A compound for targeted degradation of HDAC7, its preparation method and application

Abstract

The present invention discloses a compound represented by formula (I), its optical isomers and pharmaceutically acceptable salts thereof. The compound obtained by the present invention has a good inhibitory effect on the secretion of inflammatory factors IL-6 and TNF-α in macrophages.

Description

Technical Field

[0001] The present invention belongs to the field of drugs, and particularly relates to a compound for targeted degradation of HDAC7, a preparation method thereof, and an application thereof. Background Art

[0002] Histone deacetylases (HDACs) are important components of epigenetic factors. Together with histone acetyltransferases (HATs), they jointly regulate the dynamic balance of histone acetylation and deacetylation and play a key role in the process of gene transcription regulation. When the positive charge of lysine residues on the surface of histones is acetylated, the compact chromatin will become loose, bind to RNA polymerase II, and promote gene expression. The role of HDAC is to restore the positive charge on the lysine side chain on the surface of histones, make the chromosome structure become compact again, and then make it difficult for RNA polymerase II to bind, resulting in the inhibition of gene expression.

[0003] Similar to other class I and class IV HDACs, class IIa HDACs are zinc-dependent hydrolases. However, different from nuclear-localized class I HDACs, due to a unique adaptor domain at the N-terminus of class IIa HDACs, they can shuttle between the nucleus and cytoplasm in response to certain signaling pathways and may have other biological functions besides deacetylation. In addition, since the tyrosine catalytic residue in its enzyme active center is replaced by histidine, its deacetylation activity is extremely low, the biological substrates are not clear, and it may be a pseudo-enzyme with non-catalytic activity. Class IIa HDACs include HDAC4, 5, 7, and 9 and are involved in various physiological and pathological processes. Research shows that HDAC4 mainly binds to other proteins to form a transcriptional repressor complex. For example, in apoptotic cells, HDAC4 acts on apoptosis-related factors to promote cell apoptosis; HDAC5 plays a regulatory function in skeletal muscle, osteoblast differentiation, and angiogenesis. For example, the deletion of the HDAC5 gene will lead to corresponding myocardial hypertrophy; HDAC7 is involved in cell growth, differentiation and apoptosis, angiogenesis, endothelial cell migration and other processes and is closely related to autoimmune diseases; HDAC9 mainly plays a key role in adipocyte differentiation, myocardial development, and immune and metabolic processes. Generally speaking, class IIa HDACs are closely related to the occurrence and development of various human diseases. The increase in their expression levels will affect disease-related processes such as tumors, neurodegenerative diseases, inflammation, and metabolic disorders. Research shows that inhibiting class IIa HDACs can effectively inhibit the proliferation of melanoma cells and breast cancer cells and also affect macrophage differentiation.

[0004] Based on the important roles played by class IIa HDACs in many biological processes, the development of small molecule inhibitors to interfere with their functions is of great significance for the treatment of related diseases. However, studies have found that knocking out HDAC4 and 5 genes can lead to cardiac hypertrophy, and currently, the small molecule inhibitor TMP269 of class IIa HDACs cannot achieve selective interference with each subtype (HDAC4, 5, 9), and there are corresponding cardiac toxicity problems. Therefore, developing a treatment strategy that specifically inhibits HDAC7 or 9 is an urgent problem to be solved. In addition, our research has found that silencing HDAC7 and 9 can inhibit the secretion of inflammatory factors IL-6 and TNF-α in macrophages, while the inhibitor TMP269 does not have this effect, which suggests that the anti-inflammatory activity of inhibiting HDAC7 or 9 may not depend on its enzymatic function. Based on the potential role of HDAC7 and 9 in inflammatory responses, we believe that developing specific interfering molecules against them is expected to become an effective treatment means for inflammatory diseases.

[0005] Targeted protein degradation technology is expected to achieve specific interference with HDAC7 and 9 by inducing the formation of new protein-protein interaction surfaces. In addition, since the degrader molecules remove the protein itself, they can simultaneously inhibit all functions of its enzymatic and non-enzymatic activities. Therefore, we used the class IIa HDAC inhibitor TMP269 as the target head, designed and synthesized a series of degrader molecules, and found that the active molecules could significantly degrade HDAC7 protein in macrophages, but had no significant effect on the expression of other class IIa HDAC proteins (HDAC4, 5, and 9). Moreover, the molecules with degrading activity could significantly inhibit the secretion of inflammatory factors IL-6 and TNF-α in macrophages, showing great therapeutic potential in autoimmune diseases and inflammatory responses. At present, the degrading activity of HDCA7 degrader needs to be improved, and the related pharmacological mechanisms still need to be explored. Summary of the Invention

[0006] The object of the present invention is to provide a compound that targets the degradation of HDAC7, its optical isomers, and its pharmaceutically acceptable salts, which have a good inhibitory effect on the secretion of inflammatory factors IL-6 and TNF-α in macrophages.

[0007] To achieve the above object, the present invention provides a compound represented by formula (Ⅰ), its optical isomers, and its pharmaceutically acceptable salts:

[0008]

[0009] Wherein,

[0010] R1 is selected from

[0011] X and Y each independently selected from N, CH;

[0012] n is selected from 0, 1, 2;

[0013] R2 is selected from H, C 1-3 alkyl;

[0014] L1 is selected from

[0015] L2 is selected from C 1-3 alkyl,

[0016] R3 is selected from CH3, CF3;

[0017] R4 and R6 are each independently selected from H, CN;

[0018] R5 and R7 are each independently selected from H, C 1-6 alkyl, C 3-6 cycloalkyl, wherein C 1-6 alkyl and C 3-6 cycloalkyl may be substituted by one or more halogens;

[0019] L3 is selected from

[0020] X1 and X5 are each independently selected from

[0021] X2 and X4 are each independently selected from a bond,

[0022] X3 is selected from a bond,

[0023] X2, X3 and X4 are not all selected from a bond;

[0024] y is an integer from 0 - 10, z is an integer from 1 - 5, and p is an integer from 1 - 3;

[0025] L4 is selected from

[0026] Preferably, R1 is selected from

[0027] Preferably, R3 is selected from trifluoromethyl; R4 is selected from H, R5 is selected from trifluoromethyl;

[0028] In some embodiments of the present invention, in the compound, R1 is selected from Furthermore, R1 is selected from

[0029] In some embodiments of the present invention, in the compound, R2 is selected from H, methyl.

[0030] In some embodiments of the present invention, in the said compound, L1 is selected from

[0031] Preferably, L2 is selected from methylene,

[0032] In some embodiments of the present invention, in the said compound, L2 is selected from

[0033] In some embodiments of the present invention, the said compound has the compound shown in formula (Ⅱ), its optical isomers and pharmaceutically acceptable salts thereof:

[0034]

[0035] In some embodiments of the present invention, in the said compound, the structural unit is selected from

[0036]

[0037] The structure of the said compound is shown as follows:

[0038]

[0039] Or

[0040]

[0041] Preferably, z is 3, 4, 5; in some embodiments, z is 4. Preferably, y is 5, 6; in some embodiments, y is 5. In some embodiments of the present invention, the said compound is:

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] Preferably, the pharmaceutically acceptable salts are the following salts of the said compound: hydrochloride, trifluoroacetate, mesylate, malate, citrate, tosylate, L-tartrate, D-tartrate.

[0048] Preferably, the pharmaceutically acceptable salts are:

[0049]

[0050]

[0051] The names are as follows:

[0052] N-((1-(2-(1-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (1)

[0053] N-((1-(4-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)butyl)-1H-1,2,3-triazol-4-yl)butyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (2)

[0054] N-((1-(4-(1-(7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl)-1H-1,2,3-triazol-4-yl)butyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (3)

[0055] N-((1-((1-(7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)heptyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (4)

[0056] N-((1-(2-(1-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (5)

[0057] N-((1-(6-(1-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)hexyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (6)

[0058] N-((1-(2-(1-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)propyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (7)

[0059] N-((1-(2-(1-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (8)

[0060] N-((1-(2-(1-(6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (9)

[0061] N-((1-(2-(1-(7-(4-(4-((2,6-dioxopiperidin-3-yl)amino)phenyl)piperidin-1-yl)-7-oxoheptyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (10)

[0062] N-((2R)-1-(benzyl(2-(1-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)amino)propan-2-yl)-4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (11)

[0063] N-((1-(2-(1-(6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)benzamide (12)

[0064] N1-((1-(2-(1-(6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-N3-hydroxyisophthalamide (13)

[0065] N-((1-(2-(1-(6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(2-oxo-1,3-dioxol-4-yl)benzamide (14)

[0066] (E)-N-((1-(2-(1-(6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(4,4,4-trifluorobut-2-enoyl)benzamide (15)

[0067] N-(6-(1-(6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)-2-(4-phenylthiazol-2-yl)hexyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (16)

[0068] N-((2-(2-(2-(1-(3-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)ethoxy)ethyl)-4-(4-phenylthiazol-2-yl)tetrahydro-2H-pyran-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (17)

[0069] N-((1-(12-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)dodecyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (18)

[0070] N-((1-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (19)

[0071] N-((1-(2-(4-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)phenoxy)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (20)

[0072] N-((1-(1-(4-(4-((2,6-dioxopiperidin-3-yl)amino)phenyl)piperidin-1-yl)-2-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (21)

[0073] (E)-N-(2-(1-(2-(1-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)ethyl)-3-(4,4,4-trifluorobut-2-enoyl)benzamide (22)

[0074] N-((3R,4S)-1-(2-(1-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)ethyl)-3-(4-phenylthiazol-2-yl)piperidin-4-yl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (23)

[0075] N-((1-(2-(4-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-5-oxopentyl)piperazin-1-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (24)

[0076] N-((1-(5-(4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3-oxopropyl)piperazin-1-yl)pentyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (25)

[0077] N-((1-(2-(4-(2-(4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino-3-oxopropyl)piperazin-1-yl)ethyl)piperazin-1-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (26)

[0078] (2S,4R)-1-((S)-3,3-dimethyl-2-(2-(2-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)ethoxy)acetamido)butanoyl)-4-hydroxy-N-((S)-1-(4-(4)-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (27)

[0079] (2S,4R)-1-((S)-3,3-dimethyl-2-(2-(2-(2-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)ethoxy)ethoxy)acetamido)butanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (28)

[0080] (2S,4R)-1-((S)-1-((S)-3,3-dimethyl-2-(2-(2-(2-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)ethoxy)ethoxy)acetylamino)butanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (29)

[0081] (2S,4R)-1-((S)-2-(tert-butyl)-4-oxo-17-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15-tetraoxa-3-azapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (30)

[0082] (2S,4R)-1-((S)-2-(tert-butyl)-4-oxo-20-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15,18-pentaoxa-3-azaeicosanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (31)

[0083] N-((1-(((2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl)-L-valyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (32)

[0084] N-((1-(4-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-3-methylbutanamido)butanoyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (33)

[0085] N-((1-(6-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-3-methylbutanamido)hexanoyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (34)

[0086] N-((1-(8-((S)-2-((2S,3R)-3-Amino-2-hydroxy-4-phenylbutanamido)-3-methylbutanamido)octanoyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (35)

[0087] N-((1-(11-((S)-2-((2S,3R)-3-Amino-2-hydroxy-4-phenylbutanamido)-3-methylbutanamido)undecanoyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (36)

[0088] N-((1-(2-(2-((S)-2-((2S,3R)-3-Amino-2-hydroxy-4-phenylbutanamido)-3-methylbutanamido)ethoxy)acetyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (37)

[0089] N-((1-((11S,14S,15R)-15-Amino-14-hydroxy-11-isopropyl-10,13-dioxa-16-phenyl-3,6-dioxa-9,12-diazapentadecanoyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (38)

[0090] N-((1-((14S,17S,18R)-18-Amino-17-hydroxy-14-isopropyl-13,16-dioxa-19-phenyl-3,6,9-trioxa-12,15-diazadec-19-anoyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (39)

[0091] N-((1-((17S,20S,21R)-21-Amino-20-hydroxy-17-isopropyl-16,19-dioxa-22-phenyl-3,6,9,12-tetraoxa-15,18-diazadocosanoyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (40)

[0092] N-((1-(6-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-3-methylbutanamido)hexyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (41)

[0093] (2S,4S)-N-(2,6-difluorophenyl)-1-((R)-3,3-dimethyl-2-((R)-2-(methylamino)propanamido)butanoyl)-4-(6-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)hexanamido)pyrrolidine-2-carboxamide (42)

[0094] (2S,4S)-N-(2,6-difluorophenyl)-1-((R)-3,3-dimethyl-2-((R)-2-(methylamino)propanamido)butanoyl)-4-(8-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)octanamido)pyrrolidine-2-carboxamide (43)

[0095] (2S,4S)-N-(2,6-difluorophenyl)-1-((R)-3,3-dimethyl-2-((R)-2-(methylamino)propanamido)butanoyl)-4-(2-(2-(2-(4-(4-phenylthiazol-2-yl))-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)ethoxy)ethoxy)acetamido)pyrrolidine-2-carboxamide (44)

[0096] (2S,4S)-N-(2,6-difluorophenyl)-1-((R)-3,3-dimethyl-2-((R)-2-(methylamino)propanamido)butanoyl)-4-(2-(2-(2-(2-(4-(4-phenylthiazol-2-yl))-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)ethoxy)ethoxy)ethoxy)acetamido)pyrrolidine-2-carboxamide (45)

[0097] (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-17-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15-tetraoxa-3-azapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (46)

[0098] (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-17-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15-tetraoxa-3-azapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide trifluoroacetate (47)

[0099] (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-17-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15-tetraoxa-3-azapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide methanesulfonate (48)

[0100] (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-17-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15-tetraoxa-3-azapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (±)-malate (49)

[0101] (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-17-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15-tetraoxa-3-azapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide citrate (50)

[0102] (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-17-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15-tetraoxa-3-azapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide p-toluenesulfonate (51)

[0103] (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-17-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15-tetraoxa-3-azapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide L-tartrate (52)

[0104] (2S,4R)-1-((S)-2-(tert-Butyl)-4-oxo-17-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-6,9,12,15-tetraoxa-3-azapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide D-tartrate (53).

[0105] In some embodiments of the present invention, there is provided a pharmaceutical composition, wherein the pharmaceutical composition contains a therapeutically effective amount of any one of the above-mentioned compounds or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0106] In some embodiments of the present invention, there is provided the use of any one of the above-mentioned compounds or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a medicament for preventing and / or treating HDAC7 abnormality-related diseases.

[0107] In some embodiments of the present invention, there is provided the use of any one of the above-mentioned compounds or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in preventing and / or treating HDAC7 abnormality-related diseases.

[0108] In some embodiments of the present invention, the use of any one of the above-mentioned compounds or a pharmaceutically acceptable salt thereof or a pharmaceutical composition, wherein the diseases are selected from metabolic diseases, inflammatory diseases, and autoimmune diseases.

[0109] In some embodiments of the present invention, the use of any of the above-mentioned compounds or their pharmaceutically acceptable salts or pharmaceutical compositions, wherein the inflammatory diseases include rheumatoid arthritis, multiple sclerosis, osteoporosis, osteoarthritis, inflammatory bowel disease, etc.

[0110] Definitions and Explanations

[0111] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indefinite or unclear in the absence of a specific definition, but should be understood in its ordinary meaning. When a trade name appears in this text, it is intended to refer to the corresponding product or its active ingredient.

[0112] It should be understood that the substitutions and combinations of substitutions described herein, whether or not explicitly stated, refer to substitutions that conform to the valence of the substituted member. For example, substitution applied to a carbon member refers to the tetravalence of C; when applied to a nitrogen member, it refers to the trivalence of N; when generally represented as carrying a positive charge, it refers to the four bonds of a nitrogen member. The valence-allowed options are part of the art.

[0113] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals, without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0114] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, which are prepared from compounds having specific substituents discovered in the present invention and relatively non-toxic acids or bases. When a compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When a compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of an acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, where the inorganic acids include, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, hydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, where the organic acids include, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and similar acids; also salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can thus be converted into either base or acid addition salts.

[0115] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing acid radicals or basic groups by conventional chemical methods. Generally, the preparation method of such salts is to react these compounds in the form of free acids or bases with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of both.

[0116] The term "isomer" means that the compounds of the present invention can exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomer- or diastereoisomer-enriched mixtures, all of which mixtures are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and their mixtures are included within the scope of the present invention.

[0117] Unless otherwise specified, "(D)" or "(+)" indicates dextrorotatory, "(L)" or "(-)" indicates levorotatory, and "(DL)" or "(±)" indicates racemic.

[0118] Unless otherwise specified, with a solid wedge bond and a dashed wedge bond Indicates the absolute configuration of a stereocenter, using straight solid lines and straight dashed lines to indicate the relative configuration of a stereocenter, using a wavy line to indicate a wedge-shaped solid line or a wedge-shaped dashed line or using a wavy line to indicate a straight solid line and a straight dashed line

[0119] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by substituents, which may include deuterium and variants of hydrogen, provided that the valence of the particular atom is normal and the resulting compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary based on what is chemically achievable.

[0120] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0 - 2 R's, the group may optionally be substituted with up to two R's, and each R has independent options in each case. In addition, combinations of substituents and / or their variants are permitted only if such combinations result in a stable compound.

[0121] When one of the variables is selected from a bond, it means that the two groups it connects are directly linked. For example, when L represents a bond in A - L - Z, it means the structure is actually A - Z.

[0122] When it is not specified which atom of an enumerated substituent is bonded to the group being substituted, such a substituent can be bonded through any of its atoms. For example, a phenyl group as a substituent can be connected to the group being substituted through any carbon atom on the benzene ring; a substituent drawn with a bond connecting to a central ring to form a ring system (as shown) represents substitution at any one of the substitutable positions on that ring system.

[0123] Unless otherwise specified, the term "alkyl" is used to denote a straight-chain or branched-chain saturated hydrocarbon group, which can be monosubstituted (e.g., -CH2F) or polysubstituted (e.g., -CF3), and can be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), etc.

[0124] Unless otherwise specified, cycloalkyl includes any stable cyclic or polycyclic hydrocarbon group, any carbon atom of which is saturated and which may be mono-substituted or multi-substituted and may be monovalent, divalent or polyvalent. Examples of such cycloalkyl groups include, but are not limited to, cyclopropyl, norbornyl, [2.2.2]bicyclooctane, [4.4.0]bicyclodecane, etc.

[0125] Unless otherwise specified, the term "halogen" by itself or as part of another substituent refers to a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom.

[0126] Unless otherwise specified, a numerical range represents all integers including the numbers at both ends of the range. Unless otherwise specified, the integers from 0 to 10 represent 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; the integers from 1 to 5 represent 1, 2, 3, 4, 5; the integers from 1 to 3 represent 1, 2, 3; C 1-3 Alkyl represents C1, C2, C3 alkyl; C 1-6 Alkyl represents C1, C2, C3, C4, C5, C6 alkyl; C 3-6 Cycloalkyl represents C3, C4, C5, C6 cycloalkyl, and so on.

[0127] In the present invention, when the listed groups are divalent (e.g., L1, L2, X1, X2, X3, X4, X5) and the specific positions at which the two linking bonds are connected to the compound are not specified, the connecting positions of the two linking bonds of such divalent groups in the compound can be interchanged. For example, X3 is selected from It can be the C of the alkynyl group connected to X2, or it can be the N on the piperidine ring connected to X2.

[0128] The compounds provided by the present invention have good inhibitory effects on the secretion of inflammatory factors IL-6 and TNF-α in macrophages. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 To screen the chain length of the compound and determine the most effective linker chain length.

[0130] Figure 2 To explore the influence of the position of the triazole relative to the target head on the degradation activity.

[0131] Figure 3 To modify around compound 5 and compare the degradation activity with compound 5.

[0132] Figure 4 To modify around compound 5 and compare the degradation activity with compound 5.

[0133] Figure 5To screen the degradation activity of compounds with VHL as the E3 ubiquitin ligase ligand.

[0134] Figure 6 To verify the degradation effect of compounds 5 and 30 on HDAC7 protein in macrophages.

[0135] Figure 7 To investigate the effects of TMP269 (HDAC7 inhibitor), compound 5, and compound 30 on the level of TNF-α secreted by macrophages.

[0136] Figure 8 To investigate the effects of TMP269 (HDAC7 inhibitor), compound 5, and compound 30 on the level of IL-6 secreted by macrophages.

[0137] Figure 9 To investigate the effects of TMP269 (HDAC7 inhibitor), compound 30, and dexamethasone on the level of TNF-α secreted by LPS induction in experimental animals.

[0138] Figure 10 To investigate the effects of TMP269 (HDAC7 inhibitor), compound 30, and dexamethasone on the level of IL-6 secreted by LPS induction in experimental animals. Detailed implementation methods

[0139] The present invention will be described in detail below through examples, but this does not mean any adverse limitation to the present invention. The present invention has been described in detail herein, and its specific implementation methods have also been disclosed. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific implementation methods of the present invention without departing from the spirit and scope of the present invention.

[0140] In the examples, the experimental methods without specific conditions are the conventional methods and conditions well-known in the art, or are operated according to the conditions recommended by the instrument manufacturers.

[0141] Example 1. Synthesis of Intermediate 1a

[0142]

[0143] Step 1: In a 250 mL round-bottom flask, dissolve 3-cyanobenzoic acid 1a-1 (5 g, 34 mmol) in 100 mL of ethanol. Add 8-hydroxyquinoline (24.65 mg, 0.17 mmol) and stir to dissolve. Dissolve hydroxylamine hydrochloride (4.76 g, 68 mmol) in 20 mL of water and add it to the above solution. Then dissolve sodium carbonate (5.8 g, 54.4 mmol) in 20 mL of water and slowly add it to the reaction solution. Place the reaction system in a reflux at 90 °C and stir vigorously for 4 h. After the reaction is completed, cool to room temperature, concentrate under reduced pressure. Pour the reaction solution into 20 mL of water, adjust the pH value to about 3 with 1N hydrochloric acid solution. After white solid precipitates, filter by suction. Wash the filter cake 3 times with water and then 3 times with acetone. Dry the obtained crude product to get 5.13 g of white solid, namely intermediate 1a-2, with a yield of 84%. ESI(M+H) + = 181.

[0144] Step 2: Dissolve intermediate 1a-2 (5.13 g, 28.3 mmol) in 50 mL of anhydrous pyridine. After cooling to 0 °C, add trifluoroacetic anhydride (11.8 mL, 85 mmol) dropwise. Slowly warm to room temperature and then heat to 50 °C to continue the reaction for 3 h. After cooling the reaction solution to room temperature, pour it into 50 mL of ice water. Adjust the pH value to about 4 with 1.5N hydrochloric acid solution, and then extract 3 times with ethyl acetate. Combine the organic layers, wash 2 times with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The obtained crude product is purified by silica gel column chromatography to get 2.12 g of white solid (intermediate 1a), with a yield of 29%, ESI(M-H) - = 257.

[0145] Example 2. Synthesis of intermediate 1b

[0146]

[0147] Step 1: Dissolve 2-bromoacetophenone 1b-1 (5 g, 25.13 mmol) and 2-cyanothioacetamide (2.5 g, 25.13 mmol) in 50 mL of ethanol. After heating the reaction system to 80 °C, stir vigorously for 4 h. After the reaction is completed, cool the reaction solution to room temperature, adjust the pH value to greater than 7 with ammonia water, extract 2 times with ethyl acetate, wash with water, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate. The obtained crude product is purified by silica gel column chromatography to get 3.9 g of orange-yellow solid (intermediate 1b-2), with a yield of 78%. ESI(M+H) + = 201.

[0148] Step 2: Under nitrogen protection, dissolve intermediate 1b-2 (3.9 g, 19.5 mmol) in 10 mL of anhydrous N,N-dimethylformamide (DMF). Add sodium hydride (2.4 g, 100 mmol) portionwise under an ice bath. After activation for 30 min, add dropwise a DMF solution (5 mL) of N,N-bis(2-chloroethyl)carbamic acid tert-butyl ester (4.21 mL, 20 mmol). Raise the temperature to 80 °C and react for 3 h. Quench the reaction system with water under an ice bath, extract with 50 mL of ethyl acetate. Wash the organic phase once with water and then with saturated sodium chloride, dry over anhydrous sodium sulfate, concentrate. The obtained crude product is purified by silica gel column chromatography to give 3.29 g of a yellow liquid (intermediate 1b-3), with a yield of 46%. ESI((M- t Bu)+H) + = 314.

[0149] Step 3: Under nitrogen protection, dissolve lithium aluminum hydride (1.32 g, 34.8 mmol) in 30 mL of anhydrous tetrahydrofuran (THF). Dissolve intermediate 1b-3 (3.29 g, 8.7 mmol) in 10 mL of anhydrous THF and add it to the above suspension under an ice bath. Raise the temperature to room temperature and react for 1 h. After monitoring the completion of the reaction by TLC, quench it by adding 1.32 mL of H2O, 1.32 mL of 15% NaOH solution and 3.96 mL of H2O successively under an ice bath. Stir well for 10 min, filter through diatomaceous earth, extract the filtrate 3 times with ethyl acetate, dry over anhydrous sodium sulfate, concentrate under reduced pressure to obtain 2.13 g of a crude product (intermediate 1b), which is directly used in the next step without further purification. ESI(M+H) + = 374.

[0150] Example 3. Synthesis of intermediate 1d

[0151]

[0152] Step 1: Dissolve intermediate 1a (1.2 g, 4.62 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) (887 mg, 4.62 mmol) and 1-hydroxybenzotriazole monohydrate (HOBt) (771 mg, 5.04 mmol) in anhydrous dichloromethane (10 mL). Add dropwise N,N-diisopropylethylamine (DIPEA) (2.2 mL, 12.6 mmol) under an ice bath. After stirring for 10 min, slowly add a dichloromethane solution (5 mL) of intermediate 1b (1.56 g, 4.2 mmol). Stir at room temperature overnight. After completion of the reaction, pour it into 20 mL of water, extract the reaction solution 3 times with dichloromethane, combine the organic phases, wash 2 times with saturated sodium chloride, dry over anhydrous sodium sulfate, and evaporate to dryness. The obtained crude product is purified by silica gel column chromatography to give 1.84 g of a white solid (intermediate 1c), with a yield of 71%.1 H-NMR (400 MHz, DMSO-d6) δ 8.79 (t, J = 6.4 Hz, 1H), 8.43 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.08 (s, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 7.3 Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 3.83 (d, J = 13.5 Hz, 2H), 3.56 (d, J = 6.3 Hz, 2H), 2.94 (s, 2H), 2.26 (d, J = 14.2 Hz, 2H), 1.92–1.80 (m, 2H), 1.37 (s, 9H). ESI(M+H) + = 614。

[0153] Step 2: Intermediate 1c (1.84 g, 3 mmol) was dissolved in 2 mL of dichloromethane, and 2 mol / L hydrogen chloride-ethyl acetate solution (15 mL, 30 mmol) was added. After reacting at room temperature for 2 h, the solution was concentrated by rotary evaporation to obtain 1.73 g of a white solid, namely Intermediate 1d. ESI(M+H) + = 514。

[0154] Example 4. Synthesis of Intermediate 1A

[0155]

[0156] Step 1: At 0 °C, raw material 1A-1 3-butyn-1-ol (2 g, 28.6 mmol) was dissolved in 20 mL of dichloromethane. Triethylamine (12 mL, 85.8 mmol) and 4-dimethylaminopyridine (349 mg, 2.86 mmol) were added. Finally, a dichloromethane solution of p-toluenesulfonyl chloride (6.6 g, 34.3 mmol) was slowly added dropwise. After the reaction mixture was warmed to room temperature and reacted for 2 h, when the raw material 1A-1 was completely reacted as monitored by TLC, 60 mL of water was poured into the reaction solution. After extracting with dichloromethane three times, the organic phases were combined and concentrated under reduced pressure. The product was separated and purified by column chromatography to obtain 5.2 g of a yellow liquid (Intermediate 1A-2) with a yield of 81%. 1 H-NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 7.9 Hz, 2H), 4.04 (t, J = 6.2 Hz, 2H), 2.88 (t, J = 2.7 Hz, 1H), 2.52 (td, J = 6.2, 2.7 Hz, 2H), 2.42 (s, 3H). ESI(M+H) + = 225。

[0157] Step 2: Under nitrogen protection, dissolve intermediate 1d (100 mg, 0.195 mmol) in 1 mL of anhydrous DMF solution, slowly add potassium carbonate (80.7 mg, 0.585 mmol), heat to 40 °C for activation for 10 min, and add dropwise the DMF solution (1 mL) of intermediate 1A-2 to the reaction system, and stir at 40 °C overnight. Pour the reaction solution into 10 mL of water, extract with ethyl acetate three times, combine the organic phases, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 67.5 mg of yellow liquid (intermediate 1A), with a yield of 61%. 1 1H-NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.44 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 8.08 (s, 2H), 7.93 (d, J = 7.6 Hz, 2H), 7.81–7.60 (m, 1H), 7.44–7.36 (m, 2H), 7.35–7.27 (m, 1H), 3.54 (s, 2H), 3.03–2.63 (m, 4H), 2.43–2.16 (m, 5H), 2.26–1.91 (m, 4H). ESI(M+H) + = 566.

[0158] Example 5. Synthesis of intermediate 1B

[0159]

[0160] Step 1: Dissolve starting material 1B-a1 o-iodobenzoic acid (8 g, 32.2 mmol) and sodium periodate (7.24 g, 33.8 mmol) in 30% aqueous acetic acid solution (50 mL), raise the temperature to 120 °C and reflux the reaction in the dark for 4 h, add 150 mL of ice water to quench the reaction system, and continue to stir in an ice bath for 1 hour to fully precipitate the product. Filter under dark conditions, wash the filter cake with ice water and acetone in sequence, and dry to obtain 7.63 g of white powdery solid (intermediate 1B-a), with a yield of 90%. 1 1H-NMR (400 MHz, DMSO-d6) δ 8.05 (s, 1H), 8.01 (d, J = 7.5 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.70 (t, J = 7.3 Hz, 1H).

[0161] Step 2: Under nitrogen protection, dissolve raw material 1B-1 (2.2 g, 8.0 mmol) in 20 mL of anhydrous DMF, slowly add sodium carbonate (5.1 g, 48 mmol), heat to 100 °C for activation for 15 min, and add dropwise a DMF solution of 3-bromopropene (10 mL) to the reaction system. Keep stirring at 100 °C overnight. Add 50 mL of ethyl acetate to dilute the reaction solution, wash the organic phase 3 times with 150 mL of water, wash 2 times with saturated sodium chloride solution, dry over anhydrous sodium sulfate, concentrate under reduced pressure. The obtained crude product is purified by silica gel column chromatography to obtain 1.3 g of white solid (intermediate 1B-2), with a yield of 53%. ESI(M+H) + = 315.

[0162] Step 3: Dissolve intermediate 1B-2 (314 mg, 1.0 mmol) in 0.2 mL of dichloromethane, add oxidant 1B-a (158 mg, 0.6 mmol), stir evenly, slowly add dropwise trimethylsilyl azide (0.29 mL, 2.2 mmol) and deionized water (0.036 mL, 2.0 mmol), and react overnight in the dark at room temperature. After monitoring the reaction completion by TLC, add dichloromethane and saturated sodium bicarbonate solution for extraction, and extract the aqueous phase with dichloromethane 2 more times. Combine the organic phases, concentrate under reduced pressure, and purify by silica gel column chromatography to obtain 186 mg of white solid (intermediate 1B), with a yield of 52%. 1 1H-NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 7.78 (dd, J = 8.5, 7.3 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H), 4.24 (t, J = 6.1 Hz, 2H), 3.53 (t, J = 6.7 Hz, 2H), 3.03–2.75 (m, 1H), 2.62–2.47 (m, 2H), 2.06–1.93 (m, 3H). ESI(M+NH4) + = 375.

[0163] Example 6. Synthesis of target compound 1

[0164]

[0165] Intermediate 1A (60 mg, 0.106 mmol) was dissolved in 3 mL of tert-butanol and 1.5 mL of dichloromethane. Intermediate 1B (45.5 mg, 0.127 mmol) and sodium ascorbate (21 mg, 0.106 mmol) were added, and the mixture was stirred thoroughly. Copper(II) sulfate pentahydrate (15.9 mg, 0.064 mmol) was dissolved in 1.5 mL of water and then added dropwise to the above reaction system. The reaction was stirred at room temperature for 2 h. After the reaction was completed, 10 mL of water and 5 mL of dichloromethane were added for extraction. The aqueous phase was extracted with dichloromethane two more times. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography to give 50 mg of a white solid (Target Compound 1) with a yield of 51%. 1 1H-NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 8.76 (t, J = 6.4 Hz, 1H), 8.44 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 6.3 Hz, 2H), 7.92 (d, J = 7.1 Hz, 2H), 7.88 (s, 1H), 7.79 (dd, J = 8.5, 7.2 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.46 (dd, J = 7.9, 5.8 Hz, 2H), 7.38 (t, J = 7.6 Hz, 2H), 7.28 (t, J = 7.4 Hz, 1H), 5.10 (dd, J = 12.9, 5.4 Hz, 1H), 4.52 (t, J = 6.8 Hz, 2H), 4.16 (t, J = 6.0 Hz, 2H), 3.55 (d, J = 6.3 Hz, 2H), 2.96–2.83 (m, 3H), 2.81–2.72 (m, 2H), 2.63–2.50 (m, 4H), 2.37–2.14 (m, 6H), 2.06–1.93 (m, 3H). ESI (M+H) + = 923.

[0166] Example 7. Synthesis of Target Compound 2

[0167]

[0168] Intermediate 2A (3-butyn-1-ol was replaced with 5-hexyn-1-ol) and 2B (3-bromopropene was replaced with 4-bromo-1-butene) were synthesized by referring to the methods of Reference Example 4 and Example 5. Referring to the synthesis method of the target compound in Reference Example 6, Intermediate 2A (60 mg, 0.106 mmol) was used instead of Intermediate 1A, and Intermediate 2B (44.5 mg, 0.127 mmol) was used instead of Intermediate 1B to obtain 48.7 mg of Target Compound 2 with a yield of 51%. 1H-NMR(400 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.79 (s, 1H), 8.44 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H), 8.06 (d, J = 7.3 Hz, 2H), 7.92 (d, J = 7.3 Hz, 2H), 7.85 (s, 1H), 7.83–7.77 (m, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.46 (dd, J = 14.3, 7.9 Hz, 2H), 7.38 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H), 4.41 (t, J = 6.9 Hz, 2H), 4.21 (t, J = 6.2 Hz, 2H), 3.54 (s, 2H), 2.96–2.81 (m, 2H), 2.65–2.54 (m, 5H), 2.45–2.23 (m, 3H), 2.09–1.94 (m, 5H), 1.81–1.66 (m, 3H), 1.64–1.47 (m, 5H), 1.41–1.27 (m, 1H). ESI(M+H) + = 965。

[0169] Example 8. Synthesis of Target Compound 3

[0170]

[0171] Synthesize intermediate 3B by referring to the method of Reference Example 5 (replace 3-bromopropene with 7-bromo-1-heptene). Refer to the synthesis method of the target compound in Reference Example 6, use intermediate 2A (60 mg, 0.106 mmol) instead of intermediate 1A, and intermediate 3B (44 mg, 0.106 mmol) instead of intermediate 1B to obtain 36 mg of target compound 3, with a yield of 34%. 1H-NMR(400 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.81 (t, J = 6.4 Hz, 1H), 8.44 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.10–8.04 (m, 2H), 7.92 (d, J = 7.0 Hz, 2H), 7.82 (s, 1H), 7.78 (dd, J = 8.5, 7.3 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.44–7.35 (m, 3H), 7.29 (t, J = 7.3 Hz, 1H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H), 4.27 (t, J = 7.0 Hz, 2H), 4.16 (t, J = 6.4 Hz, 2H), 3.55 (d, J = 6.3 Hz, 2H), 3.07–2.98 (m, 2H), 2.94–2.81 (m, 1H), 2.68–2.52 (m, 4H), 2.37 (d, J = 13.3 Hz, 2H), 2.16–1.97 (m, 3H), 1.83–1.67 (m, 4H), 1.60–1.49 (m, 4H), 1.45–1.31 (m, 4H), 1.26–1.19 (m, 6H). ESI(M+H) + = 1007。

[0172] Example 9. Synthesis of target compound 4

[0173]

[0174] Synthesize intermediate 3A by referring to the method of Reference Example 4 (replace intermediate 1A-2 with 3-bromopropyne). Refer to the synthesis method of the target compound in Reference Example 6, use intermediate 3A (50 mg, 0.091 mmol) instead of intermediate 1A, and intermediate 3B (35 mg, 0.091 mmol) instead of intermediate 1B as raw materials to obtain 44 mg of target compound 4 with a yield of 50%. 1H-NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.75 (t, 1H), 8.43 (t, J = 1.8 Hz, 1H), 8.17 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 6.4 Hz, 2H), 7.96 (s, 1H), 7.91 (d, J = 7.0 Hz, 2H), 7.78 (dd, J = 8.5, 7.2 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 (t, J = 7.3 Hz, 1H), 5.12–5.05 (m, 1H), 4.29 (t, J = 6.9 Hz, 2H), 4.16 (t, J = 6.3 Hz, 2H), 3.64–3.43 (m, 4H), 2.94–2.68 (m, 3H), 2.63–2.50 (m, 3H), 2.30 (d, J = 13.6 Hz, 2H), 2.21–2.12 (m, 1H), 2.05–1.94 (m, 3H), 1.80–1.68 (m, 4H), 1.46–1.26 (m, 6H). ESI (M+H) + = 965。

[0175] Example 10. Synthesis of Target Compound 5

[0176]

[0177] Synthesize 4B by referring to the method of Reference Example 5 (replace 3-bromopropene with 6-bromo-1-hexene). Referring to the synthesis method of the target compound in Reference Example 6, using intermediate 1A (100 mg, 0.177 mmol) as the raw material and intermediate 4B (77.7 mg, 0.195 mmol) instead of intermediate 1B, 55.8 mg of target compound 5 was obtained, with a yield of 33%. 1H-NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.74 (t, J = 6.4 Hz, 1H), 8.43 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.04 (s, 2H), 7.92 (d, J = 7.0 Hz, 2H), 7.82 (s, 1H), 7.77 (dd, J = 8.5, 7.3 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.44–7.34 (m, 3H), 7.31–7.26 (m, 1H), 5.07 (dd, J = 12.9, 5.4 Hz, 1H), 4.27 (t, J = 7.0 Hz, 2H), 4.15 (t, J = 6.3 Hz, 2H), 3.55 (d, J = 5.9 Hz, 2H), 2.96–2.79 (m, 3H), 2.78–2.71 (m, 2H), 2.64–2.51 (m, 2H), 2.48–2.44 (m, 1H), 2.31 (d, J = 12.2 Hz, 2H), 2.24–2.07 (m, 2H), 2.07–1.92 (m, 3H), 1.86–1.74 (m, 2H), 1.75–1.66 (m, 2H), 1.56–1.38 (m, 2H), 1.35–1.19 (m, 3H). ESI (M+H) + = 965。

[0178] Example 11. Synthesis of Target Compound 6

[0179]

[0180] Intermediate 4A was synthesized by referring to the method of Reference Example 4 (3-butyn-1-ol was replaced with 7-octyn-1-ol). Referring to the synthesis method of the target compound in Reference Example 6, using Intermediate 4A (60 mg, 0.097 mmol) instead of Intermediate 1A and Intermediate 1B (42 mg, 0.116 mmol) as raw materials, 35 mg of the target compound 6 was obtained with a yield of 37%. 1H-NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 8.82 (s, 1H), 8.44 (t, J = 1.7 Hz, 1H), 8.19 (d, 1H), 8.07 (d, J = 6.5 Hz, 2H), 7.93 (d, 2H), 7.86 (s, 1H), 7.80 (t, J = 8.5, 7.2 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.47 (t, J = 7.9, 5.7 Hz, 2H), 7.38 (t, 2H), 7.30 (t, 1H), 5.10 (dd, J = 12.9, 5.4 Hz, 1H), 4.52 (t, J = 6.8 Hz, 2H), 4.18 (t, J = 6.0, 5.3 Hz, 2H), 3.61–3.51 (m, 2H), 3.16–2.94 (m, 2H), 2.97–2.83 (m, 2H), 2.64–2.51 (m, 6H), 2.45–2.26 (m, 6H), 2.22–2.00 (m, 4H), 1.60–1.38 (m, 6H). ESI (M+H) + = 979。

[0181] Example 12. Synthesis of Target Compound 7

[0182]

[0183] Synthesize intermediate 5B by referring to the method of Reference Example 5 (replace 3-bromopropene with 2-(allyloxy)ethyl 4-methylbenzenesulfonate). Refer to the synthesis method of the target compound in Reference Example 6. Using intermediate 1A (50 mg, 0.088 mmol) as the raw material and intermediate 5B (37.3 mg, 0.093 mmol) instead of intermediate 1B, 32 mg of target compound 7 was obtained with a yield of 38%. 1H-NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 8.76 (t, J = 6.2 Hz, 1H), 8.43 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 5.3 Hz, 2H), 7.92 (d, J = 7.1 Hz, 2H), 7.82–7.76 (m, 2H), 7.68 (t, J = 7.8 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.3 Hz, 1H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 (t, J = 7.3 Hz, 1H), 5.07 (dd, J = 12.8, 5.4 Hz, 1H), 4.36–4.31 (m, 4H), 3.75–3.72 (m, 2H), 3.54 (d, J = 6.3 Hz, 2H), 3.43 (t, J = 6.0 Hz, 3H), 2.92–2.80 (m, 3H), 2.76–2.70 (m, 2H), 2.62–2.53 (m, 2H), 2.32 (d, J = 12.7 Hz, 2H), 2.26–2.09 (m, 2H), 2.07–1.93 (m, 6H). ESI (M+H) + = 967。

[0184] Example 13. Synthesis of Target Compound 8

[0185]

[0186] Synthesize intermediate 6B by referring to the method of Reference Example 5. Referring to the synthesis method of the target compound in Reference Example 6, using intermediate 1A (34 mg, 0.067 mmol) as the raw material and intermediate 6B (50 mg, 0.08 mmol) instead of intermediate 1B, 37 mg of target compound 8 was obtained with a yield of 57%. 1H-NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 8.78 (t, 1H), 8.44 (t, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 6.4 Hz, 2H), 7.93 (d, J = 7.0 Hz, 2H), 7.85 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.40–7.35 (m, 3H), 7.32–7.26 (m, 2H), 5.12 (dd, J = 12.9, 5.4 Hz, 1H), 4.28 (t, J = 6.9 Hz, 2H), 4.12 (t, J = 6.4 Hz, 2H), 3.56 (d, J = 5.9 Hz, 2H), 3.02–2.74 (m, 5H), 2.62–2.54 (m, 2H), 2.38–2.31 (m, 2H), 2.13–1.93 (m, 4H), 1.81–1.68 (m, 4H), 1.48–1.37 (m, 3H), 1.30–1.24 (m, 4H). ESI (M+H) + = 965。

[0187] Example 14. Synthesis of Target Compound 9

[0188]

[0189] Intermediate 7B was synthesized by referring to the method of Reference Example 5. According to the synthesis method of the target compound in Reference Example 6, using intermediate 1A (50 mg, 0.088 mmol) as the raw material and intermediate 7B (37.3 mg, 0.093 mmol) instead of intermediate 1B, 30 mg of target compound 9 was obtained with a yield of 36%. 1H-NMR(400MHz, DMSO-d6) δ 11.02 (s, 1H), 8.87 (s, 1H), 8.44 (s, 1H), 8.18 (d, J = 7.8, 1.5 Hz, 1H), 8.10 (s, 2H), 7.93 (d, 2H), 7.88 (s, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 7.5 Hz, 2H), 7.33–7.22 (m, 2H), 6.91 (d, J = 7.4 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H), 5.57 (t, J = 5.5 Hz, 1H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.32–4.09 (m, 4H), 3.63–3.28 (m, 10H), 3.10–3.05 (m, 2H), 3.02–2.80 (m, 4H), 2.67–2.57 (m, 2H), 2.43–2.24 (m, 3H), 2.05–1.99 (m, 1H), 1.82–1.72 (m, 2H), 1.58–1.49 (m, 2H), 1.39–1.32 (m, 2H). ESI(M+H) + = 950。

[0190] Example 15. Synthesis of Target Compound 10

[0191]

[0192] Step 1: Dissolve raw material 8B-1 (867 mg, 3.14 mmol) in 13 mL of anhydrous DMF. Add DIPEA (1.64 mL, 9.42 mmol) and 3-bromopiperidine-2,6-dione (500 mg, 2.62 mmol) at room temperature, and then heat the mixture to 60 °C and continue the reaction for 9 h. After monitoring the completion of the reaction by TLC, add 50 mL of ethyl acetate to dilute the reaction solution, wash the organic phase with 100 mL of water three times and with saturated sodium chloride solution twice, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The obtained crude product is purified by silica gel column chromatography to obtain 254 mg of white solid (intermediate 8B-2), with a yield of 25%. 11H-NMR (400 MHz, Chloroform-d) δ 8.11 (s, 1H), 7.06 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 8.5 Hz, 2H), 4.68 (d, J = 3.6 Hz, 1H), 4.39–4.13 (m, 2H), 4.05 (dt, J = 12.4, 4.2 Hz, 1H), 3.01–2.69 (m, 4H), 2.63–2.49 (m, 2H), 1.90 (qd, J = 13.3, 4.7 Hz, 1H), 1.82–1.72 (m, 2H), 1.62–1.49 (m, 2H), 1.48 (s, 9H). ESI ((M - t Bu)+H) + = 332. Referring to the synthesis method in Step 2 of Reference Example 3, replace Intermediate 1c with Intermediate 8B-2 (254 mg, 0.656 mmol) to obtain 225 mg of a gray solid (Intermediate 8B-3). ESI (M + H) + = 288.

[0193] Step 2: Dissolve 7-bromoheptanoic acid (500 mg, 2.4 mmol) in anhydrous DMF (5 mL), slowly add sodium azide (219 mg, 3.36 mmol), and warm to 80 °C and stir overnight. After completion of the reaction, slowly add 20 mL of water, extract the reaction solution 3 times with ethyl acetate, combine the organic phases, wash 2 times with saturated sodium chloride, dry over anhydrous sodium sulfate, evaporate to dryness, and purify the obtained crude product by silica gel column chromatography to obtain 309 mg of a colorless liquid (Intermediate 8B-5) with a yield of 75%. ESI (M - H) - = 170.

[0194] Step 3: Dissolve Intermediate 8B-5 (90 mg, 0.523 mmol), EDCI (97 mg, 0.628 mmol), and HOBt (104 mg, 0.68 mmol) in anhydrous DMF (2 mL), add DIPEA (0.55 mL, 3.14 mmol) dropwise under ice bath conditions, stir for 10 min, and then slowly add a DMF solution (1 mL) of Intermediate 8B-3 (150 mg, 0.523 mmol), and stir at room temperature overnight. After completion of the reaction, pour into 10 mL of water, extract the reaction solution 3 times with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, evaporate to dryness, and purify the obtained crude product by silica gel column chromatography to obtain 130 mg of Intermediate 8B with a yield of 56%. ESI (M + H) + = 441.

[0195] Step 4: Referring to the synthetic method of the target compound in Example 6, using intermediate 1A (44 mg, 0.078 mmol) as the raw material and intermediate 8B (34 mg, 0.078 mmol) instead of intermediate 1B, 33 mg of the target compound 10 was obtained with a yield of 42%. 1 H-NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.80 (s, 1H), 8.45 (t, J = 1.8 Hz, 1H), 8.19 (d, J = 7.8, 1.4 Hz, 1H), 8.07 (d, J = 6.7 Hz, 2H), 7.93 (d, 2H), 7.85 (s, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.38 (dd, J = 8.3, 6.8 Hz, 2H), 7.29 (t, 1H), 6.93 (d, 2H), 6.60 (d, J = 8.6 Hz, 2H), 5.68 (d, J = 7.5 Hz, 1H), 4.50 (d, J = 12.7 Hz, 1H), 4.26 (t, J = 6.7 Hz, 3H), 3.90 (d, J = 13.2 Hz, 1H), 3.56 (d, J = 4.7 Hz, 2H), 3.01 (t, J = 12.0 Hz, 2H), 2.87–2.67 (m, 4H), 2.62–2.51 (m, 5H), 2.35 (d, J = 12.5 Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H), 2.16–1.92 (m, 4H), 1.93–1.55 (m, 6H), 1.51–1.26 (m, 8H). ESI(M+H) + = 1006.

[0196] Example 16. Synthesis of target compound 11

[0197]

[0198] Step 1: Add 3A molecular sieve (600 mg) and raw material (S)-2-N-Boc-1,2-propanediamine hydrochloride (5A-1) (500 mg, 2.87 mmol) into a 50 mL round-bottom flask. After evacuating and filling with nitrogen, add anhydrous ether and stir for 10 min. Then add benzaldehyde 5A-2 (292 μL, 2.87 mmol), and stir the reaction mixture thoroughly at room temperature for 18 h. After the reaction is completed, filter the mixture, wash the filter cake with ether three times, and collect the synthetic filtrate. Concentrate the filtrate under reduced pressure to obtain 737 mg of crude product, which can be directly used in the next step without further purification. Dissolve the above crude product (737 mg, 2.81 mmol) in 10 mL of anhydrous ethanol, cool the reaction system to 0 °C, and slowly add sodium borohydride (160 mg, 4.21 mmol) portionwise. Keep the reaction at 0 °C for 3 h. After monitoring the completion of the reaction of the raw material by TLC, concentrate the reaction mixture under reduced pressure, dissolve it in 10 mL of methanol, filter, and concentrate the filtrate under reduced pressure. The obtained crude product is purified by silica gel column chromatography to obtain 420 mg of intermediate 5A-3 with a yield of 55% (two steps). ESI(M+H) + = 265.

[0199] Step 2: Under nitrogen protection, dissolve intermediate 1A-2 (596 mg, 2.66 mmol) in 5 mL of anhydrous acetonitrile, and successively add K2CO3 (611 mg, 4.43 mmol), KI (123 mg, 0.74 mmol) and intermediate 5A-3 (390 mg, 1.48 mmol). Heat the reaction mixture to 85 °C and reflux with stirring overnight. Concentrate the reaction mixture under reduced pressure, dissolve it in 5 mL of dichloromethane, filter, and concentrate the filtrate to dryness and then purify it by silica gel column chromatography. Dissolve the obtained product (379 mg, 1.2 mmol) in 1 mL of dichloromethane, add 2 mol / L hydrogen chloride-ethyl acetate solution (6 mL, 12 mmol), react at room temperature for 2 h and then concentrate to dryness to obtain 270 mg of intermediate 5A-4 with a yield of 84% (two steps). ESI(M+H) + = 217.

[0200] Step 3: Refer to the synthesis method of intermediate 1c in Example 3, use intermediate 1a (220 mg, 0.85 mmol) as the raw material, and replace intermediate 1b with intermediate 5A-4 to obtain 260 mg of intermediate 5A with a yield of 67%. ESI(M+H) + = 457.

[0201] Step 4: Refer to the synthesis method of the target compound in Example 6, replace intermediate 1A with intermediate 5A-4 (40 mg, 0.088 mmol), and replace intermediate 1B with intermediate 4B (35 mg, 0.088 mmol) to obtain 25 mg of target compound 11 with a yield of 33%. 1H-NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 8.38 (d, J = 8.2 Hz, 1H), 8.15 (d, J = 8.5 Hz, 2H), 8.04 (d, J = 8.5 Hz, 2H), 7.80 (dd, 1H), 7.73 (s, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H), 7.38–7.33 (m, 1H), 7.26–7.21 (m, 4H), 5.07 (dd, J = 12.9, 5.3 Hz, 1H), 4.29–4.24 (m, 1H), 4.22 (t, 2H), 4.16 (t, J = 6.4 Hz, 2H), 3.71–3.59 (m, 2H), 2.92–2.59 (m, 8H), 2.03–1.99 (m, 1H), 1.74–1.63 (m, 5H), 1.53–1.42 (m, 4H), 1.10 (d, J = 6.6 Hz, 3H). ESI(M+H) + = 856。

[0202] Example 17. Synthesis of Target Compound 12

[0203]

[0204] Step 1: Intermediate 6A-1 (acetic anhydride replaced trifluoroacetic anhydride) was obtained by referring to the synthesis method of Intermediate 1a in Example 1. Referring to the synthesis method of Intermediate 1d in Example 3, using Intermediate 6A-1 (100 mg, 0.85 mmol) instead of Intermediate 1a and Intermediate 1b (317 mg, 0.85 mmol) as raw materials, 260 mg of Intermediate 6A-2 was obtained with a yield of 67% (two steps). ESI(M+H) + = 460。

[0205] Step 2: Intermediate 6A was synthesized by referring to the method in Example 4 (Intermediate 1d was replaced by Intermediate 6A-2). Referring to the synthesis method of the target compound in Example 6, using Intermediate 6A (34 mg, 0.073 mmol) instead of Intermediate 1A and Intermediate 4B (35 mg, 0.088 mmol) instead of Intermediate 1B, 26 mg of target compound 12 was obtained with a yield of 39%. ESI(M+H) + = 911。

[0206] Example 18. Synthesis of Target Compound 13

[0207]

[0208] Step 1: Refer to the specific experimental steps for synthesizing Intermediate 1d in Example 3. Use monomethyl isophthalate 7A-1 (263 mg, 1.46 mmol) to replace Intermediate 1a, and Intermediate 1b (545 mg, 1.46 mmol) as the raw materials to obtain 586 mg of Intermediate 7A-2 with a yield of 92% (two steps). ESI(M+H) + = 436.

[0209] Step 2: Refer to the synthesis method in Step 2 of Example 4. Use Intermediate 7A-2 (586 mg, 1.35 mmol) to replace Intermediate 1d to obtain 426 mg of Intermediate 7A-3 with a yield of 65%. ESI(M+H) + = 488.

[0210] Step 3: Add hydroxylamine hydrochloride (1.2 g, 17.5 mmol) to a 50 mL round-bottom flask, dissolve it with 5 mL of methanol. Dissolve potassium hydroxide (1.0 g, 18.4 mmol) in 5 mL of methanol and add it to the above reaction system under an ice bath. After stirring well for 30 min, filter. Add Intermediate 7A-3 (426 mg, 0.9 mmol) to the filtrate and stir evenly. Raise the reaction system to 55 °C and react for 1 h. Concentrate under reduced pressure, adjust the pH value to less than 4 with 1N hydrochloric acid solution, extract with ethyl acetate three times, combine the organic phases, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 125 mg of Intermediate 7A with a yield of 29%. ESI(M+H) + = 489.

[0211] Step 4: Refer to the synthesis method of the target compound in Example 6. Use Intermediate 7A (80 mg, 0.163 mmol) to replace Intermediate 1A, and Intermediate 4B (69 mg, 0.172 mmol) to replace Intermediate 1B as the raw materials to obtain 50 mg of the target compound 13 with a yield of 35%. 1H-NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.56 (t, J = 6.3 Hz, 1H), 8.22 (s, 1H), 8.19 (t, J = 1.8 Hz, 1H), 8.04 (s, 1H), 7.94 (d, J = 7.0 Hz, 2H), 7.89 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 7.78 (dd, J = 8.5, 7.3 Hz, 1H), 7.52–7.46 (m, 2H), 7.41 (t, J = 7.4 Hz, 3H), 7.31 (t, J = 7.3 Hz, 1H), 5.07 (dd, J = 12.8, 5.4 Hz, 1H), 4.27 (t, J = 7.0 Hz, 2H), 4.16 (t, J = 6.3 Hz, 2H), 3.76–3.60 (m, 2H), 3.52 (d, J = 6.3 Hz, 2H), 2.93–2.80 (m, 3H), 2.74 (t, J = 7.7 Hz, 2H), 2.62–2.51 (m, 2H), 2.49–2.44 (m, 1H), 2.30 (d, J = 12.8 Hz, 2H), 2.16 (t, J = 10.5 Hz, 2H), 2.06–1.91 (m, 3H), 1.85–1.64 (m, 4H), 1.50–1.40 (m, 2H), 1.34–1.19 (m, 2H). ESI (M+H) + = 888.

[0212] Example 19. Synthesis of Target Compound 14

[0213]

[0214] Dissolve compound 13 (10 mg, 11.3 μmol) in 700 μL of dichloromethane, add N,N'-carbonyldiimidazole (2.7 mg, 16.9 μmol), and stir the reaction at room temperature for 2 h. Concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 3.5 mg of target compound 14 with a yield of 34%. ESI (M+H) + = 913.

[0215] Example 20. Synthesis of Target Compound 15

[0216]

[0217] Step 1: Under nitrogen protection, dissolve trifluoroacetaldehyde methyl hemiacetal (317 mg, 2.44 mmol) in 5 mL of anhydrous THF. Dropwise add pyrrolidine (141 μL, 1.71 mmol) at room temperature, stir well for 30 min, add the raw material 3-acetylbenzoic acid (9A-1) (400 mg, 2.44 mmol), and raise the temperature to 80 °C for reflux reaction overnight. After the reaction is completed, add 10 mL of ethyl acetate to dilute the reaction solution, extract the organic phase with saturated sodium bicarbonate solution 3 times, adjust the pH value of the aqueous phase to about 1 with 1N hydrochloric acid solution, then extract with ethyl acetate 3 times. Combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 419 mg of white solid (intermediate 9A-2) with a yield of 66%. ESI(M+H) + = 263. Refer to the synthesis method of intermediate 1d in Reference Example 3, use intermediate 9A-2 instead of intermediate 1a, and use intermediate 1b as the raw material to obtain intermediate 9A-3 with a yield of 80% (two steps). ESI(M+H) + = 518.

[0218] Step 2: Refer to the method of Reference Example 4 to synthesize intermediate 9A. Refer to the synthesis method of the target compound in Reference Example 6, use intermediate 4B as the raw material to obtain intermediate 9A-4 with a yield of 32%. ESI(M+H) + = 969. Dissolve intermediate 9A-4 (35 mg, 0.0368 mmol) in 2 mL of toluene, add p-toluenesulfonic acid monohydrate (4.9 mg, 0.0257 mmol) and magnesium sulfate (40.9 mg, 0.341 mmol), heat to 120 °C for reflux reaction for 24 h. After cooling to room temperature, filter, concentrate the filtrate under reduced pressure, and purify by silica gel column chromatography to obtain the target compound 15. ESI(M+H) + = 951.

[0219] Example 21. Synthesis of Target Compound 16

[0220]

[0221] Refer to the specific experimental operation steps for synthesizing intermediate 1b in Reference Example 2. After obtaining intermediate 10A-2, refer to the synthesis method of intermediate 1c in Reference Example 3 (using intermediate 10A-2 and intermediate 1a as raw materials) to obtain intermediate 10A. Refer to the synthesis method of the target compound in Reference Example 6, use intermediate 10A (30 mg, 0.057 mmol) instead of intermediate 1A, and use intermediate 4B (25.1 mg, 0.063 mmol) as the raw material to obtain 21.9 mg of target compound 16 with a yield of 42%. 1H-NMR (400 MHz, Chloroform-d) δ 8.51 (s, 2H), 8.20 (d, J = 7.8 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.90–7.80 (m, 3H), 7.65 (t, 1H), 7.58–7.51 (m, 1H), 7.45–7.40 (m, 2H), 7.38–7.28 (m, 4H), 7.17 (d, J = 8.6 Hz, 1H), 4.93 (dd, J = 12.2, 5.2 Hz, 1H), 4.30 (t, 2H), 4.13 (td, J = 7.2, 6.8, 2.4 Hz, 2H), 3.98–3.77 (m, 2H), 3.52–3.31 (m, 1H), 2.99–2.77 (m, 2H), 2.76–2.67 (m, 3H), 2.01–1.79 (m, 7H), 1.77–1.67 (m, 2H), 1.61–1.44 (m, 6H). ESI (M+H) + = 924.

[0222] Example 22. Synthesis of Target Compound 17

[0223]

[0224] Step 1: Under nitrogen protection, dissolve intermediate 1b-2 (5 g, 25 mmol) in 50 mL of anhydrous THF. After cooling to 0 °C, add sodium hydride (3 g, 75 mmol) portionwise, and keep it activated at 0 °C for 20 min. Dissolve (2-bromoethoxy)-tert-butyldimethylsilane (12 g, 50 mmol) in 15 mL of anhydrous THF and add it to the above reaction system. Raise the temperature to room temperature and continue the reaction for 3 h. Quench the reaction by adding 100 mL of saturated sodium chloride solution, extract with ethyl acetate three times, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Purify the obtained crude product by silica gel column chromatography. Dissolve the purified intermediate (2 g, 3.876 mmol) in 3.8 mL of tetrahydrofuran, add acetic acid (11.5 mL, 201.6 mmol) and 3.84 mL of water, and stir the reaction overnight at room temperature. Neutralize acetic acid by adding saturated sodium bicarbonate solution, extract the aqueous phase with ethyl acetate three times, combine the organic phases, and concentrate under reduced pressure to obtain 830 mg of intermediate 11A-1. ESI (M+H) + = 289.

[0225] Step 2: Dissolve intermediate 11A-1 (4.4 g, 15.28 mmol) in 75 mL of acetonitrile. Subsequently, add copper(I) trifluoromethanesulfonate CuOTf (162 mg, 0.764 mmol), 2,2'-bipyridine BPy (120 mg, 0.764 mmol), 2,2,6,6-tetramethylpiperidine 1-oxyl TEMPO (119 mg, 0.764 mmol), and N-methylimidazole NMI (171 mg, 1.53 mmol). Stir the reaction under atmospheric pressure at room temperature overnight. After monitoring the completion of the reaction by TLC, dilute with water and extract with dichloromethane three times. Combine the organic phases, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 510 mg of intermediate 11A-2 with a yield of 12%. ESI(M+H) + = 287.

[0226] Step 3: Dissolve intermediate 11A-2 (510 mg, 1.78 mmol) in 4 mL of toluene. Add triethyl phosphonoacetate (519 mg, 2.32 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene DBU (299 mg, 1.96 mmol), and heat the reaction to 80 °C overnight. After the reaction is completed, add 10 mL of water to the reaction solution for dilution, extract with dichloromethane three times, combine the organic phases, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 279 mg of intermediate 11A-3 with a yield of 44%. ESI(M+H) + = 357.

[0227] Step 4: Refer to the specific experimental method in Step 3 of Example 2, and use intermediate 11A-3 (279 mg, 0.78 mmol) instead of intermediate 1b-3 to obtain 188 mg of the reduction product. Dissolve this intermediate (188 mg, 0.59 mmol) in 2 mL of dichloromethane, add di-tert-butyl dicarbonate (141.5 mg, 0.65 mmol) and triethylamine (98 μL, 0.708 mmol) at 0 °C, and raise the temperature to room temperature for reaction for 3 h. Add 10 mL of dichloromethane and 20 mL of water for extraction, extract the aqueous phase with dichloromethane two more times, combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify by silica gel column chromatography to obtain 106 mg of intermediate 11A-4 with a yield of 32% (two steps). ESI(M+H) + = 419.

[0228] Step 5: Refer to the synthetic method of intermediate 1A-2 in Example 4, and use intermediate 11A-4 (106 mg, 0.254 mmol) instead of raw material 1A-1 to obtain 119 mg of intermediate 11A-5 with a yield of 82%. ESI(M+H) + = 573.

[0229] Step 6: Under nitrogen protection, dissolve raw material 3-butyn-1-ol (25 mg, 0.358 mmol) in 1 mL of anhydrous N,N-dimethylformamide (DMF). Add sodium hydride (16 mg, 0.403 mmol) portionwise under an ice bath. After activation for 30 min, add dropwise a DMF solution (1 mL) of intermediate 11A-5 (119 mg, 0.208 mmol). Raise the temperature to room temperature and react for 3 h. Quench the reaction system with water under an ice bath. Add 10 mL of ethyl acetate for extraction. Wash the organic phase once with water and then with saturated sodium chloride solution. Dry over anhydrous sodium sulfate and concentrate. Purify the obtained crude product by silica gel column chromatography. Then, deprotect the amino group according to the method in Step 2 of Example 3 to obtain 47 mg of intermediate 11A-6 with a yield of 61% (two steps). ESI(M+H) + = 371.

[0230] Step 7: Refer to the experimental method in Step 1 of Example 3. Using intermediate 1a (33 mg, 0.127 mmol) as the raw material and replacing intermediate 1b with intermediate 11A-6 (47 mg, 0.127 mmol), 25 mg of intermediate 11A is obtained with a yield of 32%. Refer to the synthesis method of the target compound in Example 6. Using intermediate 11A (25 mg, 0.041 mmol) to replace intermediate 1A and intermediate 1B (16 mg, 0.043 mmol) as the raw material, 18 mg of target compound 17 is obtained with a yield of 45%. 1 1H-NMR (400 MHz, Chloroform-d) δ 8.67 (d, J = 59.3 Hz, 1H), 8.48 (d, J = 1.9 Hz, 1H), 8.21–8.03 (m, 2H), 7.97 (d, 1H), 7.87 (d, J = 7.2, 1.7 Hz, 2H), 7.78–7.69 (m, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.54–7.49 (m, 1H), 7.46 (d, J = 1.6 Hz, 1H), 7.40–7.30 (m, 4H), 7.14–7.09 (m, 1H), 4.91 (dd, 1H), 4.74–4.60 (m, 2H), 4.27–3.86 (m, 7H), 3.78–3.40 (m, 4H), 3.08–2.94 (m, 2H), 2.89–2.57 (m, 3H), 2.56–2.41 (m, 2H), 2.07–1.98 (m, 3H), 1.78–1.55 (m, 4H). ESI(M+H) + = 968.

[0231] Example 23. Synthesis of Target Compound 18

[0232]

[0233] Step 1: Refer to the synthetic method in the literature (literature source: https: / / doi.org / 10.1002 / ejoc.201501522). Intermediate 12A-2 was prepared from raw material 12A-1 (1,12-dodecanediol). Under nitrogen protection, raw material 1B-1 (250 mg, 0.91 mmol) and intermediate 12A-2 (349 mg, 1.0 mmol) were dissolved in 3 mL of anhydrous DMF. Sodium bicarbonate (153 mg, 1.82 mmol) and potassium iodide (151 mg, 0.91 mmol) were slowly added. The reaction system was heated to 70 °C and reacted for 24 h. 10 mL of ethyl acetate was added to dilute the reaction solution. The organic phase was washed 3 times with saturated sodium bicarbonate solution, 2 times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 329 mg of a crude product, a yellowish-green solid (intermediate 12A-3). Without further purification, it was directly used for the next step. ESI(M+H) + = 459.

[0234] Step 2: Refer to the synthetic method in Step 1 of Example 4. Intermediate 12A-3 (329 mg, 0.72 mmol) was used instead of raw material 1A-1 to obtain 147 mg of intermediate 12A-4 with a yield of 26% (two steps). ESI(M+H) + = 613.

[0235] Step 3: Under nitrogen protection, raw material 1d (60 mg, 0.109 mmol) was dissolved in 1 mL of anhydrous DMF. DIPEA (95 μL, 0.545 mmol) and KI (21.7 mg, 0.131 mmol) were added. Subsequently, a solution of intermediate 12A-4 (80 mg, 0.131 mmol) in anhydrous DMF (1 mL) was added dropwise. The reaction was stirred at room temperature overnight. 10 mL of water was added to the reaction system. The organic phase was extracted 3 times with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 29 mg of a crude product, a yellow solid (target compound 18), with a yield of 28%. 1H-NMR (400 MHz, Chloroform-d) δ 8.51 (t, J = 1.8 Hz, 1H), 8.21 (dt, J = 7.8, 1.4 Hz, 1H), 7.99 (dt, J = 7.8, 1.5 Hz, 1H), 7.90–7.83 (m, 2H), 7.65 (dd, J = 8.5, 7.3 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.48 (s, 1H), 7.43 (d, J = 7.3 Hz, 1H), 7.38–7.25 (m, 3H), 7.20 (d, J = 8.5 Hz, 1H), 4.94 (dd, J = 12.4, 5.1 Hz, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.86 (d, J = 5.6 Hz, 2H), 2.92–2.64 (m, 7H), 2.50–2.28 (m, 4H), 2.15–2.06 (m, 3H), 1.86 (dt, J = 14.6, 6.7 Hz, 2H), 1.55–1.45 (m, 4H), 1.44–1.22 (m, 14H). ESI(M+H) + = 954。

[0236] Example 24. Synthesis of Target Compound 19

[0237]

[0238] Referring to the synthetic method of the target compound in Reference Example 23, using 2,2'-(oxybis(ethane-2,1-diyl))bis(oxy)bis(1-ethanol) (13A-1) as the starting material instead of the starting material 12A-1, the specific experimental procedure was the same as that in Example 23, and 14.4 mg of the target compound 19 was obtained. 11H-NMR (400 MHz, Chloroform-d) δ 8.50 (t, J = 1.8 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 6.8 Hz, 2H), 7.65 (t, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.49 (s, 1H), 7.44 (d, J = 7.3 Hz, 1H), 7.40–7.29 (m, 3H), 7.24 (d, J = 8.3 Hz, 1H), 4.94 (dd, J = 12.0, 5.4 Hz, 1H), 4.32 (t, J = 5.1 Hz, 2H), 3.92 (t, J = 4.6 Hz, 2H), 3.90–3.86 (m, 2H), 3.83–3.74 (m, 4H), 3.72–3.50 (m, 6H), 3.28–3.01 (m, 2H), 2.98–2.68 (m, 6H), 2.63–2.44 (m, 2H), 2.38–2.22 (m, 3H), 2.19–2.08 (m, 1H). ESI (M+H) + = 946。

[0239] Example 25. Synthesis of the target compound 20

[0240]

[0241] Step 1: Under nitrogen protection, dissolve the raw material 4-benzyloxyphenol 14A-1 (1.35 g, 6.74 mmol) in 5 mL of anhydrous DMF. After adding K2CO3 (2.48 g, 18.39 mmol) and activating for 10 min, dropwise add a DMF solution (5 mL) of the raw material 2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 4-methylbenzenesulfonate 14A-2 (2.2 g, 6.13 mmol). Heat the reaction system to 70 °C and stir overnight. After monitoring the completion of the reaction by TLC, add 30 mL of water to the reaction system, extract with ethyl acetate three times, combine the organic phases, wash twice with saturated sodium chloride solution, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 1.12 g of intermediate 14A-3 with a yield of 47%. ESI (M+H) + = 388。

[0242] Step 2: Under nitrogen protection, dissolve the obtained intermediate 14A-3 (1.12 g, 2.89 mmol) in 10 mL of methanol. After adding 10% palladium on carbon (207 mg), replace the hydrogen, and raise the reaction system to 50 °C and stir thoroughly. After monitoring the completion of the reaction by TLC, filter, and concentrate the filtrate under reduced pressure to obtain intermediate 14A-4, which is directly used in the next step without further purification.

[0243] Step 3: Referring to the synthesis method of Step 1 in this example, using intermediate 14A-4 (740 mg, 2.49 mmol) instead of 14A-1 and ethylene glycol ditosylate (1.0 g, 2.72 mmol) instead of 14A-2, 366 mg of intermediate 14A-5 was obtained with a yield of 30%. ESI ((M- t Bu)+H) + = 440.

[0244] Step 4: Referring to the synthesis method of Step 1 in this example, using intermediate 1d (110 mg, 0.214 mmol) instead of 14A-1 and intermediate 14A-5 (127 mg, 0.257 mmol) instead of 14A-2, after purifying the obtained crude product by silica gel column chromatography, 2 mol / L hydrogen chloride-ethyl acetate solution was added to remove the Boc protection to obtain 115 mg of intermediate 14A-6. The yield was 73%. ESI (M+H) + = 737.

[0245] Step 5: Under nitrogen protection, dissolve intermediate 14A-6 (60 mg, 0.081 mmol) in 1 mL of anhydrous thionyl chloride, add DIPEA (85 μL, 0.488 mmol) and activate for 10 min, then dropwise add a solution of raw material 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione 14B (25 mg, 0.081 mmol) in thionyl chloride (1 mL), heat to 110 °C and stir the reaction overnight. After monitoring the completion of the reaction by TLC, add 10 mL of water to the reaction system, extract with ethyl acetate three times, combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 26 mg of the target compound 20 with a yield of 32%. 1H-NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.77 (t, J = 6.4 Hz, 1H), 8.43 (t, J = 1.8 Hz, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 4.6 Hz, 2H), 7.92 (d, J = 7.0 Hz, 2H), 7.69–7.66 (m, 2H), 7.56 (dd, J = 8.6, 7.1 Hz, 1H), 7.37 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.03 (d, J = 7.0 Hz, 1H), 6.82–6.80 (m, 4H), 5.05 (dd, J = 12.7, 5.4 Hz, 1H), 4.01–3.95 (m, 4H), 3.75–3.73 (m, 2H), 3.67 (t, J = 5.4 Hz, 2H), 3.54 (d, J = 6.3 Hz, 2H), 3.49–3.47 (m, 2H), 2.92–2.84 (m, 3H), 2.62–2.58 (m, 2H), 2.34–2.29 (m, 2H), 2.26–2.17 (m, 2H), 2.03–1.94 (m, 4H), 1.64–1.60 (m, 1H). ESI(M+H) + = 993。

[0246] Example 26. Synthesis of Target Compound 21

[0247]

[0248] Step 1: Dissolve the raw material 8B-3 (50 mg, 0.174 mmol) in 1 mL of anhydrous DMF, add DIPEA (90.8 μL, 0.522 mmol) and tert-butyl bromoacetate (26.4 μL, 0.183 mmol), and continue stirring the reaction overnight at room temperature. After monitoring the completion of the reaction by TLC, add 10 mL of water to the reaction solution, extract the organic phase with ethyl acetate 3 times, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 42.4 mg of a white solid with a yield of 61%. ESI(M+H) + = 402. Dissolve this intermediate in 0.24 mL of dichloromethane, slowly add trifluoroacetic acid (81.2 μL, 1.06 mmol), react at room temperature for 4 h, spin dry after monitoring the complete reaction of the raw materials by TLC to obtain intermediate 9B-1. ESI(M+H) + = 346。

[0249] Step 2: Referring to the synthesis method of Intermediate 1d in Example 3, using Intermediate 15A-1 (53 mg, 0.172 mmol) instead of Intermediate 1a and Intermediate 1d (70 mg, 0.136 mmol) instead of Intermediate 1b, 51.6 mg of Intermediate 15A-2 was obtained with a yield of 54% (two steps). ESI(M+H) + = 703.

[0250] Step 3: Dissolve Intermediate 9B-1 (36.4 mg, 0.106 mmol), EDCI (15.5 mg, 0.081 mmol) and HOBt (13.5 mg, 0.088 mmol) in anhydrous DMF (0.5 mL). Under ice bath conditions, add DIPEA (0.1 mL, 0.588 mmol) dropwise. After stirring for 10 min, slowly add a DMF solution (0.5 mL) of Intermediate 15A-2 (51.6 mg, 0.074 mmol). Stir at room temperature overnight. After the reaction is completed, pour it into 10 mL of water, extract the reaction solution with ethyl acetate 3 times, combine the organic phases, dry over anhydrous sodium sulfate, evaporate to dryness, and purify the obtained crude product by silica gel column chromatography to obtain the target compound 21. 1 1H-NMR (400 MHz, Chloroform-d) δ 8.48 (t, J = 1.7 Hz, 1H), 8.26–8.16 (m, 2H), 7.99 (dt, J = 7.9, 1.5 Hz, 1H), 7.90–7.84 (m, 2H), 7.61–7.50 (m, 3H), 7.40–7.28 (m, 3H), 7.07 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 8.2 Hz, 2H), 4.88–4.56 (m, 1H), 4.20 (s, 2H), 4.15–4.04 (m, 1H), 3.99 (dd, J = 13.5, 6.8 Hz, 1H), 3.95–3.85 (m, 1H), 3.80–3.54 (m, 14H), 3.52–3.38 (m, 3H), 3.29–2.95 (m, 2H), 2.91–2.68 (m, 2H), 2.62–2.17 (m, 6H), 2.13–1.71 (m, 9H). ESI(M+H) + = 1030.

[0251] Example 27. Synthesis of Target Compound 22

[0252]

[0253] Step 1: Dissolve lithium diisopropylamide (7.75 mL, 15.5 mmol) in 10 mL of anhydrous tetrahydrofuran, lower the temperature to -78 °C, add an anhydrous THF solution (10 mL) of raw material 16A-1 methyl 1-Boc-4-piperidinecarboxylate (2.5 g, 10.3 mmol), stir well for 30 min, then dropwise add an anhydrous THF solution (10 mL) of bromoacetonitrile (2.47 g, 20.6 mmol), slowly raise the temperature to room temperature and continue the reaction for 5 h. After monitoring the completion of the reaction by TLC, concentrate under reduced pressure, add 50 mL of ethyl acetate for dilution and extract with 1N hydrochloric acid solution, extract the aqueous phase with ethyl acetate twice more, combine the organic phases, spin dry, and purify the obtained crude product by silica gel column chromatography to obtain 1.25 g of intermediate 16A-2 with a yield of 43%. ESI(M+H) + = 283.

[0254] Step 2: Dissolve intermediate 16A-2 (1.25 g, 4.42 mmol) in 15 mL of ammonia water solution, stir and react at room temperature overnight. Concentrate under reduced pressure to obtain 850 mg of intermediate 16A-3 with a yield of 72%. ESI(M+H) + = 268.

[0255] Step 3: Dissolve intermediate 16A-3 (850 mg, 3.18 mmol) in 10 mL of anhydrous THF, add Lawesson's reagent (1.29 g, 3.18 mmol), reflux and stir the reaction for 6 h. Concentrate under reduced pressure, add 10 mL of ethyl acetate and 20 mL of water for extraction, extract the aqueous phase with ethyl acetate twice more, combine the organic phases, wash with saturated sodium bicarbonate solution twice, dry over anhydrous sodium sulfate, spin dry, and purify the obtained crude product by silica gel column chromatography to obtain 504 mg of intermediate 16A-4 with a yield of 56%. ESI(M+H) + = 284.

[0256] Step 4: Refer to the experimental method of Step 1 in Example 2, use intermediate 16A-4 (504 mg, 1.78 mmol) to replace raw material 2-cyanothioacetamide, to obtain 498 mg of intermediate 16A-5 with a yield of 73%. ESI(M+H) += 384. Referring to the specific experimental method in Step 3 of Reference Example 2, using intermediate 16A-5 (498 mg, 1.3 mmol) to replace intermediate 1b-3, 265 mg of intermediate 16A-6 was obtained with a yield of 53%. Referring to the method in Reference Example 3 (using intermediate 9A-2 to replace intermediate 1a; using intermediate 16A-6 to replace intermediate 1b), 312 mg of intermediate 16A-7 was obtained with a yield of 86% (two steps). Referring to the method in Reference Example 4 (using intermediate 16A-7 to replace intermediate 1d) to synthesize intermediate 16A, referring to the synthesis method of the target compound in Reference Example 6, using intermediate 4B and intermediate 16A as raw materials, intermediate 16A-8 was obtained with a yield of 36%. ESI(M+H) + = 983. Referring to the synthesis method of target compound 15 in Reference Example 20, using intermediate 16A-8 to replace intermediate 9A-4, target compound 22 was obtained. ESI(M+H) + = 965.

[0257] Example 28. Synthesis of Target Compound 23

[0258]

[0259] Step 1: Dissolve raw material 17A-1 (R)-(+)-1-phenylethylamine (2.47 g, 20 mmol) in 10 mL of methanol and 10 mL of tetrahydrofuran, add acetic acid (1.14 mL, 20 mmol) and N-Boc-4-piperidone-3-carboxylic acid methyl ester (5 g, 19.4 mmol), and heat under reflux for 3 h. Dilute with 50 mL of ethyl acetate, extract with saturated sodium hydroxide solution, wash the organic phase with saturated sodium chloride solution, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 6.2 g of intermediate 17A-2 with a yield of 89%. ESI(M+H) + = 361.

[0260] Step 2: Dissolve sodium borohydride (630 mg, 16.7 mmol) in 45 mL of anhydrous THF, slowly add trifluoroacetic acid (3.71 mL, 49.8 mmol) at 0 °C, lower the temperature to -45 °C, add an acetonitrile solution (16 mL) of intermediate 17A-2 (3 g, 8.3 mmol), and continue stirring for 1 h. After monitoring the completion of the reaction by TLC, quench with 25% ammonium chloride solution in an ice bath, extract 3 times with ethyl acetate, wash once with water, wash once with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and evaporate to dryness. Purify the obtained crude product by silica gel column chromatography to obtain 2.6 g of intermediate 17A-3 with a yield of 87%. ESI(M+H) + = 363.

[0261] Step 3: Referring to the experimental method in Step 2 of Example 25, using intermediate 17A-3 (2.6 g, 7.2 mmol) instead of intermediate 14A-3, 1.65 g of intermediate 17A-4 was obtained with a yield of 89%. ESI(M+H) + = 259. Intermediate 17A-6 was obtained by referring to the methods in Step 2 and Step 3 of Example 27. Intermediate 17A-7 was obtained by referring to the experimental method in Step 1 of Example 2. Intermediate 17A-8 was obtained by referring to the method in Example 3. Intermediate 17A was synthesized by referring to the method in Example 4. Referring to the synthesis method of the target compound in Example 6, using intermediate 4B as the raw material, the target compound 23 was obtained. ESI(M+H) + = 951.

[0262] Example 29. Synthesis of target compound 24

[0263]

[0264] Step 1: Dissolve intermediate 1d (150 mg, 0.273 mmol) in 2 mL of anhydrous 1,4-dioxane, add triethylamine (189 μL, 1.365 mmol), and then add a dioxane solution (0.5 mL) of 2-bromoethanol (47 mg, 0.382 mmol). Heat under reflux overnight. After monitoring the completion of the reaction by TLC, concentrate under reduced pressure, add 10 mL of ethyl acetate and 20 mL of water for extraction, and extract the aqueous phase with ethyl acetate twice more. Combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product by silica gel column chromatography to obtain 63 mg of intermediate 18A-1 with a yield of 41%. ESI(M+H) + = 558. Intermediate 18A-2 was obtained by referring to the method in Step 1 of Example 4. ESI(M+H) + = 712.

[0265] Step 2: Dissolve the raw material 5-bromovaleric acid (600 mg, 3.33 mmol) in 4 mL of anhydrous dichloromethane, add oxalyl chloride (0.56 mL, 6.66 mmol) and anhydrous DMF (1 drop) dropwise under ice bath, and react at room temperature for 2 h. Concentrate under reduced pressure, and drop it into an anhydrous tetrahydrofuran solution (15 mL) of the raw material 10B-1 pomalidomide (455 mg, 1.67 mmol). Heat under reflux for 4 h. Spin dry, add 4 mL of ethyl acetate to dissolve it completely, then add 16 mL of petroleum ether. Filter after white solid precipitates, and dry the obtained solid to obtain intermediate 10B-2 with a yield of 96%. ESI(M+H) + = 436.

[0266] Step 3: Under nitrogen protection, dissolve 1-Boc-piperazine (74.4 mg, 0.4 mmol) in 5 mL of anhydrous DMF. Sequentially add DIPEA (348 μL, 2.0 mmol) and potassium iodide (132.8 mg, 0.8 mmol). After activation for 5 min, add intermediate 10B-2 (350 mg, 0.8 mmol), and stir the reaction overnight at room temperature. Add 10 mL of ethyl acetate to dilute the reaction solution, wash the organic phase 3 times with saturated sodium bicarbonate solution, wash 2 times with saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Purify the obtained crude product by silica gel column chromatography, and obtain 167 mg of intermediate 10B-3 according to the method in Step 2 of Example 3, with a yield of 77% (two steps). ESI(M+H) + = 442.

[0267] Step 4: Refer to the method in Step 3 of this example. Use intermediate 10B-3 (30 mg, 0.068 mmol) to replace 1-Boc-piperazine, and intermediate 18A-2 (40 mg, 0.057 mmol) to replace intermediate 10B-2 to obtain 12.8 mg of target compound 24, with a yield of 23%. ESI(M+H) + = 981.

[0268] Example 30. Synthesis of target compound 25

[0269]

[0270] Refer to the synthetic method of the target compound in Example 29. Use 5-bromopentanol to replace 2-bromoethanol, and 3-bromopropionic acid to replace 5-bromovaleric acid to obtain target compound 25. ESI(M+H) + = 995.

[0271] Example 31. Synthesis of target compound 26

[0272]

[0273] Refer to the synthetic method in Step 1 of Example 29 to obtain tert-butyl 4-(2-(phenylsulfonyl)ethyl)piperazine-1-carboxylate; obtain intermediate 26-1 according to Step 3 of Example 29; obtain target compound 26 according to Step 4 of Example 29. ESI(M+H) + = 1065.

[0274] Example 32. Synthesis of target compound 27

[0275]

[0276] Step 1: Under nitrogen protection, dissolve intermediate 1d (150 mg, 0.292 mmol) in 1 mL of anhydrous DMF. After adding K2CO3 (121 mg, 0.877 mmol) and activating for 10 min, dropwise add a DMF solution (1 mL) of starting material 19A-1 (193 mg, 0.585 mmol). Heat the mixture to 60 °C and stir the reaction overnight. After monitoring the completion of the reaction by TLC, add 10 mL of water to the reaction system, extract with ethyl acetate three times, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Purify the obtained crude product by silica gel column chromatography to obtain 137 mg of the intermediate with a yield of 70%. ESI(M+H) + = 672. Dissolve the obtained intermediate (137 mg, 0.204 mmol) in 0.15 mL of dichloromethane, slowly add trifluoroacetic acid (152 μL, 2.04 mmol), and react at room temperature for 4 h. After monitoring the complete reaction of the starting material by TLC, evaporate to dryness to obtain intermediate 19A-2. ESI(M+H) + = 616.

[0277] Step 2: Dissolve intermediate 19A-2 (125 mg, 0.203 mmol), EDCI (38 mg, 0.244 mmol), and HOBt (44 mg, 0.285 mmol) in 1 mL of anhydrous DMF. Under ice bath conditions, dropwise add DIPEA (353 μL, 2.033 mmol), stir for 10 min, and then slowly add a DMF solution (0.5 mL) of intermediate 10B (90 mg, 0.203 mmol). Stir at room temperature overnight. After the reaction is completed, pour the reaction mixture into 10 mL of water, extract the reaction solution with ethyl acetate three times, combine the organic phases, dry over anhydrous sodium sulfate, and evaporate to dryness. Purify the obtained crude product by silica gel column chromatography to obtain 36 mg of the target compound 27 with a yield of 17%. 1H-NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.81 (s, 1H), 8.47–8.40 (m, 2H), 8.19 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 7.9 Hz, 2H), 7.93 (d, J = 7.1 Hz, 2H), 7.74–7.65 (m, 2H), 7.44–7.40 (m, 3H), 7.39–7.33 (m, 3H), 7.29 (t, J = 7.3 Hz, 1H), 5.15 (s, 1H), 4.96–4.84 (m, 1H), 4.54 (d, J = 9.6 Hz, 1H), 4.44 (t, J = 8.3 Hz, 1H), 4.28 (s, 1H), 4.18–4.08 (m, 1H), 3.97 (s, 2H), 3.67–3.53 (m, 6H), 2.45 (s, 3H), 2.42–2.30 (m, 2H), 2.25–1.97 (m, 4H), 1.83–1.73 (m, 1H), 1.68–1.59 (m, 1H), 1.51–1.43 (m, 2H), 1.39–1.33 (m, 4H), 0.93 (s, 9H). ESI (M+H) + = 1042.

[0278] Example 33. Synthesis of Target Compound 28

[0279]

[0280] Referring to the synthesis method of the target compound in Reference Example 32, 20A-1 was used instead of 19A-1 to obtain the target compound 28. 1H-NMR(400MHz, DMSO-d6) δ 8.99 (s, 1H), 8.90 (s, 1H), 8.50–8.44 (m, 2H), 8.19 (d, J = 7.8 Hz, 1H), 8.15–8.09 (m, 2H), 7.94 (d, J = 7.0 Hz, 2H), 7.69 (t, J = 7.8 Hz, 1H), 7.42–7.30 (m, 8H), 4.91–4.86 (m, 1H), 4.54 (d, J = 9.6 Hz, 1H), 4.43 (t, J = 8.3 Hz, 1H), 4.28 (s, 1H), 3.95 (s, 2H), 3.78–3.72 (m, 2H), 3.63–3.53 (m, 9H), 3.26–3.10 (m, 2H), 2.98–2.74 (m, 2H), 2.44 (s, 3H), 2.37–2.29 (m, 2H), 2.10–2.03 (m, 1H), 1.79–1.72 (m, 1H), 1.48–1.38 (m, 1H), 1.35 (d, J = 7.0 Hz, 3H), 1.30–1.21 (m, 3H), 0.91 (s, 9H). ESI(M+H) + = 1086.

[0281] Example 34. Synthesis of Target Compound 29

[0282]

[0283] Referring to the synthesis method of the target compound in Reference Example 32, using 21A-1 instead of 19A-1, the target compound 29 was obtained. 1 H-NMR(400MHz, DMSO-d6) δ 8.96 (s, 2H), 8.48 (d, J = 7.8 Hz, 2H), 8.27–8.06 (m, 3H), 7.95 (d, J = 7.5 Hz, 2H), 7.70 (t, J = 7.7 Hz, 1H), 7.50–7.28 (m, 8H), 4.96–4.83 (m, 1H), 4.54 (d, J = 9.4 Hz, 1H), 4.44 (t, J = 8.1 Hz, 1H), 4.30 (s, 1H), 3.96 (s, 2H), 3.81–3.71 (m, 2H), 3.65–3.49 (m, 13H), 3.27–3.14 (m, 2H), 3.09–2.80 (m, 2H), 2.45 (s, 3H), 2.40–2.28 (m, 2H), 2.12–2.01 (m, 1H), 1.82–1.71 (m, 1H), 1.50–1.24 (m, 7H), 0.93 (s, 9H). ESI(M+H) + = 1130.

[0284] Example 35. Synthesis of Target Compound 30

[0285]

[0286] Referring to the synthesis method of the target compound in Reference Example 32, replace 19A-1 with 22A-1 to obtain the target compound 30. 1 H-NMR(400MHz,DMSO-d6)δ8.97(s,1H),8.77(t,J=6.5Hz,1H),8.53–8.41(m,2H),8.30(s,1H),8.18(d,J=7.7Hz,1H),8.06(d,J=10.1Hz,2H),7.92(d,J=7.6Hz,2H),7.67(t,J=7.8Hz,1H),7.45–7.34(m,6H),7.29(t,J=7.3Hz,1H),4.90(t,J=7.2Hz,1H),4.54(d,J=9.6Hz,1H),4.45(t,J=8.2Hz,1H),4.28(s,1H),3.95(s,2H),3.67–3.43(m,18H),2.84(d,J=11.2Hz,2H),2.45(s,5H),2.35–2.15(m,4H),2.11–1.94(m,3H),1.83–1.72(m,1H),1.52–1.20(m,4H),0.93(s,9H). ESI(M+H) + =1174。

[0287] Example 36. Synthesis of Target Compound 31

[0288]

[0289] Referring to the synthesis method of the target compound in Reference Example 32, replace 19A-1 with 23A-1 to obtain the target compound 31. 1H-NMR(400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.85 (s, 1H), 8.50–8.42 (m, 2H), 8.20 (d, J = 7.8 Hz, 1H), 8.14–8.06 (m, 2H), 7.94 (d, J = 6.9 Hz, 2H), 7.69 (t, J = 7.8 Hz, 1H), 7.44–7.28 (m, 8H), 4.94–4.86 (m, 1H), 4.54 (d, J = 9.5 Hz, 1H), 4.44 (t, J = 8.1 Hz, 1H), 4.28 (s, 1H), 4.00–3.94 (m, 4H), 3.61–3.48 (m, 21H), 2.45 (s, 3H), 2.43–2.32 (m, 2H), 2.24–2.09 (m, 2H), 2.08–2.03 (m, 1H), 1.81–1.73 (m, 1H), 1.59–1.45 (m, 4H), 1.37 (d, J = 7.1 Hz, 3H), 1.33–1.29 (m, 2H), 0.93 (s, 9H). ESI(M+H) + = 1218。

[0290] Example 37. Synthesis of Target Compound 32

[0291]

[0292] Step 1: Dissolve the raw material (S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanoic acid 11B-1 (150 mg, 0.487 mmol) and potassium carbonate (80.1 mg, 0.584 mmol) in 6.6 mL. Add a solution of di-tert-butyl dicarbonate (134 μL, 0.584 mmol) in tetrahydrofuran / water (1:1) (1.65 mL) at 0 °C, and raise the reaction system to room temperature and react for 7 h. Add 10 mL of ethyl acetate and 20 mL of water for extraction, and extract the aqueous phase with ethyl acetate twice more. Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 175 mg of a white solid, which is Intermediate 11B, with a yield of 88%. Without further purification, directly use it for the next step. ESI(M+H) + = 409。

[0293] Step 2: Dissolve raw material N-Boc-glycine 24A-1 (18.8 mg, 0.107 mmol), EDCI (20.6 mg, 0.107 mmol) and HOBt (17.9 mg, 0.117 mmol) in 0.5 mL of anhydrous DMF. Under ice bath condition, add DIPEA (136 μL, 0.78 mmol) dropwise. After stirring for 10 min, slowly add the DMF solution (0.5 mL) of intermediate 1d (50 mg, 0.098 mmol), and stir at room temperature overnight. After the reaction is completed, pour it into 10 mL of water, extract the reaction solution with ethyl acetate for 3 times, combine the organic phases, dry over anhydrous sodium sulfate, spin dry, and purify the obtained crude product by silica gel column chromatography to obtain 66.6 mg of white solid. Add 2 mol / L hydrogen chloride-ethyl acetate solution (0.5 mL, 1.0 mmol), react at room temperature for 2 h and then spin dry to obtain 47.9 mg of intermediate 24A-2, with a yield of 86% (two steps). ESI(M+H) + = 571.

[0294] Step 3: Refer to the experimental method of Step 2 in this example, use intermediate 11B (37.7 mg, 0.092 mmol) instead of 24A-1, and intermediate 24A-2 (47.9 mg, 0.084 mmol) instead of intermediate 1d to obtain the target compound 32, with a yield of 50% (two steps). 1 1H-NMR (400 MHz, DMSO-d6) δ 8.82 (t, J = 6.4 Hz, 1H), 8.43 (s, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.16–8.04 (m, 4H), 8.00 (d, J = 5.4 Hz, 2H), 7.93 (d, J = 7.2 Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.34–7.21 (m, 4H), 4.34 (q, J = 7.8 Hz, 1H), 4.13 (d, J = 13.3 Hz, 1H), 4.06–3.97 (m, 2H), 3.93 (t, J = 4.4 Hz, 1H), 3.84 - 3.76 (m, 4H), 3.58 (d, J = 6.6 Hz, 3H), 3.16 (t, J = 10.8 Hz, 1H), 3.04–2.81 (m, 3H), 2.46–2.20 (m, 2H), 2.05–1.77 (m, 2H), 1.69–1.59 (m, 1H), 1.56–1.47 (m, 2H), 0.87 (t, 6H). ESI(M+H) + = 861.

[0295] Example 38. Synthesis of target compound 33

[0296]

[0297] Referring to the synthesis method of the target compound in Example 37, using intermediate 1d as the raw material and replacing 24A-1 with raw material 25A-1, the target compound 33 was obtained. 1 H-NMR(400MHz,DMSO-d6)δ8.80(t,J=6.1Hz,1H),8.43(s,1H),8.18(d,J=7.6Hz,1H),8.14–8.08(m,2H),8.06(d,J=7.7Hz,1H),7.98–7.91(m,3H),7.68(t,J=7.7Hz,1H),7.38(t,J=7.5Hz,2H),7.33–7.26(m,6H),4.26–4.11(m,2H),3.95(d,J=3.1Hz,1H),3.78(d,J=13.3Hz,1H),3.60–3.56(m,3H),3.54–3.45(m,2H),3.14–3.00(m,4H),2.93–2.78(m,3H),2.29(q,J=15.3,13.1Hz,4H),1.93–1.80(m,2H),1.63–1.54(m,3H),1.52–1.43(m,2H),0.85(t,J=9.7,6.3Hz,6H). ESI(M+H) + =889。

[0298] Example 39. Synthesis of target compound 34

[0299]

[0300] Referring to the synthesis method of the target compound in Example 37, using intermediate 1d as the raw material and replacing 24A-1 with raw material 26A-1, the target compound 34 was obtained. 1H-NMR(400 MHz, DMSO-d6) δ 8.81 (t, J = 6.4 Hz, 1H), 8.43 (s, 1H), 8.18 (d, J = 7.7 Hz, 1H), 8.10–8.04 (m, 3H), 8.02–7.97 (m, 3H), 7.93 (d, J = 7.5 Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 7.5 Hz, 2H), 7.34–7.27 (m, 4H), 4.27–4.14 (m, 2H), 3.99 (d, J = 3.5 Hz, 1H), 3.81 (d, J = 10.8 Hz, 1H), 3.14 (t, J = 10.6 Hz, 1H), 3.02–2.81 (m, 6H), 2.36–2.23 (m, 5H), 1.93–1.80 (m, 2H), 1.55–1.16 (m, 13H), 0.86 (t, 6H). ESI(M+H) + = 917.

[0301] Example 40. Synthesis of Target Compound 35

[0302]

[0303] Referring to the synthetic method of the target compound in Reference Example 37, using intermediate 1d as the raw material and replacing 24A-1 with raw material 27A-1, target compound 35 was obtained. 1 H-NMR(400 MHz, DMSO-d6) δ 8.81 (t, J = 6.2 Hz, 1H), 8.43 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.09–7.98 (m, 6H), 7.93 (d, J = 7.1 Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 7.5 Hz, 2H), 7.34–7.28 (m, 4H), 4.26–4.15 (m, 2H), 4.00 (d, J = 3.3 Hz, 1H), 3.83 (d, J = 13.4 Hz, 1H), 3.59–3.48 (m, 11H), 3.19–2.79 (m, 8H), 2.37–2.23 (m, 4H), 1.91–1.77 (m, 2H), 1.61–1.41 (m, 6H), 0.86 (t, J = 9.5, 6.4 Hz, 6H). ESI(M+H) + = 945.

[0304] Example 41. Synthesis of Target Compound 36

[0305]

[0306] Reference Example 37. Synthesis method of the target compound. Using intermediate 1d as the raw material and replacing 24A-1 with raw material 28A-1, the target compound 36 was obtained. ESI(M+H) + = 987.

[0307] Example 42. Synthesis of target compound 37

[0308]

[0309] Reference Example 37. Synthesis method of the target compound. Using intermediate 1d as the raw material and replacing 24A-1 with raw material 29A-1, the target compound 37 was obtained. ESI(M+H) + = 905.

[0310] Example 43. Synthesis of target compound 38

[0311]

[0312] Reference Example 37. Synthesis method of the target compound. Using intermediate 1d as the raw material and replacing 24A-1 with raw material 30A-1, the target compound 38 was obtained. ESI(M+H) + = 949.

[0313] Example 44. Synthesis of target compound 39

[0314]

[0315] Reference Example 37. Synthesis method of the target compound. Using intermediate 1d as the raw material and replacing 24A-1 with raw material 31A-1, the target compound 39 was obtained. ESI(M+H) + = 993.

[0316] Example 45. Synthesis of target compound 40

[0317]

[0318] Reference Example 37. Synthesis method of the target compound. Using intermediate 1d as the raw material and replacing 24A-1 with raw material 32A-1, the target compound 40 was obtained. ESI(M+H) + = 1037.

[0319] Example 46. Synthesis of target compound 41

[0320]

[0321] Refer to the synthesis method of Intermediate 19A-2 in Step 1 of Example 32. Using Intermediate 1d as the raw material and replacing 19A-1 with Raw Material 32A-1, Intermediate 32A-2 is obtained. Then, according to the synthesis method of the target compound in Step 3 of Example 37, the target compound 41 is obtained. 1 H-NMR(400MHz,DMSO-d6)δ8.42(s,1H),8.17–8.06(m,3H),7.96(d,J=7.7Hz,3H),7.90(d,J=7.6Hz,2H),7.66(t,J=7.7Hz,1H),7.38–7.21(m,7H),6.65(d,J=5.8Hz,1H),4.24–4.15(m,1H),3.99–3.91(m,1H),3.56–3.36(m,9H),3.11–2.62(m,7H),2.39–2.28(m,2H),1.72–1.07(m,12H),1.01(t,J=7.0Hz,1H),0.82(t,J=7.8Hz,6H). ESI(M+H) + =903。

[0322] Example 47. Synthesis of Target Compound 42

[0323]

[0324] Step 1: Dissolve L-tert-leucine methyl ester hydrochloride (4.45 g, 24.6 mmol) of Raw Material 12B-1 in 42 mL of dichloromethane, add Boc-N-methyl-L-alanine (5 g, 24.6 mmol) of Raw Material 12B-2, stir evenly, and successively add N-methylmorpholine (5.4 mL, 49.2 mmol), EDCI (5.67 g, 29.5 mmol), and HOBt (4.52 g, 29.5 mmol) under ice bath. Warm up to room temperature and stir the reaction overnight. Pour the reaction solution into 50 mL of saturated sodium bicarbonate solution for extraction. Wash the organic phase with 1N hydrochloric acid solution, then wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. The obtained crude product is purified by silica gel column chromatography to obtain 3.28 g of a pale yellow liquid with a yield of 40%. Dissolve the obtained intermediate (3.28 g, 9.9 mmol) in 24 mL of tetrahydrofuran and 3 mL of methanol, add an aqueous solution (3 mL) of lithium hydroxide (548 mg, 22.8 mmol), stir the reaction overnight at room temperature, adjust the pH to about 3 with 1N hydrochloric acid solution, extract with ethyl acetate 3 times, combine the organic phases, and spin dry to obtain 2.9 g of Intermediate 12B-3 with a yield of 92%. ESI(M+H) + =317。

[0325] Step 2: Referring to the synthesis method of Intermediate 1A-2 in Step 1 of Example 4, using raw material 12B-4N-Boc-trans-4-hydroxy-L-proline methyl ester (8 g, 32.6 mmol) to replace Intermediate 1A-1, 9.6 g of yellow liquid was obtained with a yield of 74%. According to the synthesis method of Intermediate 12B-3 in Step 1 of this example, the above-obtained intermediate (9.6 g, 24 mmol) was hydrolyzed under alkaline conditions to obtain Intermediate 12B-5 (8.92 g, yield 97%). ESI(M+H) + = 386.

[0326] Step 3: Dissolve Intermediate 12B-5 (8.92 g, 23.17 mmol) and raw material 2,6-difluoroaniline (2.62 mL, 24.33 mmol) in 100 mL of dichloromethane, add N,N'-dicyclohexylcarbodiimide (5 g, 24.33 mmol), and react at room temperature overnight. Filter, spin-dry the filtrate, and purify it by silica gel column chromatography to obtain 9.84 g of white solid with a yield of 86%. Referring to the method in Step 2 of Example 3, deprotect the amino group of this white solid to obtain 7.54 g of Intermediate 12B-6, ESI(M+H) + = 397.

[0327] Step 4: Referring to the specific experimental method in Step 1 of Example 3, using Intermediate 12B-3 (2.9 g, 9.18 mmol) to replace Intermediate 1a, and Intermediate 12B-6 (4.36 g, 11.01 mmol) to replace Intermediate 1b, 2.9 g of Intermediate 12B-7 was obtained with a yield of 46%. ESI(M+H) + = 695.

[0328] Step 5: Dissolve Intermediate 12B-7 (2.9 g, 4.18 mmol) in 30 mL of anhydrous DMF, add sodium azide (353 mg, 5.43 mmol), raise the temperature to 80 °C and react overnight. Cool to room temperature, pour into 100 mL of ice water, extract 3 times with methyl tert-butyl ether, combine the organic phases, concentrate under reduced pressure to obtain 2.17 g of Intermediate 12B-8 with a yield of 92%. Without further purification, directly use it in the next step. ESI(M+H) + = 566.

[0329] Step 6: Under nitrogen protection, dissolve the obtained Intermediate 12B-8 (2.17 g, 3.84 mmol) in 25 mL of methanol, add 10% palladium on carbon (217 mg), then replace the hydrogen, and stir evenly at room temperature. After monitoring the reaction completion by TLC, filter, concentrate the filtrate under reduced pressure, and purify it by silica gel column chromatography to obtain 1.5 g of white solid (Intermediate 12B) with a yield of 72%. ESI(M+H) + = 540.

[0330] Step 7: Referring to the synthetic method of the target compound in Example 32, replace 19A-1 with 33A-1 to obtain the target compound 42. 1 H-NMR(400MHz,DMSO-d6)δ9.93(s,1H),8.74(t,J=6.4Hz,1H),8.43(s,1H),8.18(d,J=6.4Hz,1H),8.04(d,J=6.7Hz,3H),7.92(d,J=7.1Hz,2H),7.85(d,J=9.3Hz,1H),7.67(t,J=7.8Hz,1H),7.39–7.26(m,4H),7.14(t,J=8.1Hz,2H),4.56–4.50(m,1H),4.44(d,J=9.3Hz,1H),4.36–4.25(m,1H),4.15–4.05(m,1H),3.53(d,J=6.4Hz,2H),3.23(t,J=9.3Hz,1H),3.01–2.91(m,1H),2.78–2.66(m,2H),2.33–2.26(m,2H),2.19–2.12(m,5H),2.07–1.99(m,4H),1.98–1.88(m,3H),1.82–1.73(m,1H),1.51–1.43(m,2H),1.39–1.33(m,2H),1.23–1.21(m,2H),1.11(d,J=6.9Hz,3H),0.91(s,9H). ESI(M+H) + =1049。

[0331] Example 48. Synthesis of the target compound 43

[0332]

[0333] Referring to the synthetic method of the target compound in Example 32, replace 19A-1 with 34A-1 to obtain the target compound 43. 1H-NMR(400 MHz, DMSO-d6) δ 9.94 (s, 1H), 8.73 (t, J = 6.4 Hz, 1H), 8.43 (s, 1H), 8.17 (d, J = 7.7 Hz, 1H), 8.04 (d, J = 11.2 Hz, 3H), 7.91 (d, J = 7.7 Hz, 2H), 7.86 (d, J = 9.4 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.38–7.26 (m, 4H), 7.13 (t, J = 8.1 Hz, 2H), 4.54 (t, J = 8.6 Hz, 1H), 4.44 (d, J = 9.3 Hz, 1H), 4.36–4.27 (m, 1H), 4.15–4.06 (m, 1H), 3.53 (d, J = 6.0 Hz, 2H), 3.24 (t, J = 9.3 Hz, 1H), 3.00–2.93 (m, 1H), 2.75–2.69 (m, 2H), 2.29 (d, J = 12.5 Hz, 2H), 2.18–2.12 (m, 5H), 2.07–1.91 (m, 7H), 1.83–1.74 (m, 1H), 1.49–1.43 (m, 2H), 1.38–1.33 (m, 2H), 1.23–1.19 (m, 6H), 1.11 (d, J = 6.8 Hz, 3H), 0.92 (s, 9H). ESI(M+H) + = 1077。

[0334] Example 49. Synthesis of Target Compound 44

[0335]

[0336] Referring to the synthesis method of Intermediate 1d in Reference Example 3, using Intermediate 20A-2 instead of Intermediate 1a and Intermediate 12B instead of Intermediate 1b, the target compound 44 was obtained. 1H-NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.72 (s, 1H), 9.05–8.99 (m, 1H), 8.66 (d, J = 7.6 Hz, 1H), 8.45 (s, 1H), 8.23–8.14 (m, 4H), 7.94 (d, J = 7.1 Hz, 2H), 7.70 (t, J = 7.9 Hz, 1H), 7.39–7.30 (m, 4H), 7.13 (t, J = 8.1 Hz, 2H), 4.61–4.58 (m, 1H), 4.43–4.41 (m, 1H), 4.15–4.10 (m, 1H), 4.09–4.05 (m, 1H), 4.00–3.92 (m, 2H), 3.87–3.84 (m, 2H), 3.75 (t, J = 4.7 Hz, 1H), 3.62–3.53 (m, 4H), 3.48–3.36 (m, 3H), 3.18–3.14 (m, 2H), 2.95–2.86 (m, 2H), 2.46–2.39 (m, 5H), 1.36 (d, J = 6.8 Hz, 3H), 1.34–1.19 (m, 6H), 0.97 (s, 9H). ESI (M+H) + = 1081。

[0337] Example 50. Synthesis of Target Compound 45

[0338]

[0339] Referring to the synthesis method of Intermediate 1d in Reference Example 3, using Intermediate 21A-2 instead of Intermediate 1a and Intermediate 12B instead of Intermediate 1b, the target compound 45 was obtained. 1H-NMR(400 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.70 (s, 1H), 9.02 (t, J = 6.3 Hz, 1H), 8.66 (d, J = 7.8 Hz, 1H), 8.44 (s, 1H), 8.21–8.14 (m, 4H), 7.93 (d, J = 8.5 Hz, 2H), 7.69 (t, J = 7.8 Hz, 1H), 7.40–7.29 (m, 4H), 7.13 (t, J = 8.2 Hz, 2H), 4.64–4.61 (m, 2H), 4.45–4.38 (m, 2H), 4.08–3.96 (m, 2H), 3.89–3.86 (m, 2H), 3.76 (t, J = 5.0 Hz, 1H), 3.57–3.45 (m, 11H), 3.22–3.12 (m, 2H), 2.95–2.85 (m, 2H), 2.46–2.38 (m, 5H), 1.35 (d, J = 6.8 Hz, 3H), 1.32–1.18 (m, 6H), 0.97 (s, 9H). ESI(M+H) + = 1125.

[0340] Example 51. Synthesis of Target Compound 46

[0341]

[0342] Compound 30 (10 mg, 8.5 μmol) was dissolved in 2 mol / L hydrogen chloride-ethyl acetate solution (4.3 μL, 8.5 μmol), and the reaction was stirred overnight at room temperature. A white solid precipitated out. After centrifugation, 9.8 mg of white solid was obtained, which was the target compound 46 with a yield of 95%, ESI(M+H) + = 1174.

[0343] Example 52. Synthesis of Target Compound 47

[0344]

[0345] Compound 30 (10 mg, 8.5 μmol) and trifluoroacetic acid (1 mg, 8.5 μmol) were dissolved in 650 μL of absolute ethanol, and the reaction was stirred overnight at room temperature. A white solid precipitated out. After centrifugation, 10.3 mg of white solid was obtained, which was the target compound 47 with a yield of 94%, ESI(M+H) + = 1174.

[0346] Example 53. Synthesis of Target Compound 48

[0347]

[0348] Referring to the experimental procedure of Example 52, trifluoroacetic acid was replaced with methanesulfonic acid to obtain the target compound 48, ESI(M+H) + = 1174.

[0349] Example 54. Synthesis of Target Compound 49

[0350]

[0351] Referring to the experimental procedure of Example 52, trifluoroacetic acid was replaced with (±)-malic acid to obtain the target compound 49, ESI(M+H) + = 1174.

[0352] Example 55. Synthesis of Target Compound 50

[0353]

[0354] Referring to the experimental procedure of Example 52, trifluoroacetic acid was replaced with citric acid to obtain the target compound 50, ESI(M+H) + = 1174.

[0355] Example 56. Synthesis of Target Compound 51

[0356]

[0357] Referring to the experimental procedure of Example 52, trifluoroacetic acid was replaced with p-toluenesulfonic acid to obtain the target compound 51, ESI(M+H) + = 1174.

[0358] Example 57. Synthesis of Target Compound 52

[0359]

[0360] Referring to the experimental procedure of Example 52, trifluoroacetic acid was replaced with L-tartaric acid to obtain the target compound 52, ESI(M+H) + = 1174.

[0361] Example 58. Synthesis of Target Compound 53

[0362]

[0363] Referring to the experimental procedure of Example 52, trifluoroacetic acid was replaced with D-tartaric acid to obtain the target compound 53, ESI(M+H) + = 1174.

[0364] Example 59. Biological Activity Evaluation

[0365] I. HDAC7 Enzyme Activity Detection

[0366] Instrument: ELISA reader TECAN SPARK (TECAN, Switzerland)

[0367] Materials: HDAC7 protein, purified from SF9 cells; substrate Ac-Leu-Lys(TFAc)-AMC

[0368] Sample preparation: Compounds were dissolved in DMSO, stored at low temperature, serially diluted, and the concentration of DMSO in the final system was controlled within the range that does not affect enzyme activity detection. The positive compound used in the experiment was TMP269.

[0369] Determination method: 100 nM of HDAC7 protein and 50 μM of substrate were dissolved in kinase reaction buffer (500 mM NaCl, 50 mM Tris, pH 8.0). 0.3 μM and 3 μM of the compounds were added to the reaction system (100 μL) respectively, and the drug-free group, positive compound (TMP269, 0.3 μM) and blank control group were set up. Each sample was set with 2 replicates at each concentration. After complete dissolution, the system was transferred to a 96-well plate, and then 100 μL of 10 mg / ml trypsin was added and incubated at 37 °C. The fluorescence value was detected by an ELISA reader (absorbance at 460 nm, excitation at 390 nm) to indicate the release of AMC. The inhibition rate of sample enzyme activity was calculated from the sample readings, and the calculation formula was: (RFU of drug-free - RFU of compound) / RFU of drug-free × 100%.

[0370] Table 1 Inhibition rate of compounds on HDAC7 enzyme activity

[0371]

[0372]

[0373] Experimental results: The compounds of the present invention have a moderate inhibitory effect on the enzyme activity of HDAC7.

[0374] II. Detection of the degradation effect of compounds on HDAC7 by Western blot

[0375] Using compound TMP269 as the negative control, the degradation of HDAC7 protein on HEK293 cells by compounds was determined by Western blot, and the two compounds with the best degradation effects were selected to verify their ability to degrade HDAC7 protein on RAW264.7 (mouse mononuclear macrophages) cells.

[0376] Experimental materials:

[0377] Cell lines: HEK293 cells, mouse mononuclear macrophages (RAW264.7)

[0378] Culture medium: DMEM

[0379] HEK293: DMEM + 10% Hyclone serum

[0380] RAW264.7: DMEM + 10% Gibco serum

[0381] Drug preparation method: Dissolve the compound in DMSO to make a 50 mM stock solution, and dilute it to the corresponding concentration according to a certain ratio.

[0382] 1. In vitro culture and drug administration of HEK293 cells

[0383] (1) In vitro culture of HEK293 cells:

[0384] Culture the selected HEK293 cells in a 37°C constant temperature incubator containing 5% CO2. The culture conditions are DMEM + 10% Hyclone serum. Passage the cells when the cell density reaches 70 - 90% for subsequent experiments.

[0385] (2) Seeding cells on plates: Digest the cells and add the cell suspension to a 1.5 mL Eppendorf tube. Centrifuge at 1,000 rpm for 5 min, resuspend the cells with 2 mL of culture medium and count. Seed the cell suspension in a 24-well plate, with 200,000 cells per well, and place it for 8 - 12 h until all the cells adhere to the plate.

[0386] (3) Drug administration to cells: Dilute the stock solution of the compound with DMEM + 10% Hyclone serum to 50 mM, and make the final concentrations 1 μM and 5 μM respectively. Incubate in a 37°C cell culture incubator containing 5% CO2 for 24 h, using the DMSO group as the blank control.

[0387] 2. In vitro culture and drug administration of RAW264.7 cells

[0388] (1) In vitro culture of RAW264.7 cells:

[0389] Culture the selected RAW264.7 cells in a 37°C constant temperature incubator containing 5% CO2. The culture conditions are DMEM + 10% Gibco serum. Passage the cells (RAW264.7 cells need to be gently scraped off with a scraper and cannot be digested with trypsin) when the cell density reaches 70 - 90% for subsequent experiments.

[0390] (2) Seeding cells: Digest the cells, add the cell suspension into a 1.5 mL Eppendorf tube, centrifuge at 1,000 rpm for 5 min, resuspend the cells with 2 mL of culture medium and count them. Seed the cell suspension into a 24-well plate, with 200,000 cells in each well, and place it for 8 - 12 h until all the cells adhere to the wall.

[0391] (3) Treating cells with drugs: Dilute the stock solution of the compound with a concentration of 50 mM with DMEM + 10% Hyclone serum to make the final concentrations 1 μM and 5 μM respectively, and incubate them in a cell culture incubator at 37°C with 5% CO2 for 24 h, using the DMSO group as the blank control.

[0392] 3. Cell lysis

[0393] At the end of the action time, collect the cells and wash them once with PBS; add the corresponding volume of 4% SDS according to the cell amount to lyse the cells, and sonicate until the cells are no longer viscous; centrifuge at 12,000 rpm at room temperature for 30 min; take the supernatant and transfer it to a new EP tube for protein quantification.

[0394] 4. Protein quantification

[0395] Dilute the 2 mg / mL BSA standard product by half to obtain the concentrations used for the standard curve, which are 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.0625 mg / mL in turn; calculate according to 200 μL of solution A and 4 μL of solution B in the BCA quantification kit for each sample, and take the corresponding volumes of solution A and solution B (volume ratio 50:1) and mix them evenly; take 10 μL of BSA with different concentrations and samples respectively and add them into a 96-well plate, then add 200 μL of the evenly mixed solution A and solution B, gently tap to mix evenly and place it in the dark at 37°C for reaction for 30 min; after the reaction is completed, measure the absorbance value at 562 nm, and calculate the protein concentration of the sample using the standard curve.

[0396] 5. Western Blot

[0397] (1) Preparation of protein samples

[0398] Take 20 μg of protein and add a certain amount of 6× Loading Buffer according to the volume to make the final concentration 1× Loading Buffer; heat and denature at 95°C for 10 min, wait for it to cool and then centrifuge and mix evenly for the Western Blot experiment, and store the remaining samples at -80°C.

[0399] (2) Gel preparation

[0400] a. Separating gel: Select the concentration of the separating gel to be prepared according to the molecular weight of the target protein. First, assemble the gel cassette: clamp the thick and thin glass plates tightly and keep the bottom surface flat. Add the separating gel reagents to the centrifuge tube in sequence and vortex to mix well. After mixing, add the separating gel between the two glass plates, and then add an appropriate amount of isopropanol on the upper layer of the gel surface and wait for it to solidify.

[0401] b. Stacking gel: Discard the isopropanol on the upper layer of the separating gel and wash the remaining residue with triple-distilled water. Prepare the stacking gel and vortex to mix well. After mixing, add the stacking gel between the two glass plates, insert the gel comb, and wait for it to solidify.

[0402] (3) Electrophoresis

[0403] a. Preparation: Install the prepared gel cassette into the electrophoresis tank and place it in the electrophoresis apparatus. Add the diluted 1×Tricine buffer to the middle electrophoresis tank and add 1×Running buffer to the outer tank, and let it stand for several minutes.

[0404] b. Loading: Vortex the sample to mix well, pipette a certain amount of the sample (5 - 10 μL) for loading. After loading, turn on the electrophoresis apparatus, first set it to the constant voltage mode, electrophorese at 70 V for 10 min and confirm that the current is normal. After the sample enters the separating gel, adjust the voltage to 130 V, and stop electrophoresis when the sample runs to the appropriate position.

[0405] (4) Transfer and blocking

[0406] a. Transfer: Add a sponge, thick filter paper, and thin filter paper to the transfer cassette in sequence. Take the gel: Use a gel spatula to remove the stacking gel block at the edge and carefully remove the separating gel, place it in the center of the filter paper. Apply the membrane: Stick the PVDF membrane pre-activated with methanol from one side to the gel and expel the air bubbles. Then add thin filter paper, thick filter paper, and sponge in sequence and clamp the transfer cassette, and assemble the transfer cassette in sequence. Add an appropriate amount of transfer buffer to the transfer tank, add an appropriate amount of water to the outer layer of the transfer tank and place an ice pack to keep the transfer tank in ice water to prevent a large amount of heat from being released during the transfer process and affecting the transfer effect.

[0407] b. Blocking: After the transfer is completed, open the transfer cassette, remove the gel and PVDF membrane (if the transfer is successful, it can be seen that the protein Marker has been completely transferred to the PVDF membrane). Then cut the PVDF membrane according to the molecular weight of the target protein and place the membrane in 5% skim milk and block at room temperature for 60 min.

[0408] (5) Incubate with antibodies

[0409] a. Incubate with primary antibody: After blocking, discard the milk and wash the PVDF membrane 3 times with TBST (15 min, 5 min, 5 min), then add an appropriate amount of primary antibody (usually diluted at a ratio of primary antibody: antibody diluent = 1:1000), and incubate overnight on a shaker at 4°C.

[0410] b. Incubate the secondary antibody: Recover the primary antibody and wash it 3 times with TBST (15 min, 5 min, 5 min), then add the secondary antibody (usually prepared by mixing secondary antibody: 5% skim milk = 1:5000), and incubate at room temperature on a horizontal shaker for 60 min.

[0411] (6) Exposure

[0412] a. Wash the membrane: Discard the secondary antibody and wash it 3 times with TBST (15 min, 5 min, 5 min).

[0413] b. Exposure: Prepare the ECL luminescent solution: Solution A: Solution B = 1:1. First, blot the residual TBST on the strip with filter paper, then use forceps to pick up the strip onto the exposure board, add the ECL chromogenic solution dropwise to cover the strip, and let it stand for about 1 min. Put the exposure board into the AI800 exposure machine and select an appropriate time for exposure.

[0414] The calculation method of the degradation rate is as follows: Quantitatively analyze the exposure results through Image J software, that is, first calculate the gray value of the exposure strip through Image J software, then divide the gray value of the target protein by the gray value of the internal reference protein for normalization processing. The calculation formula is R 样本 = A 目的蛋白 / A 内参蛋白 × 100% (A is the gray value, R is the degradation rate).

[0415] Table 2 Results of the degradation of HDAC7 by compounds in HEK293 cells

[0416]

[0417] Experimental results: Table 2 and the attached Figures 1 - 5 The results shown indicate that the compounds of the present invention have a certain degradation effect on HDAC7 in HEK293 cells, and compounds 5 and 30 have the best effect on degrading HDAC7.

[0418] Table 3 Results of the degradation of HDAC7 by compounds in RAW264.7 cells

[0419]

[0420]

[0421] Experimental results: Table 3 and the attached Figure 6 The results show that compounds 5 and 30, which have the optimal degradation activity on HDAC7 in HEK293 cells, also show a significant effect on degrading HDAC7 protein in RAW264.7 cells.

[0422] III. Detection of cytokines secreted by macrophages by enzyme-linked immunosorbent assay (ELISA)

[0423] An enzyme-labeled instrument TECAN SPARK (TECAN, Switzerland) was used to detect the effects of compounds on the cytokines IL-6 and TNF-α secreted by macrophages.

[0424] The enzyme-linked immunosorbent assay (ELISA) adopts the principle of solid-phase reaction of antigen or antibody: the antigen or antibody bound to the surface of the solid-phase carrier has immunological activity, and the enzyme-labeled antigen or antibody retains both its immunological activity and enzyme activity. During the determination, the test sample reacts with the antigen or antibody on the surface of the solid-phase carrier. The antigen-antibody complex formed on the solid-phase carrier is separated from other substances in the liquid by washing, and then the enzyme-labeled antigen or antibody is added, and it binds to the solid-phase carrier through reaction (the amount of enzyme on the solid phase is in a certain proportion to the amount of the substance to be detected in the specimen). Finally, after adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to form a colored product, and the amount of the product is directly related to the amount of the substance to be detected in the specimen. Therefore, qualitative or quantitative analysis can be carried out according to the depth of the color.

[0425] Experimental materials:

[0426] Cell line: mouse mononuclear macrophage (RAW264.7)

[0427] Culture medium: DMEM

[0428] RAW264.7: DMEM + 10% Gibco serum

[0429] Drug preparation method: The compound was dissolved in DMSO to prepare a 50 mM stock solution and diluted to the corresponding concentration according to a certain ratio.

[0430] 1. Administration of drugs to RAW264.7 cells and sample collection

[0431] The stock solution of the compound at 50 mM was diluted with DMEM + 10% Hyclone serum to a final concentration of 5 μM, and incubated in a cell culture incubator at 37°C with 5% CO2 for 12 h, with the DMSO group as the blank control. Then 10 ng / mL LPS was added to each well and continued to act for 24 h. At the end of the experiment, the cell supernatant was collected as the experimental sample.

[0432] 2. Detection of cytokines secreted by macrophages by enzyme-linked immunosorbent assay (ELISA)

[0433] (1) Dilute 10× counting buffer to 1× according to the required incubation volume (the diluent is deionized water), and prepare 1× Cap Ab (the diluent is 1× counting buffer). Then add 100 μL / well of 1× Cap Ab and incubate overnight at 4°C.

[0434] (2) Discard the primary antibody and wash three times with 100 μL / well of PBST for 2 min each time, then centrifuge to dry.

[0435] Add 1× blocking solution, 200 μL / well (the diluent is deionized water), and incubate at room temperature for 1 h.

[0436] (3) Discard the blocking solution and wash three times with 100 μL / well of PBST for 2 min each time, then centrifuge to dry.

[0437] (4) Prepare the standard curve, let it stand in the 4°C refrigerator for 20 min, add the corresponding sample 100 μL / well (the sample diluent is 1× blocking solution), set 2 parallel wells for each sample, and place at room temperature for 2 h (or incubate overnight at 4°C).

[0438] (5) Discard the sample and wash three times with 100 μL / well of PBST for 2 min each time, then centrifuge to dry.

[0439] (6) Add 100 μL / well of 1× Det Ab (the sample diluent is 1× blocking solution) and incubate at room temperature for 1 h.

[0440] (7) Discard the secondary antibody and wash four times with 100 μL / well of PBST for 2 min each time, then centrifuge to dry.

[0441] (8) Add 100 μL / well of Streptavidin-HRP (the diluent is 1× blocking solution) and incubate at room temperature for 0.5 h.

[0442] (9) Discard the HRP and wash 20 times with 100 μL / well of double-distilled water, then centrifuge to dry.

[0443] (10) Add 80 μL / well of TMB (mouse) and incubate for 15 min.

[0444] (11) Add 80 μL / well of 1 M H2SO4 to terminate the reaction.

[0445] (12) Wipe the bottom of the plate clean and measure the absorbance with an enzyme-linked immunosorbent assay reader (450 nm; Reference: 570 nm)

[0446] Calculate the concentration of the corresponding cytokine based on the absorbance value, and then compare the changes in the corresponding cytokine with those of the control group.

[0447] Result calculation: Draw a standard curve based on the OD values and concentrations of the standard products, and obtain the standard curve formula (a linear function of OD values and concentrations). Calculate the sample concentration (C administration group) according to the standard curve formula, and calculate the secretion expression level of the administration group relative to the blank group: C administration group / C blank group.

[0448] Table 4 Effects of Compounds on the Secretion Levels of TNF-α and IL-6 in Macrophages

[0449] Compound number Relative TNF-α secretion level Relative IL-6 secretion level Blank drug administration group 1.00 1.00 5 0.56 0.42 30 0.50 0.34 TMP269 0.88 1.00

[0450] Experimental results: The results in Table 4 show that the compounds of the present invention can significantly reduce the secretion of TNF-α by macrophages (see Appendix Figure 7 ) and the level of IL-6 (see Appendix Figure 8 ), and have potential therapeutic effects on related inflammatory diseases. The HDAC7 inhibitor TMP269 has little effect on the levels of TNF-α and IL-6 (see Appendix Figure 7 and 8 ).

[0451] IV. Animal Experiments on the Anti-inflammatory Effects of Compounds Targeting the Degradation of HDAC7

[0452] Experimental subjects:

[0453] ICR mice, 6 - 8 weeks old, male

[0454] Experimental methods:

[0455] 1. Administer the compound 6 hours in advance by tail vein injection; administer dexamethasone by gavage;

[0456] 2. Induce inflammation with LPS at a dose of 10 mg / kg by intraperitoneal injection;

[0457] 3. After 6 hours, collect blood from the mouse's eye socket, let it stand at room temperature for 4 - 6 hours until the blood is stratified, centrifuge at 4000 rpm for 30 minutes, and take the supernatant (100 - 300 μL);

[0458] Drug administration groups:

[0459]

[0460]

[0461] Note: Group 1 is the blank group, Group 2 is the LPS-induced group, Group 3 is the dexamethasone positive control group; Group 4 is the group using Compound 30; Group 5 is the TMP269 control group.

[0462] Detection indicators:

[0463] Serum cytokines: Detect the contents of TNF-α and IL-6 in the serum.

[0464] (ELISA experiment - The operation steps are the same as above, and the result calculation method is the same as above)

[0465] Table 5 Effects of the compound on the secretion levels of TNF-α and IL-6 in mice

[0466] Compound number Relative TNF-α secretion level Relative IL-6 secretion level Blank group 0.05 0.03 LPS-induced group 1 1 Positive control group 0.06 0.08 TMP269 0.35 0.38 30 0.07 0.03

[0467] Experimental results: The compound of the present invention can significantly reduce the levels of TNF-α and IL-6 secreted by LPS induction in mice. Its effect is comparable to that of the clinical anti-inflammatory drug dexamethasone. Therefore, it has a good therapeutic effect on related inflammatory diseases. The HDAC7 inhibitor TMP269 has little effect on the levels of TNF-α and IL-6 (attached Figure 9 and attached Figure 10 ).

Claims

1. A compound, its optical isomers and pharmaceutically acceptable salts thereof, wherein, The structure of the said compound is shown in the following formula: Or z is 4 or 5; y is selected from integers from 0 to 10.

2. The compound, its optical isomers and pharmaceutically acceptable salts thereof according to claim 1, wherein, The compound is selected from:

3. The compound, its optical isomers and pharmaceutically acceptable salts thereof according to any one of claims 1 to 2, wherein, The pharmaceutically acceptable salts are the following salts of the said compound: hydrochloride, trifluoroacetate, mesylate, malate, citrate, tosylate, L-tartrate, D-tartrate.

4. The compound, its optical isomers and pharmaceutically acceptable salts thereof according to claim 1, wherein, The pharmaceutically acceptable salt is:

5. A pharmaceutical composition, wherein, The said pharmaceutical composition contains a therapeutically effective amount of the compound or its pharmaceutically acceptable salt as described in any one of claims 1-4, and one or more pharmaceutically acceptable carriers, diluents or excipients.

6. Use of the compound or its pharmaceutically acceptable salt according to any one of claims 1 - 4 or the pharmaceutical composition according to claim 5 in the preparation of a drug for preventing and / or treating HDAC7 - associated diseases.

7. The use according to claim 6, wherein, The said diseases are selected from metabolic diseases, inflammatory diseases, autoimmune diseases.

8. The use according to claim 7, wherein, The said inflammatory diseases include one or more of rheumatoid arthritis, multiple sclerosis, osteoporosis, osteoarthritis, inflammatory bowel disease.

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