A c-kit (v559d) / pdgfr beta dual-target allosteric inhibitor, preparation method thereof, pharmaceutical composition and application thereof

Abstract

The application discloses a c-Kit (V559D) / PDGFRbeta double-target allosteric inhibitor, a preparation method, a pharmaceutical composition and application thereof, and belongs to the medical field. The inhibitor comprises a compound as shown in a general formula (I) or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, can effectively overcome all EGFR-TKI drug resistance caused by non-genetic drug resistance mechanisms, has an anti-tumor and anticancer effect, can accurately locate specific target proteins, reduces the influence on other non-target proteins, thereby reducing off-target effects and potential toxic side effects, and improves safety and tolerability. The structure of the general formula (I) is as follows.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a class of compounds that are dual-target allosteric inhibitors of c-Kit(V559D) / PDGFRβ and their application in the preparation of antitumor drugs. Background Technology

[0002] PDGFR, or platelet-derived growth factor receptor, is an important member of the PDGFR signaling pathway and belongs to the type III tyrosine protein kinase family. PDGFR includes two types, PDGFRα and PDGFRβ. Upon binding to a ligand, it undergoes dimerization of receptor subunits, activation of the tyrosine kinase domain, and autophosphorylation, triggering a cascade of reactions in different signal transduction pathways. It plays a crucial role in events closely related to growth and proliferation, such as cell mitosis, cytoskeleton rearrangement, and drug-tactic selection. Simultaneously, PDGFR binding to its ligand can directly enhance the degradation of intercellular adhesion molecules, promoting tumor invasion and metastasis. c-Kit, also a type III receptor tyrosine kinase, is a classic proto-oncogene. c-Kit and its ligand SCF are closely related to the occurrence and progression of human cancers. c-Kit gene mutations are associated with gastrointestinal stromal tumors, small cell lung cancer, non-small cell lung cancer, leukemia, glioblastoma, mastocytosis, and acute myeloid leukemia. V559D mutation is the most common primary gain-of-function mutation of c-Kit. Therefore, inhibiting c-Kit (V559D) can effectively suppress the activity of V559D-mutated c-Kit. Activated c-Kit promotes the survival, proliferation, differentiation, and metastasis of tumor cells. c-Kit phosphorylation triggers multiple signal transduction pathways, including the JAK / STAT, RAS / MAPK, PI3K, PLCγ, and SRC pathways. In summary, developing selective inhibitors targeting PDGFRβ and c-Kit holds promise for providing effective therapeutic drugs for various tumors with over-activation or expression of PDGFRβ and c-Kit.

[0003] In EGFR mutations, inhibitors can suppress the transcription of c-Kit and PDGFRβ, but these inhibitions easily lead to EGFR-TKI resistance. Currently, inhibitors that simultaneously target c-Kit and PDGFRβ are all multi-target kinase inhibitors, exerting their anti-tumor effects by acting on the ATP or endogenous ligand binding sites (orthogonal sites) of the kinases. Inhibitors such as imatinib, sorafenib, dasatinib, and anlotinib have achieved significant efficacy in clinical cancer treatment. Unfortunately, due to the high homology of the ATP binding sites of type III receptor tyrosine kinases, these inhibitors have poor selectivity. Multi-target inhibitors such as dasatinib and anlotinib can inhibit not only multiple type III receptor tyrosine kinases but also other types of receptor tyrosine kinases to varying degrees. Due to their insufficient selectivity, they inevitably cause toxic damage to the hematopoietic, metabolic, endocrine, neuropsychiatric, cardiovascular, respiratory, and digestive systems. Highly selective inhibitors can act more precisely on specific target proteins, reducing the impact on other non-target proteins, thereby decreasing off-target effects and potential toxic side effects, and improving safety and tolerability. However, to date, no highly selective c-Kit / PDGFRβ dual-target inhibitors have been reported. Therefore, there is a need to develop highly selective c-Kit / PDGFRβ dual-target inhibitors to explore the feasibility of simultaneously and selectively targeting both c-Kit and PDGFRβ to develop novel anti-tumor drugs to overcome EGFR-TKI resistance in lung cancer. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor, its preparation method, pharmaceutical composition, and applications.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] A c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor, selected from compounds of general formula (I) or their pharmaceutically acceptable salts, hydrates, solvates or prodrugs, wherein the compound of general formula (I) has the following structural formula:

[0007]

[0008] In general formula (I), ring A is selected from substituted or unsubstituted 6-membered saturated or unsaturated heterocycles, benzene rings and / or 5-membered saturated or unsaturated heterocycles or 5-6-membered saturated / or unsaturated heterocycles with 5-6-membered saturated / or unsaturated heterocycles.

[0009] R1 is selected from at least one of C1-C7 alkyl, C2-C6 heteroalkyl, C1-C6 haloalkyl, C3-C6 unsaturated aliphatic hydrocarbon group, C3-C7 cycloalkyl, saturated aliphatic heterocycloalkyl, substituted phenyl, benzene ring chain hydrocarbon group with different substitutions, heterocyclic aryl, and heterocyclic aromatic hydrocarbon group.

[0010] R2 is selected from hydrogen, fluorine, methyl, ethyl, methoxy, and cyano;

[0011] R3 is selected from at least one of C2-C7 alkyl acyl, C2-C7 haloalkyl acyl, saturated aliphatic alkyl acyl, C4-C8 saturated cycloalkyl acyl, saturated aliphatic cycloalkyl acyl, benzoyl with different substitutions, and heterocyclic aromatic acyl.

[0012] Furthermore, in the general formula (I), ring A is selected from benzene rings, furan rings, thiophene rings, pyrrole rings, thiazole rings, imidazole rings, oxazole rings, pyridine rings, pyrimidine rings, pyridazine rings, pyrazine rings, and purine rings. Furthermore, the general formula (I) is selected from compounds with the following structural formulas:

[0013]

[0014]

[0015] This invention also discloses the application of a c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor for the treatment of diseases associated with c-Kit and PDGFRβ, including cancer, pulmonary interstitial fibrosis, and neurodegenerative diseases.

[0016] Furthermore, the cancer is selected from EGFR-TKI resistant non-small cell lung cancer, ovarian cancer cells, human T lymphoma cells, human synovial sarcoma cells, human glioma cells, and gastrointestinal stromal tumor cells.

[0017] This invention also discloses a pharmaceutical composition comprising a c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor. Specifically, the pharmaceutical composition comprises any one or more of the following: a c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor and / or a pharmaceutically acceptable salt of said inhibitor, and / or a solvate of said inhibitor.

[0018] This invention also discloses a method for preparing a c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor, characterized by comprising the following steps:

[0019] (1) At room temperature, thionyl chloride (b) was added to a dichloromethane solution of bromoaromatic formic acid (a) and N,N-dimethylformamide (N,N-dimethylformamide), and the mixture was heated to 50°C and reacted for 5-8 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed to obtain intermediate compound c. The molar ratio of bromoaromatic formic acid (a), thionyl chloride (b), and N,N-dimethylformamide was 1:5:0.1.

[0020] (2) At 0℃, a dichloromethane solution of intermediate compound c is added dropwise to a dichloromethane solution of triethylamine and organic amine d. The mixture is heated to room temperature and reacted for 1-3 hours. After the reaction is completed, saturated brine and dichloromethane are added for extraction. The organic layer is collected and concentrated by column chromatography to obtain intermediate compound e. The molar ratio of intermediate compound c, organic amine and triethylamine is 1.5:1:2.

[0021] (3) Add [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride to intermediate compounds e and f, materials 2-aminopyridine-5-boronic acid pinacol ester and potassium carbonate V 1,4-二氧六环 V 水 In a solution with a ratio of 4:1, the reaction was carried out at 100℃ for 1.5 h. Then, intermediate compound e was added and the reaction continued for 0.5 h to 2.5 h. After the reaction was completed, the solvent was removed, and dichloromethane and methanol were added to dissolve the crude product. The product was then filtered, and the filtrate was concentrated and column chromatography was used to obtain the corresponding intermediate compound g. The molar ratio of intermediate compounds e and f to pinacol ester 2-aminopyridine-5-boronic acid, potassium carbonate, and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride was 1:1.5:2:0.1.

[0022] (4) At 50°C, the acyl chlorides of different substitutions in material h are added in batches to a tetrahydrofuran solution of sodium hydride and intermediate compound g. After reacting for 3-6 h, the mixture is cooled to 0°C, quenched with methanol, extracted with saturated brine and dichloromethane, and the organic layer is collected. The target inhibitor compound i of the present invention is obtained by concentrated column chromatography. The molar ratio of the acyl chlorides of different substitutions in material h, intermediate compound g and sodium hydride is 2:1:2.

[0023] The specific synthesis steps are as follows:

[0024]

[0025] Furthermore, the organic amine mentioned in step (2) includes any one of benzylamine, ethylamine, propylamine, butylamine, cyclopropylmethylamine, isopropylamine, isobutylamine, isopentylamine, 2,2-difluoroethylamine, neopentylamine, and 2,2,2-trifluoroethylamine.

[0026] The present invention relates to a c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor, its preparation method, pharmaceutical composition, and application. The beneficial effects are as follows: the inhibitor of the present invention can effectively overcome all EGFR-TKI resistance caused by non-genetic resistance mechanisms; at the same time, it has anti-tumor and anti-cancer effects, can accurately locate specific target proteins, reduce the impact on other non-target proteins, thereby reducing off-target effects and potential toxic side effects, and improving safety and tolerability. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the effects of the i-7 and i-17 compounds of the present invention on the tumor cell cycle. In the figure, (A) i-7 induces G2 / M phase arrest in A549 cells; (B) i-17 induces G2 / M phase arrest in H1975-OR cells; (C) i-17 affects the level of G2 / M phase-related regulatory proteins in H1975-OR cells.

[0029] Figure 2 This is a schematic diagram illustrating the effects of the i-7 and i-17 compounds of the present invention on tumor cell apoptosis. In the diagram, (A) i-7 induces apoptosis in A549 cells. (B) i-17 induces apoptosis in H1975-OR cells. (C) The effect of i-17 on the levels of apoptosis-related regulatory proteins in H1975-OR cells;

[0030] Figure 3 This is a schematic diagram of the kinase inhibitory activity and binding activity of the i-17 compound of the present invention. In the figure, (A) Kinase Profiler TM The kinase inhibitory activity screening platform tested the inhibitory activity of i-17 against 278 kinases related to tumor development and progression. (B) KINOMEscan TM A competitive binding screening platform was used to screen the binding effects of i-17 on 468 kinases;

[0031] Figure 4 This is a schematic diagram illustrating the effects of the i-17 compound of this invention on biochemical indicators related to the liver, kidneys, and heart;

[0032] Figure 5 This is a schematic diagram illustrating the inhibitory activity of the i-17 compound of this invention against hERG;

[0033] Figure 6This is a schematic diagram illustrating the transcriptional regulation of AURKB by the i-7 compound of this invention and its downstream Histone H3.

[0034] Figure 7 This is a schematic diagram illustrating the inhibition of A549 cell xenograft growth in nude mice by the i-7 compound of this invention. In the diagram, (A) shows the tumor size in different groups, (B) shows the tumor volume growth in different groups, (C) shows the tumor weight in different groups, and (D) shows that i-7 has no significant effect on mouse body weight. Experimental data are expressed as mean ± standard deviation (n = 5; *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant compared to the control group).

[0035] Figure 8 This is a schematic diagram illustrating the inhibition of A549 cell xenograft growth in nude mice by the i-17 compound of this invention. In the diagram, (A) shows the tumor size in different groups, (B) shows the tumor volume growth in different groups, (C) shows the tumor weight in different groups, and (D) shows that i-17 has no significant effect on mouse body weight. Experimental data are expressed as mean ± standard deviation (n = 5; *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant compared to the control group). Detailed Implementation

[0036] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The term "alkyl" as used in this invention, unless otherwise specified, refers to a straight-chain or branched hydrocarbon chain containing 1 to 7 carbon atoms. Examples of "alkyl" as used herein include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, isopentyl, etc.

[0038] As used in this invention, the term "heteroalkyl" refers to an atom with at least one heteroatom selected from oxygen, nitrogen, and sulfur as a saturated alkyl group, examples of which include, but are not limited to, methoxymethyl, methylaminomethyl, methylthiomethyl, methoxyethyl, etc.

[0039] As used in this invention, the term "halogenated alkyl" refers to an alkyl group with a halogen at the end of its carbon chain, wherein the alkyl group is as defined above. Examples of "halogenated alkyl" as used herein include, but are not limited to, fluoromethyl, fluoroethyl, fluoropropyl, fluorobutyl, difluoromethyl, difluoroethyl, difluoropropyl, trifluorofluoromethyl, trifluoroethyl, and trifluoropropyl.

[0040] The term "unsaturated aliphatic hydrocarbon group" as used in this invention refers to a straight-chain or branched alkenyl or alkynyl group containing 3-6 carbon atoms with double or triple bonds. Non-limiting examples of unsaturated aliphatic hydrocarbon groups include allyl, 2-propenyl, propynyl, etc.

[0041] The term "cycloalkyl" as used in this invention refers to a carbon ring of a saturated alkane chain of 3-7 atoms, examples of which include, but are not limited to, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclopropylpropyl, etc.

[0042] As used in this invention, the term "saturated alicyclic hydrocarbon group" refers to a hydrocarbon group with at least one heteroatom selected from oxygen, nitrogen, and sulfur as a ring atom, and the ring can be a saturated monocyclic alicyclic group or a saturated bicyclic alicyclic group. Examples include, but are not limited to, tetrahydrofuran ring, tetrahydrofuranmethyl, tetrahydropyrrole ring, tetrahydropyrrolemethyl, tetrahydrothiophene ring, tetrahydrothiophenemethyl, tetrahydrofuranethyl, morpholineethyl, etc.

[0043] As used in this invention, the term "differently substituted phenyl" refers to a monocyclic group with 6-8 carbon atoms and a fully conjugated π-electron system. Non-limiting examples of differently substituted phenyl groups include: phenyl, p-methylphenyl, p-fluorophenyl, 3-methoxyphenyl, etc.

[0044] The term "alkanes with different substituted benzene rings" as used in this invention refers to alkane chains with different substituents on the benzene ring, including but not limited to benzyl, p-methylbenzyl, p-fluorobenzyl, phenethyl, etc.

[0045] As used in this invention, the term "heterocyclic aryl" refers to an unsaturated carbon ring with 5-10 ring atoms, wherein one or more carbon atoms are replaced by heteroatoms such as oxygen, nitrogen, sulfur, etc. The heterocyclic aryl ring can be monocyclic or bicyclic, i.e., formed by the fusion of two rings. Specific heterocyclic aryl groups can be: furanyl, thiophene, pyrrole, pyridinyl, pyrimidinyl, pyrazinyl, etc.

[0046] The term "heterocyclic aromatic hydrocarbon group" as used in this invention refers to an alkyl chain of 6-12 unsaturated carbon rings, wherein one or more carbon atoms are replaced by heteroatoms such as oxygen, nitrogen, sulfur, etc. The heterocyclic aromatic ring can be monocyclic or bicyclic. Specific heterocyclic aryl groups can be: furanylmethyl, thiophenemethyl, pyrrolemethyl, pyridinemethyl, pyrimidinemethyl, furanylethyl, thiopheneethyl, pyridineethyl, pyrimidineethyl, indoleethyl, etc.

[0047] As used in this invention, the term "alkanoyl" refers to a straight or branched carbonyl chain containing 2 to 7 carbon atoms, examples of which include, but are not limited to, acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanyl, isopropylcarboxyl, tert-butylcarboxyl, etc.

[0048] The term "haloalkylyl" as used in this invention refers to a carbonyl chain with a halogen on the carbon chain, wherein the carbonyl chain is as defined above. Examples of "haloalkylyl" as used herein include, but are not limited to, fluoroacetyl, fluoropropionyl, fluorobutyryl, difluoroacetyl, difluoropropionyl, chloroacetyl, chloropropionyl, etc.

[0049] As used in this invention, the term "saturated aliphatic alkyl acyl" refers to a straight or branched carbonyl chain with at least one heteroatom selected from oxygen, nitrogen, and sulfur as a saturated alkyl acyl group. Examples include, but are not limited to, ethylcarbamoyl, propylcarbamoyl, propylcarbamoyl, ethylcarbamoyl, propylcarbamoyl, isopropylcarbamoyl, etc.

[0050] The term "saturated cycloalkanoyl" as used in this invention refers to a formyl, acetyl, or propionyl group of a saturated carbon ring with 4-8 atoms, including but not limited to cyclopropanoyl, cyclobutanoyl, cyclopentanoyl, cyclohexanoyl, cyclopropanoylacetyl, cyclobutanoylacetyl, cyclopentanoylacetyl, etc.

[0051] The term "saturated alicyclic alkyl acyl" as used in this invention refers to an acyl group of a saturated cyclic group with at least one heteroatom selected from oxygen, nitrogen, and sulfur as a ring atom, examples of which include, but are not limited to, tetrahydrofuran carboxyl, tetrahydropyrrole carboxyl, thiocyclic butane carboxyl, tetrahydrofuran acetyl, etc.

[0052] The term "different substituted benzoyl group" as used in this invention refers to a formyl group with different substituents on the benzene ring, including but not limited to benzoyl group, p-methoxybenzoyl group, p-fluorobenzoyl group, 2-fluorobenzoyl group, etc.

[0053] As used in this invention, the term "heterocyclic aromatic acyl" refers to a formyl group, acetyl group, etc., of an unsaturated carbon ring, wherein one or more carbon atoms are replaced by heteroatoms such as oxygen, nitrogen, sulfur, etc. The heterocyclic aromatic ring can be monocyclic or bicyclic, i.e., formed by the fusion of two rings. Specific heterocyclic aromatic acyl groups can be: furanoyl, thiopheneformyl, pyridineformyl, pyrimidineformyl, furanoacetyl, thiopheneacetyl, pyridineacetyl, etc.

[0054] The term "halogen" as used in this invention refers to fluorine, chlorine, bromine, or iodine.

[0055] In this invention, "pharmaceutically acceptable salts" include alkali metal salts, alkaline earth metal salts, other metal salts, inorganic alkali salts, organic alkali salts, inorganic acid salts, lower alkyl sulfonates, aryl sulfonates, organic acid salts, and amino acid salts.

[0056] Example 1: 3-Bromobenzoyl chloride (c-1)

[0057]

[0058] Thionyl chloride (b) (5.92 g, 49.750 mmol) was added to a solution of 3-bromobenzoic acid (a) (2 g, 9.950 mmol) and N,N-dimethylformamide (72.70 mg, 0.995 mmol) in dichloromethane (40 mL). The mixture was reacted at 50 °C for 5 hours until the turbidity cleared. After cooling to room temperature, the solvent was removed under vacuum to obtain the crude product 3-bromobenzoyl chloride (c-1), which required no further purification.

[0059] Example 2: 4-Bromopyridyl chloride (c-2)

[0060]

[0061] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 4-bromopyridinecarboxylic acid.

[0062] Example 3: 5-Bromonicotinyl chloride (c-3)

[0063]

[0064] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 5-bromonicotinic acid.

[0065] Example 4: 2-Bromoisonicotinyl chloride (c-4)

[0066]

[0067] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 2-bromoisonicotinic acid.

[0068] Example 5: 6-Bromopyridyl chloride (c-5)

[0069]

[0070] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 6-bromopyridinecarboxylic acid.

[0071] Example 6: 5-Bromo-2-fluorobenzoyl chloride (c-6)

[0072]

[0073] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 5-bromo-2-fluorobenzoic acid.

[0074] Example 7: 3-Bromo-5-fluorobenzoyl chloride (c-7)

[0075]

[0076] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 3-bromo-5-fluorobenzoic acid.

[0077] Example 8: 3-Bromo-4-fluorobenzoyl chloride (c-8)

[0078]

[0079] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 3-bromo-4-fluorobenzoic acid.

[0080] Example 9: 3-Bromo-2-fluorobenzoyl chloride (c-9)

[0081]

[0082] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 3-bromo-2-fluorobenzoic acid.

[0083] Example 10: 3-Bromo-4-methylbenzoyl chloride (c-10)

[0084]

[0085] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 3-bromo-4-methylbenzoic acid.

[0086] Example 11: 3-Bromo-2-methylbenzoyl chloride (c-11)

[0087]

[0088] The procedure is the same as in Example 1, except that 3-bromobenzoic acid is replaced with 3-bromo-2-methylbenzoic acid.

[0089] Example 12: N-Benzyl-3-bromobenzamide (e-1)

[0090]

[0091] At 0 °C, a solution of 3-bromobenzoyl chloride (c-1) (1.536 g, 6.999 mmol) in dichloromethane (10 mL) was added dropwise to a solution of benzylamine (d) (500 mg, 4.666 mmol) and triethylamine (943 mg, 9.332 mmol) in dichloromethane (25 mL), and stirred for 10 minutes. The reaction was then heated to 30 °C and reacted for 1 hour (TLC monitoring was used to monitor the reaction progress). After the reaction was complete, the mixture was extracted with dichloromethane (3 × 40 mL) and saturated brine (30 mL), and the organic layer was collected. The mixture was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum. The solution was purified by silica gel column chromatography using dichloromethane as the eluent to give N-benzyl-3-bromobenzoamide (e-1) (1.232 g, 4.26 mmol), a white solid with a yield of 91%. ESI-MS: m / z 291.1 [M+H] + .

[0092] Example 13: 3-Bromo-N-ethylbenzamide (e-2)

[0093]

[0094] The procedure was the same as in Example 12, except that benzylamine was replaced with ethylamine, resulting in a yield of 82%. ESI-MS: m / z 229.1 [M+H] + .

[0095] Example 14: 3-Bromo-N-propylbenzamide (e-3)

[0096]

[0097] The procedure was the same as in Example 12, except that benzylamine was replaced with propylamine, resulting in a yield of 85%. ESI-MS: m / z 243.2 [M+H] + .

[0098] Example 15: 3-Bromo-N-propylbenzamide (e-4)

[0099]

[0100] The procedure was the same as in Example 12, except that benzylamine was replaced with butylamine, resulting in a yield of 84%. ESI-MS: m / z 257.2 [M+H] + .

[0101] Example 16: 3-Bromo-N-cyclopropylmethylbenzamide (e-5)

[0102]

[0103] The procedure was the same as in Example 12, except that benzylamine was replaced with cyclopropylmethylamine, with a yield of 81%. ESI-MS: m / z 255.2 [M+H] + .

[0104] Example 17: 3-Bromo-N-isopropylbenzamide (e-6)

[0105]

[0106] The procedure was the same as in Example 12, except that benzylamine was replaced with isopropylamine, with a yield of 80%. ESI-MS: m / z 243.1 [M+H] + .

[0107] Example 18: 3-Bromo-N-isobutylbenzamide (e-7)

[0108]

[0109] The procedure was the same as in Example 12, except that benzylamine was replaced with isobutylamine, resulting in a yield of 83%. ESI-MS: m / z 257.1 [M+H] + .

[0110] Example 19: 3-Bromo-N-isopentylbenzamide (e-8)

[0111]

[0112] The procedure was the same as in Example 12, except that benzylamine was replaced with isoamylamine, resulting in a yield of 83%. ESI-MS: m / z 271.2 [M+H] + .

[0113] Example 20: 3-Bromo-N-(2,2-difluoroethyl)benzamide (e-9)

[0114]

[0115] The procedure was the same as in Example 12, except that benzylamine was replaced with 2,2-difluoroethylamine, with a yield of 81%. ESI-MS: m / z 265.1 [M+H] + .

[0116] Example 21: 3-Bromo-N-neopentylbenzamide (e-10)

[0117]

[0118] The procedure was the same as in Example 12, except that benzylamine was replaced with neopentylamine, resulting in a yield of 81%. ESI-MS: m / z 271.2 [M+H] + .

[0119] Example 22: 3-Bromo-N-(2,2,2-trifluoroethyl)benzamide (e-11)

[0120]

[0121] The procedure was the same as in Example 12, except that benzylamine was replaced with 2,2,2-trifluoroethylamine, with a yield of 81%. ESI-MS: m / z 283.1 [M+H] + .

[0122] Example 23: 5-Bromo-2-fluoro-N-isopentylbenzamide (e-12)

[0123]

[0124] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 5-bromo-2-fluorobenzoyl chloride. The yield was 84%, and the ESI-MS result was m / z 289.2 [M+H]. + .

[0125] Example 24: 3-Bromo-5-fluoro-N-isopentylbenzamide (e-13)

[0126]

[0127] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 3-bromo-5-fluorobenzoyl chloride. The yield was 82%, and the ESI-MS result was m / z 289.1 [M+H]. + .

[0128] Example 25: 3-Bromo-4-fluoro-N-isopentylbenzamide (e-14)

[0129]

[0130] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 3-bromo-4-fluorobenzoyl chloride. The yield was 81%, and the ESI-MS result was m / z 288.9 [M+H]. + .

[0131] Example 26: 3-Bromo-2-fluoro-N-isopentylbenzamide (e-15)

[0132]

[0133] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 3-bromo-2-fluorobenzoyl chloride. The yield was 81%, and the ESI-MS result was m / z 288.1 [M+H]. + .

[0134] Example 27: 3-Bromo-N-isopentyl-4-methylbenzamide (e-16)

[0135]

[0136] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 3-bromo-4-methylbenzoyl chloride. The yield was 83%, and the ESI-MS result was m / z 285.2 [M+H]. + .

[0137] Example 28: 3-Bromo-N-isopentyl-2-methylbenzamide (e-17)

[0138]

[0139] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 3-bromo-2-methylbenzoyl chloride. The yield was 82%, and the ESI-MS result was m / z 285.1 [M+H].+ .

[0140] Example 29: 4-Bromo-N-isopentylpyridine amide (e-18)

[0141]

[0142] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 4-bromopyridyl chloride. The yield was 81%, and the ESI-MS result was m / z 272.1 [M+H]. + .

[0143] Example 30: 5-Bromo-N-isopentylnicotinamide (e-19)

[0144]

[0145] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 5-bromonicotinyl chloride. The yield was 84%, and the ESI-MS result was m / z 272.2 [M+H]. + .

[0146] Example 31: 2-Bromo-N-isopentylisonicotinamide (e-20)

[0147]

[0148] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 2-bromoisondinochloride. The yield was 83%, and the ESI-MS result was m / z 272.1 [M+H]. + .

[0149] Example 32: 6-Bromo-N-isopentylpyridine amide (e-21)

[0150]

[0151] The procedure was the same as in Example 12, except that benzylamine was replaced with isopentylamine and 3-bromobenzoyl chloride was replaced with 6-bromopyridyl chloride. The yield was 81%, and the ESI-MS result was m / z 272.3 [M+H]. + .

[0152] Example 33: 2-Bromo-N-(Cyclopropylmethyl)isonicotinamide (e-22)

[0153]

[0154] The procedure was the same as in Example 12, except that benzylamine was replaced with cyclopropylmethylamine and 3-bromobenzoyl chloride was replaced with 2-bromoisondinochloride. The yield was 81%, and the ESI-MS result was m / z 256.1 [M+H]. + .

[0155] Example 34: 2-Bromo-N-isobutylisonicotinamide (e-23)

[0156]

[0157] The procedure was the same as in Example 12, except that benzylamine was replaced with isobutylamine and 3-bromobenzoyl chloride was replaced with 2-bromoisondinochloride. The yield was 84%, and the ESI-MS result was m / z 258.0 [M+H]. + .

[0158] Example 35: 2-Bromo-N-(2,2-difluoroethyl)isonicotinamide (e-24)

[0159]

[0160] The procedure was the same as in Example 12, except that benzylamine was replaced with 2,2-difluoroethylamine and 3-bromobenzoyl chloride was replaced with 2-bromoisonitinyl chloride. The yield was 81%, and the ESI-MS result was m / z 266.1 [M+H]. + .

[0161] Example 36: 2-Bromo-N-neopentylisonicotinamide (e-25)

[0162]

[0163] The procedure was the same as in Example 12, except that benzylamine was replaced with neopentylamine and 3-bromobenzoyl chloride was replaced with 2-bromoisondinochloride. The yield was 84%, and the ESI-MS result was m / z 272.1 [M+H]. + .

[0164] Example 37: 2-Bromo-N-(2,2,2-trifluoroethyl)isonicotinamide (e-26)

[0165]

[0166] The procedure was the same as in Example 12, except that benzylamine was replaced with 2,2,2-trifluoroethylamine and 3-bromobenzoyl chloride was replaced with 2-bromoisonitinyl chloride. The yield was 82%, and the ESI-MS result was m / z 284.0 [M+H]. + .

[0167] Example 38: 3-(6-aminopyridin-3-yl)-N-benzylbenzamide (g-1)

[0168]

[0169] Potassium carbonate (476 mg, 3.446 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (126 mg, 0.172 mmol) were added to a solution of N-benzyl-3-bromobenzamide (e-1) (500 mg, 1.723 mmol) and 2-aminopyridine-5-boronic acid pinacol ester (f) (569 mg, 2.584 mmol) in 1,4-dioxane (20 mL). The mixture was then heated to 100 °C and reacted for 1.5 hours. Add water (5 mL), continue the reaction for 2 hours (monitor progress by TLC), cool to room temperature, remove solvent under vacuum, dissolve the crude product in dichloromethane and methanol, filter to remove potassium carbonate, remove solvent under vacuum again, and precipitate the crude product by silica gel column chromatography using dichloromethane:methanol = 50:1 as eluent to give 3-(6-aminopyridin-3-yl)-N-benzylbenzamide (g-1) (408 mg, 1.344 mmol), white solid, yield 78%, ESI-MS: m / z 304.5 [M+H] + .

[0170] Example 39: 3-(6-aminopyridin-3-yl)-N-ethylbenzamide (g-2)

[0171]

[0172] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-ethylbenzamide, with a yield of 71%. ESI-MS: m / z 242.3 [M+H] + .

[0173] Example 40: 3-(6-aminopyridin-3-yl)-N-propylbenzamide (g-3)

[0174]

[0175] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-propylbenzamide, with a yield of 73%. ESI-MS: m / z 256.4 [M+H] + .

[0176] Example 41: 3-(6-aminopyridin-3-yl)-N-butylbenzamide (g-4)

[0177]

[0178] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-butylbenzamide, with a yield of 71%. ESI-MS: m / z 270.2 [M+H] + .

[0179] Example 42: 3-(6-aminopyridin-3-yl)-N-(cyclopropylmethyl)benzamide (g-5)

[0180]

[0181] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-cyclopropylmethylbenzamide, with a yield of 77%. ESI-MS: m / z 268.3 [M+H] + .

[0182] Example 43: 3-(6-aminopyridin-3-yl)-N-isopropylbenzamide (g-6)

[0183]

[0184] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-isopropylbenzamide, with a yield of 75%. ESI-MS: m / z 256.2 [M+H] + .

[0185] Example 44: 3-(6-aminopyridin-3-yl)-N-isobutylbenzamide (g-7)

[0186]

[0187] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-isobutylbenzamide, with a yield of 72%. ESI-MS: m / z 270.3 [M+H] + .

[0188] Example 45: 3-(6-aminopyridin-3-yl)-N-isopentylbenzamide (g-8)

[0189]

[0190] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-isopentylbenzamide, with a yield of 78%. ESI-MS: m / z 284.4 [M+H] + .

[0191] Example 46: 3-(6-aminopyridin-3-yl)-N-(2,2-difluoroethyl)benzamide (g-9)

[0192]

[0193] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-(2,2-difluoroethyl)benzamide, with a yield of 81% and ESI-MS: m / z 278.3 [M+H]. + .

[0194] Example 47: 3-(6-aminopyridin-3-yl)-N-neopentylbenzamide (g-10)

[0195]

[0196] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-neopentylbenzamide, with a yield of 76%. ESI-MS: m / z 284.4 [M+H] + .

[0197] Example 48: 3-(6-aminopyridin-3-yl)-N-(2,2,2-trifluoroethyl)benzamide (g-11)

[0198]

[0199] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-(2,2,2-trifluoroethyl)benzamide, with a yield of 82% and ESI-MS: m / z 295.3 [M+H]. + .

[0200] Example 49: 5-(6-aminopyridin-3-yl)-2-fluoro-N-isopentylbenzamide (g-12)

[0201]

[0202] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 5-bromo-2-fluoro-N-isopentylbenzamide, with a yield of 71%. ESI-MS: m / z 302.3 [M+H] + .

[0203] Example 50: 3-(6-aminopyridin-3-yl)-5-fluoro-N-isopentylbenzamide (g-13)

[0204]

[0205] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-5-fluoro-N-isopentylbenzamide, with a yield of 72%. ESI-MS: m / z 302.1 [M+H] + .

[0206] Example 51: 3-(6-aminopyridin-3-yl)-4-fluoro-N-isopentylbenzamide (g-14)

[0207]

[0208] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-4-fluoro-N-isopentylbenzamide, with a yield of 75%. ESI-MS: m / z 302.3 [M+H] + .

[0209] Example 52: 3-(6-aminopyridin-3-yl)-2-fluoro-N-isopentylbenzamide (g-15)

[0210]

[0211] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-2-fluoro-N-isopentylbenzamide, with a yield of 72%. ESI-MS: m / z 302.3 [M+H] + .

[0212] Example 53: 3-(6-aminopyridin-3-yl)-N-isopentyl-4-methylbenzamide (g-16)

[0213]

[0214] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-isopentyl-4-methylbenzamide, with a yield of 70%. ESI-MS: m / z 298.4 [M+H] + .

[0215] Example 54: 3-(6-aminopyridin-3-yl)-N-isopentyl-2-methylbenzamide (g-17)

[0216]

[0217] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 3-bromo-N-isopentyl-2-methylbenzamide, with a yield of 68% and ESI-MS resolution of m / z 298.3 [M+H]. + .

[0218] Example 55: 6-Amino-N-isopentyl-[3,4'-bipyridine]-2'-carboxamide (g-18)

[0219]

[0220] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 4-bromo-N-isopentylpyridine amide, with a yield of 78%. ESI-MS: m / z 284.3 [M+H] + .

[0221] Example 56: 6'-Amino-N-isopentyl-[3,3'-bipyridine]-5-carboxamide (g-19)

[0222]

[0223] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 5-bromo-N-isopentylnicotinamide, with a yield of 76%. ESI-MS: m / z 284.4 [M+H] + .

[0224] Example 57: 6'-Amino-N-isopentyl-[2,3'-bipyridine]-4-carboxamide (g-20)

[0225]

[0226] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 2-bromo-N-isopentylisonicotinamide, with a yield of 74%. ESI-MS: m / z 284.2 [M+H] + .

[0227] Example 58: 6'-Amino-N-isopentyl-[2,3'-bipyridine]-6-carboxamide (g-21)

[0228]

[0229] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 6-bromo-N-isopentylpyridine amide, with a yield of 78%. ESI-MS: m / z 284.3 [M+H] + .

[0230] Example 59: 6'-Amino-N-(cyclopropylmethyl)-[2,3'-bipyridine]-4-carboxamide (g-22)

[0231]

[0232] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 2-bromo-N-(cyclopropylmethyl)isonicotinamide, with a yield of 77% and ESI-MS resolution of m / z 269.3 [M+H]. + .

[0233] Example 60: 6'-Amino-N-isobutyl-[2,3'-bipyridine]-4-carboxamide (g-23)

[0234]

[0235] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 2-bromo-N-isobutylisonicotinamide, with a yield of 80%. ESI-MS: m / z 271.2 [M+H] + .

[0236] Example 61: 6'-Amino-N-(2,2-difluoroethyl)-[2,3'-bipyridine]-4-carboxamide (g-24)

[0237]

[0238] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 2-bromo-N-(2,2-difluoroethyl)isonicotinamide, with a yield of 81% and ESI-MS resolution of m / z 279.2 [M+H]. + .

[0239] Example 62: 6'-Amino-N-neopentyl-[2,3'-bipyridine]-4-carboxamide (g-25)

[0240]

[0241] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 2-bromo-N-neopentylisonicotinamide, with a yield of 73% and ESI-MS resolution of m / z 285.3 [M+H]. + .

[0242] Example 63: 6'-Amino-N-(2,2,2-trifluoroethyl)-[2,3'-bipyridine]-4-carboxamide (g-26)

[0243]

[0244] The procedure was the same as in Example 38, except that N-benzyl-3-bromobenzamide was replaced with 2-bromo-N-(2,2,2-trifluoroethyl)isonicotinamide, with a yield of 84% and ESI-MS resolution of m / z 297.1 [M+H]. + .

[0245] Example 64: N-Benzyl-3-(6-Butylamyridin-3-yl)benzamide (i-1)

[0246]

[0247] At 50°C, n-butyryl chloride (h) (351 mg, 3.296 mmol) was added in portions to a tetrahydrofuran (25 ml) solution of sodium hydride (79 mg, 3.296 mmol) and product g-1 (500 mg, 1.648 mmol) from Example 38. The reaction was carried out for 3-6 hours (TLC monitoring of completion). After the reaction was completed, the mixture was cooled to 0°C, quenched with methanol, and extracted with dichloromethane (3 × 20 ml) and saturated brine (20 ml). The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum. The mixture was purified by silica gel column chromatography with dichloromethane:methanol = 100:1 as the eluent to give N-benzyl-3-(6-butamidopyridin-3-yl)benzamide (i-1) (462 mg, 1.236 mmol), a white solid with a yield of 75% and a melting point of 190-193°C. 1 HNMR (600MHz, DMSO-d6) δ10.58 (s, 1H), 9.19 (t, J = 6.0 Hz, 1H), 8.70 (d, J = 2. 5Hz,1H),8.24–8.18(m,2H),8.15(dd,J=8.7,2.5Hz,1H),7.91–7.85(m,2H), 7.58(t,J=7.8Hz,1H),7.37–7.29(m,4H),7.27–7.22(m,1H),4.52(d,J=6.0 Hz, 2H), 2.39 (t, J = 7.4Hz, 2H), 1.61 (h, J = 7.4Hz, 2H), 0.90 (t, J = 7.4Hz, 3H). 13 C NMR(151MHz,DMSO-d6)δ172.42,166.15,151.77,146.02,139.67,137.05,136.43,135.13,130.46,129.40 ,129.25,128.46,127.39,126.94,126.82,125.00,113.38,42.81,38.10,18.52,13.71.HRMS(ESI):calcd for C 23 H 23 N3O2Na[M+Na] + m / z,396.1683; found,396.1676.

[0248] Example 65: N-Benzyl-3-(6-pentamidopyridin-3-yl)benzamide (i-2)

[0249]

[0250] The procedure is the same as in Example 64, except that n-butyryl chloride is replaced with valeryl chloride. It is a white solid with a yield of 78% and a melting point of 203-206°C. 1 H NMR (600MHz, DMSO-d6) δ10.57(s,1H),9.19(t,J=6.0Hz,1H),8.70(d,J=2.5Hz,1 H),8.23–8.17(m,2H),8.15(dd,J=8.7,2.5Hz,1H),7.92–7.85(m,2H),7.58(t,J =7.7Hz,1H),7.37–7.29(m,4H),7.28–7.21(m,1H),4.52(d,J=6.0Hz,2H),2.41( t,J=7.4Hz,2H),1.60–1.54(m,2H),1.31(h,J=7.4Hz,2H),0.89(t,J=7.3Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ172.57,166.15,151.78,146.02,139.67,137.05,136.43,135.13,130.45,129.41,12 9.25,128.47,127.39,126.94,126.81,125.00,113.37,42.81,35.92,27.22,21.89,13.88.HRMS(ESI):calcd for C 24 H 25 N3O2Na[M+Na] + m / z,410.1839; found,410.1834.

[0251] Example 66: N-Benzyl-3-(6-(cyclohexanecarboxamide)pyridin-3-yl)benzamide (i-3)

[0252]

[0253] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with cyclohexyl chloride, with a yield of 69% and a melting point of 180-183°C. 1 HNMR(600MHz,DMSO-d6)δ10.52(s,1H),9.19(t,J=6.0Hz,1H),8.72(d,J=2.5Hz,1H),8.24–8.19(m,2H),8.15(dd,J=8.7,2.6Hz,1H),7.92–

[0254] 7.86(m,2H),7.58(t,J=7.7Hz,1H),7.36–7.32(m,4H),7.27–7.22(m,1H),4.53(d,J=6.0Hz,2H),2.56–2.51(m,1H) ,1.83–1.79(m,2H),1.76–1.71(m,2H),1.66–1.61(m,1H),1.44–1.37(m,2H),1.29–1.25(m,1H),1.25–1.16(m,2H). 13 C NMR(151MHz,DMSO-d6)δ175.38,165.95,151.86,145.91,139.62,136.94,136.23,135.06,130.26,129.24,12 9.07,128.34,127.28,126.80,126.70,124.88,113.26,44.30,42.70,29.07,25.41,25.20.HRMS(ESI):calcd for C 26 H 27 N3O2Na[M+Na] + m / z,436.1996; found,436.1989.

[0255] Example 67: 3-(6-acetamidopyridin-3-yl)-N-isopentylbenzamide (i-4)

[0256]

[0257] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with acetyl chloride, and product g-1 from Example 38 was replaced with product g-8 from Example 45. The yield was 66%, and the melting point was 171-174°C. 1 H NMR(600MHz,Chloroform-d)δ8.64(s,1H),8.49(d,J=2.4Hz,1H),8.26(d,J=8.6Hz,1H),8.00(s,1H),7.90(dd,J=8.6,2.4Hz,1H),7.71(d,J=7.8Hz,1H),7. 64(d,J=7.8Hz,1H),7.49(t,J=7.7Hz,1H),6.36–6.30(m,1H),3.52–3.47(m,2 H),2.22(s,3H),1.72–1.65(m,1H),1.55–1.50(m,2H),0.95(d,J=6.6Hz,6H). 13CNMR(151MHz,Chloroform-d)δ169.00,167.37,151.07,146.01,138.05,137.05,135.89,132 .05,129.64,129.37,125.90,125.77,114.00,38.66,26.11,24.85,22.62.HRMS(ESI):calcd for C 19 H 23 N3O2Na[M+Na] + m / z,348.1683; found,348.1675.

[0258] Example 68: N-Isopentyl-3-(6-propamidopyridin-3-yl)benzamide (i-5)

[0259]

[0260] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with propionyl chloride, and product g-1 from Example 38 was replaced with product g-8 from Example 45. The yield was 71%, and the melting point was 189-192°C. 1 H NMR(600MHz,Chloroform-d)δ8.51–8.47(m,1H),8.36(s,1H),8.29(d,J=8.6Hz,1H) ,8.01–7.97(m,1H),7.91(dd,J=8.6,2.4Hz,1H),7.73–7.69(m,1H),7.67–7.63(m,1H ),7.49(t,J=7.7Hz,1H),6.34–6.25(m,1H),3.52–3.47(m,2H),2.45(q,J=7.5Hz,2H) ,1.72–1.65(m,1H),1.55–1.50(m,2H),1.25(t,J=7.5Hz,3H),0.95(d,J=6.6Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ172.62,167.34,151.07,146.05,138.09,137.03,135.89,131.96 ,129.62,129.37,125.89,125.72,113.91,38.66,30.91,26.11,22.62,9.50.HRMS(ESI):calcd for C 20 H 25 N3O2Na[M+Na] + m / z,362.1839; found,362.1831.

[0261] Example 69: 3-(6-Butyramidopyridin-3-yl)-N-isopentylbenzamide (i-6)

[0262]

[0263] The operation was the same as in Example 64, except that product g-1 from Example 38 was replaced with product g-8 from Example 45, with a yield of 75% and a melting point of 172-175°C. 1 H NMR(600MHz,Chloroform-d)δ8.49(d,J=2.4Hz,1H),8.34(s,1H),8.29(d,J=8.6Hz,1H),8.01–7 .97(m,1H),7.91(dd,J=8.6,2.4Hz,1H),7.71(dt,J=7.8,1.4Hz,1H),7.65(dt,J=7.8,1.4Hz,1H ),7.49(t,J=7.7Hz,1H),6.36–6.23(m,1H),3.53–3.47(m,2H),2.39(t,J=7.4Hz,2H),1.77(h,J =7.4Hz,2H),1.72–1.65(m,1H),1.55–1.50(m,2H),1.00(t,J=7.4Hz,3H),0.95(d,J=6.7Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ171.90,167.34,151.03,146.02,138.08,137.04,135.90,131.99,12 9.62,129.37,125.90,125.71,113.94,39.78,38.66,26.11,22.62,18.94,13.85.HRMS(ESI):calcd forC 21 H 27 N3O2Na[M+Na] + m / z,376.1996; found,376.1987.

[0264] Example 70: N-Isopentyl-3-(6-pentamidopyridin-3-yl)benzamide (i-7)

[0265]

[0266] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with valeryl chloride, and product g-1 from Example 38 was replaced with product g-8 from Example 45. The yield was 79%, and the melting point was 119-122°C. 1HNMR(600MHz,DMSO-d6)δ10.59(s,1H),8.70(d,J=2.5Hz,1H),8.54(t,J=5.6Hz ,1H),8.21(d,J=8.7Hz,1H),8.15(d,J=2.5Hz,1H),8.14–8.12(m,1H),7.87–7. 81(m,2H),7.55(t,J=7.7Hz,1H),3.33–3.29(m,2H),2.42(t,J=7.4Hz,2H),1.6 5–1.55(m,3H),1.44(q,J=7.1Hz,2H),1.32(h,J=7.4Hz,2H),0.93–0.88(m,9H). 13 C NMR (151MHz, DMSO-d6) δ172.38,165.76,151.70,145.91,136.85,136.29,135.47,130.37,129.14,128. 84,126.56,124.79,113.22,38.19,37.55,35.82,27.11,25.38,22.49,21.81,13.78.HRMS(ESI):calcd for C 22 H 30 N3O2[M+H] + m / z,368.2333; found,368.2333.

[0267] Example 71: 3-(6-(cyclopropanecarboxamide)pyridin-3-yl)-N-isopentylbenzamide (i-8)

[0268]

[0269] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with cyclopropionyl chloride, and product g-1 from Example 38 was replaced with product g-8 from Example 45. The yield was 77%, and the melting point was 199-202°C. 1H NMR(600MHz,Chloroform-d)δ8.66(s,1H),8.51(d,J=2.4Hz,1H),8.27(d,J=8.6Hz,1H),8.00–7 .96(m,1H),7.90(dd,J=8.6,2.4Hz,1H),7.70(dt,J=7.7,1.4Hz,1H),7.65(dt,J=7.8,1.4Hz,1H) ,7.50(t,J=7.7Hz,1H),6.30–6.19(m,1H),3.53–3.46(m,2H),1.74–1.64(m,J=6.6Hz,1H),1.63– 1.58(m,1H),1.53(q,J=7.1Hz,2H),1.15–1.10(m,2H),0.96(d,J=6.6Hz,6H),0.92–0.89(m,2H). 13 C NMR(151MHz,Chloroform-d)δ172.59,167.34,151.14,145.97,138.11,137.07,135.91,131.84,12 9.62,129.39,125.88,125.68,113.98,38.68,38.66,26.12,22.63,16.03,8.66.HRMS(ESI):calcd for C 21 H 25 N3O2Na[M+Na] + m / z,374.1839; found,374.1831.

[0270] Example 72: 3-(6-(cyclobutanecarboxamide)pyridin-3-yl)-N-isopentylbenzamide (i-9)

[0271]

[0272] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with cyclobutyryl chloride, and product g-1 from Example 38 was replaced with product g-8 from Example 45. The yield was 78%, and the melting point was 193-196°C. 1H NMR(600MHz,Chloroform-d)δ8.48(d,J=2.4Hz,1H),8.30(d,J=8.6Hz,1H),8.09(s,1H),8.00–7.9 6(m,1H),7.90(dd,J=8.6,2.4Hz,1H),7.73–7.68(m,1H),7.66–7.62(m,1H),7.49(t,J=7.7Hz,1H) ,6.36–6.27(m,1H),3.51–3.47(m,2H),3.26–3.17(m,1H),2.43–2.36(m,2H),2.27–2.21(m,2H),2 .05–1.98(m,1H),1.95–1.88(m,1H),1.74–1.64(m,1H),1.55–1.50(m,2H),0.95(d,J=6.6Hz,6H). 13 CNMR(151MHz,Chloroform-d)δ173.79,167.33,151.06,146.02,138.07,137.01,135.89,131.94,12 9.60,129.36,125.91,125.68,113.85,40.94,38.65,26.10,25.30,22.62,18.13.HRMS(ESI):calcd for C 22 H 27 N3O2Na[M+Na] + m / z,388.1996; found,388.1986.

[0273] Example 73: 3-(6-(cyclopentanamide)pyridin-3-yl)-N-isopentylbenzamide (i-10)

[0274]

[0275] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with cyclopentyl chloride, and product g-1 from Example 38 was replaced with product g-8 from Example 45. The yield was 79%, and the melting point was 169-172°C. 1H NMR (600MHz, DMSO-d6) δ10.59(s,1H),8.70(d,J=2.5Hz,1H),8.54(t,J=5.6H z,1H),8.22(d,J=8.7Hz,1H),8.17–8.10(m,2H),7.88–7.79(m,2H),7.55(t,J =7.7Hz,1H),3.33–3.29(m,2H),3.00–2.92(m,1H),1.89–1.82(m,2H),1.75– 1.60(m,5H),1.58–1.51(m,2H),1.44(q,J=7.1Hz,2H),0.91(d,J=6.6Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ175.48,165.76,151.81,145.87,136.84,136.26,135.47,130.33,129.13,1 28.82,126.55,124.76,113.28,44.83,38.19,37.56,30.07,25.81,25.38,22.49.HRMS(ESI):calcd for C 23 H 30 N3O2[M+H] + m / z,380.2333; found,380.2333.

[0276] Example 74: 3-(6-(cyclohexanecarboxamide)pyridin-3-yl)-N-isopentylbenzamide (i-11)

[0277]

[0278] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with cyclohexyl chloride, and product g-1 from Example 38 was replaced with product g-8 from Example 45. The yield was 80%, and the melting point was 163-166°C. 1 H NMR(600MHz,Chloroform-d)δ8.49(d,J=2.4Hz,1H),8.30(d,J=8.6Hz,1H),8.17(s,1 H),8.01–7.94(m,1H),7.91(dd,J=8.6,2.4Hz,1H),7.74–7.68(m,1H),7.68–7.62(m,

[0279] 1H),7.49(t,J=7.7Hz,1H),6.30–6.21(m,1H),3.55–3.43(m,2H),2.33–2.24(m,1H),1.99–1.95(m,2H ),1.88–1.80(m,2H),1.74–1.64(m,2H),1.59–1.49(m,4H),1.33–1.23(m,3H),0.95(d,J=6.6Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ174.97,167.32,151.14,146.01,138.11,137.02,135.91,131.95,

[0280] 129.62,129.38,125.90,125.67,113.93,46.64,38.68,38.65,29.62,26.11,25.77,25.72,22.63.HRMS(ESI):calcd for C 24 H 31 N3O2Na[M+Na] + m / z,416.2309; found,416.2300.

[0281] Example 75: 3-(6-Benzamidepyridin-3-yl)-N-ethylbenzamide (i-12)

[0282]

[0283] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-2 from Example 39. The yield was 83%, and the melting point was 204-207°C. 1 HNMR (600MHz, DMSO-d6) δ10.97(s,1H),8.80(d,J=2.5Hz,1H),8.62(t,J=5.6Hz,1H),8.34(d,J=8.4Hz,1H),8.24(dd,J=8.7,2.5Hz,1H),8. 19(t,J=1.9Hz,1H),8.10–8.03(m,2H),7.92–7.85(m,2H),7.64–7.56(m,2H),7.56–7.49(m,2H),3.36–3.31(m,2H),1.16(t,J=7.2Hz,3H). 13C NMR(151MHz,DMSO-d6)δ166.11,165.71,151.79,145.94,136.80,136.33,135.51,134.05,132.04, 131.02,129.19,128.99,128.41,128.10,126.68,124.95,114.55,34.17,14.86.HRMS(ESI):calcd for C 21 H 20 N3O2[M+H] + m / z,346.1550; found,346.1549.

[0284] Example 76: 3-(6-benzamidepyridin-3-yl)-N-propylbenzamide (i-13)

[0285]

[0286] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-3 from Example 40. The yield was 82%, and the melting point was 147-150°C. 1 HNMR(600MHz,DMSO-d6)δ10.98(s,1H),8.80(t,J=1.9Hz,1H),8.61(t,J=5.7 Hz,1H),8.34(d,J=8.7Hz,1H),8.24(dt,J=8.6,2.1Hz,1H),8.20(d,J=1.9Hz ,1H),8.07(d,J=7.8Hz,2H),7.93–7.85(m,2H),7.65–7.57(m,2H),7.53(t,J =7.4Hz,2H),3.30–3.25(m,2H),1.57(h,J=7.4Hz,2H),0.92(t,J=7.4Hz,3H). 13 CNMR(151MHz,DMSO-d6)δ166.10,165.89,151.78,145.94,136.79,136.33,135.53,134.04,132.03,13 1.02,129.18,128.98,128.40,128.09,126.70,124.97,114.54,41.09,22.46,11.55.HRMS(ESI):calcd for C 22 H 22 N3O2[M+H] + m / z,360.1707; found,360.1702.

[0287] Example 77: 3-(6-benzamidepyridin-3-yl)-N-butylbenzamide (i-14)

[0288]

[0289] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-4 from Example 41. The yield was 79%, and the melting point was 187-190°C. 1 HNMR(600MHz,DMSO-d6)δ10.97(s,1H),8.79(d,J=2.5Hz,1H),8.59(t,J=5.6Hz, 1H),8.34(d,J=8.7Hz,1H),8.23(dd,J=8.7,2.5Hz,1H),8.19(t,J=1.9Hz,1H),8. 09–8.03(m,2H),7.92–7.84(m,2H),7.63–7.56(m,2H),7.56–7.49(m,2H),3.33– 3.28(m,2H),1.54(p,J=7.4Hz,2H),1.35(h,J=7.4Hz,2H),0.91(t,J=7.4Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ166.09,165.84,151.78,145.94,136.79,136.33,135.53,134.04,132.03,131.02 ,129.18,128.97,128.40,128.09,126.70,124.96,114.54,38.99,31.32,19.74,13.79.HRMS(ESI):calcd for C 23 H 24 N3O2[M+H] + m / z,374.1863; found,374.1863.

[0290] Example 78: 3-(6-benzamidepyridin-3-yl)-N-(cyclopropylmethyl)benzamide (i-15)

[0291]

[0292] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-5 from Example 42. The yield was 85%, and the melting point was 188-191°C. 1HNMR(600MHz,DMSO-d6)δ10.97(s,1H),8.80(d,J=2.5Hz,1H),8.72(t,J=5.7Hz ,1H),8.34(d,J=8.6Hz,1H),8.24(dd,J=8.7,2.5Hz,1H),8.22–8.19(m,1H),8. 09–8.04(m,2H),7.92–7.87(m,2H),7.63–7.57(m,2H),7.53(t,J=7.6Hz,2H),3 .19(t,J=6.2Hz,2H),1.11–1.03(m,1H),0.48–0.42(m,2H),0.28–0.23(m,2H). 13 C NMR(151MHz,DMSO-d6)δ166.11,165.87,151.79,145.95,136.81,136.35,135.43,134.05,132.04,13 1.03,129.19,129.05,128.41,128.09,126.76,125.03,114.55,43.67,11.07,3.43.HRMS(ESI):calcd for C 23 H 22 N3O2[M+H] + m / z,372.1707; found,372.1707.

[0293] Example 79: 6'-benzoamide-N-(cyclopropylmethyl)-[2,3'-bipyridine]-4-carboxamide (i-16)

[0294]

[0295] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-22 from Example 59. The yield was 79%, and the melting point was 158-161°C. 1H NMR (600MHz, DMSO-d6) δ11.06 (s, 1H), 9.17 (d, J = 2.4Hz, 1H), 8.97 (t, J = 5.6Hz, 1H),8.82(d,J=5.0Hz,1H),8.58(dd,J=8.8,2.5Hz,1H),8.41–8.35(m,2H),8.0 9–8.04(m,2H),7.75(dd,J=5.0,1.5Hz,1H),7.63–7.59(m,1H),7.55–7.50(m,2 H),3.23–3.19(m,2H),1.10–1.03(m,1H),0.49–0.45(m,2H),0.29–0.25(m,2H). 13 C NMR (151MHz, DMSO) δ166.17,164.46,154.29,152.96,150.42,146.37,142.82,136.27,133.95, 132.10,129.73,128.41,128.12,120.42,117.33,114.22,43.84,10.88,3.49.HRMS(ESI):calcd for C 22 H 21 N4O2[M+H] + m / z,373.1659; found,373.1659.

[0296] Example 80: 3-(6-benzamidepyridin-3-yl)-N-isopentylbenzamide (i-17)

[0297]

[0298] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-8 from Example 45. The yield was 81%, and the melting point was 168-171°C. 1 HNMR(600MHz,Chloroform-d)δ8.85(s,1H),8.53–8.46(m,2H),8.01–7.97(m,2H),7.95(d,J=7.0Hz,2H),7.72(d,J=7.8Hz,1H),7.67(d,J=8.1Hz ,1H),7.58(t,J=7.4Hz,1H),7.53–7.49(m,3H),6.23(s,1H),3.53–3.49 (m,2H),1.74–1.66(m,1H),1.54(q,J=7.0Hz,2H),0.97(d,J=6.6Hz,6H). 13C NMR (151MHz, CDCl3) δ167.30,165.89,151.15,146.17,138.02,137.18,135.96,134.26,132.53,132.31, 129.67,129.43,129.03,127.41,126.00,125.71,114.16,38.69,38.67,26.13,22.65.HRMS(ESI):calcd for C 24 H 25 N3O2Na[M+Na] + m / z,410.1839; found,410.1830.

[0299] Example 81: 3-(6-(4-fluorobenzamide)pyridin-3-yl)-N-isopentylbenzamide (i-18)

[0300]

[0301] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with p-fluorobenzoyl chloride, and product g-1 from Example 38 was replaced with product g-8 from Example 45. The yield was 83%, and the melting point was 194-197°C. 1 H NMR (600MHz, DMSO-d6) δ11.02(s,1H),8.79(d,J=2.5Hz,1H),8.56(t,J=5.6Hz ,1H),8.31(d,J=8.7Hz,1H),8.23(dd,J=8.7,2.5Hz,1H),8.19–8.17(m,1H),8. 17–8.10(m,2H),7.91–7.84(m,2H),7.58(t,J=7.7Hz,1H),7.40–7.31(m,2H),3 .34–3.30(m,2H),1.67–1.60(m,1H),1.48–1.43(m,2H),0.91(d,J=6.6Hz,6H). 13C NMR(151MHz,DMSO-d6)δ165.78,165.03,164.38(165.20,163.55,d,J=249.9Hz ),151.74,145.93,136.77,136.34,135.52,131.07,130.92(130.95,130.89,d, J=9.2Hz),130.53(130.54,130.52,d,J=3.0Hz),129.18,128.98,126.70,124. 96,115.36(115.43,115.29,d,J=21.9Hz),114.55,38.20,37.58,25.39,22.50. 19 F NMR(565MHz,DMSO-d6)δ-108.12.HRMS(ESI):calcd for C 24 H 25 FN3O2[M+H] + m / z,406.1925; found,406.1925.

[0302] Example 82: 6-benzoamide-N-isopentyl-[3,4'-bipyridine]-2'-carboxamide (i-19)

[0303]

[0304] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-18 from Example 55. The yield was 85%, and the melting point was 149-152°C. 1 H NMR(600MHz,Chloroform-d)δ9.16(s,1H),8.58(d,J=5.0Hz,1H),8.54(d,J=8.7Hz,1H),8.51(d,J=2.5Hz,1H),8.41(d,J=1.8Hz,1H),8.10–8.03 (m,2H),7.96–7.92(m,2H),7.60–7.55(m,2H),7.52–7.47(m,2H),3.53–3 .48(m,2H),1.76–1.66(m,1H),1.57–1.52(m,2H),0.96(d,J=6.6Hz,6H). 13C NMR(151MHz,Chloroform-d)δ166.16,164.09,152.58,151.00,148.88,146.40,146.23,137.06,134.18, 132.60,129.30,129.00,127.49,123.09,119.51,114.31,38.58,37.90,25.97,22.60.HRMS(ESI):calcd for C 23 H 25 N4O2[M+H] + m / z,389.1972; found,389.1971.

[0305] Example 83: 6'-benzoamide-N-isopentyl-[3,3'-bipyridine]-5-carboxamide (i-20)

[0306]

[0307] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-19 from Example 56. The yield was 87%, and the melting point was 242-245°C. 1 H NMR(600MHz,DMSO-d6)δ11.02(s,1H),9.10(d,J=2.2Hz,1H),8.99(d,J=2.0Hz,1H),8 .86(d,J=2.5Hz,1H),8.72(t,J=5.6Hz,1H),8.51(t,J=2.2Hz,1H),8.36(d,J=8.7Hz, 1H),8.31(dd,J=8.7,2.5Hz,1H),8.10–8.02(m,2H),7.64–7.57(m,1H),7.56–7.49(m ,2H),3.36–3.32(m,2H),1.69–1.60(m,1H),1.49–1.44(m,2H),0.92(d,J=6.6Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ166.20,164.42,152.33,149.41,147.58,146.22,136.60,133.97,132.31,13 2.10,132.01,130.18,128.42,128.12,127.98,114.56,38.07,37.60,25.35,22.47.HRMS(ESI):calcd for C 23 H 25 N4O2[M+H] +m / z,389.1972; found,389.1971.

[0308] Example 84: 6'-benzoamide-N-isopentyl-[2,3'-bipyridine]-4-carboxamide (i-21)

[0309]

[0310] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-20 from Example 57. The yield was 87%, and the melting point was 166-169°C. 1 H NMR(600MHz,Chloroform-d)δ9.04(s,1H),8.83(d,J=2.5Hz,1H),8.73–8.67(m,1H),8.44–

[0311] 8.39(m,1H),8.32(dd,J=8.7,2.5Hz,1H),8.01–7.99(m,1H),7.92–7.89(m,2H),7.57–7.51(m,1H),7.49–7.45 (m,3H),6.77(t,J=5.6Hz,1H),3.49–3.45(m,2H),1.70–1.62(m,1H),1.54–1.49(m,2H),0.93(d,J=6.6Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ166.07,165.75,155.33,152.20,150.57,146.67,143.15,136.95,134.09,1 32.55,130.53,128.96,127.40,119.48,117.62,113.87,38.80,38.42,26.07,22.56.HRMS(ESI):calcdfor C 23 H 25 N4O2[M+H] + m / z,389.1972; found,389.1972.

[0312] Example 85: 6'-benzoamide-N-isopentyl-[2,3'-bipyridine]-6-carboxamide (i-22)

[0313]

[0314] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-21 from Example 58. The yield was 89%, and the melting point was 143-146°C. 1 H NMR(600MHz,Chloroform-d)δ9.07(s,1H),8.85(d,J=2.5Hz,1H),8.55(d,J=8.6Hz, 1H),8.36(dd,J=8.6,2.5Hz,1H),8.17(d,J=7.6Hz,1H),8.09(t,J=6.0Hz,1H),7.98–

[0315] 7.94(m,2H),7.94–7.90(m,1H),7.81–7.78(m,1H),7.60–7.54(m,1H),7.52–

[0316] 7.48(m,2H),3.55–3.51(m,2H),1.77–1.68(m,1H),1.59–1.55(m,2H),0.97(d,J=6.6Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ165.99,164.07,152.97,152.34,150.29,146.61,138.58,136.98,134.17, 132.56,130.36,129.00,127.44,122.41,121.11,113.97,38.68,37.95,26.14,22.64.HRMS(ESI):calcd for C 23 H 25 N4O2[M+H] + m / z,389.1972; found,389.1971.

[0317] Example 86: 3-(6-benzamidepyridin-3-yl)-N-isopentyl-4-methylbenzamide (i-23)

[0318]

[0319] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-16 from Example 53. The yield was 74%, and the melting point was 162-165°C. 1HNMR(600MHz,DMSO-d6)δ10.92(s,1H),8.34(d,J=2.4Hz,1H),8.33–8.27(m,2H),8.08–8.03(m,2H),7.84(dd,J=8.5,2.5Hz,1H),7.64–7.58 (m,1H),7.57–7.50(m,2H),7.37–7.27(m,3H),3.28–3.24(m,2H),2.22(s,3H),1.69–1.61(m,1H),1.44–1.39(m,2H),0.91(d,J=6.6Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ169.16,166.13,151.20,147.58,139.31,138.73,138.44,134.11,132.55,132.29,13 2.03,130.46,128.43,128.07,126.48,125.79,114.05,38.05,37.18,25.33,22.46,17.23.HRMS(ESI):calcd for C 25 H 28 N3O2[M+H] + m / z,402.2176; found,402.2176.

[0320] Example 87: 3-(6-benzamidepyridin-3-yl)-N-isopentyl-2-methylbenzamide (i-24)

[0321]

[0322] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-17 from Example 54. The yield was 73%, and the melting point was 152-155°C. 1H NMR(600MHz,Chloroform-d)δ8.95(s,1H),8.44(d,J=8.5Hz,1H),8.19(d,J=2.3Hz,1H),7. 96–7.92(m,2H),7.71(dd,J=8.5,2.3Hz,1H),7.67(dd,J=7.9,1.9Hz,1H),7.62(d,J=1.9Hz ,1H),7.59–7.55(m,1H),7.52–7.48(m,2H),7.33(d,J=7.9Hz,1H),6.26(t,J=5.6Hz,1H),3 .49–3.44(m,2H),2.29(s,3H),1.71–1.63(m,1H),1.53–1.48(m,2H),0.93(d,J=6.6Hz,6H). 13 C NMR (151MHz, Chloroform-d)δ

[0323] 167.10,166.00,150.70,147.76,139.49,139.17,138.05,134.28,133.04,132.86,132.47,130.9 2,128.99,128.55,127.38,126.40,113.62,38.65,38.53,26.06,22.61,20.58.HRMS(ESI):calcd for C 25 H 28 N3O2[M+H] + m / z,402.2176; found,402.2176.

[0324] Example 88: 5-(6-benzamidopyridin-3-yl)-2-fluoro-N-isopentylbenzamide (i-25)

[0325]

[0326] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-12 from Example 49. The yield was 69%, and the melting point was 164-167°C. 1H NMR(600MHz,Chloroform-d)δ9.04(s,1H),8.47(d,J=8.6Hz,1H),8.39(s,1H),8.28(dd,J=7.3,2.5Hz,1H),7.96–7.92(m,3H) ,7.61–7.54(m,2H),7.49(t,J=7.6Hz,2H),7.22–7.16(m,1H),6.81–6.71(m,1H),3.54–3.50(m,2H),1.74–1.64(m,1H),1.56–

[0327] 1.51(m,2H),0.96(d,J=6.7Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ166.02,162.91,160.39(161.22,159.57,d,J=248.2Hz),151.21, 146.03,136.94,134.39(134.40,134.38,d,J=3.1Hz),134.33,132.45,131.22(131.23,131.21, d,J=2.7Hz),131.17,130.35(130.36,130.34,d,J=2.5Hz),128.95,127.43,121.85(121.89,12 1.81, d, J = 12.3Hz), 116.93 (117.01, 116.84, d, J = 25.7Hz), 114.14, 38.61, 38.43, 26.06, 22.59. 19 FNMR(565MHz,Chloroform-d)δ-115.75.HRMS(ESI):calcd for C 24 H 25 FN3O2[M+H] + m / z,406.1925; found,406.1925.

[0328] Example 89: 3-(6-benzamidopyridin-3-yl)-5-fluoro-N-isopentylbenzamide (i-26)

[0329]

[0330] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-13 from Example 50. The yield was 71%, and the melting point was 187-190°C. 1H NMR(600MHz,Chloroform-d)δ8.98(s,1H),8.47(d,J=8.6Hz,1H),8.41(s,1H),7.96–7.91(m,3H),7.73(s,1H),7.58(t,J=7.5Hz,1H),7.50(t,J=7.6Hz, 2H),7.43(d,J=8.6Hz,1H),7.33(d,J=9.0Hz,1H),6.35(t,J=5.8Hz,1H),3.5 1–3.46(m,2H),1.72–1.65(m,1H),1.56–1.50(m,2H),0.95(d,J=6.6Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ166.13,166.05,163.19(164.02,162.37,d,J=248.5Hz),151.60, 146.17,140.09(140.12,140.06,d,J=7.9Hz),138.00(138.02,137.98,d,J=7.0Hz),137.06,134 .19,132.60,131.03,129.03,127.43,121.15(121.16,121.14,d,J=2.5Hz),116.43(116.51,11 6.36, d, J=22.6Hz), 114.15, 113.34 (113.42, 113.27, d, J=22.8Hz), 38.78, 38.57, 26.10, 22.61. 19 F NMR(565MHz,DMSO-d6)δ-112.16.HRMS(ESI):calcd for C 24 H 25 FN3O2[M+H] + m / z,406.1925; found,406.1925.

[0331] Example 90: 3-(6-benzamidopyridin-3-yl)-4-fluoro-N-isopentylbenzamide (i-27)

[0332]

[0333] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-14 from Example 51. The yield was 74%, and the melting point was 150-153°C. 1H NMR(600MHz,Chloroform-d)δ8.92(s,1H),8.46(d,J=8.6Hz,1H),8.44–8.40(m,1H),7.95–

[0334] 7.91(m,3H),7.88(dd,J=7.4,2.3Hz,1H),7.76–7.72(m,1H),7.60–7.53(m,1H),7.50(t,J=7.8Hz,2H),7.23–7.1 7(m,1H),6.34(t,J=5.8Hz,1H),3.49–3.45(m,2H),1.72–1.64(m,1H),1.54–1.49(m,2H),0.94(d,J=6.6Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ166.30,165.99,161.59(162.43,160.75,d,J=253.8Hz),151.24,147.72(147.7 3,147.71,d,J=3.7Hz),138.91(138.92,138.90,d,J=3.1Hz),134.17,132.54,131.74(131.75,131.73,d,J=3. 5Hz) 129.61 (129.62, 129.59, d, J = 4.2Hz), 129.01, 128.37 (128.40, 128.34, d, J = 9.1Hz), 127.39, 127.09, 125. 65(125.70,125.60,d,J=14.3Hz),116.60(116.68,116.53,d,J=23.4Hz),113.84,38.71,38.62,26.09,22.61. 19 F NMR(565MHz,DMSO-d6)δ-114.96.HRMS(ESI):calcd for C 24 H 25 FN3O2[M+H] + m / z,406.1925; found,406.1925.

[0335] Example 91: 3-(6-benzamidopyridin-3-yl)-2-fluoro-N-isopentylbenzamide (i-28)

[0336]

[0337] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-15 from Example 52. The yield was 74%, and the melting point was 173-176°C. 1 H NMR(600MHz,Chloroform-d)δ9.00(s,1H),8.50(d,J=8.6Hz,1H),8.36(t,J=1.8Hz,1 H),8.07(td,J=7.5,1.9Hz,1H),7.97–7.93(m,2H),7.91(dt,J=8.6,1.9Hz,1H),7.60–

[0338] 7.54(m,1H),7.52–7.47(m,3H),7.33(t,J=7.7Hz,1H),6.75–6.65(m,1H),3. 53–3.49(m,2H),1.74–1.64(m,1H),1.55–1.50(m,2H),0.95(d,J=6.7Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ166.00,163.21(163.22,163.21,d,J=2.8Hz),157.54(158.37,156.72,d,J=248.5Hz),1 51.30,147.80(147.81,147.78,d,J=3.2Hz),139.11(139.12,139.10,d,J=2.8Hz),134.20,133.55(133.57,133.54,d ,J=4.1Hz),132.51,131.92(131.93,131.91,d,J=2.4Hz),129.00,127.42,127.18,126.12(126.18,126.07,d,J=17.2 Hz), 125.17 (125.18, 125.15, d, J = 3.8 Hz), 122.53 (122.57, 122.49, d, J = 13.1 Hz), 113.87, 38.61, 38.41, 26.04, 22.57. 19 F NMR(565MHz,DMSO-d6)δ-120.46.HRMS(ESI):calcd forC 24 H 25 FN3O2[M+H] + m / z,406.1925; found,406.1925.

[0339] Example 92: 3-(6-benzamidepyridin-3-yl)-N-isopropylbenzamide (i-29)

[0340]

[0341] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-6 from Example 43. The yield was 78%, and the melting point was 231-234°C. 1 HNMR(600MHz,DMSO-d6)δ10.96(s,1H),8.79(s,1H),8.38–8.32(m,2H),8.24(d,J=8.7Hz,1H),8.18(s,1H),8.06(d ,J=7.4Hz,2H),7.90–7.85(m,2H),7.63–7.57(m,2H),7.53(t,J=7.6Hz,2H),4.17–4.12(m,1H),1.22–1.18(m,6H). 13 C NMR(151MHz,DMSO-d6)δ166.12,165.11,151.77,145.97,136.76,136.38,135.63,134.05,132.06, 131.08,129.12,128.99,128.42,128.10,126.83,125.03,114.55,41.15,22.42.HRMS(ESI):calcd for C 22 H 22 N3O2[M+H] + m / z,360.1707; found,360.1706.

[0342] Example 93: 3-(6-Benzamidepyridin-3-yl)-N-isobutylbenzamide (i-30)

[0343]

[0344] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-7 from Example 44. The yield was 75%, and the melting point was 195-198°C. 1HNMR (600MHz, DMSO-d6) δ10.96(s,1H),8.80(d,J=2.5Hz,1H),8.62(t,J=5.8Hz,1H),8.34(d,J=8.6Hz,1H),8.24(dd,J=8.6,2.5Hz,1H),8.20(t,J=1. 8Hz,1H),8.09–8.03(m,2H),7.91–7.85(m,2H),7.63–7.56(m,2H),7.53(t, J=7.6Hz,2H),3.15–3.11(m,2H),1.91–1.84(m,1H),0.91(d,J=6.7Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ166.10,166.03,151.78,145.95,136.81,136.35,135.58,134.05,132.04,131 .05,129.18,128.99,128.41,128.09,126.74,125.01,114.55,46.84,28.20,20.30.HRMS(ESI):calcd for C 23 H 24 N3O2[M+H] + m / z,374.1863; found,374.1863.

[0345] Example 94: 6'-benzoamide-N-isobutyl-[2,3'-bipyridine]-4-carboxamide (i-31)

[0346]

[0347] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-23 from Example 60. The yield was 80%, and the melting point was 171-174°C. 1 H NMR (600MHz, DMSO-d6) δ11.05(s,1H),9.16(d,J=2.5Hz,1H),8.85(t,J=5.8Hz,1H),8.81(d,J=5.0Hz,1H),8.57(dd,J=8.7,2.5Hz,1H),8.42–8.33( m,2H),8.09–8.05(m,2H),7.75–7.71(m,1H),7.63–7.59(m,1H),7.53(t,J =7.7Hz,2H),3.17–3.13(m,2H),1.92–1.85(m,1H),0.92(d,J=6.7Hz,6H). 13C NMR(151MHz,DMSO-d6)δ166.16,164.64,154.27,152.95,150.40,146.37,142.96,136.26,133.95 ,132.09,129.73,128.41,128.12,120.42,117.33,114.21,46.89,28.13,20.27.HRMS(ESI):calcd for C 22 H 23 N4O2[M+H] + m / z,375.1816; found,375.1815.

[0348] Example 95: 3-(6-benzamidepyridin-3-yl)-N-(2,2-difluoroethyl)benzamide (i-32)

[0349]

[0350] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-9 from Example 46. The yield was 80%, and the melting point was 207-210°C. 1 HNMR(600MHz, DMSO-d6)δ10.98(s,1H),9.02(t,J=5.9Hz,1H),8.81(d,J=2.5Hz,1H),8.34(d,J=8.6Hz,1H),8.27–8.22(m,2H),8.10–8.03(m, 2H),7.95(dt,J=7.8,1.4Hz,1H),7.91(dt,J=7.8,1.4Hz,1H),7.65–7. 58(m,2H),7.53(t,J=7.7Hz,2H),6.27–6.06(m,1H),3.77–3.70(m,2H). 13 C NMR (151MHz, DMSO-d6) δ166.66,166.11,151.86,145.96,136.94,136.34,134.38,134.04,132.04,130.84,129.58,129.36,128. 40,128.10,126.90,125.15,114.60(116.20,114.60,113.01,t,J=240.3Hz), 114.54,41.67(41.85,41.67,41.50,t,J=26.5Hz). 19 F NMR (565MHz, DMSO-d6)δ

[0351] -121.37.HRMS(ESI):calcd for C 21 H 18 F2N3O2[M+H] + m / z,382.1362; found,382.1362.

[0352] Example 96: 6'-benzoylamino-N-(2,2-difluoroethyl)-[2,3'-bipyridine]-4-carboxamide (i-33)

[0353]

[0354] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-24 from Example 61. The yield was 82%, and the melting point was 177-180°C. 1 H NMR (600MHz, DMSO-d6) δ11.06(s,1H),9.26(t,J=5.9Hz,1H),9.20–9.13(m,1H),8.84(d,J=5.0Hz,1H),8.57(dd,J=8.7,2.5Hz,1H),8.43–8 .36(m,2H),8.10–8.04(m,2H),7.76(dd,J=5.0,1.5Hz,1H),7.63–7.59(m,1H),7.53(t,J=7.7Hz,2H),6.29–6.09(m,1H),3.81–3.74(m,2H). 13 C NMR(151MHz,DMSO-d6)δ166.18,165.35,154.42,153.03,150.58,146.37,141.82,136.26,133.95,132.10,129.59,128.41, 128.13, 120.43, 117.37, 114.45 (116.05, 114.45, 112.86, t, J = 240.2Hz), 114.23, 41.63 (41.81, 41.63, 41.46, t, J = 26.0Hz). 19 F NMR(565MHz,DMSO-d6)δ-121.43.HRMS(ESI):calcd for C 20 H 17 F2N4O2[M+H] + m / z,383.1314; found,383.1314.

[0355] Example 97: 3-(6-Benzamidepyridin-3-yl)-N-neopentylbenzamide (i-34)

[0356]

[0357] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-10 from Example 47. The yield was 78%, and the melting point was 183-186°C. 1 H NMR (600MHz, DMSO-d6) δ10.96(s,1H),8.80(d,J=2.5Hz,1H),8.50(t,J=6.4Hz,1H),8.34(d,J=8.6Hz,1H),8.24(dd,J=8.6,2.5Hz,1H), 8.20(t,J=1.9Hz,1H),8.10–8.04(m,2H),7.91–7.86(m,2H),7.63–7.57(m,2H),7.55–7.50(m,2H),3.16(d,J=6.4Hz,2H),0.92(s,9H). 13 C NMR(151MHz,DMSO-d6)δ166.46,166.09,151.77,145.96,136.78,136.36,135.78,134.05,132.02,131 .08,129.12,128.97,128.40,128.08,126.86,125.15,114.54,50.14,32.75,27.55.HRMS(ESI):calcd for C 24 H 26 N3O2

[0358] [M+H] + m / z,388.2020; found,388.2020.

[0359] Example 98: 6'-benzoamide-N-neopentyl-[2,3'-bipyridine]-4-carboxamide (i-35)

[0360]

[0361] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-25 from Example 62. The yield was 82%, and the melting point was 203-206°C. 1H NMR (600MHz, DMSO-d6) δ11.05(s,1H),9.17(d,J=2.5Hz,1H),8.81(d,J=5.0Hz,1H),8.73(t,J=6.4Hz,1H),8.58(dd,J=8.7,2.5Hz,1H),8. 40–8.35(m,2H),8.10–8.04(m,2H),7.74(dd,J=5.0,1.5Hz,1H),7.63–7.59(m,1H),7.55–7.50(m,2H),3.17(d,J=6.4Hz,2H),0.93(s,9H). 13 C NMR(151MHz,DMSO-d6)δ166.16,165.13,154.23,152.94,150.33,146.39,143.18,136.28,133.96 ,132.08,129.75,128.40,128.12,120.56,117.46,114.21,50.27,32.74,27.51.HRMS(ESI):calcd for C 23 H 25 N4O2

[0362] [M+H] + m / z,389.1972; found,389.1972.

[0363] Example 99: 3-(6-benzamidepyridin-3-yl)-N-(2,2,2-trifluoroethyl)benzamide (i-36)

[0364]

[0365] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-11 from Example 48. The yield was 82%, and the melting point was 215-218°C. 1 H NMR (600MHz, DMSO-d6) δ10.98(s,1H),9.25(t,J=6.3Hz,1H),8.81(d,J=2.5Hz,1H),8.34(d,J=8.7Hz,1H),8.27–8.23(m,2H),8.1 0–8.04(m,2H),7.97(dt,J=7.8,1.5Hz,1H),7.92(dt,J=7.8,1.5Hz,1H),7.66–7.57(m,2H),7.56–7.48(m,2H),4.19–4.13(m,2H). 13C NMR (151MHz, DMSO) δ166.67,166.12,151.89,145.99,137.03,136.37,134.04,134.01,132.05,130.79,129.84,129.44,128.41,128.1 0,127.02,125.25,124.90(127.67,125.82,123.97,122.12,q,J=279.8Hz),114.54,40.24(40.57,40.35,40.13,39.91,q,J=33.4Hz). 19 F NMR(565MHz,DMSO-d6)δ-70.34.HRMS(ESI):calcd for C 21 H 17 F3N3O2[M+H] + m / z,400.1267; found,400.1267.

[0366] Example 100: 6'-benzoamide-N-(2,2,2-trifluoroethyl)-[2,3'-bipyridine]-4-carboxamide (i-37)

[0367]

[0368] The procedure was the same as in Example 64, except that n-butyryl chloride was replaced with benzoyl chloride, and product g-1 from Example 38 was replaced with product g-26 from Example 63. The yield was 82%, and the melting point was 202-204°C. 1 HNMR (600MHz, DMSO-d6) δ11.06(s,1H),9.50(t,J=6.3Hz,1H),9.17(d,J=2.5Hz,1H),8.86(d,J=5.0Hz,1H),8.58(dd,J=8.7,2.5Hz,1H),8. 42(s,1H),8.39(d,J=8.7Hz,1H),8.09–8.04(m,2H),7.78(d,J=5.0Hz,1H),7.61(t,J=7.6Hz,1H),7.53(t,J=7.6Hz,2H),4.23–4.17(m,2H). 13C NMR(151MHz,DMSO-d6)δ166.19,165.47,154.52,153.06,150.65,146.41,141.44,136.29,133.95,132.11,129.54,128.41,128.13, 124.76 (127.53, 125.68, 123.83, 121.98, q, J = 279.8Hz), 120.47, 117.43, 114.23, 40.06 (40.40, 40.17, 39.95, 39.73, q, J = 33.7Hz). 19 F NMR(565MHz,DMSO-d6)δ-70.24.HRMS(ESI):calcd for C 21 H 16 F3N4O2[M+H] + m / z,401.1220; found,401.1220.

[0369] I. Antitumor activity test

[0370] The CCK8 (Cell Counting Kit-8) method was used for assay. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the half-maximal inhibitory rate (IC50) was calculated. 50 The value can indirectly reflect the number of viable cells. A549 cells (human non-small cell lung cancer cells resistant to gefitinib and osimertinib and with KRAS mutations), OVK18 (ovarian cancer cells), PC9-GR cells (gefitinib-resistant non-small cell lung cancer cells), H1975-OR cells (osimertinib-resistant non-small cell lung cancer cells), PF382 cells (human T lymphoma cells highly expressing aurora kinase B), SW982 (human synovial sarcoma cells), and T98G (human glioma cells) were identified as tumor cell lines highly expressing PDGFR. CIST-T1 was identified as a gastrointestinal stromal tumor cell line co-expressing PDGFR and C-kit. The cells were seeded into 96-well plates at a density of 3000-5000 cells / well and cultured in RPMI 1640 or DMEM complete medium containing 10% penicillin-streptomycin-amphotericidal B for 24 h at 37°C and 5% CO2. The control group received the corresponding volume of DMSO, while the experimental groups received different concentrations of compounds i-1 to i-37, with three replicates for each concentration. After 72 h of incubation, 10 μL of CCK 8 reagent was added to each well, and the cells were cultured for another 2 h. The absorbance at 450 nm was measured, and the IC50 was calculated. 50 The results are shown in Table 1:

[0371] Table 1. Inhibitory activity of compounds against the proliferation of tumor cells with high expression of c-Kit (V559D) or PDGFRβ, or resistance to EGFR-TKI.

[0372]

[0373]

[0374]

[0375] As shown in Table 1, compounds i-1 to i-37 exhibited strong inhibitory activity against various tumor cells, including A549 cells, OVK18 cells, PC9-GR cells, H1975-OR cells, PF382 cells, SW982 cells, T98G cells, and CIST-T1 cells, which were either highly expressed with c-Kit (V559D) or PDGFRβ or resistant to EGFR-TKIs. The IC50 values ​​of most compounds were... 50 The values ​​are all less than 100 nM. Among them, the IC50 values ​​of compounds i-15, i-17, i-21, i-30, and i-34 are... 50 The values ​​are all less than 10 nM.

[0376] II. Effects of the compounds of this invention on the cell cycle of tumor cells

[0377] Based on the binding properties of PI dyes to intracellular DNA and RNA, fluorescence intensity was detected using flow cytometry to reflect changes in DNA content, thereby distinguishing different phases of the cell cycle, including G1 / G0, S, and G2 / M phases. Tumor cells in the logarithmic growth phase were seeded in 6-well plates at a seeding density of 2 × 10⁶ cells / well. 5 Cells were cultured in 2 mL of DMEM complete medium containing 10% penicillin-streptomycin-amphotericidal B at 37°C and 5% CO2 for 24 h. Then, 1 mL of different concentrations of the drug (i-7 compound and i-17 compound) prepared in the medium was added and incubated for 24 h. After drug treatment, cells were collected, washed 2-3 times with pre-chilled PBS, and then fixed overnight in pre-chilled 70% ice-cold ethanol at 4°C. The supernatant was discarded after centrifugation, and the cells were washed twice with PBS. 100 μL of RNase A (100 μg / mL) and 400 μL of LPI (50 μg / mL) were added, and the cells were incubated at room temperature in the dark for 30 min. The cells were then analyzed by flow cytometry. The results are shown below. Figure 1 As shown. From Figure 1 It can be seen that compounds i-7 and i-17 in this invention inhibit the proliferation and growth of tumor cells by inducing G2 / M phase arrest. Western blot analysis results show that i-17 induces G2 / M phase arrest by mediating the expression levels of cell cycle-related proteins such as CHK1, Cyclin B1, and c-Myc.

[0378] III. Effects of the compounds of this invention on apoptosis of tumor cells

[0379] Flow cytometry was used to detect the cells using the Annexin V-FITC / PI kit. A549 or H1975OR cells in logarithmic growth phase were seeded in 6-well plates at a seeding density of 1 × 10⁶ cells / well. 5 Cells were cultured in 2 mL of DMEM complete medium containing 10% penicillin-streptomycin-amphotericidal B at 37°C and 5% CO2 for 24 h. Then, 1 mL of different concentrations of the drug (i-7 compound and i-17 compound) prepared in the medium was added and incubated for 48 h. After drug treatment, cells were collected, washed 2-3 times with pre-chilled PBS, and resuspended in 1× binding buffer. 10 μL of Annexin V-FITC and 10 μL of LPI were added, and the cells were incubated at room temperature in the dark for 20 min. Flow cytometry analysis was performed, and the results are shown below. Figure 2 As shown, from Figure 2 It can be seen that compounds i-7 and i-17 of the present invention can induce tumor cell apoptosis in a dose-dependent manner. In addition, i-17 also induces apoptosis in H1975-OR cells by upregulating the pro-apoptotic proteins p53 and cleaved-PARP, while downregulating the protein level of the anti-apoptotic protein Bcl-2.

[0380] IV. Confirmation of the target of compound i-17 of the present invention

[0381] Using KINOMEscan TM Competitive combination screening platform and KinaseProfiler TM The kinase inhibitory activity screening platform measures the kinase inhibitory activity and target binding activity of i-17. (KinaseProfiler) TM The results of the kinase inhibitory activity screening platform test are as follows: Figure 3 As shown, 1.0 μM i-17 exhibited weak kinase inhibitory activity against CLK1 / 2 / 4, but showed no significant kinase inhibitory activity against any of the other 275 kinases (including c-Kit and PDGFRβ). i-17 at 40 nM showed strong cell growth inhibition and apoptosis-inducing activity, indicating that CLK1 / 2 / 4 are not the main targets for i-17's antitumor activity. Furthermore, these results suggest that i-17 may not exert its antitumor activity by acting on the ortho-conformal sites of kinases. To investigate whether i-17 exerts its antitumor activity by acting on the allosteric sites of kinases, KINOMEscan was used. TMA competitive binding screening platform was used to investigate the binding interactions of i-17 with 486 kinases. 1 μM of i-17 selectively bound to c-Kit (V559D) and PDGFRβ. Figure 3 (A and B), given KinaseProfiler TM Kinase inhibition activity screening showed that i-17 did not inhibit the kinase activity of c-Kit(V559D) and PDGFRβ, suggesting that this compound may exert its antitumor effect by selectively acting on the allosteric pockets of c-Kit(V559D) and PDGFRβ. To further determine its selectivity, the Kd values ​​of compound i-17 against c-Kit(V559D), PDGFRβ, c-Kit, PDGFRα, and CLK4 were determined. The results showed that the Kd values ​​of i-17 against the above five targets were 98.20 nM, 116.50 nM, 1908 nM, 5788 nM, and 6496 nM, respectively. Figure 3 (C and D). The above results indicate that i-17 is a highly selective c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor.

[0382] V. Inhibitory activity of the compounds of this invention against Kit (V559D) and PDGFRβ

[0383] Using KINOMEscan TM The inhibitory activities of the synthesized compounds i-1 to i-7 against Kit(V559D) and PDGFRβ were determined using a competitive binding screening platform. The results are shown in Table 2. Compounds at 1.0 μM exhibited varying inhibitory activities against both Kit(V559D) and PDGFRβ. Compounds i-15, i-17, i-21, i-30, and i-36 all achieved inhibition rates exceeding 90% against both Kit(V559D) and PDGFRβ. Compounds i-7, i-13, i-16, i-27, and i-34 all showed inhibition rates exceeding 80%.

[0384] Table 2. Inhibitory activity of the compounds in this invention against Kit (V559D) and PDGFRβ.

[0385]

[0386]

[0387] VI. Safety Evaluation

[0388] 6.1 The effects of i-17 at a dose of 20 mg / kg for three consecutive weeks on biochemical indicators related to cardiac, hepatic, and renal function in mice were investigated. (See attached image.) Figure 4As shown, i-17 had no significant effect on liver function indicators ALT and AST, kidney function indicators CREA and UREA, and cardiac function indicators CK-MB and LDH. These results indicate that i-17 has good safety at an effective dose achieving a tumor inhibition rate of 93.75%.

[0389] 6.2. Inhibition of hERG potassium channels is a key reason for QT prolongation and cardiotoxicity in several multi-target inhibitors targeting c-Kit and PDGFRβ orthotopic sites, such as imatinib and anlotinib. Whole-cell patch-clamp techniques were used to investigate the inhibitory activity of i-17 against hERG. (See attached image.) Figure 5 As shown, 30 μM i-17 showed no significant inhibitory activity, suggesting that acting on the c-Kit and PDGFRβ allosteric sites may have a safe and efficient antitumor effect.

[0390] VII. Antitumor effect of compound i-7 of the present invention

[0391] A549 cells treated with compound i-7 were analyzed using RNA-seq, QRT-PCR, and Western blot methods. (See attached image.) Figure 6 As shown, i-7 can inhibit the transcription of AURKB and reduce its protein level, thereby inhibiting the downstream signaling pathway to exert anti-tumor effects.

[0392] Nude mouse xenograft tumor models of H1975-OR cells and A549 cells were constructed to investigate the in vivo antitumor activity of compounds i-7 and i-17 of this invention. (See attached image) Figure 7 As shown, compound i-7 exhibits good growth inhibitory activity against A549 cell xenograft tumors in nude mice. The tumor inhibition rates of i-7 at 30 mg / kg and 15 mg / kg were 76.60% and 51.11%, respectively. Furthermore, there was no significant change in mouse body weight during the experiment, indicating good tolerability. (See attached image.) Figure 8 As shown, i-17 exhibited strong inhibitory activity against the growth of H1975-OR cell xenografts. The tumor inhibition rates of 5 mg / kg i-17 were 77.45%, and those of 10 mg / kg i-17 were 93.75%, demonstrating good in vivo efficacy. Therefore, i-17 can effectively inhibit the growth of EGFR-TKI-resistant xenografts. The lack of significant changes in mouse body weight during the experiment indicates good tolerability.

[0393] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0394] Finally, it should be noted that the embodiments disclosed in this invention are merely preferred embodiments of this invention and are only used to illustrate the technical solutions of this invention, not to limit it. Although this invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this invention.

Claims

1. A c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor, characterized in that: Selected from compounds of general formula (I) or pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof, wherein the compounds of general formula (I) have the following structural formula: ； In general formula (I), ring A is selected from the benzene ring; R1 is selected from C1-C7 alkyl, C1-C6 haloalkyl, and C3-C7 cycloalkyl; R2 is selected from hydrogen, fluorine, methyl, ethyl, methoxy, and cyano; R3 is selected from at least one of C2-C7 alkyl acyl, C2-C7 haloalkyl acyl, saturated aliphatic alkyl acyl, C4-C8 saturated cycloalkyl acyl, saturated aliphatic cycloalkyl acyl, benzoyl with different substitutions, and heterocyclic aromatic acyl.

2. The c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor according to claim 1, characterized in that: The general formula (I) is selected from the following structural compounds: 。 3. The application of the c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor according to claims 1-2, characterized in that: This is used to prepare drugs for treating diseases associated with c-Kit and PDGFRβ, including cancer, pulmonary interstitial fibrosis, and neurodegenerative diseases.

4. The application of the c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor according to claim 3, characterized in that: The cancers mentioned are selected from EGFR-TKI resistant non-small cell lung cancer, ovarian cancer cells, human T lymphoma cells, human synovial sarcoma cells, human glioma cells, and gastrointestinal stromal tumor cells.

5. A pharmaceutical composition comprising at least one compound as described in claims 1-2.

6. The composition according to claim 5, characterized in that: Includes any one or more of the compounds of claims 1-2 and / or pharmaceutically acceptable salts of the compounds, and / or solvates of the compounds.

7. A method for preparing the c-Kit(V559D) / PDGFRβ dual-target allosteric inhibitor according to any one of claims 1-2, characterized in that: Includes the following steps: (1) At room temperature, thionyl chloride (b) was added to a dichloromethane solution of bromoaromatic formic acid (a) and N,N-dimethylformamide (n), and the mixture was heated to 50°C and reacted for 5-8 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed to obtain intermediate compound c. The molar ratio of bromoaromatic formic acid (a), thionyl chloride (b), and N,N-dimethylformamide was 1:5:0.

1. (2) At 0℃, a dichloromethane solution of intermediate compound c is added dropwise to a dichloromethane solution of triethylamine and organic amine d. The mixture is heated to room temperature and reacted for 1-3 hours. After the reaction is completed, saturated brine and dichloromethane are added for extraction. The organic layer is collected and concentrated by column chromatography to obtain intermediate compound e. The molar ratio of intermediate compound c, organic amine and triethylamine is 1.5:1:

2. (3) Add [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride to intermediate compounds e and f, materials 2-aminopyridine-5-boronic acid pinacol ester and potassium carbonate V 1,4-二氧六环 V 水 In a solution with a ratio of 4:1, the reaction was carried out at 100℃ for 1.5 h. Then, intermediate compound e was added and the reaction was continued for 0.5 h to 2.5 h. After the reaction was completed, the solvent was removed, and dichloromethane and methanol were added to dissolve the crude product. The product was then filtered, and the filtrate was concentrated and column chromatography was used to obtain the corresponding intermediate compound g. The molar ratio of intermediate compound e to f, 2-aminopyridine-5-boronic acid pinacol ester, potassium carbonate, and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride, was 1:1.5:2:0.

1. (4) At 50°C, the acyl chlorides of different substitutions in material h are added in batches to a solution of sodium hydride and intermediate compound g in tetrahydrofuran. After reacting for 3-6 h, the mixture is cooled to 0°C, quenched with methanol, extracted with saturated brine and dichloromethane, and the organic layer is collected. The target inhibitor compound i of the present invention is obtained by concentrated column chromatography. The molar ratio of the acyl chlorides of different substitutions in material h, intermediate compound g and sodium hydride is 2:1:

2. The specific synthesis steps are as follows: 。

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