the definition of each group in the general formula (I) is the same as the definition in the specification.

Description

Aromatic amide derivatives and their preparation method and use Technical Field

[0001] The present invention relates to an aromatic amide derivative, a preparation method thereof, a pharmaceutical composition containing the derivative, and use of the derivative as a therapeutic agent, in particular as a KIF18A inhibitor. Background Art

[0002] Kinesin molecules are motor proteins that use microtubules as tracks and play an important role in organelle migration, tissue and organ development, signal transduction, mitosis, meiosis, and other processes. Various microtubule-associated proteins (MAPs) in the kinesin-8 family regulate microtubule dynamic instability by affecting microtubule polymerization and depolymerization. KIF18A, a member of the kinesin-8 family, can move toward the positive pole using microtubules as tracks and prefers to bind to longer microtubules. Its activity is length-dependent and affects the length of the spindle, ensuring the timely and smooth alignment of sister chromosomes. Its functions are very similar and conserved across species.

[0003] KIF18A is a molecular motor protein that moves along microtubules toward the plus-end of microtubules. It regulates chromosome midplate assembly by influencing the dynamic instability of microtubule ends, thus functioning during mitosis. During anaphase, the protein is ubiquitinated and degraded, ensuring precise chromosome segregation during mitosis and enabling the successful completion of mitosis and cytokinesis. During early mitosis, KIF18A localization to the plus-end of microtubules near the kinetochore is essential for its function. This localization depends not only on the motor activity of its N-terminus but also on the microtubule-binding tail domain. KIF18A is also subject to reversible phosphorylation / dephosphorylation, but how these post-translational modifications regulate KIF18A function remains understudied. The estrogen receptor ERɑ binds to KIF18A and promotes its transcription, but whether KIF18A is regulated by other transcription factors remains unclear. Therefore, further research is needed into the mechanisms regulating KIF18A's gene transcription. During meiosis, cells lacking KIF18A will be unable to complete meiosis, which will lead to sperm formation disorders and testicular dysplasia in male animals.

[0004] Studies have found that KIF18A protein is highly expressed in a variety of cancers, including but not limited to hepatocellular carcinoma, glioblastoma, colon cancer, breast cancer, lung cancer, bile duct cancer, pancreatic cancer, prostate cancer, bladder cancer, head cancer, neck cancer, cervical cancer, ovarian cancer, synovial sarcoma, and rhabdomyosarcoma. This suggests that KIF18A is closely associated with the occurrence and progression of tumors and could serve as a target for molecular diagnosis and treatment of various tumors. KIF18A expression is associated with the progression of clinical colorectal cancer. Studies have shown that KIF18A can induce Akt phosphorylation, and knocking out KIF18A in mice significantly promotes cell apoptosis. It is speculated that KIF18A promotes the occurrence and progression of colorectal cancer by activating the PI3K-Akt signaling pathway. KIF18A is also highly expressed in human breast cancer cells, and its overexpression is associated with breast tumor grade, migration, and prognosis. Studies in breast cancer cells have revealed that overexpression of KIF18A leads to the formation of multinucleated cells, while low expression significantly reduces cell proliferation both in vitro and in vivo. This is attributed to KIF18A stabilizing microtubules at their ends and inactivating the PI3K-Akt signaling pathway, leading to apoptosis. Furthermore, KIF18A is upregulated at both the transcriptional and translational levels in lung adenocarcinoma, and abnormal KIF18A expression correlates with clinical and pathological malignancy. KIF18 gene mutations have been observed in lung adenocarcinoma, and its expression is also regulated by DNA copy number. KIF18A knockout inhibits lung adenocarcinoma cell proliferation in vitro and in vivo, inducing apoptosis and G2 / M arrest. Genes that are overexpressed alongside KIF18A are concentrated in cell cycle signaling pathways, thus further investigation of the mechanisms of action of KIF18A in tumors is of great clinical significance.

[0005] No new KIF18A inhibitors have been marketed, and currently only Amgen's AMG-650 has entered Phase I clinical trials. As a cutting-edge research area, KIF18A remains a promising target for further investigation, necessitating continued study of its mechanism of action and the development of new inhibitors.

[0006] Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides an aromatic amide derivative represented by general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts:

[0008] in:

[0009] R A Select from -OR B 、-NR B R C 、-S(=O) r R B 、-C(=O)R 5、-C(=O)OR 5 、-NHC(=O)R 5 、-NHC(=O)OR 5 、-C(=O)NR 6 R 7 、-CH2OR 5 、-CH2NR 6 R 7 , 5-10 membered aryl, 5-10 membered heteroaryl or 6-14 membered fused ring, wherein the aryl, heteroaryl or fused ring is optionally further substituted by one or more R a replace;

[0010] R B are the same or different and are independently selected from deuterated alkyl, 5-10 membered aryl, 5-10 membered heteroaryl or 6-14 membered fused ring, wherein the aryl, heteroaryl or fused ring is optionally further replaced by one or more R a replace;

[0011] R C is selected from a hydrogen atom or an alkyl group;

[0012] R a are the same or different and are each independently selected from halogen, hydroxy, cyano, alkyl, cycloalkyl or alkoxy; wherein the alkyl, cycloalkyl or alkoxy is optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, alkyl or alkoxy;

[0013] R 1 Selected from hydrogen, cyano, halogen, alkyl, hydroxyl, cycloalkyl, heterocyclic, aryl, heteroaryl, -OR 5 、-C(=O)R 5 、-C(=O)OR 5 、-NHC(=O)R 5 、-NHC(=O)OR 5 、-NR 6 R 7 、-C(=O)NR 6 R 7 、-CH2NHC(=O)OR 5 、-CH2NR 6 R 7 or -S(=O) r R 5 wherein the alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent;

[0014] L1 is selected from a bond, -C 1-6 Alkylene-, -C 0-4 Alkylene-NR c S(=O)(=NH)-C 0-4 Alkylene-, -C 0-4 Alkylene-SC 0-4 Alkylene-, -C 0-4 Alkylene-S(=O)-C 0-4 Alkylene-, -C 0-4 Alkylene-SO2-C 0-4 Alkylene-, -C 0-4 Alkylene-S(=O)(=NH)-C 0-4 Alkylene-, -C 0-4 Alkylene-NR c SO2-C 0-4 Alkylene-, -C 0-4 Alkylene-SO2NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-OC 0-4 Alkylene-, -C 0-4 Alkylene-NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-NR c SO2NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-NR c C(O)NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-C(O)NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-NR c C(O)-C 0-4 Alkylene-, -C 0-4 Alkylene-PC 0-4 Alkylene-, -C 0-4 Alkylene-P(=O)2-C0-4 Alkylene, -C 0-4 Alkylene-C(=O)-C 0-4 Alkylene- or -C 0-4 Alkylene-C(=N(OH))-C 0-4 Alkylene-, wherein the -C 1-6 Alkylene- or -C 0-4 Alkylene - optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, cycloalkyl or alkoxy;

[0015] R c is selected from a hydrogen atom or an alkyl group;

[0016] R 2 are the same or different and are each independently selected from halogen, hydroxy, cyano, alkyl, cycloalkyl or alkoxy; wherein the alkyl, cycloalkyl or alkoxy is optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, alkyl or alkoxy;

[0017] L2 is selected from in Indicates that the group and the general formula (I) The attachment site; Indicates that the group and the general formula (I) The attachment site;

[0018] R 3 are each independently selected from a hydrogen atom or an alkyl group, wherein the alkyl group is optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano or alkoxy; R 3 Preferably a hydrogen atom;

[0019] R 4 Selected from cyano, halogen, alkyl, hydroxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR 5 、-C(=O)R 5 、-C(=O)OR 5 、-NHC(=O)R 5 、-NHC(=O)OR 5 、-NR 6 R 7 、-C(=O)NR 6 R 7 、-CH2NHC(=O)OR 5 、-CH2NR 6 R 7 or -S(O) r R 5wherein the alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent;

[0020] R 5 Each is independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the heterocyclic group, the aryl group or the heteroaryl group is optionally further substituted by one or more hydroxyl groups, halogen groups, nitro groups, cyano groups, alkyl groups, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyl groups, heterocyclic groups, aryl groups, heteroaryl groups, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent;

[0021] R 6 and R 7 are each independently selected from hydrogen, hydroxy, halogen, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR9 C(=O)R 10 substituted by a substituent;

[0022] Or, R 6 and R 7 Together with the atoms to which they are attached, they form a 4- to 8-membered heterocyclic group, wherein the 4- to 8-membered heterocyclic group contains one or more N, O, or S(O)r, and the 4- to 8-membered heterocyclic group is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclic group, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent;

[0023] R 8 、R 9 and R 10 Each is independently selected from a hydrogen atom, an alkyl group, an amino group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the heterocyclic group, the aryl group or the heteroaryl group is optionally further substituted with one or more substituents selected from a hydroxyl group, a halogen group, a nitro group, an amino group, a cyano group, an alkyl group, an alkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, a carboxyl group or a carboxylate group;

[0024] m is 0, 1 or 2; and

[0025] r is independently 0, 1 or 2.

[0026] A preferred embodiment of the present invention is a compound of formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts, which is a compound of formula (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts:

[0027] Wherein: Ring A is selected from a 5- to 10-membered aryl group, a 5- to 10-membered heteroaryl group, or a 6- to 14-membered fused ring;

[0028] R a are the same or different, each independently selected from halogen, hydroxy, cyano, C1-C3 alkyl, cyclopropyl or methoxy;

[0029] n is 0, 1 or 2;

[0030] L1, L2, R 1 、R 2 、R 4 and m are as defined in the general formula (I).

[0031] A preferred embodiment of the present invention is a compound of formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein R A Selected from -OCD3, -C(=O)OH, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)CH3, -CH2OH, -CH2OCH3,

[0032] A preferred embodiment of the present invention is a compound of formula (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein n is 0 or 1.

[0033] A preferred embodiment of the present invention is a compound of formula (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein Selected from the following groups:

[0034] A preferred embodiment of the present invention is a compound of general formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein m is 0.

[0035] A preferred embodiment of the present invention is a compound of formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein:

[0036] L1 is selected from a bond, -NR c -C 0-4 Alkylene-, -NR c SO2-C 0-4 Alkylene-, -SO2NR c -C 0-4 Alkylene-, -NR c SO2NR c -, -S(=O)(=NH)-, -NR c S(=O)(=NH)-, -C 1-4 Alkylene-, -S(=O)-, -O-, -C(=O)-, -C(=O)NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-SO2-C 0-4 Alkylene-, -C=N(OH)- or -NR c -C(=O)-, wherein the -C 0-4Alkylene- or -C 1-4 Alkylene - optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, cyclopropyl or methoxy;

[0037] R c are each independently selected from a hydrogen atom or a methyl group.

[0038] A preferred embodiment of the present invention is a compound of formula (I) or (II) or a stereoisomer, tautomer or a pharmaceutically acceptable salt thereof, wherein L1 is selected from a bond, -NHSO2CH2CH2-, -SO2NHCH2CH2-, -SO2-, -CH2SO2-, -NHSO2-, -SO2NH-, -NHC(CH3)2CH2-, -C(=O)NHCH2CH2-, -C(=O)NHC(CH3)2CH2-, -C(=O)N(CH3)CH2CH2-, -CH(CH3)(OH)CH2-, -NHSO2CH(CH3)CH2-, -SO2NHC(CH3)2CH2-, -C(=O)NH-, -NHCH2CH2 or -CH2SO2CH2CH2-.

[0039] A preferred embodiment of the present invention is a compound of formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein R 1 is selected from hydrogen atom, hydroxyl, alkyl, heterocyclic, cycloalkyl or heteroaryl, wherein the alkyl, heterocyclic, cycloalkyl or heteroaryl is optionally further substituted by one or more substituents selected from hydroxyl or alkyl.

[0040] A preferred embodiment of the present invention is a compound of formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein for

[0041] A preferred embodiment of the present invention is a compound of formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein L2 is selected from in Indicates that the group and the general formula (I) The attachment site; Indicates that the group and the general formula (I) The attachment site;

[0042] R 3 A hydrogen atom.

[0043] A preferred embodiment of the present invention is a compound of formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein R 4Select from -OR b 、-NHR b , 3-6 membered cycloalkyl or 3-6 membered heterocyclic group; wherein the cycloalkyl or heterocyclic group is optionally further substituted by one or more substituents selected from halogen, alkyl, and hydroxy;

[0044] R b It is selected from alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocyclic group, wherein the alkyl, cycloalkyl or heterocyclic group is optionally further substituted by one or more halogens.

[0045] A preferred embodiment of the present invention is a compound of formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, wherein R 4 is halogen, methyl, difluoromethyl, trifluoromethyl, methoxy, trifluoroethoxy,

[0046] In a preferred embodiment of the present invention, the compound described by the general formula is selected from:

[0047] or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof.

[0048] Note: If there is a discrepancy between a drawn structure and the name given for that structure, the drawn structure will be given greater weight.

[0049] Furthermore, the present invention provides a pharmaceutical composition comprising an effective dose of a compound of formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier, excipient or a combination thereof.

[0050] The present invention provides a use of a compound of general formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, or a pharmaceutical composition thereof in the preparation of a KIF18A inhibitor.

[0051] The present invention also provides a use of a compound of formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, or a pharmaceutical composition thereof, in the preparation of a medicament for treating a disease mediated by KIF18A, wherein the disease mediated by KIF18A is preferably cancer; wherein the disease mediated by KIF18A is selected from hepatocellular carcinoma, glioblastoma, colon cancer, breast cancer, lung cancer, bile duct cancer, pancreatic cancer, prostate cancer, bladder cancer, head cancer, neck cancer, cervical cancer, ovarian cancer, synovial sarcoma, rhabdomyosarcoma, colorectal cancer and lung adenocarcinoma.

[0052] The present invention further provides use of a compound of general formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, or a pharmaceutical composition thereof in the preparation of a drug for treating cancer.

[0053] The present invention provides a use of a compound of general formula (I) or (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts, or a pharmaceutical composition thereof, in the preparation of a medicament for treating hepatocellular carcinoma, glioblastoma, colon cancer, breast cancer, lung cancer, bile duct cancer, pancreatic cancer, prostate cancer, bladder cancer, head cancer, neck cancer, cervical cancer, ovarian cancer, synovial sarcoma, rhabdomyosarcoma, colorectal cancer and lung adenocarcinoma.

[0054] Detailed Description of the Invention

[0055] Unless otherwise stated, some of the terms used in the specification and claims of the present invention are defined as follows:

[0056] "Alkyl" when used as a group or a part of a group refers to a group comprising C1-C 20 A straight chain or branched aliphatic hydrocarbon group. Preferably C1-C 10 Alkyl, more preferably C1-C6 alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl can be substituted or unsubstituted.

[0057] “C α-β"Alkylene" refers to an aliphatic hydrocarbon group containing a minimum of α and a maximum of β carbon atoms in a branched or linear relationship, having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms, wherein α and β represent integers, the designation of C0 alkylene represents a direct bond, and C 1-6 Examples of alkylene include, but are not limited to, methylene, 1,1-ethylene, 1,2-ethylene, 1,1-propylene, 1,2-propylene, 1,3-propylene, 1,4-butylene, and the like. α-β The alkylene group may be substituted or unsubstituted.

[0058] "Cycloalkyl" refers to a non-aromatic cyclic alkyl group in which one or more of the ring atoms is a carbon atom, including monocyclic, polycyclic, fused, bridged and spirocyclic rings, preferably a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic or tricyclic ring, containing zero, one or more double bonds within the ring, but no ring has a completely conjugated π electron aromatic system. Examples of "cycloalkyl" include, but are not limited to, cyclopropyl, cyclopentyl, cyclobutyl, The cycloalkyl group may be substituted or unsubstituted.

[0059] "Spiroalkyl" refers to a polycyclic group with 5 to 18 members, two or more cyclic structures, and one carbon atom (called spiro atom) shared between the monocyclic rings, containing 0, 1 or more double bonds in the ring, but no ring has a completely conjugated π electron aromatic system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. According to the number of spiro atoms shared between the rings, the spiroalkyl group is divided into single spiro, double spiro or multiple spiroalkyl groups, preferably single spiro and double spiroalkyl groups, preferably 4 / 5 members, 4 / 6 members, 5 / 5 members or 5 / 6 members. Non-limiting examples of "spiroalkyl" include, but are not limited to, spiro[4.5]decyl, spiro[4.4]nonyl, spiro[3.5]nonyl, spiro[2.4]heptyl.

[0060] "Fused cycloalkyl" refers to a 5- to 18-membered, all-carbon polycyclic group containing two or more cyclic structures sharing a pair of carbon atoms. One or more rings may contain 0, 1, or more double bonds, but no ring has a completely conjugated π electron aromatic system. It is preferably 6- to 12-membered, and more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic, or polycyclic fused cycloalkyl groups, preferably bicyclic or tricyclic, and more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl groups. Non-limiting examples of "fused cycloalkyl" include, but are not limited to, bicyclo[3.1.0]hexyl, bicyclo[3.2.0]hept-1-enyl, bicyclo[3.2.0]heptyl, decahydronaphthyl, or tetradecahydrophenanthrenyl.

[0061] "Bridged cycloalkyl" refers to an aromatic system with 5 to 18 members, containing two or more cyclic structures that share two non-directly connected carbon atoms. One or more rings may contain zero, one, or more double bonds, but none of the rings have completely conjugated π electrons. Preferably, it is 6 to 12 members, more preferably 7 to 10 members. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, bridged cycloalkyl groups can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cycloalkyl" include, but are not limited to: (1s,4s)-bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, (1s,5s)-bicyclo[3.3.1]nonyl, bicyclo[2.2.2]octyl, and (1r,5r)-bicyclo[3.3.2]decyl.

[0062] "Heterocyclyl," "heterocycloalkyl," "heterocycle," or "heterocyclic" are used interchangeably herein to refer to a non-aromatic heterocyclic group in which one or more of the ring atoms is selected from nitrogen, oxygen, or S(O) t (wherein t is selected from 0,1 or 2) heteroatoms, including monocycle, polycycle, condensed ring, bridged ring and spirocycle.Preferably there are 5 to 7 yuan of monocycles or 7 to 10 yuan of bicyclic or tricyclic rings, which can include 1,2 or 3 atoms selected from nitrogen, oxygen and / or sulphur.The example of " heterocyclic radical " includes but is not limited to morpholinyl, oxetanyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazine-2-one, 8-oxa-3-aza-bicyclo [3.2.1] octyl, piperazinyl, hexahydropyrimidine.

[0063] The heterocyclic group may be substituted or unsubstituted.

[0064] "Spiro heterocyclyl" refers to a polycyclic group with 5 to 18 members, two or more ring structures, and one atom shared between the rings, containing one or more double bonds in the ring, but no ring has a completely conjugated π electron aromatic system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) t (wherein t is selected from 0, 1 or 2) heteroatoms, and the remaining ring atoms are carbon. Preferably it is 6 to 14 members, more preferably 7 to 10 members. According to the number of shared spiro atoms between rings, spiroalkyl is divided into monospiro heterocyclic group, bispiro heterocyclic group or polyspiro heterocyclic group, preferably monospiro heterocyclic group and bispiro heterocyclic group. More preferably, it is 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan monospiro heterocyclic group. Non-limiting examples of "spiro heterocyclic group" include but are not limited to: 1,7-dioxaspiro[4.5]decyl, 2-oxa-7-azaspiro[4.4]nonyl, 7-oxaspiro[3.5]nonyl, 5-oxaspiro[2.4]heptyl,

[0065] "Fused heterocyclic group" refers to an all-carbon polycyclic group containing two or more ring structures sharing a pair of atoms, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π-electron aromatic system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) t (wherein t is selected from 0, 1 or 2) heteroatom, and the remaining ring atoms are carbon. Preferably it is 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of "fused heterocyclic groups" include, but are not limited to: octahydropyrrolo[3,4-c]pyrrolyl, octahydro-1H-isoindolyl, 3-azabicyclo[3.1.0]hexyl, octahydrobenzo[b][1,4]dioxin.

[0066] "Bridged heterocyclic group" refers to a 5- to 14-membered, 5- to 18-membered polycyclic group containing two or more ring structures that share two atoms that are not directly connected to each other, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π-electron aromatic system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) t (wherein t is selected from 0, 1 or 2) heteroatoms, and the remaining ring atoms are carbon. Preferably 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged heterocyclic groups" include but are not limited to: 2-azabicyclo [2.2.1] heptyl, 2-azabicyclo [2.2.2] octyl, 2-azabicyclo [3.3.2] decyl.

[0067] "Aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be joined together in a fused manner. The term "aryl" includes monocyclic or bicyclic aromatic groups, such as phenyl, naphthyl, tetrahydronaphthyl aromatic groups. Preferably, aryl is C6-C 10 The aryl group is more preferably phenyl and naphthyl, and most preferably naphthyl. The aryl group may be substituted or unsubstituted.

[0068] "Heteroaryl" refers to an aromatic 5- to 6-membered monocyclic or 8- to 10-membered bicyclic ring which may contain 1 to 4 atoms selected from nitrogen, oxygen and / or sulfur. Examples of "heteroaryl" include, but are not limited to, furanyl, pyridinyl, 2-oxo-1,2-dihydropyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, benzodioxolyl, benzothienyl, benzimidazolyl, indolyl, isoindolyl, 1,3-dioxo-isoindolyl, quinolinyl, indazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, isothiazolyl, 1H-1,2,4-triazolyl, 4H-1,2,4-triazolyl, pyridinyl, pyridinyl- 2(1H)-onyl, pyrimidinyl, pyrazin-2(1H)-onyl, pyrimidin-4(3H)-onyl, pyrimidin-2(1H)-onyl, pyridazin-3(2H)-onyl, 1H-indolyl, 1H-benzo[d]imidazolyl, 1H-pyrrolo[2,3-c]pyridinyl, 3H-imidazo[4,5-c]pyridinyl, isoquinolinyl, quinazolinyl, 2H-isoindolyl, furo[3,2-b]pyridinyl, furo[2,3-c]pyridinyl, thieno[2,3-c]pyridinyl, benzofuranyl, benzo[b]thienyl, 1H-pyrrolo[3,2-b]pyridinyl, 2H-pyrrolo[3,4-c]pyridinyl.

[0069] Heteroaryl groups can be substituted or unsubstituted.

[0070] "Fused ring" refers to a polycyclic group in which two or more cyclic structures share a pair of atoms, wherein at least one ring has a completely conjugated π-electron aromatic system, and at least one ring may contain one or more double bonds, but at least one ring does not have a completely conjugated π-electron aromatic system, wherein the ring atoms are selected from 0, one or more selected from nitrogen, oxygen or S(O) r (wherein r is selected from 0, 1 or 2) heteroatoms, and the remaining ring atoms are carbon. The fused ring preferably includes a bicyclic or tricyclic fused ring, wherein the bicyclic fused ring is preferably a fused ring of an aryl or heteroaryl group and a monocyclic heterocyclyl or monocyclic cycloalkyl group. Preferably, it includes 6 to 14 members, more preferably 8 to 10 members. Examples of "fused rings" include but are not limited to:

[0071] "Alkoxy" refers to a group (alkyl-O-). Alkyl is defined herein. C1-C6 alkoxy groups are preferred. Examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, and tert-butoxy.

[0072] "Nitro" refers to a -NO2 group.

[0073] "Hydroxy" refers to an -OH group.

[0074] "Halogen" refers to fluorine, chlorine, bromine and iodine.

[0075] "Amino" refers to -NH2.

[0076] "Cyano" refers to -CN.

[0077] "Benzyl" refers to -CH2-phenyl.

[0078] "Carboxyl" refers to -C(=O)OH.

[0079] "Carboxylate" refers to a -C(=O)O-alkyl group or a -C(=O)O-cycloalkyl group, wherein alkyl and cycloalkyl are as defined above.

[0080] "Hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

[0081] "Aminoalkyl" refers to an alkyl group substituted with an amino group, wherein alkyl is as defined above.

[0082] "Haloalkyl" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

[0083] "Haloalkoxy" refers to an alkoxy group substituted with a halogen group, wherein alkoxy is as defined above.

[0084] "DMSO" refers to dimethyl sulfoxide.

[0085] "BOC" refers to tert-butoxycarbonyl.

[0086] "Bn" refers to benzyl.

[0087] "THP" refers to 2-tetrahydropyranyl.

[0088] "TFA" refers to trifluoroacetic acid.

[0089] "Ts" refers to p-toluenesulfonyl.

[0090] "Leaving group", or leaving group, is an atom or functional group that breaks away from a larger molecule in a chemical reaction. It is a term used in nucleophilic substitution reactions and elimination reactions. In a nucleophilic substitution reaction, the reactant attacked by the nucleophile is called the substrate, and the atom or group of atoms that breaks away from the substrate molecule with a pair of electrons is called the leaving group. Groups that easily accept electrons and have a strong ability to withstand negative charges are good leaving groups. The smaller the pKa of the conjugate acid of the leaving group, the easier it is for the leaving group to break away from other molecules. The reason is that when the pKa of its conjugate acid is smaller, the corresponding leaving group does not need to bind to other atoms, and the tendency to exist as an anion (or an electrically neutral leaving group) is enhanced. Common leaving groups include but are not limited to halogens, methylsulfonyl, -OTs or -OH.

[0091] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0092] As used herein, "substituted" or "substituted", unless otherwise specified, means that a group may be substituted by one or more groups selected from the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, =O, OR 5 、-C(=O)R 5 、-C(=O)OR 5 、-NHC(=O)R 5 、-NHC(=O)OR 5 、-NR 6 R 7 、-C(=O)NR 6 R 7 、-CH2NHC(=O)OR 5 、-CH2NR 6 R 7 or -S(O) r R 5 substituted by a substituent;

[0093] R 5Each is independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the heterocyclic group, the aryl group or the heteroaryl group is optionally further substituted by one or more hydroxyl groups, halogen groups, nitro groups, cyano groups, alkyl groups, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyl groups, heterocyclic groups, aryl groups, heteroaryl groups, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent;

[0094] R 6 and R 7 are each independently selected from hydrogen, hydroxy, halogen, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent;

[0095] Or, R 6 and R 7 Together with the atoms to which they are attached, they form a 4- to 8-membered heterocyclic group, wherein the 4- to 8-membered heterocyclic group contains one or more N, O, or S(O)r, and the 4- to 8-membered heterocyclic group is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclic group, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent;

[0096] R 8 、R 9 and R 10 Each is independently selected from a hydrogen atom, an alkyl group, an amino group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the heterocyclic group, the aryl group or the heteroaryl group is optionally further substituted with one or more substituents selected from a hydroxyl group, a halogen group, a nitro group, an amino group, a cyano group, an alkyl group, an alkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, a carboxyl group or a carboxylate group;

[0097] r is independently 0, 1 or 2.

[0098] The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers and geometric (conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention.

[0099] Unless otherwise indicated, structures depicted herein also encompass all isomers (e.g., diastereoisomers, enantiomers, and atropisomers, and geometric (conformational) isomeric forms of such structures; for example, R and S configurations at various asymmetric centers, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, individual stereoisomers as well as enantiomeric mixtures, diastereomeric mixtures, and geometric (conformational) isomeric mixtures of the present compounds are within the scope of the invention.

[0100] "Pharmaceutically acceptable salts" refer to salts of the above compounds that retain their original biological activity and are suitable for pharmaceutical use. Pharmaceutically acceptable salts of the compounds represented by general formula (I) may be metal salts or amine salts formed with suitable acids.

[0101] A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredients and thereby exerting their biological activity.

[0102] Synthesis method of the compound of the present invention

[0103] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0104] The present invention provides a method for preparing a compound of general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts, the method comprising:

[0105] The compound of formula (IA) undergoes a condensation reaction with the compound (IB), and optionally further undergoes a substitution reaction to obtain the compound of formula (I)

[0106] in:

[0107] L2 is selected from

[0108] R 3 selected from hydrogen atoms;

[0109] Y is selected from hydroxy or chlorine;

[0110] L1, R 1 、R 2 、R 4 、R A and m are as defined in the general formula (I). DETAILED DESCRIPTION

[0111] The present invention is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present invention.

[0112] Example

[0113] The examples provide the preparation of representative compounds represented by formula (I) and related structural identification data. It must be noted that the following examples are used to illustrate the present invention rather than to limit the present invention. 1 H NMR spectra were obtained using a Bruker instrument (400 MHz), and chemical shifts are expressed in ppm using tetramethylsilane as an internal standard (0.00 ppm). 1 H NMR notation: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of a doublet, dt = doublet of a triplet. Coupling constants, when given, are given in Hz.

[0114] Mass spectra were obtained using LC / MS, and the ionization method could be ESI or APCI.

[0115] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specification of the silica gel plate used in thin layer chromatography (TLC) is 0.15mm~0.2mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm~0.5mm.

[0116] Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0117] In the following examples, unless otherwise indicated, all temperatures are in degrees Celsius. Unless otherwise indicated, various starting materials and reagents are commercially available or synthesized according to known methods. Commercially available raw materials and reagents are used directly without further purification, unless otherwise indicated. Commercial manufacturers include but are not limited to Aldrich Chemical Company, ABCR GmbH & Co.KG, Acros Organics, Guangzan Chemical Technology Co., Ltd. and Jingyan Chemical Technology Co., Ltd.

[0118] CD3OD: deuterated methanol.

[0119] CDCl3: deuterated chloroform.

[0120] DMSO-d6: deuterated dimethyl sulfoxide.

[0121] Argon atmosphere means that the reaction bottle is connected to an argon balloon with a capacity of about 1 L.

[0122] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0123] The compound was purified using silica gel column chromatography and reverse phase column chromatography, with the eluent system selected from: A: petroleum ether and ethyl acetate; B: dichloromethane and methanol; C: dichloromethane: ethyl acetate; and D: aqueous trifluoroacetic acid and acetonitrile. The volume ratio of the solvents varied depending on the polarity of the compound and could be adjusted by adding a small amount of an acidic or alkaline reagent, such as acetic acid or triethylamine.

[0124] Example 1

[0125] N-(4-carbamoyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0126] N-(4-Carbamoyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0127] first step

[0128] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzonitrile

[0129] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzonitrile

[0130] 2-Fluoro-4-nitrobenzonitrile 1a (1 g, 6.02 mmol, commercially available), 4,4-difluoropiperidine 1b (802.13 mg, 6.62 mmol, commercially available), and potassium carbonate (2.50 g, 18.06 mmol) were dissolved in N,N-dimethylformamide (5 mL), heated to 80°C, and stirred for 4 hours. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (30 mL x 2). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was dissolved in ethyl acetate, and petroleum ether was added to precipitate a solid, which was filtered and dried to afford 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzonitrile 1c (1.0 g) in a 62.16% yield.

[0131] MS m / z(ESI):268.1[M+1]

[0132] Step 2

[0133] 4-amino-2-(4,4-difluoropiperidin-1-yl)benzonitrile

[0134] 4-Amino-2-(4,4-difluoropiperidin-1-yl)benzonitrile

[0135] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzonitrile 1c (1.0 g, 3.74 mmol) was dissolved in methanol (10 mL), and 10% palladium on carbon (119.47 mg, 1.12 mmol) was added. The atmosphere was replaced with hydrogen three times, and the mixture was stirred at room temperature for 6 hours. The mixture was filtered and concentrated under reduced pressure. The residue was isolated and purified by silica gel column chromatography (eluent: System A) to obtain 4-amino-2-(4,4-difluoropiperidin-1-yl)benzonitrile 1d (850 mg) in a 95.74% yield.

[0136] MS m / z(ESI):238.1[M+1]

[0137] Step 3

[0138] N-(4-cyano-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0139] N-(4-cyano-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0140] 2-(6-Azaspiro[2.5]octan-6-yl)-4-iodo-benzoic acid 1e (662.44 mg, 1.85 mmol, prepared by the method disclosed in WO2020132648) and 2-amino-6-(4,4-difluoropiperidin-1-yl)benzonitrile 1d (400 mg, 1.69 mmol) were dissolved in 1,4-dioxane (1 mL), and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (1.42 g, 5.06 mmol) and N-methylimidazole (415.2 The reaction mixture was stirred for 3 hours at 100 ° C., and water (20 mL) was added. The mixture was extracted with ethyl acetate (30 mL × 2). The combined organic phase was washed with saturated sodium chloride (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column chromatography (eluent: System A) to give N-(4-cyano-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamide 1f (700 mg) in a yield of 72.03%.

[0141] MS m / z(ESI):576.2[M+1]

[0142] Step 4

[0143] N-(4-cyano-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0144] N-(4-Cyano-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0145] To a solution of N-(4-cyano-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamide 1f (500 mg, 867.43 μmol) and ethanesulfonamide 1g (94.68 mg, 867.43 μmol, commercially available) in N,N-dimethylformamide (5 mL) were added sarcosine (77.28 mg, 867.43 μmol), cuprous iodide (82.60 mg, 433.71 μmol) and potassium phosphate (920.64 mg, 4.34 mmol). The mixture was stirred at 100 ° C under N2 atmosphere for 16 hours. Mass spectrometry showed that the reaction was complete. The reaction solution was filtered and concentrated, and the residue was separated by preparative liquid phase (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(4-cyano-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 1h (235 mg) with a yield of 47.35%.

[0146] MS m / z(ESI):558.6[M+1]

[0147] 1H NMR(400MHz,)δ11.83(s,1H),10.19(s,1H),7.82–7.68(m,3H),7.50–7.42(m,1H),7.16(d,J=2.0Hz,1H),7.03(dd,J=8.5,2.1Hz,1H),3 .28(d,J=6.6Hz,4H),3.20(q,J=7.3Hz,2H),2.96(t,J=5.2Hz,4H),2.22–2.12(m,4H),1.51(s,4H),1.21(t,J=7.3Hz,3H),0.34(s,4H).

[0148] Step 5

[0149] N-(4-carbamoyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0150] N-(4-Carbamoyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0151] N-(4-cyano-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 1h (200 mg, 358.65 μmol) was dissolved in dimethyl sulfoxide (1 mL). An aqueous solution of ethanol (1 mL) and sodium hydroxide (28.69 mg, 717.29 μmol) was added. Hydrogen peroxide (0.5 mL, 25%) was added dropwise and the mixture was reacted at room temperature for 2 hours. Mass spectrometry showed that the reaction was complete. Water (20 mL) was added and extracted with ethyl acetate (30 mL × 2). The organic phases were combined, washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column chromatography (eluent: System A) to give N-(4-carbamoyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 1 (143.1 mg) in a yield of 67.65%.

[0152] MS m / z(ESI):576.3[M+1]

[0153] 1H NMR(400MHz)δ11.81(s,1H),10.17(s,1H),8.18(s,1H),7.82(d,J=8.4Hz,1H),7.71(d, J=8.4Hz,1H),7.62(d,J=2.0Hz,1H),7.52(dd,J=8.4,1.9Hz,1H),7.41(s,1H),7.17(d, J=2.1Hz,1H),7.04(dd,J=8.5,2.1Hz,1H),3.20(q,J=7.3Hz,2H),3.05(t,J=5.5Hz,4H),2.9 8(d,J=10.7Hz,4H),2.19(d,J=14.1Hz,4H),1.54(s,4H),1.21(t,J=7.3Hz,3H),0.35(s,4H).

[0154] Example 2

[0155] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoic acid

[0156] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoic acid

[0157] first step

[0158] methyl 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzoate

[0159] Methyl 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzoate

[0160] 4,4-Difluoropiperidine 1b (500 mg, 4.13 mmol) and methyl 2-fluoro-4-nitrobenzoate 2a (822.00 mg, 4.13 mmol) were dissolved in dimethyl sulfoxide (6 mL). Triethylamine (417.70 mg, 4.13 mmol) was added and stirred at 120°C for 8 hours. Mass spectrometry indicated the reaction was complete. Water (10 mL) was added and the mixture was extracted with ethyl acetate (30 mL x 2). The combined organic phases were washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to give methyl 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzoate 2b (872 mg) in a yield of 70.36%.

[0161] MS m / z(ESI):301.1[M+1]

[0162] Step 2

[0163] methyl 4-amino-2-(4,4-difluoropiperidin-1-yl)benzoate

[0164] Methyl 4-amino-2-(4,4-difluoropiperidin-1-yl)benzoate

[0165] Methyl 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzoate 2b (500 mg, 1.67 mmol) was dissolved in methanol (10 mL) at room temperature. Palladium on carbon (606.73 mg, 5.00 mmol, 10%) was added and stirred under a hydrogen atmosphere for 16 hours. Mass spectrometry indicated the reaction was complete. The mixture was filtered, the filtrate was dried, and directly used in the next step to afford methyl 4-amino-2-(4,4-difluoropiperidin-1-yl)benzoate 2c (400 mg) in an 88.87% yield.

[0166] MS m / z(ESI):271.2[M+1]

[0167] Step 3

[0168] methyl

[0169] 2-(4,4-difluoropiperidin-1-yl)-4-(4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoate

[0170] Methyl 2-(4,4-difluoropiperidin-1-yl)-4-(4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoate

[0171] 2-(6-Azaspiro[2.5]octan-6-yl)-4-iodo-benzoic acid 1e (500 mg, 1.35 mmol) was dissolved in dichloromethane (1 mL), and oxalyl chloride (170.97 mg, 1.35 mmol) was added dropwise. The mixture was stirred at room temperature for 15 minutes. The reaction system was concentrated under reduced pressure. A solution of methyl 4-amino-2-(4,4-difluoropiperidin-1-yl)benzoate 2c (364.04 mg, 1.35 mmol) in dichloromethane (1 mL) was added dropwise to the reaction flask. The mixture was stirred at room temperature for 15 minutes. The reaction mixture was concentrated under reduced pressure. After stirring for 30 minutes, mass spectrometry showed that the reaction was complete. Water (10 mL) was added, and the mixture was extracted with ethyl acetate (30 mL × 2). The combined organic phases were washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated and purified by silica gel column chromatography (eluent: System A) to obtain methyl 2-(4,4-difluoropiperidin-1-yl)-4-(4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoate 2d (800 mg) in a yield of 97.46%.

[0172] MS m / z(ESI):610.1[M+1]

[0173] Step 4

[0174] methyl

[0175] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)

[0176] benzoate

[0177] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoic acid

[0178] Methyl ester

[0179] Methyl 2-(4,4-difluoropiperidin-1-yl)-4-(4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoate 2d (800 mg, 1.31 mmol) and ethanesulfonamide 1 g (214.91 mg, 1.97 mmol) were dissolved in N,N-dimethylformamide (10 mL), and potassium phosphate (557.28 mg, 2.63 mmol), cuprous iodide (145.75 mg, 656.33 μmol), and sarcosine (116.95 mg, 1.31 mmol) were added. The mixture was heated to 110 ° C under argon protection for three hours. Water (10 mL) was added, extracted with ethyl acetate (30 mL×2), the combined organic phases were washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was separated by preparative liquid phase separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give the product methyl 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonyl)-2-(6-azaspiro[2.5]octane-6-yl)benzamido)benzoate 2e (160 mg) with a yield of 20.64%.

[0180] MS m / z(ESI):591.1[M+1]

[0181] Step 5

[0182] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)

[0183] benzoic acid

[0184] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoic acid

[0185] Methyl 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoate 2e (100 mg, 169.30 μmol) was dissolved in methanol (1 mL) and water (1 mL). Sodium hydroxide (20.32 mg, 507.89 μmol) was added and the mixture was stirred at room temperature for 16 hours. Mass spectrometry showed that the reaction was complete. The mixture was extracted with ethyl acetate (30 mL × 2), and the organic phases were combined, washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated by preparative liquid phase separation (separation column AKZONOBEL Kromasil; 250 × 21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA + H2O, mobile phase B: CH3CN) to give 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonyl)-2-(6-azaspiro[2.5]octane-6-yl)benzamido)benzoic acid 2 (80 mg) with a yield of 74.73%.

[0186] MS m / z(ESI):576.9[M+1]

[0187] Example 3

[0188] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)-N-methylbenzamide

[0189] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)-N-methylbenzamide

[0190] To a mixture of 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoic acid 2 (15 mg, 26.01 μmol) and methylamine (807.86 μg, 26.01 μmol) in 1,4-dioxane (1 mL) were added 1-hydroxybenzotriazole (3.51 mg, 26.01 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.99 mg, 26.01 μmol). The mixture was stirred at room temperature for 1 hour. Mass spectrometry showed that the reaction was complete. The reaction mixture was concentrated, and the residue was separated by preparative liquid chromatography (AKZONOBEL Kromasil column; 250 × 21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA + H₂O, mobile phase B: CH₃CN) to afford 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)-N-methylbenzamide 3 (10 mg) in a 64.93% yield. MS m / z (ESI): 590.0 [M+1].

[0191] Example 4

[0192] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)-N,N-dimethylbenzamide

[0193] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)-N,N-dimethylbenzamide

[0194] To a mixture of 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoic acid 2 (15 mg, 26.01 μmol) and dimethylamine (781.81 μg, 17.34 μmol) in 1,4-dioxane (1 mL) were added tetramethyl chlorouronium hexafluorophosphate (4.87 mg, 17.34 μmol) and N-methylimidazole (1.42 mg, 17.34 μmol). The mixture was stirred at room temperature for 1 hour. Mass spectrometry showed that the reaction was complete. The reaction solution was concentrated, and the residue was separated by preparative liquid phase (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)-N,N-dimethylbenzamide 4 (8 mg) with a yield of 76.18%.

[0195] MS m / z(ESI):604.2[M+1]

[0196] 1H NMR(400MHz)δ11.82(s,1H),7.83(d,J=8.5Hz,1H),7.58(d,J=1.9Hz,1H),7.46–7.35(m,1H),7.24–7.11(m,2H),7.03(dd,J=8.5,2.1Hz,1H ),3.18(d,J=7.3Hz,2H),2.98(d,J=11.0Hz,8H),2.87(s,3H),2.79(s,3H),2.12–1.97(m,4H),1.55(s,3H),1.25–1.17(m,4H),0.35(s,4H).

[0197] Example 5

[0198] N-(4-acetyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0199] N-(4-Acetyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0200] first step

[0201] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)-N-methoxy-N-methylbenzamide

[0202] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)-N-methoxy-N-methylbenzamide

[0203] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoic acid 2 (100 mg, 173.41 μmol) was dissolved in N,N-dimethylformamide (0.5 mL), and tetramethylchlorouronium hexafluorophosphate (97.31 mg, 346.83 μmol) and N-methylimidazole (28.47 mg, 346.83 μmol) were added. After stirring at room temperature for 1 hour, free N-methoxymethylamine (16.92 mg, 173.41 μmol) was added dropwise, and stirred at room temperature for 1 hour. Mass spectrometry showed that the reaction was complete. Water (10 mL) was added, and the mixture was extracted with ethyl acetate (30 mL × 2). The combined organic phases were washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated and purified by silica gel column chromatography (eluent: System A) to give 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamido)-N-methoxy-N-methylbenzamide 5a (100 mg) in a yield of 93.05%.

[0204] MS m / z(ESI):620.1[M+1]

[0205] Step 2

[0206] N-(4-acetyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0207] N-(4-Acetyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0208] 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)-N-methoxy-N-methylbenzamide 5a (100 mg, 161.36 μmol) was dissolved in tetrahydrofuran (2 mL). Methyllithium (1.6 M in diethyl ether) (3.55 mg, 161.36 μmol) was added at 0°C and stirred at room temperature for 1 hour. Mass spectrometry indicated the reaction was complete. The reaction was quenched by adding water, and the product was separated by preparative liquid phase (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(4-acetyl-3-(4,4-difluoropiperidin-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 5 (8 mg) with a yield of 8.63%.

[0209] MS m / z(ESI):574.9[M+1]

[0210] 1H NMR (400MHz, DMSO-d6) δ13.05(s,1H),7.70(d,J=8.7Hz,1H),7.57(d,J=8.1 Hz,2H),7.49(d,J=8.5Hz,1H),6.83(d,J=2.2Hz,1H),6.73–6.62(m,1H),3. 07(d,J=5.8Hz,4H),2.92(d,J=5.5Hz,4H),2.72(q,J=7.4Hz,2H),2.57(s,3 H), 2.18 (d, J = 16.3Hz, 4H), 1.23 (s, 4H), 1.12 (t, J = 7.3Hz, 3H), 0.39 (s, 4H).

[0211] Example 6

[0212] N-(3-(4,4-difluoropiperidin-1-yl)-4-(hydroxymethyl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0213] N-(3-(4,4-difluoropiperidin-1-yl)-4-(hydroxymethyl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0214] Methyl 2-(4,4-difluoropiperidin-1-yl)-4-(4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamido)benzoate 2e (50 mg, 84.65 μmol) was dissolved in tetrahydrofuran (1 mL), and lithium borohydride (1.84 mg, 84.65 μmol) was added dropwise. The mixture was heated to 75°C and stirred for 6 hours. After quenching with water, the mixture was separated by preparative liquid phase separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5μm, 20mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to obtain N-(3-(4,4-difluoropiperidin-1-yl)-4-(hydroxymethyl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 6 (5 mg) with a yield of 10.50%.

[0215] MS m / z(ESI):563.2[M+1]

[0216] Example 7

[0217] N-(3-(4,4-difluoropiperidin-1-yl)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0218] N-(3-(4,4-difluoropiperidin-1-yl)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0219] first step

[0220] 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid

[0221] 4-(Ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid

[0222] 2-(6-Azaspiro[2.5]octan-6-yl)-4-iodobenzoic acid 1e (1.0 g, 2.80 mmol) and 1 g of ethanesulfonamide (458.36 mg, 4.20 mmol) were dissolved in N,N-dimethylformamide (10 mL). Cuprous iodide (266.60 mg, 1.40 mmol), sarcosine (249.43 mg, 2.80 mmol), and potassium phosphate (3.73 g, 14.00 mmol) were added. Argon was bubbled through the mixture for 5 minutes, and the temperature was raised to 110°C, followed by stirring for 6 hours. The temperature was cooled to room temperature, and the reaction mixture was poured into 200 mL of ice water. The pH was adjusted to 6 with dilute hydrochloric acid (2 M). Dichloromethane was added for extraction (200 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated and purified by silica gel column chromatography (eluent: System A) to give 4-(ethylsulfonyl)-2-(6-azaspiro[2.5]octane-6-yl)benzoic acid 7a (0.75 g) in a yield of 79.16%.

[0223] MS m / z(ESI):339.4[M+1]

[0224] Step 2

[0225] 2-fluoro-4-nitrobenzoyl chloride

[0226] 2-Fluoro-4-nitrobenzoyl chloride

[0227] 2-Fluoro-4-nitrobenzoic acid 7b (5.00 g, 27.01 mmol, commercially available) was dissolved in dichloromethane (60 mL). One drop of N,N-dimethylformamide was added. Oxalyl chloride (3.43 g, 27.01 mmol) was added portionwise under ice-cooling. The atmosphere was replaced with nitrogen three times. The reaction was allowed to proceed at 25°C for 1 hour to yield a yellow suspension. Mass spectrometry confirmed the complete reaction. The reaction solution was concentrated to dryness under reduced pressure to yield 2-fluoro-4-nitrobenzoyl chloride 7c (5.60 g), which was used directly in the next reaction.

[0228] MS m / z(ESI):200.0[M+1]

[0229] Step 3

[0230] N-(2,2-dimethoxyethyl)-2-fluoro-4-nitrobenzamide

[0231] N-(2,2-Dimethoxyethyl)-2-fluoro-4-nitrobenzamide

[0232] 2-Fluoro-4-nitrobenzoyl chloride 7c (5.60 g, 27.51 mmol, crude) was dissolved in tetrahydrofuran (60 mL). Sodium bicarbonate (6.93 g, 82.53 mmol) and 2,2-dimethoxyethane-1-amine 7d (4.34 g, 41.27 mmol) were added, and the atmosphere was purged with nitrogen three times. The reaction was incubated at 25°C for 18 hours to yield a yellow suspension. The reaction was complete. The reaction mixture was poured into 200 mL of water and extracted with ethyl acetate (60 mL x 2). The combined organic phases were washed with saturated sodium chloride solution (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford the crude product, which was isolated and purified by silica gel column chromatography (eluent: System A) to afford N-(2,2-dimethoxyethyl)-2-fluoro-4-nitrobenzamide 7e (6.3 g) in an 84.12% yield.

[0233] 1H NMR (400MHz, DMSO) δ8.73(s,1H),8.20(dd,J=9.8,2.1Hz,1H),8.14(dd,J=8.4,2.1Hz,1H ),7.81(dd,J=8.4,7.1Hz,1H),4.51(t,J=5.8Hz,1H),3.38(t,J=5.8Hz,2H),3.31(s,6H).

[0234] Step 4

[0235] 2-(2-fluoro-4-nitrophenyl)oxazole

[0236] 2-(2-Fluoro-4-nitrophenyl)oxazole

[0237] N-(2,2-Dimethoxyethyl)-2-fluoro-4-nitrobenzamide 7e (200.00 mg, 0.73 mmol) was added to a solution of Eaton's reagent (1.75 g, 7.35 mmol) at 0°C. The atmosphere was purged with nitrogen three times, and the reaction was quickly transferred to 145°C for 2 hours to yield a black suspension. Mass spectrometry confirmed the complete reaction of the starting material. The reaction solution was poured into water (100 mL) and extracted with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford the crude product, which was isolated and purified by silica gel column chromatography (eluent: System A) to afford 2-(2-fluoro-4-nitrophenyl)oxazole 7f (105.00 mg) in a 68.66% yield.

[0238] MS m / z(ESI):209.0[M+1]

[0239] Step 5

[0240] 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)oxazole

[0241] 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)oxazole

[0242] 2-(2-Fluoro-4-nitrophenyl)oxazole 7f (125.00 mg, 0.60 mmol) was dissolved in dimethyl sulfoxide (2 mL) at room temperature. Potassium carbonate (249.00 mg, 1.80 mmol) and 4,4-difluoropiperidine 1b (145.28 mg, 1.20 mmol) were added. The atmosphere was purged with nitrogen three times. The reaction was continued at 120°C for 56 hours to yield a yellow suspension. Mass spectrometry confirmed the complete reaction of the starting material. The reaction solution was poured into water (100 mL) and extracted with ethyl acetate (50 mL x 2). The combined organic phase was washed with a saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product, which was separated and purified by silica gel column chromatography (eluent: System A) to give 7 g (92.00 mg) of 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)oxazole in a yield of 49.53%.

[0243] MS m / z(ESI):310.2[M+1]

[0244] Step 6

[0245] 3-(4,4-difluoropiperidin-1-yl)-4-(oxazol-2-yl)aniline

[0246] 3-(4,4-difluoropiperidin-1-yl)-4-(oxazol-2-yl)aniline

[0247] At room temperature, 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)oxazole 7g (50.00 mg, 0.16 mmol) was dissolved in ethyl acetate (1 mL) solution, palladium carbon (1.72 mg, 0.02 mmol) was added, and hydrogen was replaced three times. The reaction was allowed to react at room temperature for 18 hours under a hydrogen atmosphere to obtain a black suspension. Mass spectrometry showed that the raw material reaction was complete and the product was generated. The reaction solution was filtered, washed with ethyl acetate, and the organic phases were combined and concentrated to dryness under reduced pressure to obtain 3-(4,4-difluoropiperidin-1-yl)-4-(oxazole-2-yl)aniline 7h (44.00 mg). The crude product was used directly in the next step.

[0248] MS m / z(ESI):280.2[M+1]

[0249] Step 7

[0250] N-(3-(4,4-difluoropiperidin-1-yl)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0251] N-(3-(4,4-difluoropiperidin-1-yl)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0252] At room temperature, 3-(4,4-difluoropiperidin-1-yl)-4-(oxazol-2-yl)aniline 7h (40.00 mg, 0.16 mmol) was dissolved in N,N-dimethylformamide (1 mL). N,N-diisopropylethylamine (81.44 mg, 0.63 mmol), 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (53.32 mg, 0.16 mmol), and (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (123.21 mg, 0.24 mmol) were added sequentially. The atmosphere was purged with nitrogen three times. The reaction was allowed to proceed at 25°C for 18 hours to yield a yellow suspension. Mass spectrometry confirmed the complete reaction of the starting material. The reaction solution was poured into water (100 mL) and extracted with ethyl acetate (40 mL x 2). The combined organic phases were washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 7 (19.95 mg) with a yield of 21.12%.

[0253] MS m / z(ESI):600.2[M+1]

[0254] 1H NMR (400MHz, DMSO) δ11.87(s,1H),10.19(s,1H),8.22(d,J=0.6Hz,1H),7.85(dd,J=17.1 ,8.4Hz,2H),7.65(d,J=1.6Hz,1H),7.58(dd,J=8.4,1.8Hz,1H),7.38(d,J=0.6Hz,1H),7 .17(d,J=1.9Hz,1H),7.05(dd,J=8.5,2.0Hz,1H),3.20(q,J=7.3Hz,2H),3.08–3.03(m,4 H), 2.98 (t, J = 5.0Hz, 4H), 2.15 (m, 4H), 1.55 (m, 4H), 1.21 (t, J = 7.2Hz, 3H), 0.35 (s, 4H).

[0255] Example 8

[0256] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0257] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0258] first step

[0259] 4-(2-bromo-4-nitrophenyl)pyridine

[0260] 4-(2-Bromo-4-nitrophenyl)pyridine

[0261] At room temperature, 2-bromo-1-iodo-4-nitrobenzene 8a (500 mg, 1.52 mmol, commercially available) was added to toluene (5 mL), ethanol (1 mL), and water (2 mL). Pyridine-4-boronic acid 8b (281 mg, 2.29 mmol, commercially available), tetrakis(triphenylphosphine)palladium (176 mg, 0.152 mmol), and sodium carbonate (323 mg, 3.05 mmol) were added to the reaction solution. The atmosphere was purged with nitrogen three times and the reaction was continued at 100°C for 18 hours. The mixture was filtered and concentrated to dryness under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to obtain 4-(2-bromo-4-nitrophenyl)pyridine 8c (200 mg) in a 47.0% yield.

[0262] MS m / z(ESI):279.0[M+1]

[0263] Step 2

[0264] 4-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine

[0265] 4-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine

[0266] At room temperature, 4-(2-bromo-4-nitrophenyl)pyridine 8c (100 mg, 0.358 mmol) and 4,4-difluoropiperidine 1b (86.8 mg, 0.717 mmol) were added to 1,4-dioxane (3 mL). Palladium acetate (8.04 mg, 0.0358 mmol), cesium carbonate (233.49 mg, 0.717 mmol), and 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (41.5 mg, 0.0717 mmol) were then added to the reaction mixture. The atmosphere was purged with nitrogen three times and the reaction was continued at 100°C for 18 hours. The mixture was concentrated to dryness under reduced pressure, and the crude product was isolated and purified by silica gel column chromatography (eluent: System A) to give 4-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine 8d (50.0 mg) in a 43.70% yield.

[0267] MS m / z(ESI):320.2[M+1]

[0268] Step 3

[0269] 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-4-yl)aniline

[0270] 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-4-yl)aniline

[0271] 4-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine 8d (50.0 mg, 0.157 mmol) was added to methanol (2 mL) at room temperature, followed by 10% palladium on carbon (16.7 mg, 0.157 mmol). The atmosphere was replaced with hydrogen three times, and the reaction was continued at 25°C for 4 hours. The mixture was concentrated to dryness under reduced pressure to provide 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-4-yl)aniline 8e (45.0 mg) in a 99.33% yield.

[0272] MS m / z(ESI):290.2[M+1]

[0273] Step 4

[0274] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0275] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0276] At room temperature, 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-4-yl)aniline 8e (45.0 mg, 0.156 mmol) and 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (63.2 mg, 0.187 mmol) were added to N,N-dimethylformamide (2 mL). (7-Azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (122 mg, 0.233 mmol) and N,N-diisopropylethylamine (40.2 mg, 0.311 mmol) were added to the reaction solution. The atmosphere was replaced with nitrogen three times and the reaction was continued at 60°C for 3 hours. The mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 8 (3.00 mg) in a yield of 4.75%.

[0277] MS m / z(ESI):610.2[M+1]

[0278] 1H NMR (400MHz, DMSO-d6) δ11.84 (s, 1H), 10.19 (s, 1H), 8.67 (d, J = 5.2Hz, 2H), 7.91 –7.80(m,3H),7.67–7.44(m,1H),7.59–7.56(m,1H),7.42–7.37(m,1H),7.18(d, J=2.0Hz,1H),7.07–7.03(m,1H),3.23–3.18(m,2H),3.00–2.96(m,4H),2.94–2. 89(m,4H),1.61–1.52(m,4H),1.24–1.22(m,4H),1.22–1.19(m,3H),0.36(s,4H).

[0279] Example 9

[0280] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0281] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0282] first step

[0283] 3-(2-bromo-4-nitrophenyl)pyridine

[0284] 3-(2-Bromo-4-nitrophenyl)pyridine

[0285] 2-Bromo-1-iodo-4-nitrobenzene 8a (500 mg, 1.52 mmol, commercially available) was added to toluene (5 mL), ethanol (1 mL), and water (2 mL) at room temperature. Pyridine-3-boronic acid 9a (469 mg, 2.29 mmol, commercially available), tetrakis(triphenylphosphine)palladium (176 mg, 0.152 mmol), and sodium carbonate (323 mg, 3.05 mmol) were then added. The atmosphere was purged with nitrogen three times and the reaction was continued at 100°C for 18 hours. The mixture was filtered and concentrated to dryness under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to afford 3-(2-bromo-4-nitrophenyl)pyridine 9b (150 mg) in a 35.25% yield.

[0286] MS m / z(ESI):279.0[M+1]

[0287] Step 2

[0288] 3-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine

[0289] 3-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine

[0290] 3-(2-Bromo-4-nitrophenyl)pyridine 9b (150 mg, 0.537 mmol) and 4,4-difluoropiperidine 1b (130.20 mg, 1.07 mmol) were added to 1,4-dioxane (3 mL) at room temperature. Palladium acetate (12.1 mg, 0.0538 mmol), cesium carbonate (350 mg, 1.07 mmol), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (62.2 mg, 0.107 mmol) were then added to the reaction mixture. The atmosphere was purged with nitrogen three times and the reaction was continued at 100°C for 18 hours. The mixture was concentrated to dryness under reduced pressure, and the crude product was isolated and purified by silica gel column chromatography (eluent: System A) to give 3-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine 9c (50.0 mg) in a 29.14% yield.

[0291] MS m / z(ESI):320.0[M+1]

[0292] Step 3

[0293] 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-3-yl)aniline

[0294] 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-3-yl)aniline

[0295] 3-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine 9c (50.0 mg, 0.157 mmol) was added to methanol (2 mL) at room temperature, followed by 10% palladium on carbon (16.7 mg, 0.157 mmol). The atmosphere was replaced with hydrogen three times and the reaction was continued at 25°C for 4 hours. The mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to afford 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-3-yl)aniline 9d (45.0 mg) in a 99.33% yield.

[0296] MS m / z(ESI):290.2[M+1]

[0297] Step 4

[0298] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0299] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0300] At room temperature, 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-3-yl)aniline 9d (45.0 mg, 0.156 mmol) and 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (63.2 mg, 0.187 mmol) were added to N,N-dimethylformamide (2 mL), followed by (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (122 mg, 0.233 mmol) and N,N-diisopropylethylamine (40.2 mg, 0.311 mmol). The atmosphere was replaced with nitrogen three times and the reaction was continued at 60°C for 3 hours. The mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 9 (5.00 mg) with a yield of 5.27%.

[0301] MS m / z(ESI):610.2[M+1]

[0302] 1H NMR (400MHz, DMSO-d6) δ11.84(s,1H),10.24(s,1H),8.82(d,J=2.0Hz,1H),8.54–8.50(m,1H),8.1 4–8.17(m,1H),7.85(d,J=8.4Hz,1H),7.64(d,J=2.0Hz,1H),7.58–7.54(m,1H),7.48–7.44(m,1H) ,7.32(d,J=8.0Hz,1H),7.18(d,J=2.0Hz,1H),7.07–7.02(m,1H),3.22–3.17(m,2H),3.02–2.96(m ,4H),2.91–2.85(m,4H),1.95–1.84(m,4H),1.63–1.51(m,4H),1.21(t,J=7.2Hz,3H),0.36(s,4H).

[0303] Example 10

[0304] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0305] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0306] first step

[0307] 2-(2-fluoro-4-nitrophenyl)pyridine

[0308] 2-(2-Fluoro-4-nitrophenyl)pyridine

[0309] At room temperature, 2-(2-fluoro-4-nitrophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 10a (800 mg, 3.00 mmol, commercially available) was added to 1,4-dioxane (10 mL) and water (1 mL). 2-bromopyridine 10b (710 mg, 4.49 mmol, commercially available), tetrakis(triphenylphosphine)palladium (346 mg, 0.300 mmol), and potassium carbonate (828 mg, 5.99 mmol) were then added. The atmosphere was purged with nitrogen three times and the reaction was continued at 100°C for 18 hours. The mixture was filtered and concentrated to dryness under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to give 2-(2-fluoro-4-nitrophenyl)pyridine 10c (500 mg) in a 76.50% yield. MS m / z (ESI): 280.0 [M+1]

[0310] Step 2

[0311] 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine

[0312] 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine

[0313] 2-(2-Fluoro-4-nitrophenyl)pyridine 10c (300 mg, 1.37 mmol) was added to dimethyl sulfoxide (3 mL) at room temperature, followed by triethylamine (278 mg, 2.75 mmol) and 4,4-difluoropiperidine 1b (666 mg, 5.50 mmol). The atmosphere was purged with nitrogen three times, and the reaction was continued at 100°C for 18 hours. The mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to obtain 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine 10d (150 mg) in a 34.17% yield.

[0314] MS m / z(ESI):320.0[M+1]

[0315] Step 3

[0316] 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yl)aniline

[0317] 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yl)aniline

[0318] 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)pyridine 10d (130 mg, 0.407 mmol) was added to anhydrous methanol (2 mL) at room temperature, followed by 10% palladium on carbon (43.3 mg, 0.407 mmol). The atmosphere was replaced with hydrogen three times and the reaction was continued at 25°C for 4 hours. The mixture was concentrated to dryness under reduced pressure to provide 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yl)aniline 10e (110 mg) in a 93.38% yield.

[0319] MS m / z(ESI):290.2[M+1]

[0320] Step 4

[0321] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0322] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0323] At room temperature, 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yl)aniline 10e (110 mg, 0.380 mmol) and 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (154 mg, 0.456 mmol) were added to N,N-dimethylformamide (2 mL), followed by (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (297 mg, 0.570 mmol) and N,N-diisopropylethylamine (98.3 mg, 0.760 mmol). The atmosphere was replaced with nitrogen three times and the reaction was continued at 60°C for 3 hours. The mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 10 (5.00 mg) with a yield of 2.16%.

[0324] MS m / z(ESI):610.4[M+1]

[0325] 1H NMR (400MHz, DMSO-d6) δ11.89 (s, 1H), 8.67–8.64 (m, 1H), 8.11 (d, J = 8.0Hz, 1H), 7.86–7.81(m,2H),7.62–7.59(m,1H),7.58–7.54(m,2H),7.31–7.28(m,1H),7.1 6(d,J=2.0Hz,1H),7.06–7.01(m,1H),3.21–3.15(m,2H),3.01–2.96(m,4H),2.9 5–2.89(m,4H),1.58(s,4H),1.28–1.23(m,4H),1.20–1.18(m,3H),0.36(s,4H).

[0326] Examples 11-19 were synthesized according to the synthesis method of Examples 9-10 of the present invention, and their structures and characterization data are shown in the following table:

[0327] Example 20

[0328] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0329] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0330] first step

[0331] 1-(2-fluoro-4-nitrophenyl)-1H-imidazole

[0332] 1-(2-Fluoro-4-nitrophenyl)-1H-imidazole

[0333] To a solution of 1,2-difluoro-4-nitrobenzene 20a (2.5 g, 15.7 mmol, commercially available) in N,N-dimethylformamide (20 mL) were added imidazole (1.18 g, 17.3 mmol) and potassium carbonate (6.5 g, 47.1 mmol) at 25°C. The mixture was reacted at 70°C for 18 hours. The mixture was poured into water (100 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 1-(2-fluoro-4-nitrophenyl)-1H-imidazole 20b (2.00 g) with a yield of 61.4%. The crude product was used directly in the next reaction.

[0334] MS m / z(ESI):208.0[M+1]

[0335] Step 2

[0336] 1-(2-(1H-imidazol-1-yl)-5-nitrophenyl)-4,4-difluoropiperidine

[0337] To a solution of 1-(2-fluoro-4-nitrophenyl)-1H-imidazole 20b (2.00 g, 9.65 mmol) in dimethyl sulfoxide (20 mL) at 25°C were added potassium carbonate (4.00 g, 29.0 mmol) and 4,4-difluoropiperidine 1b (3.51 g, 29.0 mmol). The mixture was reacted at 130°C for 18 hours. The mixture was poured into water (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to obtain 1-(2-(1H-imidazol-1-yl)-5-nitrophenyl)-4,4-difluoropiperidine 20c (800 mg) in a 26.9% yield.

[0338] MS m / z(ESI):309.2[M+1]

[0339] Step 3

[0340] 3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-1-yl)aniline

[0341] 3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-1-yl)aniline

[0342] To a solution of 1-(2-(1H-imidazol-1-yl)-5-nitrophenyl)-4,4-difluoropiperidine 20c (100 mg, 0.324 mmol) in methanol (3 mL) was added 10% wet palladium on carbon (30.0 mg, 0.282 mmol) at 25°C. The atmosphere was replaced with hydrogen three times and the mixture was allowed to react at 25°C for 4 hours. The mixture was filtered and concentrated under reduced pressure to afford 3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-1-yl)aniline 20d (90.0 mg), which was used directly in the next reaction.

[0343] MS m / z(ESI):279.1[M+1]

[0344] Step 4

[0345] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0346] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0347] 3-(4,4-Difluoropiperidin-1-yl)-4-(1H-imidazol-1-yl)aniline 20d (80.0 mg, 0.287 mmol) and 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (146 mg, 0.431 mmol) were added to N,N-dimethylformamide (5 mL) at 25°C, followed by (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (300 mg, 0.575 mmol) and N,N-diisopropylethylamine (149 mg, 1.15 mmol). The mixture was reacted at 65°C for 2 hours. The mixture was poured into water (50 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 20 (50.0 mg) with a yield of 29%.

[0348] MS m / z(ESI):599.4[M+1]

[0349] 1 H NMR (400MHz, DMSO-d6) δ11.79(s,1H),10.17(s,1H),8.02(s,1H),7.83(d,J=8.4Hz,1H),7. 70(d,J=2.4Hz,1H),7.60–7.50(m,2H),7.34(d,J=8.4Hz,1H),7.18(d,J=2.0Hz,1H),7.10( s,1H),7.05(dd,J=8.4,2.0Hz,1H),3.20(q,J=7.2Hz,2H),2.98(t,J=5.2Hz,4H),2.79(t,J =5.6Hz,4H),2.05–1.91(m,4H),1.55(d,J=5.2Hz,4H),1.21(t,J=7.2Hz,3H),0.35(s,4H).

[0350] Example 21

[0351] N-(3-(4,4-difluorocyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0352] N-(3-(4,4-difluorocyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0353] first step

[0354] 1-(2-bromo-4-nitrophenyl)-1H-imidazole

[0355] 1-(2-Bromo-4-nitrophenyl)-1H-imidazole

[0356] 2-Bromo-1-fluoro-4-nitrobenzene 21a (1.00 g, 4.55 mmol, commercially available) was added to N,N-dimethylformamide (10 mL) at room temperature, followed by imidazole (619 mg, 9.09 mmol) and potassium carbonate (1.26 g, 9.09 mmol). The atmosphere was purged with nitrogen three times and the reaction was continued at 100°C for 18 hours. The mixture was filtered and concentrated to dryness under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to give 1-(2-bromo-4-nitrophenyl)-1H-imidazole 21b (300 mg) in a 24.6% yield.

[0357] MS m / z(ESI):269.0[M+1]

[0358] Step 2

[0359] 1-(4',4'-difluoro-5-nitro-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-2-yl)-1H-imidazole

[0360] 1-(4',4'-difluoro-5-nitro-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-2-yl)-1H-imidazole

[0361] At room temperature, 1-(2-bromo-4-nitrophenyl)-1H-imidazole 21b (200 mg, 746.08 μmol) and 2-(4,4-difluorocyclohex-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 21c (273 mg, 1.12 mmol, commercially available) were added to 1,4-dioxane (3 mL) and water (1 mL). [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (54.6 mg, 0.0746 mmol) and sodium carbonate (158 mg, 1.49 mmol) were then added. The atmosphere was purged with nitrogen three times and the reaction was continued at 100°C for 18 hours. The mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to afford 1-(4',4'-difluoro-5-nitro-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-2-yl)-1H-imidazole 21d (130 mg) in a yield of 57.1%.

[0362] MS m / z(ESI):306.0[M+1]

[0363] Step 3

[0364] 3-(4,4-difluorocyclohexyl)-4-(1H-imidazol-1-yl)aniline

[0365] 3-(4,4-difluorocyclohexyl)-4-(1H-imidazol-1-yl)aniline

[0366] 1-(4',4'-difluoro-5-nitro-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-2-yl)-1H-imidazole 21d (150 mg, 0.491 mmol) was added to anhydrous methanol (3 mL) and acetic acid (0.5 mL) at room temperature. Palladium on carbon (10%) (26.1 mg, 0.246 mmol) was then added. The atmosphere was replaced with hydrogen three times and the reaction was continued at 25°C for 4 hours. The mixture was concentrated to dryness under reduced pressure to afford 3-(4,4-difluorocyclohexyl)-4-(1H-imidazol-1-yl)aniline 21e (50.0 mg) in a 36.9% yield.

[0367] MS m / z(ESI):278.2[M+1]

[0368] Step 4

[0369] N-(3-(4,4-difluorocyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0370] N-(3-(4,4-difluorocyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0371] At room temperature, 3-(4,4-difluorocyclohexyl)-4-(1H-imidazol-1-yl)aniline 21e (30 mg, 0.108 mmol) and 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (43.9 mg, 0.130 mmol) were added to N,N-dimethylformamide (2 mL), followed by (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (84.6 mg, 0.162 mmol) and N,N-diisopropylethylamine (28.0 mg, 0.216 mmol). The atmosphere was replaced with nitrogen three times and the reaction was continued at 60°C for 3 hours. The mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with anhydrous sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluorocyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 21 (2 mg) with a yield of 3.1%.

[0372] MS m / z(ESI):598.3[M+1]

[0373] 1 H NMR(400MHz,DMSO-d6)δ11.90(s,1H),10.17(s,1H),7.90–7.83(m,2H),7.82–7.80(m,1H),7. 77–7.75(m,1H),7.40–7.36(m,1H),7.31(d,J=8.8Hz,1H),7.19(d,J=2.0Hz,1H),7.11(t,J=1. 1Hz,1H),7.07–7.04(m,1H),3.29(s,2H),3.23–3.17(m,2H),3.00–2.96(m,4H),2.11–2.01(m, 2H),1.86–1.80(m,2H),1.71–1.66(m,2H),1.59–1.52(m,4H),1.22-1.20(m,4H),0.35(s,4H).

[0374] Example 22 was synthesized according to the synthesis method of Example 21 of the present invention, and the structure and characterization data are shown in the following table:

[0375] Example 23

[0376] N-(3-(3,3-difluorocyclobutoxy)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0377] N-(3-(3,3-difluorocyclobutyloxy)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0378] first step

[0379] 1-bromo-2-(3,3-difluorocyclobutoxy)-4-nitrobenzene

[0380] 1-Bromo-2-(3,3-difluorocyclobutyloxy)-4-nitrobenzene

[0381] 2-Bromo-5-nitrophenol 23a (2.00 g, 9.17 mmol) was dissolved in tetrahydrofuran (15 mL), and triphenylphosphine (2.78 g, 13.76 mmol) was added. After nitrogen replacement three times, 1a (1.49 g, 13.76 mmol) and diisoethyl azodicarboxylate (2.78 g, 13.76 mmol) were added dropwise in an ice bath. The mixture was allowed to react at 25°C for 18 hours. Mass spectrometry confirmed the complete reaction of the starting material and the formation of the product. The reaction solution was diluted with water (150 mL) and extracted with ethyl acetate (60 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to give 1-bromo-2-(3,3-difluorocyclobutyloxy)-4-nitrobenzene 23b (2.56 g) in a 72.4% yield. MS m / z (ESI): 308.0 / 310.0 [M+1].

[0382] Step 2

[0383] 2-(2-(3,3-difluorocyclobutoxy)-4-nitrophenyl)oxazole

[0384] 2-(2-(3,3-difluorocyclobutyloxy)-4-nitrophenyl)oxazole

[0385] At room temperature, 1-bromo-2-(3,3-difluorocyclobutyloxy)-4-nitrobenzene 23b (500.00 mg, 1.62 mmol) and 2-(tributyltinyl)oxazole 23c (871.80 mg, 2.43 mmol) were dissolved in N,N-dimethylformamide (5 mL). 1,1-Bis(diphenylphosphino)diphenylferric palladium chloride (252.56 mg, 0.32 mmol) and cuprous iodide (30.91 mg, 0.16 mmol) were added sequentially. The atmosphere was purged with nitrogen three times, and the reaction mixture was stirred at 100°C for 18 hours. The reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (40 mL x 3). The combined organic phase was washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (eluent: System A) to give 2-(2-(3,3-difluorocyclobutyloxy)-4-nitrophenyl)oxazole 23d (196.00 mg) in a yield of 40.8%.

[0386] MS m / z(ESI):297.0[M+1]

[0387] Step 3

[0388] 3-(3,3-difluorocyclobutoxy)-4-(oxazol-2-yl)aniline

[0389] 3-(3,3-difluorocyclobutyloxy)-4-(oxazol-2-yl)aniline

[0390] 2-(2-(3,3-difluorocyclobutyloxy)-4-nitrophenyl)oxazole 23d (100.00 mg, 0.34 mmol) was dissolved in a mixture of ethanol (1 mL) and water (0.5 mL). Ammonium chloride (18.06 mg, 0.34 mmol) and iron powder (18.85 mg, 0.34 mmol) were added sequentially. After nitrogen substitution three times, the reaction was stirred at 100°C for 3 hours and then cooled to room temperature. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate (20 mL x 3). The combined organic phases were concentrated under reduced pressure to afford 3-(3,3-difluorocyclobutyloxy)-4-(oxazol-2-yl)aniline 23e (62.00 mg) in a 68.9% yield.

[0391] MS m / z(ESI):267.0[M+1]

[0392] Step 4

[0393] N-(3-(3,3-difluorocyclobutoxy)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0394] N-(3-(3,3-difluorocyclobutyloxy)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0395] At room temperature, 3-(3,3-difluorocyclobutyloxy)-4-(oxazol-2-yl)aniline 23e (62.00 mg, 0.23 mmol) was dissolved in N,N-dimethylformamide (1 mL). N,N-diisopropylethylamine (120.39 mg, 0.93 mmol), 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (78.81 mg, 0.23 mmol), and (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (182.12 mg, 0.39 mmol) were added sequentially. After nitrogen substitution three times, the reaction mixture was incubated at 25°C for 18 hours. The reaction mixture was poured into water (80 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(3,3-difluorocyclobutyloxy)-4-(oxazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 23 (41.89 mg) with a yield of 30.7%.

[0396] MS m / z(ESI):587.4[M+1]

[0397] 1H NMR (400MHz, DMSO) δ11.73 (s, 1H), 10.30 (s, 1H), 8.17 (s, 1H), 7.90 (d, J = 8.4Hz, 1H), 7.82(d,J=8.4Hz,1H),7.64(dd,J=8.4,1.5Hz,1H),7.42(d,J=1.2Hz,1H),7.35(s,1H) ,7.16(d,J=1.6Hz,1H),7.03(dd,J=8.4,1.9Hz,1H),4.84(s,1H),3.27–3.16(m,4H),3 .01–2.94(m,4H),2.88-2.74(m,2H),1.54(s,4H),1.21(t,J=7.2Hz,3H),0.35(s,4H).

[0398] Example 24

[0399] N-(3-(3,3-difluorocyclobutoxy)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0400] N-(3-(3,3-difluorocyclobutyloxy)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0401] first step

[0402] 1-(2-(3,3-difluorocyclobutoxy)-4-nitrophenyl)-1H-imidazole

[0403] 1-(2-(3,3-difluorocyclobutyloxy)-4-nitrophenyl)-1H-imidazole

[0404] 1-Bromo-2-(3,3-difluorocyclobutyloxy)-4-nitrobenzene 23b (200.00 mg, 0.65 mmol) was dissolved in N,N-dimethylformamide (20 mL). Potassium phosphate (413.40 mg, 1.95 mmol), trans-(1R,2R)-N,N'-dimethyl-1,2-cyclohexanediamine (46.17 mg, 0.32 mmol), imidazole (44.20 mg, 0.65 mmol), and cuprous iodide (123.64 mg, 0.65 mmol) were added sequentially. After nitrogen substitution three times, the reaction mixture was refluxed at 100°C for 8 hours. The reaction mixture was filtered, the liquid phase was diluted with water (100 mL), and extracted with ethyl acetate (40 mL x 3). The combined organic phase was washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (eluent: System A) to give 1-(2-(3,3-difluorocyclobutyloxy)-4-nitrophenyl)-1H-imidazole 24a (119.00 mg) in a yield of 62.1%.

[0405] MS m / z(ESI):296.0[M+1]

[0406] Step 2

[0407] 3-(3,3-difluorocyclobutoxy)-4-(1H-imidazol-1-yl)aniline

[0408] 3-(3,3-difluorocyclobutyloxy)-4-(1H-imidazol-1-yl)aniline

[0409] 1-(2-(3,3-difluorocyclobutyloxy)-4-nitrophenyl)-1H-imidazole 24a (119.00 mg, 0.40 mmol) was dissolved in methanol (5 mL) and palladium on carbon (8.58 mg, 0.08 mmol) was added. The atmosphere was replaced with hydrogen three times, and the reaction was stirred at 25°C under a hydrogen atmosphere for 18 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to provide 3-(3,3-difluorocyclobutyloxy)-4-(1H-imidazol-1-yl)aniline 24b (105 mg). The crude product was used directly in the next step with a yield of 98.2%.

[0410] MS m / z(ESI):266.0[M+1]

[0411] Step 3

[0412] N-(3-(3,3-difluorocyclobutoxy)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0413] N-(3-(3,3-difluorocyclobutyloxy)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0414] At room temperature, 3-(3,3-difluorocyclobutyloxy)-4-(1H-imidazol-1-yl)aniline 24b (55.00 mg, 0.20 mmol) was dissolved in N,N-dimethylformamide (1 mL). N,N-diisopropylethylamine (107.19 mg, 0.89 mmol), 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (70.17 mg, 0.20 mmol), and (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (162.16 mg, 0.31 mmol) were added sequentially. After nitrogen substitution three times, the reaction mixture was allowed to react at 20°C for 18 hours. The mixture was poured into water (100 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(3,3-difluorocyclobutyloxy)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 24 (55.03 mg) with a yield of 45.3%.

[0415] MS m / z(ESI):587.2[M+1]

[0416] 1H NMR (400MHz, DMSO-d6) δ11.69(s,1H),10.30(s,1H),7.94(s,1H),7.82(d,J=8. 4Hz,1H),7.58(dd,J=8.6,1.9Hz,1H),7.51–7.43(m,3H),7.16(d,J=1.7Hz,1H) ,7.05(s,2H),4.79(s,1H),3.18(dd,J=14.6,7.2Hz,4H),2.97(d,J=5.0Hz,4H) ,2.79(dd,J=14.0,4.6Hz,2H),1.55(s,4H),1.21(t,J=7.2Hz,3H),0.35(s,4H).

[0417] Example 25

[0418] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0419] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0420] first step

[0421] 2-(2-fluoro-4-nitrophenyl)-1H-imidazole

[0422] 2-(2-Fluoro-4-nitrophenyl)-1H-imidazole

[0423] 2-Fluoro-4-nitrobenzonitrile 1a (1.50 g, 9.03 mmol) was dissolved in methanol (20 mL). Sodium methoxide (243.92 mg, 4.52 mmol) was added and stirred at 25°C for 4 hours. Acetic acid (1.08 g, 18.06 mmol) and imidazole (1.14 g, 10.84 mmol) were then added to the system. The mixture was stirred at 50°C for 0.5 hours and then cooled. Finally, hydrochloric acid (6 M, 10 mL) was added and refluxed at 80°C for 2 hours. After cooling, the reaction solution was diluted with water (100 mL) and extracted with dichloromethane (40 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to give 2-(2-fluoro-4-nitrophenyl)-1H-imidazole 25a (500.00 mg) in a 26.7% yield. MS m / z (ESI): 208.0 [M+1].

[0424] Step 2

[0425] 1-(2-(1H-imidazol-2-yl)-5-nitrophenyl)-4,4-difluoropiperidine

[0426] 1-(2-(1H-imidazol-2-yl)-5-nitrophenyl)-4,4-difluoropiperidine

[0427] 2-(2-Fluoro-4-nitrophenyl)-1H-imidazole 25a (200.00 mg, 0.97 mmol) was dissolved in dimethyl sulfoxide (2 mL), and triethylamine (488.46 mg, 4.83 mmol) and 4,4-difluoropiperidine 1b (584.71 mg, 4.83 mmol) were added. The atmosphere was purged with nitrogen three times, and the reaction was incubated at 125°C for 18 hours. The reaction solution was poured into water (50 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to obtain 1-(2-(1H-imidazol-2-yl)-5-nitrophenyl)-4,4-difluoropiperidine 25b (157.00 mg) in a 52.7% yield.

[0428] MS m / z(ESI):309.0[M+1]

[0429] Step 3

[0430] 3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-2-yl)aniline

[0431] 3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-2-yl)aniline

[0432] At room temperature, 1-(2-(1H-imidazol-2-yl)-5-nitrophenyl)-4,4-difluoropiperidine 25b (150.00 mg, 0.49 mmol) was dissolved in methanol (2 mL). Palladium on carbon (10.36 mg, 0.10 mmol) was added and the atmosphere was replaced with hydrogen three times. The reaction was allowed to proceed at 25°C under a hydrogen atmosphere for 5 hours. The reaction solution was filtered and washed with methanol (20 mL x 3). The combined organic phases were concentrated to dryness under reduced pressure to afford 3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-2-yl)aniline 25c (111.00 mg), which was used directly in the next reaction.

[0433] MS m / z(ESI):279.2[M+1]

[0434] Step 4

[0435] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0436] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0437] At room temperature, 3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-2-yl)aniline 25c (50.00 mg, 0.18 mmol) was dissolved in N,N-dimethylformamide (1 mL). N,N-diisopropylethylamine (92.88 mg, 0.72 mmol), 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (60.80 mg, 0.18 mmol), and (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (140.51 mg, 0.27 mmol) were added sequentially. The atmosphere was purged with nitrogen three times. The reaction mixture was allowed to react at 25°C for 18 hours. The reaction mixture was poured into water (80 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(1H-imidazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 25 (14.30 mg) with a yield of 13.3%.

[0438] MS m / z(ESI):600.4[M+1]

[0439] 1 H NMR (400MHz, DMSO-d6) δ11.82(s,1H),10.32(s,1H),7.84(d,J=8.4Hz,2H),7.68(d,J=1.8Hz,1H),7.56(dd,J=8.4,1.8Hz,1H),7.29 –6.94(m,4H),3.19(dd,J=14.6,7.2Hz,2H),3.00–2.88(m,8H),2.28-2.14(m,4H),1.56(s,4H),1.21(t,J=7.3Hz,3H),0.36(s,4H).

[0440] Example 26 was synthesized according to the synthesis method of Example 25 of the present invention. The structure and characterization data are shown in the following table:

[0441] Example 27

[0442] N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0443] N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0444] first step

[0445] 4-(2-fluoro-4-nitrophenyl)-4H-1,2,4-triazole

[0446] 4-(2-Fluoro-4-nitrophenyl)-4H-1,2,4-triazole

[0447] N,N'-Bis(dimethylaminomethylene)hydrazine dihydrochloride 27b (2.76 g, 12.8 mmol, commercially available) and p-toluenesulfonic acid (110 mg, 0.64 mmol) were added to a toluene (10 mL) solution of 2-fluoro-4-nitroaniline 27a (1 g, 6.41 mmol, commercially available) at room temperature. The reaction mixture was allowed to react at 110°C for 16 hours. The reaction mixture was then concentrated under reduced pressure. The crude product was separated by preparative liquid chromatography (AKZONOBEL Kromasil column; 250 × 21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA + H2O, mobile phase B: CH3CN) to afford 4-(2-fluoro-4-nitrophenyl)-4H-1,2,4-triazole 27c (600 mg) in a 45% yield.

[0448] MS m / z(ESI):209.0[M+1]

[0449] Step 2

[0450] 4,4-difluoro-1-(5-nitro-2-(4H-1,2,4-triazol-4-yl)phenyl)piperidine

[0451] 4,4-Difluoro-1-(5-nitro-2-(4H-1,2,4-triazol-4-yl)phenyl)piperidine

[0452] 4-(2-Fluoro-4-nitrophenyl)-4H-1,2,4-triazole 27c (300 mg, 1.44 mmol) was added to a mixture of dimethyl sulfoxide (2.5 mL) and 4,4-difluoropiperidine (2.5 mL) at room temperature. The reaction mixture was allowed to react at 120°C for 16 hours. The reaction mixture was poured into water and extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to obtain 4,4-difluoro-1-(5-nitro-2-(4H-1,2,4-triazol-4-yl)phenyl)piperidine 27d (260 mg) in a 58% yield.

[0453] MS m / z(ESI):310.0[M+1]

[0454] Step 3

[0455] 3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-4-yl)aniline

[0456] 3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-4-yl)aniline

[0457] Palladium on carbon (50.0 mg, 10%) was added to a solution of 4,4-difluoro-1-(5-nitro-2-(4H-1,2,4-triazol-4-yl)phenyl)piperidine 27d (260 mg, 0.84 mmol) in methanol (20 mL) at room temperature. The mixture was purged with a hydrogen balloon three times and allowed to react at room temperature for 2 hours. The reaction mixture was filtered, the filter cake rinsed with methanol (50 mL), and the filtrate concentrated under reduced pressure to afford 3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-4-yl)aniline 27e (220 mg) in a 94% yield.

[0458] MS m / z(ESI):280.0[M+1]

[0459] Step 4

[0460] N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0461] N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0462] O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (70.8 mg, 0.18 mmol) and N,N-diisopropylethylamine (27.7 mg, 0.21 mmol) were added to a solution of 3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-4-yl)aniline 27e (40.0 mg, 0.14 mmol) and 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (50 mg, 0.15 mmol) in N,N-dimethylformamide (1 mL) at room temperature. The reaction mixture was reacted at 25°C for 16 hours. The reaction solution was poured into water (5 mL) and extracted with ethyl acetate (5 mL×3). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated by preparative liquid phase separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to obtain N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-4-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 27 (18.6 mg) with a yield of 21%.

[0463] MS m / z(ESI):601.2[M+1]

[0464] 1 H NMR(400MHz,DMSO-d6)δ11.80(s,1H),10.22(s,1H),8.93(s,2H),7.87–7.72( m,2H),7.56(d,J=8.4,2.0Hz,1H),7.44(d,J=8.4Hz,1H),7.16(d,J=2.0Hz,1H ),7.03(d,J=8.4,2.0Hz,1H),3.24–3.11(m,2H),3.06–2.91(m,4H),2.86–2.7 2(m,4H),2.04–1.86(m,4H),1.55(s,4H),1.21(t,J=7.2Hz,3H),0.35(s,4H).

[0465] Examples 28-32 were synthesized according to the synthesis method of Examples 20-25 of the present invention, and their structures and characterization data are shown in the following table:

[0466] Example 36

[0467] N-(3-(4,4-difluoropiperidin-1-yl)-4-(isoxazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0468] N-(3-(4,4-difluoropiperidin-1-yl)-4-(isoxazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0469] first step

[0470] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzaldehyde

[0471] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzaldehyde

[0472] To a solution of 2-fluoro-4-nitro-benzaldehyde 36a (3 g, 17.74 mmol, commercially available) and 4,4-difluoropiperidine 1b (4.30 g, 35.48 mmol) in dimethyl sulfoxide (10 mL) was added triethylamine (5.39 g, 53.22 mmol). The mixture was protected with nitrogen, heated to 100°C, and stirred for 18 hours. The reaction solution was poured into water and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to give 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzaldehyde 36b (2 g) in a 41.7% yield.

[0473] MS m / z(ESI):271.2[M+1]

[0474] Step 2

[0475] (E)-2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzaldehyde oxime

[0476] (E)-2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzaldehyde oxime

[0477] To a solution of 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzaldehyde 36b (500 mg, 1.85 mmol) in tetrahydrofuran (8 mL) were added pyridine (293 mg, 3.70 mmol) and hydroxylamine hydrochloride (257 mg, 3.70 mmol). The reaction mixture was stirred at 0-25°C under nitrogen for 18 hours. The mixture was poured into water (60 mL) and extracted with ethyl acetate (60 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (100 mL) and dried over anhydrous sodium sulfate. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to afford (E)-2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzaldehyde oxime 36c (484 mg) in a 91% yield.

[0478] MS m / z(ESI):286.2[M+1]

[0479] Step 3

[0480] 3-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)isoxazole

[0481] 3-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)isoxazole

[0482] To a suspension of (E)-2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzaldehyde oxime 36c (310 mg, 1.09 mmol), calcium carbide (453 mg, 7.06 mmol), and N-chlorosuccinimide (189 mg, 1.41 mmol) in carbon tetrachloride (1.05 mL) was added water (254 mg, 14.1 mmol), and the mixture was quickly sealed. The reaction mixture was stirred at 25°C for 16 hours. The mixture was concentrated to dryness under reduced pressure, and the crude product was isolated and purified by silica gel column chromatography (eluent: System A) to afford 3-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)isoxazole 36d (124 mg) in a 36% yield.

[0483] MS m / z(ESI):310.2[M+1]

[0484] Step 4

[0485] 3-(4,4-difluoropiperidin-1-yl)-4-(isoxazol-3-yl)aniline

[0486] 3-(4,4-difluoropiperidin-1-yl)-4-(isoxazol-3-yl)aniline

[0487] To a suspension of 3-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)isoxazole 36d (60 mg, 0.194 mmol) in tetrahydrofuran (5 mL) was added wet palladium on carbon (31 mg, 0.029 mmol, 10% purity), the atmosphere was replaced with hydrogen three times, and the mixture was stirred at 25°C for 5 hours. The mixture was filtered, and the solid was washed with ethyl acetate (20 mL × 3). The filtrate was concentrated to dryness under reduced pressure to give 3-(4,4-difluoropiperidin-1-yl)-4-(isoxazol-3-yl)aniline 36e (50 mg), which was used directly in the next reaction.

[0488] MS m / z(ESI):280.2[M+1]

[0489] Step 5

[0490] N-(3-(4,4-difluoropiperidin-1-yl)-4-(isoxazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0491] N-(3-(4,4-difluoropiperidin-1-yl)-4-(isoxazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0492] To a solution of 3-(4,4-difluoropiperidin-1-yl)-4-(isoxazol-3-yl)aniline 36e (50 mg, 0.179 mmol), 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (61 mg, 0.179 mmol), and N,N-diisopropylethylamine (58 mg, 0.448 mmol) in N,N-dimethylformamide (1.5 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (102 mg, 0.269 mmol). The reaction mixture was stirred at 50°C for 3 hours. The mixture was dissolved in water (20 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure. The crude product was separated by preparative liquid chromatography (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(isoxazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 36 (27.1 mg) in a yield of 25%.

[0493] MS m / z(ESI):600.4[M+1]

[0494] 1 H NMR (400MHz, DMSO-d6) δ11.83(s,1H),10.17(s,1H),8.96(d,J=1.6Hz,1H),7.84(d,J=8.8Hz,1H),7.70(d,J=1.6Hz,1H),7.65-7.53(m,2H),7.22-7. 13(m,2H),7.05(dd,J=8.4,2.0Hz,1H),3.20(q,J=7.2Hz,2H),3.02-2.89( m,8H),2.15-2.01(m,4H),1.56(s,4H),1.21(t,J=7.2Hz,3H),0.35(s,4H).

[0495] Example 37

[0496] N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0497] N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0498] first step

[0499] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzamide

[0500] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzamide

[0501] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzonitrile 1c (500.00 mg, 1.87 mmol) was dissolved in dimethyl sulfoxide (5 mL). Potassium carbonate (129.29 mg, 0.94 mmol) and hydrogen peroxide (318.21 mg, 2.81 mmol) were added, and the atmosphere was replaced with nitrogen three times. The reaction was incubated at 25°C for 5 hours. The reaction mixture was poured into water (80 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzamide 37a (424.00 mg), which was used directly in the next step.

[0502] MS m / z(ESI):286.0[M+1]

[0503] Step 2

[0504] 4,4-difluoro-1-(5-nitro-2-(4H-1,2,4-triazol-3-yl)phenyl)piperidine

[0505] 4,4-Difluoro-1-(5-nitro-2-(4H-1,2,4-triazol-3-yl)phenyl)piperidine

[0506] To 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzamide 37a (424.00 mg, 1.49 mmol) was added N,N-dimethylformamide dimethyl acetal (2.66 g, 22.30 mmol) at room temperature. After nitrogen substitution three times, the reaction mixture was stirred at 120°C for 1 hour, cooled, concentrated under reduced pressure, and then spun down to dryness. Hydrazine hydrate (9.53 mg, 0.30 mmol) and acetic acid (4 mL) were then added, and the temperature was raised to 90°C and stirred for 1 hour. The reaction mixture was concentrated to dryness under reduced pressure, and the crude product was isolated and purified by silica gel column chromatography (eluent: System A) to afford 4,4-difluoro-1-(5-nitro-2-(4H-1,2,4-triazol-3-yl)phenyl)piperidine 37b (218.00 mg) in a 47.4% yield.

[0507] MS m / z(ESI):310.0[M+1]

[0508] Step 3

[0509] 3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-3-yl)aniline

[0510] 3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-3-yl)aniline

[0511] At room temperature, 4,4-difluoro-1-(5-nitro-2-(4H-1,2,4-triazol-3-yl)phenyl)piperidine 37b (60.00 mg, 0.19 mmol) was dissolved in methanol (3 mL). Palladium on carbon (4.13 mg, 0.04 mmol) was added, and the atmosphere was replaced with hydrogen three times. The reaction was stirred at 25°C under a hydrogen atmosphere for 18 hours. The reaction mixture was filtered, and the filter cake was washed with methanol (20 mL x 3). The filtrate was concentrated under reduced pressure to afford 3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-3-yl)aniline 37c (52.00 mg), which was used directly in the next step.

[0512] MS m / z(ESI):280.2[M+1]

[0513] Step 4

[0514] N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0515] N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0516] At room temperature, 3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-3-yl)aniline 37c (52.00 mg, 0.19 mmol) was dissolved in N,N-dimethylformamide (1 mL). N,N-diisopropylethylamine (96.25 mg, 0.74 mmol), 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (63.01 mg, 0.19 mmol), and (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (145.61 mg, 0.28 mmol) were added sequentially. The atmosphere was purged with nitrogen three times. The reaction mixture was allowed to react at 25°C for 18 hours. The reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by preparative liquid chromatography (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(4H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 37 (10.35 mg) in a yield of 9.3%.

[0517] MS m / z(ESI):600.4[M+1]

[0518] 1 H NMR(400MHz,DMSO-d6)δ13.84(s,1H),11.87(s,1H),10.40(s,1H),8.11(s,1 H),7.81(dd,J=19.7,8.5Hz,2H),7.69(s,1H),7.59(d,J=8.4Hz,1H),7.16(d ,J=2.0Hz,1H),7.04(dd,J=8.4,2.0Hz,1H),3.19(q,J=7.3Hz,2H),3.04-2.8 7(m,8H),2.24-2.09(m,4H),1.56(s,4H),1.21(t,J=7.3Hz,3H),0.35(s,4H).

[0519] Examples 38-39 were synthesized according to the synthesis method of Example 37 of the present invention, and their structures and characterization data are shown in the following table:

[0520] Example 40

[0521] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1,3,4-oxadiazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0522] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1,3,4-oxadiazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0523] first step

[0524] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzohydrazide

[0525] 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzohydrazide

[0526] To a suspension of methyl 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzoate 2b (2.00 g, 6.66 mmol) in ethanol (20 mL) was added hydrazine hydrate (5.15 g, 82.3 mmol, 80% purity), and the mixture was heated to 70°C and stirred for 6 hours. The mixture was concentrated to dryness under reduced pressure to obtain a residue. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to obtain 2-(4,4-difluoropiperidin-1-yl)-4-nitrobenzohydrazide 40a (1.40 g) in a 70% yield.

[0527] MS m / z(ESI):301.2[M+1]

[0528] Step 2

[0529] 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)-1,3,4-oxadiazole

[0530] 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)-1,3,4-oxadiazole

[0531] To a solution of 2-(4,4-difluoropiperidin-1-yl)-4-nitrophenylhydrazide 40a (200 mg, 0.666 mmol) in acetic acid (1 mL) was added triethyl orthoformate (148 mg, 0.999 mmol), and the resulting mixture was stirred at 100°C for 2 hours. The mixture was concentrated to dryness under reduced pressure, and the crude product was isolated and purified by silica gel column chromatography (eluent: System A) to provide 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)-1,3,4-oxadiazole 40b (107 mg) in a 51% yield.

[0532] MS m / z(ESI):311.2[M+1]

[0533] Step 3

[0534] 3-(4,4-difluoropiperidin-1-yl)-4-(1,3,4-oxadiazol-2-yl)aniline

[0535] 3-(4,4-difluoropiperidin-1-yl)-4-(1,3,4-oxadiazol-2-yl)aniline

[0536] To a suspension of 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenyl)-1,3,4-oxadiazole 40b (50 mg, 0.161 mmol) in methanol (10 mL) was added wet palladium on carbon (34 mg, 0.032 mmol, 10% purity), the atmosphere was replaced with hydrogen three times, and the mixture was stirred at 25°C for 3 hours. The mixture was filtered, and the solid was washed with ethyl acetate (20 mL × 3). The filtrate was concentrated to dryness under reduced pressure to give 3-(4,4-difluoropiperidin-1-yl)-4-(1,3,4-oxadiazol-2-yl)aniline 40c (40 mg) in an 88% yield.

[0537] MS m / z(ESI):281.2[M+1]

[0538] Step 4

[0539] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1,3,4-oxadiazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0540] N-(3-(4,4-difluoropiperidin-1-yl)-4-(1,3,4-oxadiazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0541] To a solution of 3-(4,4-difluoropiperidin-1-yl)-4-(1,3,4-oxadiazol-2-yl)aniline 40c (40 mg, 0.143 mmol), 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (48 mg, 0.143 mmol), and N,N-diisopropylethylamine (46 mg, 0.357 mmol) in N,N-dimethylformamide (1 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (71 mg, 0.186 mmol), and the mixture was stirred at 60°C for 16 hours. The mixture was dissolved in water (20 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(1,3,4-oxadiazol-2-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 40 (25 mg) with a yield of 29%.

[0542] MS m / z(ESI):601.4[M+1]

[0543] 1 H NMR(400MHz,DMSO-d6)δ11.90(s,1H),10.40–10.05(s,1H),9.31(s,1H),7.92(d,J=8 .4Hz,1H),7.83(d,J=8.4Hz,1H),7.74(s,1H),7.63(d,J=7.2Hz,1H),7.17(s,1H),7. 05(d,J=8.4Hz,1H),6.82–6.77(m,1H),3.24–3.16(m,2H),3.12–3.03(m,4H),3.02–2 .93(m,4H),2.23–2.10(m,4H),1.62–1.45(s,4H),1.21(t,J=7.2Hz,3H),0.35(s,4H).

[0544] Examples 41-43 were synthesized according to the synthesis method of Example 40 of the present invention, and their structures and characterization data are shown in the following table:

[0545] Example 44

[0546] N-(3-(4,4-difluoro-1-hydroxycyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0547] N-(3-(4,4-difluoro-1-hydroxycyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0548] first step

[0549] 4-bromo-3-iodo-N,N-bis(4-methoxybenzyl)aniline

[0550] 4-Bromo-3-iodo-N,N-bis(4-methoxybenzyl)aniline

[0551] To a solution of 4-bromo-3-iodoaniline 44a (900.00 mg, 3.02 mmol, commercially available) in N,N-dimethylformamide (10 mL) at 0°C was added sodium hydride (144.99 mg, 6.04 mmol). The mixture was stirred at room temperature for 0.5 hours, followed by the addition of 4-methoxybenzyl chloride (709.67 mg, 4.53 mmol) and stirring at 25°C for 1 hour. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (45 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was isolated and purified by silica gel column chromatography (eluent: System A) to afford 4-bromo-3-iodo-N,N-bis(4-methoxybenzyl)aniline 44b (1.4 g) in an 86.1% yield.

[0552] MS m / z(ESI):538 / 540[M+1]

[0553] Step 2

[0554] 1-(5-(bis(4-methoxybenzyl)amino)-2-bromophenyl)-4,4-difluorocyclohexan-1-ol

[0555] 1-(5-(bis(4-methoxybenzyl)amino)-2-bromophenyl)-4,4-difluorocyclohexan-1-ol

[0556] Under nitrogen, 4-bromo-3-iodo-N,N-bis(4-methoxybenzyl)aniline 44b (1.40 g, 2.60 mmol) was dissolved in tetrahydrofuran (20 mL). The temperature was cooled to -78°C, and isopropylmagnesium chloride-lithium chloride solution (1.13 g, 7.80 mmol) was added dropwise. After stirring for 0.5 hour, 4,4-difluorocyclohexane-1-one 44c (523.32 mg, 3.90 mmol, commercially available) dissolved in tetrahydrofuran (5 mL) was added. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was isolated and purified by silica gel column chromatography (eluent: System A) to afford 1-(5-(bis(4-methoxybenzyl)amino)-2-bromophenyl)-4,4-difluorocyclohexane-1-ol 44d (983.00 mg) in a 69.2% yield.

[0557] MS m / z(ESI):546.2 / 548.2[M+1]

[0558] Step 3

[0559] 1-(5-(bis(4-methoxybenzyl)amino)-2-(1H-imidazol-1-yl)phenyl)-4,4-difluorocyclohexan-1-ol

[0560] 1-(5-(bis(4-methoxybenzyl)amino)-2-(1H-imidazol-1-yl)phenyl)-4,4-difluorocyclohexan-1-ol

[0561] At room temperature, 1-(5-(bis(4-methoxybenzyl)amino)-2-bromophenyl)-4,4-difluorocyclohexan-1-ol 44d (983.00 mg, 1.80 mmol) was dissolved in N,N-dimethylformamide (10 mL). Potassium phosphate (1.15 g, 5.40 mmol), imidazole (122.46 mg, 1.80 mmol), trans-(1R,2R)-N,N'-dimethyl-1,2-cyclohexanediamine (127.94 mg, 0.90 mmol), and cuprous iodide (342.60 mg, 1.80 mmol) were added sequentially. The atmosphere was purged with nitrogen three times and the reaction was continued at 100°C for 4 hours. The mixture was poured into water (100 mL), allowed to stand for 10 minutes, and then filtered. The filter cake was washed with ethyl acetate (20 mL × 3) and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was separated and purified by silica gel column chromatography (eluent: System A) to give 1-(5-(bis(4-methoxybenzyl)amino)-2-(1H-imidazol-1-yl)phenyl)-4,4-difluorocyclohexan-1-ol 44e (628.00 mg) in a yield of 65.4%.

[0562] MS m / z(ESI):534.4[M+1]

[0563] Step 4

[0564] 1-(5-amino-2-(1H-imidazol-1-yl)phenyl)-4,4-difluorocyclohexan-1-ol

[0565] 1-(5-amino-2-(1H-imidazol-1-yl)phenyl)-4,4-difluorocyclohexan-1-ol

[0566] 1-(5-(bis(4-methoxybenzyl)amino)-2-(1H-imidazol-1-yl)phenyl)-4,4-difluorocyclohexan-1-ol 44e (628.00 mg, 1.18 mmol) was dissolved in ethanol (10 mL) at room temperature. Trifluoroacetic acid (5 mL) was added dropwise. The atmosphere was replaced with nitrogen three times and the reaction was continued at 70°C for 18 hours. The mixture was concentrated under reduced pressure to afford 1-(5-amino-2-(1H-imidazol-1-yl)phenyl)-4,4-difluorocyclohexan-1-ol 44f (290.00 mg), which was used directly in the next step.

[0567] MS m / z(ESI):294.2[M+1]

[0568] Step 5

[0569] N-(3-(4,4-difluoro-1-hydroxycyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0570] N-(3-(4,4-difluoro-1-hydroxycyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0571] At room temperature, 1-(5-amino-2-(1H-imidazol-1-yl)phenyl)-4,4-difluorocyclohexan-1-ol 44f (290.00 mg, 0.98 mmol) was dissolved in N,N-dimethylformamide (3 mL). N,N-diisopropylethylamine (511.12 mg, 3.95 mmol), 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (334.60 mg, 0.98 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (563.91 mg, 1.48 mmol) were added sequentially. After nitrogen substitution three times, the reaction mixture was allowed to react at 20°C for 18 hours. The mixture was poured into water (40 mL) and extracted with ethyl acetate (15 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoro-1-hydroxycyclohexyl)-4-(1H-imidazol-1-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide 44 (35.57 mg) with a yield of 5.9%.

[0572] MS m / z(ESI):614.3[M+1]

[0573] 1 H NMR (400MHz, DMSO-d6) δ11.93 (s, 1H), 10.18 (s, 1H), 8.20 (d, J = 2.4Hz, 1H), 7.88–7. 82(m,2H),7.75(s,1H),7.32(s,1H),7.20(d,J=2.0Hz,1H),7.15(d,J=8.4Hz,1H),7. 06(dd,J=8.4,2.0Hz,1H),7.02(s,1H),5.36(s,1H),3.20(q,J=7.2Hz,2H),3.03–2.9 7(m,4H),2.19–2.01(m,2H),1.81–1.55(m,10H),1.22(t,J=7.3Hz,3H),0.37(s,4H).

[0574] Example 45

[0575] N-(3-(3,3-difluorocyclobutoxy)-4-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0576] N-(3-(3,3-difluorocyclobutyloxy)-4-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0577] first step

[0578] 3-(2-fluoro-4-nitrophenyl)-1-methyl-1H-1,2,4-triazole

[0579] 3-(2-Fluoro-4-nitrophenyl)-1-methyl-1H-1,2,4-triazole

[0580] At room temperature, 2-(2-fluoro-4-nitrophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 10a (1.4 g, 5.24 mmol) and 3-bromo-1-methyl-1,2,4-triazole 45a (1.02 g, 6.29 mmol, commercially available) were dissolved in dioxane (15 mL) and water (5 mL). [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (191.79 mg, 262.11 μmol) and potassium carbonate (1.45 g, 10.48 mmol) were added. The atmosphere was replaced with argon for 5 minutes, and the temperature was raised to 90°C and stirred for 3 hours. The reaction mixture was cooled to room temperature, and water (20 mL) was added. The mixture was extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (eluent: System A) to give 3-(2-fluoro-4-nitro-phenyl)-1-methyl-1,2,4-triazole 45b (0.6 g) in a yield of 51.5%.

[0581] MS m / z(ESI):223.1[M+1]

[0582] Step 2

[0583] 2-(1-methyl-1H-1,2,4-triazol-3-yl)-5-nitrophenol

[0584] 2-(1-Methyl-1H-1,2,4-triazol-3-yl)-5-nitrophenol

[0585] To a solution of 3-(2-fluoro-4-nitrophenyl)-1-methyl-1,2,4-triazole 45b (300 mg, 1.35 mmol) in dimethyl sulfoxide (5 mL) was added a solution of sodium hydroxide (270.04 mg, 6.75 mmol) in water (1 mL). The mixture was heated to 100°C and stirred for 18 hours. Water (20 mL) was added to the reaction solution, and the pH was adjusted to 3-4 with 1M hydrochloric acid. The reaction solution was extracted with ethyl acetate (30 mL x 2). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford 2-(1-methyl-1H-1,2,4-triazol-3-yl)-5-nitrophenol 45c (0.28 g) in a 94.2% yield.

[0586] MS m / z(ESI):221.1[M+1]

[0587] Step 3

[0588] 3-(2-(3,3-difluorocyclobutoxy)-4-nitrophenyl)-1-methyl-1H-1,2,4-triazole

[0589] 3-(2-(3,3-difluorocyclobutyloxy)-4-nitrophenyl)-1-methyl-1H-1,2,4-triazole

[0590] To a solution of 2-(1-methyl-1H-1,2,4-triazol-3-yl)-5-nitrophenol 45c (300 mg, 1.36 mmol) in N,N-dimethyldimethylamine (10 mL) was added cesium carbonate (1.33 g, 4.09 mmol) and 3,3-difluorocyclobutyl trifluoromethanesulfonate 45d (325 mg, 1.36 mmol, prepared according to the published patent "WO2020176765") at room temperature and stirred at room temperature for 2 h. Water (30 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 3-(2-(3,3-difluorocyclobutyloxy)-4-nitrophenyl)-1-methyl-1H-1,2,4-triazole 45e (0.4 g), which was used directly in the next reaction.

[0591] MS m / z(ESI):311.1[M+1]

[0592] Step 4

[0593] 3-(3,3-difluorocyclobutoxy)-4-(1-methyl-1H-1,2,4-triazol-3-yl)aniline

[0594] 3-(3,3-difluorocyclobutyloxy)-4-(1-methyl-1H-1,2,4-triazol-3-yl)aniline

[0595] To a solution of 3-(2-(3,3-difluorocyclobutyloxy)-4-nitrophenyl)-1-methyl-1H-1,2,4-triazole 45e (400 mg, 1.29 mmol) in ethanol (10 mL) was added a solution of ammonium chloride (482.75 mg, 9.02 mmol) in water (2 mL) at room temperature. Iron powder (359.99 mg, 6.45 mmol) was then added. The atmosphere was replaced with argon three times, and the temperature was raised to 80°C and stirred for 2 h. The mixture was cooled to room temperature, tetrahydrofuran (30 mL) was added, stirred for 30 minutes, filtered, and concentrated under reduced pressure. Water (30 mL) was added to the residue, and the pH was adjusted to 7-8 with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 3-(3,3-difluorocyclobutyloxy)-4-(1-methyl-1H-1,2,4-triazol-3-yl)aniline 45f (0.35 g) in a yield of 96.8%.

[0596] MS m / z(ESI):281.1[M+1]

[0597] Step 5

[0598] N-(3-(3,3-difluorocyclobutoxy)-4-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0599] N-(3-(3,3-difluorocyclobutyloxy)-4-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0600] To a solution of 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (100 mg, 295.49 μmol) in acetonitrile (5 mL) were added 1-methylimidazole (72.78 mg, 886.47 μmol) and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (248.72 mg, 886.47 μmol) at room temperature. The mixture was stirred at room temperature for 1 hour. 3-(3,3-difluorocyclobutyloxy)-4-(1-methyl-1H-1,2,4-triazol-3-yl)aniline 45f (107.66 mg, 384.14 μmol) was added, and the temperature was raised to 70°C for 1 hour. After the reaction solution was concentrated, the crude product was subjected to preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% NH4HCO3+H2O, mobile phase B: CH3CN) to obtain N-(3-(3,3-difluorocyclobutyloxy)-4-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 45 (100.6 mg) with a yield of 53.9%.

[0601] MS m / z(ESI):601.3[M+1]

[0602] 1H NMR (400MHz, DMSO-d6) δ11.65(s,1H),10.17(s,1H),8.64(s,1H),7.83(d,J=8.5Hz,1H), 7.80(d,J=8.4Hz,1H),7.61(dd,J=8.5,1.9Hz,1H),7.37(d,J=1.9Hz,1H),7.18(d,J=2.1 Hz,1H),7.05(dd,J=8.5,2.1Hz,1H),4.78(s,1H),3.93(s,3H),3.21(t,J=7.7Hz,4H),2. 99(t,J=5.2Hz,4H),2.87–2.72(m,2H),1.55(s,4H),1.21(t,J=7.3Hz,3H),0.35(s,4H).

[0603] Example 46 was synthesized according to the synthesis method of Example 45 of the present invention. The structure and characterization data are shown in the following table:

[0604] Example 47

[0605] 4-(ethylsulfonamido)-N-(4-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(2,2,2-trifluoroethoxy)phenyl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0606] 4-(Ethylsulfonamido)-N-(4-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(2,2,2-trifluoroethoxy)phenyl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0607] first step

[0608] 1-methyl-3-(4-nitro-2-(2,2,2-trifluoroethoxy)phenyl)-1H-1,2,4-triazole

[0609] 1-Methyl-3-(4-nitro-2-(2,2,2-trifluoroethoxy)phenyl)-1H-1,2,4-triazole

[0610] At room temperature, 2,2,2-trifluoroethanol (175.61 mg, 1.76 mmol) was dissolved in N,N-dimethylformamide (10 mL), cooled to 0°C, and sodium hydride (108.01 mg, 2.70 mmol, 60% purity) was added. The mixture was stirred at 0°C for 30 minutes, and 3-(2-fluoro-4-nitro-phenyl)-1-methyl-1,2,4-triazole 45b (300 mg, 1.35 mmol) was added. The mixture was stirred at room temperature for 1 hour. The reaction was quenched by the addition of water (20 mL) and extracted with dichloromethane (30 mL × 2). The combined organic phases were washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford 1-methyl-3-(4-nitro-2-(2,2,2-trifluoroethoxy)phenyl)-1H-1,2,4-triazole 47a (0.38 g) in a 93.1% yield.

[0611] MS m / z(ESI):303.1[M+1]

[0612] Step 2

[0613] 4-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(2,2,2-trifluoroethoxy)aniline

[0614] 4-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(2,2,2-trifluoroethoxy)aniline

[0615] At room temperature, 1-methyl-3-(4-nitro-2-(2,2,2-trifluoroethoxy)phenyl)-1H-1,2,4-triazole 47a (350 mg, 1.16 mmol) was dissolved in ethanol (10 mL). A solution of ammonium chloride (433.65 mg, 8.11 mmol) in water (2 mL) was added, followed by the addition of iron powder (323.38 mg, 5.79 mmol). The atmosphere was replaced with argon, and the temperature was raised to 80°C. The mixture was stirred for 3 hours. The temperature was cooled to room temperature, tetrahydrofuran (30 mL) was added to the reaction solution, and the mixture was stirred for 30 minutes, filtered, and concentrated under reduced pressure. Water (20 mL) was added, and the pH was adjusted to 7-8 with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate (30 mL×3). The combined organic phases were washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 4-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(2,2,2-trifluoroethoxy)aniline 47b (0.3 g) in a yield of 95.1%.

[0616] MS m / z(ESI):273.1[M+1]

[0617] Step 3

[0618] 4-(ethylsulfonamido)-N-(4-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(2,2,2-trifluoroethoxy)phenyl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0619] 4-(Ethylsulfonamido)-N-(4-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(2,2,2-trifluoroethoxy)phenyl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0620] To a solution of 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (280 mg, 827.37 μmol) in acetonitrile (5 mL) were added 1-methylimidazole (203.79 mg, 2.48 mmol) and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (696.43 mg, 2.48 mmol) at room temperature. The mixture was stirred at room temperature for 1 hour. 4-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(2,2,2-trifluoroethoxy)aniline 47b (292.80 mg, 1.08 mmol) was added, and the temperature was raised to 80°C and stirred for 2 hours. After the reaction solution was concentrated, the crude product was separated by preparative liquid separation (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% NH4HCO3+H2O, mobile phase B: CH3CN) to obtain 4-(ethylsulfonyl)-N-(4-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(2,2,2-trifluoroethoxy)phenyl)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 47 (288 mg) with a yield of 56.1%.

[0621] MS m / z(ESI):593.2[M+1]

[0622] 1H NMR(400MHz,DMSO-d6)δ11.72(s,1H),10.18(s,1H),8.51(s,1H),7.89-7.80(m ,2H),7.77(d,J=2.0Hz,1H),7.51(dd,J=8.6,1.9Hz,1H),7.18(d,J=2.1Hz,1H), 7.05(dd,J=8.5,2.0Hz,1H),4.72(q,J=9.0Hz,2H),3.91(s,3H),3.21(q,J=7.3 Hz,2H),2.98(t,J=5.1Hz,4H),1.54(s,4H),1.21(t,J=7.3Hz,3H),0.35(s,4H).

[0623] Examples 48-54 were synthesized according to the synthesis method of Examples 45 and 47 of the present invention, and their structures and characterization data are shown in the following table:

[0624] Example 55

[0625] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yloxy)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0626] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yloxy)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0627] first step

[0628] 2-(2-bromo-4-nitrophenoxy)pyridine

[0629] 2-(2-Bromo-4-nitrophenoxy)pyridine

[0630] To a solution of 1H-pyridin-2-one 55a (358.66 mg, 3.77 mmol, commercially available) in N,N-dimethylformamide (10 mL) at 0°C, potassium tert-butoxide (529.00 mg, 4.71 mmol) and 2-bromo-1-fluoro-4-nitrobenzene 21a (500 mg, 3.14 mmol) were added. The atmosphere was purged with nitrogen three times and the temperature was slowly raised to room temperature for 18 hours. The mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (eluent: System A) to give 1-(2-bromo-4-nitrophenyl)pyridin-2(1H)-one 56a (200 mg) in a yield of 27.2% and 2-(2-bromo-4-nitrophenoxy)pyridine 55b (300 mg, 1.28 mmol, 40.76% yield) in a yield of 40.8%.

[0631] MS m / z(ESI):297.0[M+1]

[0632] HNMR of 56a

[0633] 1 H NMR (400MHz, DMSO-d6) δ8.64(d,J=2.8Hz,1H),8.41–8.33(m,1H),7.83(d,J=8.4Hz,1H),7.62–7.56(m,2H),6.58–6.50(m,1H),6.43–6.38(m,1H).

[0634] HNMR of 55b

[0635] 1 H NMR (400MHz, DMSO-d6) δ8.57(d,J=2.8Hz,1H),8.32–8.26(m,1H),8.18–8.13(m,1H),7.99–7.94(m,1H),7.51(d,J=8.8Hz,1H),7.28–7.21(m,2H).

[0636] Step 2

[0637] 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenoxy)pyridine

[0638] 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenoxy)pyridine

[0639] To a solution of 2-(2-bromo-4-nitrophenoxy)pyridine 55b (300 mg, 1.02 mmol) and 4,4-difluoropiperidine 1b (492.58 mg, 4.07 mmol) in 1,4-dioxane (3 mL) were added palladium acetate (22.82 mg, 101.66 μmol), 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (117.65 mg, 203.33 μmol), and cesium carbonate (662.49 mg, 2.03 mmol) at room temperature. The atmosphere was purged with nitrogen three times and the reaction was continued at 100°C for 18 hours. The reaction solution was concentrated under reduced pressure, and the crude product was isolated and purified by silica gel column chromatography (eluent: System A) to give 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenoxy)pyridine 55c (130 mg) in a 38.1% yield.

[0640] MS m / z(ESI):336.0[M+1]

[0641] Step 3

[0642] 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yloxy)aniline

[0643] 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yloxy)aniline

[0644] Palladium on carbon (10%) (47.61 mg, 447.35 μmol) was added to a solution of 2-(2-(4,4-difluoropiperidin-1-yl)-4-nitrophenoxy)pyridine 55c (150 mg, 447.35 μmol) in methanol (3 mL) at room temperature. The mixture was purged with a hydrogen balloon three times and then reacted at 25°C for 2 hours. The reaction mixture was filtered, the filter cake was rinsed with methanol (10 mL), and the filtrate was concentrated under reduced pressure to afford 3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yloxy)aniline 55d (130 mg) in a 95.2% yield.

[0645] MS m / z(ESI):306.2[M+1]

[0646] Step 4

[0647] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yloxy)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0648] N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yloxy)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0649] 3-(4,4-Difluoropiperidin-1-yl)-4-(pyridin-2-yloxy)aniline 55d (130 mg, 425.78 μmol) and 4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzoic acid 7a (172.91 mg, 510.94 μmol) were added to N,N-dimethylformamide (3 mL) at room temperature, followed by (7-azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate (333.00 mg, 638.67 μmol) and N,N-diisopropylethylamine (110.06 mg, 851.56 μmol). The atmosphere was purged with nitrogen three times and the reaction was continued at 65°C for 18 hours. The mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude product was separated by preparative liquid chromatography (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% TFA+H2O, mobile phase B: CH3CN) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(pyridin-2-yloxy)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 55 (130 mg) in a yield of 48.8%.

[0650] MS m / z(ESI):626.2[M+1]

[0651] 1 H NMR (400MHz, DMSO-d6) δ11.75(s,1H),10.16(s,1H),8.14–8.11(m,1H),7.85(d,J=8.4Hz,1H),7.83– 7.78(m,1H),7.63(d,J=2.4Hz,1H),7.43–7.39(m,1H),7.19(d,J=2.0Hz,1H),7.13(d,J=8.8Hz,1H), 7.11–7.08(m,1H),7.07–7.04(m,1H),6.94(d,J=8.0Hz,1H),3.24–3.17(m,2H),3.07–3.03(m,4H),3 .01–2.96(t,J=5.3Hz,4H),1.83–1.73(m,4H),1.61–1.54(m,4H),1.21(t,J=7.2Hz,3H),0.36(s,4H).

[0652] Example 56 was synthesized according to the synthesis method of Example 55 of the present invention. The structure and characterization data are shown in the following table:

[0653] Example 57

[0654] N-(3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0655] N-(3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0656] first step

[0657] 2-bromo-1-(methoxy-d3)-4-nitrobenzene

[0658] 2-Bromo-1-(methoxy-d3)-4-nitrobenzene

[0659] 2-Bromo-4-nitrophenol 57a (1.0 g, 4.59 mmol, commercially available) and potassium carbonate (1.27 g, 9.17 mmol) were dissolved in N,N-dimethylformamide (20 mL) at room temperature. Triiodomethane (997.39 mg, 6.88 mmol) was then added and stirred at room temperature for 6 hours. After concentration to dryness, the crude product was isolated and purified by silica gel column chromatography (eluent: System A) to afford 2-bromo-1-(methoxy-d3)-4-nitrobenzene 57b (1.05 g) in a 97.4% yield.

[0660] MS m / z(ESI):234.8[M+1]

[0661] Step 2

[0662] 4,4-difluoro-1-(2-(methoxy-d3)-5-nitrophenyl)piperidine

[0663] 4,4-Difluoro-1-(2-(methoxy-d3)-5-nitrophenyl)piperidine

[0664] At room temperature, 2-bromo-1-(methoxy-d3)-4-nitrobenzene 57b (1.0 g, 4.25 mmol) and 4,4-difluoropiperidine (772.99 mg, 6.38 mmol) were dissolved in toluene (20 mL), followed by the addition of tris(dibenzylideneacetone)dipalladium (194.79 mg, 212.72 μmol), R-(+)-1,1'-binaphthyl-2,2'-bisdiphenylphosphine (264.91 mg, 425.44 μmol) and sodium tert-butoxide (1.23 g, 12.76 mmol). The atmosphere was replaced with argon for 5 minutes, the temperature was raised to 100°C, and the reaction was stirred for 18 hours. Water (20 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (eluent: System A) to give 4,4-difluoro-1-(2-(methoxy-d3)-5-nitrophenyl)piperidine 57c (0.87 g) in a yield of 74.3%.

[0665] MS m / z(ESI):276.1[M+1]

[0666] Step 3

[0667] 3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)aniline

[0668] 3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)aniline

[0669] 4,4-Difluoro-1-(2-(methoxy-d3)-5-nitrophenyl)piperidine 57c (870 mg, 3.16 mmol) was dissolved in methanol (50 mL) at room temperature. 10% palladium on carbon (336.35 mg, 3.16 mmol) was then added. The atmosphere was replaced with hydrogen three times, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered under reduced pressure, and the filtrate was concentrated to afford 3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)aniline 57d (0.75 g) in a 96.7% yield.

[0670] MS m / z(ESI):245.9[M+1]

[0671] Step 4

[0672] N-(3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)phenyl)-4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0673] N-(3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)phenyl)-4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0674] At room temperature, 2-(6-azaspiro[2.5]octan-6-yl)-4-iodo-benzoic acid 1e (780 mg, 2.18 mmol) was dissolved in acetonitrile (50 mL), followed by the addition of 1-methylimidazole (537.88 mg, 6.55 mmol) and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (1.84 g, 6.55 mmol). The mixture was stirred for 1 hour, and then 3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)aniline 57d (696.32 mg, 2.84 mmol) was added. The temperature was raised to 60°C and stirred for 3 hours. The reaction mixture was concentrated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography (eluent: System A) to give N-(3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)phenyl)-4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamide 57e (0.7 g) in a yield of 54.8%.

[0675] MS m / z(ESI):585.2[M+1]

[0676] Step 5

[0677] N-(3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0678] N-(3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0679] At room temperature, N-(3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)phenyl)-4-iodo-2-(6-azaspiro[2.5]octan-6-yl)benzamide 57e (700 mg, 1.20 mmol) and ethanesulfonamide 1 g (196.09 mg, 1.80 mmol) were dissolved in N,N-dimethylformamide (5 mL), followed by the addition of cuprous iodide (114.05 mg, 598.85 μmol), 2-(methylamino)acetic acid (106.71 mg, 1.20 mmol) and tripotassium phosphate trihydrate (956.89 mg, 3.59 mmol). The atmosphere was replaced with argon three times, the temperature was raised to 110°C, and the mixture was stirred for 6 h. After the reaction solution was concentrated, it was separated by preparative liquid phase (separation column AKZONOBEL Kromasil; 250×21.2 mm ID; 5 μm, 20 mL / min; mobile phase A: 0.05% NH4CO3+H2O, mobile phase B: CH3CN) to obtain N-(3-(4,4-difluoropiperidin-1-yl)-4-(methoxy-d3)phenyl)-4-(ethylsulfonamido)-2-(6-azaspiro[2.5]octane-6-yl)benzamide 57 (360 mg) with a yield of 52.2%.

[0680] MS m / z(ESI):566.3[M+1]

[0681] 1 H NMR (400MHz, DMSO-d6) δ11.60(s,1H),10.13(s,1H),7.83(d,J=8.4Hz,1H),7.43(d,J =2.5Hz,1H),7.37(dd,J=8.7,2.4Hz,1H),7.17(d,J=2.2Hz,1H),7.04(dd,J=8.5,2.1H z,1H),6.96(d,J=8.6Hz,1H),3.19(q,J=7.3Hz,2H),3.10(t,J=5.9Hz,4H),2.96(t,J= 5.4Hz,4H),2.16-2.05(m,4H),1.60-1.47(m,4H),1.21(t,J=7.3Hz,3H),0.35(s,4H).

[0682] Example 58 was synthesized according to the synthesis method of Example 57 of the present invention. The structure and characterization data are shown in the following table:

[0683] Biological evaluation

[0684] Test Example 1: Determination of the Inhibitory Effect of the Compounds of the Invention on OVCAR-3 Cell Proliferation

[0685] The following method was used to determine the effect of the compounds of the present invention on the proliferation of OVCAR-3 cells. OVCAR-3 cells (containing the TP53 R248Q mutation) were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U penicillin, and 100 μg / mL streptomycin. Cell viability was measured by The assay was performed using the Luminescent Cell Viability Assay Kit (Promega, Cat. No. G7573).

[0686] The experimental method was performed according to the steps in the kit instructions, which are briefly described as follows: the test compound was first dissolved in DMSO to prepare a 10mM stock solution, and then diluted with culture medium to prepare the test sample. The final concentration of the compound ranged from 1000nM to 0.015nM. Cells in the logarithmic growth phase were seeded into 96-well cell culture plates at a density of 1000 cells per well and cultured overnight in a 37°C, 5% CO2 incubator. The test compound was then added and cultured for another 72 hours. After the culture was completed, 50uL of CellTiter-Glo detection solution was added to each well, shaken for 5 minutes, and then allowed to stand for 10 minutes. The luminescence value of each sample well was then read on a microplate reader using the Luminescence mode. The percentage inhibition rate of the compound at each concentration point was calculated by comparing it with the value of the control group (0.3% DMSO). Nonlinear regression analysis was then performed in GraphPad Prism 5 software using the logarithm of the compound concentration-inhibition rate to obtain the IC value of the compound for inhibiting cell proliferation. 50 Values ​​are shown in Table 1.

[0687] Table 1 IC of the compounds of the present invention on the inhibition of OVCAR-3 cell proliferation 50 data

[0688] Conclusion: The compounds of the present invention have an IC inhibitory effect on the proliferation of OVCAR-3 cells. 50 <50nM, showing good inhibitory effect.

[0689] Note: The structure of AMG 650 (prepared according to Example 4 of patent publication WO2020132648A1) is as follows:

[0690] Test Example 2: Determination of the Inhibitory Effect of the Compounds of the Invention on HT-29 Cell Proliferation

[0691] The following method was used to determine the effect of the compounds of the present invention on HT-29 cell proliferation. HT-29 cells (containing the TP53R273H mutation) were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured in McCoy's 5A medium containing 10% fetal bovine serum, 100 U penicillin, and 100 μg / mL streptomycin. Cell viability was measured by The assay was performed using the Luminescent Cell Viability Assay Kit (Promega, Cat. No. G7573).

[0692] The experimental method was performed according to the steps in the kit instructions and is briefly described as follows: the test compound was first dissolved in DMSO to prepare a 10mM stock solution, and then diluted with culture medium to prepare the test sample. The final concentration of the compound ranged from 1000nM to 0.015nM. Cells in the logarithmic growth phase were seeded into 96-well cell culture plates at a density of 1000 cells per well and cultured overnight in a 37°C, 5% CO2 incubator. The test compound was then added and cultured for another 120 hours. After the incubation period, 50uL of CellTiter-Glo detection solution was added to each well, shaken for 5 minutes, and then allowed to stand for 10 minutes. The luminescence value of each sample well was then read on a microplate reader using the Luminescence mode. The percentage inhibition rate of the compound at each concentration point was calculated by comparing it with the value of the control group (0.3% DMSO). Nonlinear regression analysis was then performed in GraphPad Prism 5 software using the logarithm of the compound concentration-inhibition rate to obtain the IC value of the compound for inhibiting cell proliferation. 50 Values ​​are shown in Table 2.

[0693] Table 2 IC of the compounds of the present invention on the inhibition of HT-29 cell proliferation 50 data

[0694] Conclusion: The compounds of the present invention have an inhibitory effect on the proliferation of HT-29 cells. 50 <50nM, showing good inhibitory effect.

[0695] Test Example 3: Test of the Inhibition of KIF18A Enzyme Activity by the Compounds of the Invention

[0696] The following method is used to determine the degree of inhibition of the compound of the present invention on the activity of recombinant human KIF18A enzyme under in vitro conditions. This method uses Promega's ADP-Glo TM Kinase Assay Kit (Cat. No. V9102). Detailed experimental procedures can be found in the kit instructions.

[0697] The experimental process is briefly described as follows: the test compound is first dissolved in DMSO to prepare a stock solution, and then serially diluted using reaction buffer A (15mM Tris, pH 7.5, 10mM MgCl2, 0.01% Pluronic F-68). The final concentration of the test compound in the reaction system ranges from 10000nM to 0.15nM; KIF18A protein and ATP working solution are prepared using reaction buffer B (15mM Tris, pH 7.5, 10mM MgCl2, 0.01% Pluronic F-68, 37.5μg / ml tubulin, 1.25μM paclitaxel). The reaction is carried out in a 384-well microplate. First, the test compound and recombinant human KIF18A protein (final concentration 100nM, expressed by GenScript) are added to the wells and incubated at room temperature for 20 minutes. Then, ATP solution (from ADP-Glo) is added to the reaction solution. TM Kinase Assay kit component V915A, final concentration 60 μM) and incubate at room temperature for 20 minutes. Subsequently, 5 μL ADP-Glo ​​Reagent was added to the reaction system and incubated at room temperature for 50 minutes. Thereafter, 10 μL Kinase Detection Reagent was added to the reaction system and incubated at room temperature for 30 minutes. After the incubation, the chemiluminescence intensity value of each well was measured in the luminescence mode on the microplate reader. The percentage inhibition rate of the compound at each concentration was calculated by comparing with the luminescence intensity value of the control group (0.1% DMSO), and the nonlinear regression analysis of the compound concentration-inhibition rate was performed using GraphPad Prism 5 software to obtain the IC value of the compound. 50 Values ​​are shown in Table 3.

[0698] Table 3 IC values ​​of the compounds of the present invention for inhibiting KIF18A enzyme activity 50 data

[0699] Conclusion: The compounds of the present invention have an IC inhibitory effect on KIF18A enzyme activity. 50 <200nM, with significant inhibitory effect.

[0700] Test Example 4: Pharmacokinetics of the Compounds of the Invention in Mice

[0701] 1. Experimental Purpose

[0702] ICR mice were used as test animals, and the LC / MS / MS method was used to determine the drug concentrations in plasma at different times after oral administration of the control compound AMG650 and the present compounds 47 and 53 to study the pharmacokinetic characteristics of the present compounds in mice.

[0703] 2. Experimental plan

[0704] 2.1 Experimental drugs and animals;

[0705] Reference compounds AMG650, compounds 47 and 53.

[0706] ICR mice, male, 20-22 g, were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd.

[0707] 2.2 Drug preparation

[0708] An appropriate amount of the test compound was weighed, and appropriate amounts of DMA, 30% HS-15, and Saline were added in sequence. The mixture was ultrasonically vortexed to mix well to prepare a 1 mg / mL dosing formulation, wherein DMSO:30% HS-15:Saline = 5:5:90 (v:v:v).

[0709] 2.3 Administration

[0710] ICR mice in each test compound injection group (9 mice in each group) were fasted overnight and then orally administered (PO, compound dosage: 10 mg / kg, administration volume: 10 mL / kg), and were fed 4 hours after administration.

[0711] 3. Operation

[0712] Approximately 0.1 mL of blood was collected intraorbitally before dosing and at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing. Whole blood samples were placed in tubes containing EDTA-K2 for anticoagulation. After collection, the blood samples were placed on ice and centrifuged at 1500 g for 10 minutes to separate the plasma. The collected plasma was stored at –40 to –20°C until analysis.

[0713] The levels of the test compounds in mouse plasma after oral administration were determined by LC-MS / MS.

[0714] 4. Pharmacokinetic parameter results

[0715] The pharmacokinetic parameters of the test compounds are shown in Table 4 below.

[0716] Table 4 Pharmacokinetic parameters of the test compounds in mice

[0717] Conclusion: Compounds 47 and 53 of the present invention have high plasma concentrations and areas under the curve, and have good pharmacokinetic properties.

[0718] Test Example 5: Efficacy test of compounds 47 and 53 of the present invention in mice

[0719] 1. Purpose of the experiment

[0720] The anti-tumor effects and safety of compounds 47 and 53 of the present invention were evaluated in the OVCAR-3 human ovarian adenocarcinoma tumor subcutaneously xenografted BALB / c nude mouse animal model.

[0721] 2. Experimental Animals

[0722] BALB / c nude mice, female, 6-7 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0723] 3. Preparation of test substances

[0724] The vehicle control group was given DMA:CrEL:5% GS=10:10:80 (v:v:v).

[0725] AMG650: Weigh an appropriate amount of AMG-650, add appropriate amounts of DMA, CrEL, and 5% GS in that order, mix thoroughly by ultrasonic vortexing, and prepare a 0.8 mg / mL dosing formulation with DMA, CrEL, and 5% GS in a ratio of 10:10:80 (v:v:v).

[0726] Compound 47: An appropriate amount of compound 47 was weighed, and appropriate amounts of DMA, CrEL, and 5% GS were added in sequence. The mixture was mixed by ultrasonic vortexing to prepare a 0.8 mg / mL dosing formulation, wherein DMA, CrEL, and 5% GS were in a ratio of 10:10:80 (v:v:v).

[0727] Compound 53: An appropriate amount of compound 53 was weighed, and appropriate amounts of DMA, CrEL, and 5% GS were added in sequence. The mixture was mixed by ultrasonic vortexing to prepare a 0.8 mg / mL dosing formulation, wherein DMA, CrEL, and 5% GS were in a ratio of 10:10:80 (v:v:v).

[0728] 4. Obtaining Tumor Mass

[0729] Select appropriate OVCAR-3 tumor-bearing animals, aseptically obtain samples, obtain 1mm*1mm*1mm OVCAR-3 tumor masses, and place them in normal saline for use in subcutaneous tumor inoculation on the right side of the back of BALB / c nude mice.

[0730] 5. Animal Inoculation and Grouping

[0731] Female BALB / c nude mice were subcutaneously inoculated with OVCAR-3 tumors measuring approximately 1 mm*1 mm*1 mm on the right side of their backs. 3 The mice were randomly divided into groups according to tumor size, with 6 mice in each group.

[0732] 6. Animal Medication and Observation

[0733] Each group of animals was given the test substance once a day at a fixed time according to the animal body weight, orally (po), starting with the first dose on the day of grouping for 35 consecutive days, and the animal body weight was recorded every day.

[0734] Group 1 (G1), solvent control group;

[0735] Groups 2-4 (G2-G3) were orally administered with AMG-650, compound 47, and compound 53, respectively, at a dose of 8 mg / kg, once a day (QD);

[0736] The formation of tumors at the inoculation site of each group of animals was observed, and the tumor volume was measured twice a week and calculated according to the following formula:

[0737] The calculation formulas for tumor volume (TV), relative tumor volume (RTV), relative tumor proliferation rate (T / C) and relative tumor inhibition rate (TGI) are as follows:

[0738] (1)TV(tumor volume)=1 / 2×a×b 2 , where a and b represent the length and width of the tumor, respectively;

[0739] (2)RTV(relative tumor volume)=V t / V0, V0 is the tumor volume measured when grouping, V t is the tumor volume at each measurement;

[0740] (3) T / C (%) = T RTV / C RTV × 100%, where T RTV is the RTV of the treatment group, C RTV is the RTV of the vehicle control group;

[0741] (4) TGI% = (1-T / C) × 100%; where T and C are the relative tumor volumes of the treatment group and the vehicle control group at a specific time point, respectively.

[0742] 7. Results

[0743] Table 5 Pharmacodynamic analysis of the test compounds in each group in the OVCAR-3 human ovarian adenocarcinoma tumor model Note: 1. Data are expressed as "mean ± standard error";

[0744] Conclusion: Under the conditions set in this experiment, in the OVCAR-3 human ovarian adenocarcinoma tumor subcutaneously xenograft BALB / c nude mouse animal model, compared with the solvent control group and AMG650, compounds 47 and 53 of the present invention showed more significant tumor growth inhibition effects at 8 mg / kg.

Claims

1. A compound represented by general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof: in: R A Select from -OR B 、-NR B R C 、-S(=O) r R B 、-C(=O)R 5 、-C(=O)OR 5 、-NHC(=O)R 5 、-NHC(=O)OR 5 、-C(=O)NR 6 R 7 、-CH2OR 5 、-CH2NR 6 R 7 , 5-10 membered aryl, 5-10 membered heteroaryl or 6-14 membered fused ring, wherein the aryl, heteroaryl or fused ring is optionally further substituted by one or more R a replace; R B are the same or different and are independently selected from deuterated alkyl, 5-10 membered aryl, 5-10 membered heteroaryl or 6-14 membered fused ring, wherein the aryl, heteroaryl or fused ring is optionally further substituted by one or more R a replace; R C is selected from a hydrogen atom or an alkyl group; R a are the same or different and are each independently selected from halogen, hydroxy, cyano, alkyl, cycloalkyl or alkoxy; wherein the alkyl, cycloalkyl or alkoxy is optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, alkyl or alkoxy; R 1 is selected from hydrogen, cyano, halogen, alkyl, hydroxyl, cycloalkyl, heterocyclic, aryl, heteroaryl, -OR 5 、-C(=O)R 5 、-C(=O)OR 5 、-NHC(=O)R 5 、-NHC(=O)OR 5 、-NR 6 R 7 、-C(=O)NR 6 R 7 、-CH2NHC(=O)OR 5 、-CH2NR 6 R 7 or -S(=O) r R 5 wherein the alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent; L1 is selected from a bond, -C 1-6 Alkylene-, -C 0-4 Alkylene-NR c S(=O)(=NH)-C 0-4 Alkylene-, -C 0-4 Alkylene-SC 0-4 Alkylene-, -C 0-4 Alkylene-S(=O)-C 0-4 Alkylene-, -C 0-4 Alkylene-SO2-C 0-4 Alkylene-, -C 0-4 Alkylene-S(=O)(=NH)-C 0-4 Alkylene-, -C 0-4 Alkylene-NR c SO2-C 0-4 Alkylene-, -C 0-4 Alkylene-SO2NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-OC 0-4 Alkylene-, -C 0-4 Alkylene-NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-NR c SO2NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-NR c C(O)NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-C(O)NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-NR c C(O)-C 0-4 Alkylene-, -C 0-4 Alkylene-PC 0-4 Alkylene-, -C 0-4 Alkylene-P(=O)2-C 0-4 Alkylene, -C 0-4 Alkylene-C(=O)-C 0-4 Alkylene- or -C 0-4 Alkylene-C(=N(OH))-C 0-4 Alkylene-, wherein the -C 1-6 Alkylene- or -C 0-4 Alkylene - optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, cycloalkyl or alkoxy; R c is selected from a hydrogen atom or an alkyl group; R 2 are the same or different and are each independently selected from halogen, hydroxy, cyano, alkyl, cycloalkyl or alkoxy; wherein the alkyl, cycloalkyl or alkoxy group is optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, alkyl or alkoxy; L2 is selected from in Indicates that the group and the general formula (I) The attachment site of Indicates that the group and the general formula (I) The attachment site of R 3 are each independently selected from a hydrogen atom or an alkyl group, wherein the alkyl group is optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano or alkoxy; R 3 Preferably a hydrogen atom; R 4 is selected from cyano, halogen, alkyl, hydroxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR 5 、-C(=O)R 5 、-C(=O)OR 5 、-NHC(=O)R 5 、-NHC(=O)OR 5 、-NR 6 R 7 、-C(=O)NR 6 R 7 、-CH2NHC(=O)OR 5 、-CH2NR 6 R 7 or -S(O) r R 5 wherein the alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent; R 5 Each is independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the heterocyclic group, the aryl group or the heteroaryl group is optionally further substituted by one or more selected from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyl, heterocyclic group, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent; R 6 and R 7 Each is independently selected from hydrogen, hydroxy, halogen, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl or heteroaryl, wherein the alkyl, alkoxy, cycloalkyl, heterocyclic, aryl or heteroaryl is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent; Or, R 6 and R 7 Together with the atoms to which they are attached, they form a 4- to 8-membered heterocyclic group, wherein the 4- to 8-membered heterocyclic group contains one or more N, O or S(O)r, and the 4- to 8-membered heterocyclic group is optionally further substituted by one or more selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclic group, aryl, heteroaryl, =O, -C(=O)R 8 、-C(=O)OR 8 、-OC(=O)R 8 、-NR 9 R 10 、-C(=O)NR 9 R 10 、-SO2NR 9 R 10 or -NR 9 C(=O)R 10 substituted by a substituent; R 8 , R 9 and R 10 Each is independently selected from a hydrogen atom, an alkyl group, an amino group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the heterocyclic group, the aryl group or the heteroaryl group is optionally further substituted by one or more substituted by a substituent selected from hydroxy, halogen, nitro, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyl or carboxylate; m is 0, 1 or 2; and r is independently 0, 1 or 2.

2. The compound according to claim 1 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, which is a compound or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof described by general formula (II): in: Ring A is selected from a 5- to 10-membered aryl group, a 5- to 10-membered heteroaryl group, or a 6- to 14-membered fused ring; R a are the same or different, each independently selected from halogen, hydroxy, cyano, C1-C3 alkyl, cyclopropyl or methoxy; n is 0, 1 or 2; L1, L2, R 1 , R 2 , R 4 and m are as defined in claim 1.

3. The compound according to claim 1 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein R A Selected from -OCD3, -C(=O)OH, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)CH3, -CH2OH, -CH2OCH3, 4 . The compound according to claim 2 , or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein n is 0 or 1.

5. The compound according to claim 2 or 4, or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein Selected from the following groups: 6 . The compound according to claim 1 , or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein m is 0.

7. The compound according to any one of claims 1 to 3, or its stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein: L1 is selected from a bond, -NR c -C 0-4 Alkylene-, -NR c SO2-C 0-4 Alkylene-, -SO2NR c -C 0-4 Alkylene-, -NR c SO2NR c -, -S(=O)(=NH)-, -NR c S(=O)(=NH)-、-C 1-4 Alkylene-, -S(=O)-, -O-, -C(=O)-, -C(=O)NR c -C 0-4 Alkylene-, -C 0-4 Alkylene-SO2-C 0-4 Alkylene-, -C=N(OH)- or -NR c -C(=O)-, wherein the -C 0-4 Alkylene- or -C 1-4 Alkylene - optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, cyclopropyl or methoxy; R c Each is independently selected from a hydrogen atom or a methyl group.

8. The compound according to claim 7 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein L1 is selected from a bond, -NHSO2CH2CH2-, -SO2NHCH2CH2-, -SO2-, -CH2SO2-, -NHSO2-, -SO2NH-, -NHC(CH3)2CH2-, -C(=O)NHCH2CH2-, -C(=O)NHC(CH3)2CH2-, -C(=O)N(CH3)CH2CH2-, -CH(CH3)(OH)CH2-, -NHSO2CH(CH3)CH2-, -SO2NHC(CH3)2CH2-, -C(=O)NH-, -NHCH2CH2 or -CH2SO2CH2CH2-.

9. The compound according to any one of claims 1 to 3, or its stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein R 1 is selected from a hydrogen atom, a hydroxyl group, an alkyl group, a heterocyclic group, a cycloalkyl group or a heteroaryl group, wherein the alkyl group, the heterocyclic group, the cycloalkyl group or the heteroaryl group is optionally further substituted by one or more substituents selected from a hydroxyl group or an alkyl group.

10. The compound according to any one of claims 1 to 3, or its stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein for 11. The compound according to any one of claims 1 to 3, or its stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein L2 is selected from in Indicates that the group and the general formula (I) The attachment site of Indicates that the group and the general formula (I) The attachment site of R 3 A hydrogen atom.

12. The compound according to any one of claims 1 to 3, or its stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein R 4 Select from -OR b 、-NHR b , 3-6 membered cycloalkyl or 3-6 membered heterocyclic group; wherein the cycloalkyl or heterocyclic group is optionally further substituted by one or more substituents selected from halogen, alkyl, hydroxyl; R b The group is selected from alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocyclic group, wherein the alkyl, cycloalkyl or heterocyclic group is optionally further substituted by one or more halogens.

13. The compound according to any one of claims 1 to 3, or its stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein R 4 is halogen, methyl, difluoromethyl, trifluoromethyl, methoxy, trifluoroethoxy, 14. The compound according to any one of claims 1 to 13 or its stereoisomer, tautomer or pharmaceutically acceptable salt, wherein the compound is:

15. A pharmaceutical composition comprising an effective dose of the compound according to any one of claims 1 to 14 or its stereoisomer, tautomer or pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier, excipient or a combination thereof.

16. Use of the compound according to any one of claims 1 to 14 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 15 in the preparation of a KIF18A inhibitor.

17. Use of the compound according to any one of claims 1 to 14 or its stereoisomer, tautomer or pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating a disease mediated by KIF18A; preferably, the disease mediated by KIF18A is cancer.

18. The method of claim 17, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, glioblastoma, colon cancer, breast cancer, lung cancer, bile duct cancer, pancreatic cancer, prostate cancer, bladder cancer, head cancer, neck cancer, cervical cancer, ovarian cancer, synovial sarcoma, rhabdomyosarcoma, colorectal cancer and lung adenocarcinoma.

19. Use of the compound according to any one of claims 1 to 14 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 15 in the preparation of a drug for treating cancer.

20. The use according to claim 19, wherein the cancer is selected from hepatocellular carcinoma, glioblastoma, colon cancer, breast cancer, lung cancer, bile duct cancer, pancreatic cancer, prostate cancer, bladder cancer, head cancer, neck cancer, cervical cancer, ovarian cancer, synovial sarcoma, rhabdomyosarcoma, colorectal cancer and lung adenocarcinoma.

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