A class of compounds targeting and degrading transcription factor KLF5 and their applications

Abstract

The present invention discloses a class of compounds that target the degradation of transcription factor KLF5 and their applications. The compounds that target the degradation of transcription factor KLF5 include compounds as shown in formula (I). The present invention also provides PROTACs compounds that target the degradation of transcription factor KLF5 as shown in formula (II). The compounds and degradation agents provided by the present invention have a significant inhibitory effect on the proliferation of various tumor cells, and representative compounds also show significant anti-tumor effects in tumor-bearing mice. Related results also show that the compounds provided by the present invention can directly target KLF5 protein, providing a new approach for the treatment of TNBC tumors. Compared with currently available KLF5 inhibitors, the present invention can directly target KLF5 and can induce KLF5 protein degradation and significantly inhibit the proliferation of various tumor cells such as triple-negative breast cancer.

Description

Field of the Invention

[0001] The present invention belongs to the technical field of anti-tumor drugs, and in particular relates to a class of compounds for targeting and degrading transcription factor KLF5 and applications thereof. Background of the Invention

[0002] Cancer is becoming a global problem. Breast cancer remains the most common cancer in women, accounting for 23% of all cancer cases. Although the mortality rate of breast cancer has been declining in many developed countries, the incidence of breast cancer is on the rise worldwide, despite continuous improvements in diagnostic technology and treatment methods. It is the leading cause of cancer death in women, causing serious social health problems and economic harm (Arch Gynecol Obstet. 2016; 293(2): 247-69.). Breast cancer is divided into luminal A, luminal B, HER2, and triple-negative (TNBC) types based on whether estrogen receptor (ERα) and progesterone receptor (PR) are expressed and whether human epidermal growth factor receptor 2 (HER2) is amplified. TNBC is highly sensitive to chemotherapy, more aggressive, has a high probability of metastasis, a high recurrence rate, and a shorter survival time after metastasis. It is the focus and difficulty of breast cancer treatment. (Breast Cancer Res Treat. 2018; 167(2): 459-468.) There is currently no treatment option targeting TNBC, so finding molecular targets for TNBC and developing new drugs remain the current research focus.

[0003] Transcription factors are key factors in transcriptional regulation and play an important role in physiological and pathological processes. Transcription factors have been widely studied in various diseases, especially cancer, and have become an important new class of drug targets. KLF5 is a member of the Krüppel-like transcription factor family and is widely expressed in various tissues, including the digestive tract, pancreas, placenta, testis, prostate, skeletal muscle, lung, bladder and uterus. As a zinc finger transcription factor, KLF5 can regulate the expression of multiple genes. Studies have shown that KLF5 is highly expressed in various tumors and is closely related to the occurrence and development of tumors (Cancer Sci. 2021; 112(6): 2097-2117.). Evidence shows that KLF5 promotes the stemness, proliferation, survival, adhesion and migration of breast cancer cells. In breast cancer, KLF5 promotes cancer cell proliferation and cell cycle by inducing the transcription of FGF-BP1, mPGES1, TNFAIP2 and Cyclin D14, inhibiting the transcription of p27 and p21. KLF5 maintains the stemness of cells by inducing the transcription of Slug7 and Nanog. Mammary gland-specific KLF5 gene knockout mice can significantly reduce the proliferation, survival and stemness of mammary epithelial cells and inhibit PyMT-induced tumorigenesis. KLF5 can also promote the proliferation of bladder cancer cells. KLF5 can bind to the Cyclin E1 gene enhancer to activate its transcription, thereby increasing the susceptibility to bladder cancer (Mol Cancer Res. 2016; 14: 1078-1086.). In addition, KLF5 is closely related to the occurrence and development of various cancers such as prostate cancer, hepatocellular carcinoma, intestinal cancer, and esophageal squamous cell carcinoma. In addition, studies have found that high expression of KLF5 is not only associated with early recurrence, but also with early death. KLF5 expression is a prognostic factor for disease-free survival and overall survival in breast cancer patients. The prognostic value of KLF5 may be related to its role in promoting cell proliferation. (Clin Cancer Res. 2006; 12(8): 2442-8.) In summary, KLF5 plays a wide range of oncogenic roles in breast cancer and other different cancers. Therefore, targeting KLF5 will bring good therapeutic benefits.

[0004] At present, the research on KLF5 inhibitors mainly focuses on the expression regulation of its downstream genes. Reported inhibitors include: mifepristone inhibits KLF5 expression in TNBC by inducing miR-153 and inhibits tumor stem cells and tumor growth (Theranostics.2016;6(4):533-44.). Metformin inhibits basal breast cancer stem cells by activating AMPK to promote PKA-GSK3β-mediated KLF5 ubiquitination and degradation (Cell Discov.2017;3:17010.); mithramycin A inhibits TNBC by inhibiting Sp1 to downregulate KLF5 (Sci Rep.2018;8(1):1138.). The RSK inhibitor JH685 inhibits the YB-1-KLF5 axis and significantly inhibits the growth of basal breast cancer cell transplants (Cell Death Differ.2022;29(6):1283-1295.). JQ1 derivative Compound 870 and PROTAC compounds inhibit the growth of basal breast cancer by targeting BRD4 to downregulate KLF5 expression (Int J Biol Sci. 2019; 15(8): 1733-1742.). CID5951923 and its two structural derivatives inhibit the proliferation of colorectal cancer epithelial cells by inhibiting EGR1 to downregulate KLF5 expression (Mol Cancer Ther. 2011; 10(11): 2043-51). ML-264 and SR18662 induce apoptosis of colorectal cancer epithelial cells and inhibit tumor growth in vivo by inhibiting EGR1 to downregulate KLF5 expression (Mol Cancer Ther. 2016; 15(1): 72-83; Mol Cancer Ther. 2019; 18(11): 1973-1984.). However, current inhibitors all indirectly inhibit KLF5, and there is still no effective way to directly target KLF5 to interfere with its function.

[0005] Proteolysis targeting chimeras (PROTACs) are a promising therapeutic strategy that utilizes endogenous proteasomes to degrade disease-associated proteins. PROTACs use a linker to connect the ligand of an E3 ubiquitin ligase to the ligand of a target protein (POI) (Science 2017; 355(6330): 1163-1167.) and promote the subsequent degradation of the POI by recruiting the E3 ubiquitin ligase near the POI. In recent years, clinical research on PROTACs has made significant progress (Nat Rev Drug Discov. 2022; 21(3): 181-200.), which has potential therapeutic significance and market development prospects for cancer patients. Compared with traditional small molecule inhibitors, PROTAC molecules work by degrading proteins, have higher selectivity and efficacy, and have shown greater advantages than traditional small molecule drugs in preclinical studies of tumor treatment.

[0006] In general, due to the current lack of targeted treatment options for TNBC, various targeted therapies are gaining increasing attention. Through continuous research and development, the present invention aims to provide a class of small molecule compounds and degraders that can target the transcription factor KLF5. These small molecule organic compounds and degraders have potential therapeutic implications and market development prospects for patients with various cancers. Summary of the Invention

[0007] The present invention found in the early screening and experiments that the anthelmintic NTZ (nitazoxanide) is an effective invasion inhibitor. k369Q NTZ exerted a potent inhibitory effect on osteoclast differentiation in both preventive and therapeutic modes. K369Q The cellular process of bone metastasis induced by NTZ. Further analysis showed that NTZ binds to the KLF5 protein. Based on this research result, in order to develop small molecule compounds and degraders with novel structures targeting KLF5, the present invention initially used NTZ as the lead compound, and used various technologies such as structure-based drug design (SBDD) to significantly change the parent nucleus structure, reduce the rigidity of the structural skeleton, and improve the drugability of the compound. In combination with the PROTACs design strategy, the present invention creatively designed and synthesized a class of small molecule compounds and degraders targeting the KLF5 protein. The compounds and degraders provided by the present invention have a significant inhibitory effect on the proliferation of various tumor cells, and representative compounds also show significant anti-tumor effects in tumor-bearing mice. Related results also show that the compounds of the present invention can directly target the KLF5 protein, providing a new approach for the treatment of TNBC tumors. Compared with currently available KLF5 inhibitors, the compounds of the present invention can directly target KLF5 and can induce KLF5 protein degradation and significantly inhibit the proliferation of various tumor cells such as triple-negative breast cancer.

[0008] The technical solution of the present invention is as follows: The present invention provides a class of small molecule compounds that target the degradation of the transcription factor KLF5, the compounds comprising a compound as represented by formula (I), a related analogue or a pharmaceutically acceptable salt, metabolite or prodrug of the compound as represented by formula (I); the analogues include stereoisomers, geometric isomers, tautomers, nitrogen oxides or solvates;

[0009]

[0010] In formula I, R1 is selected from any one or more of the following groups: halogen, C1-C4 alkyl, hydroxy, amino, ester, trifluoromethyl, cyano, C1-C4 alkoxy;

[0011] In formula I, R2 is selected from any one or more of the following groups: hydrogen, halogen, hydroxyl, cyano, trifluoromethyl, C1-C4 alkyl, C1-C4 alkoxy, morpholine, piperidine, piperazine;

[0012] The present invention also provides a class of PROTACs compounds that target the degradation of the transcription factor KLF5, comprising a compound as represented by formula (II), a related analogue or a pharmaceutically acceptable salt, metabolite or prodrug of the compound as represented by formula (II);

[0013]

[0014]

[0015] Wherein, X represents a ligand that binds to the KLF5 protein, selected from the small molecule compound that targets the degradation of the transcription factor KLF5, Z represents a ligand of the E3 ubiquitin ligase, and Y represents a linking group connecting X and Z.

[0016] As a further description of the above scheme: X is selected from the compound shown in the following structural formula;

[0017]

[0018] As a further description of the above scheme: Z is selected from the compound represented by formula (III):

[0019]

[0020] in:

[0021] A is selected from any one of CH2, C(O), SO or SO2;

[0022] B, E, and W are each independently selected from any one of CH or CF;

[0023] M is selected from any one of the following groups:

[0024] As a further description of the above scheme: the Z is selected from one or more of the following structures:

[0025]

[0026] As a further description of the above scheme: Y is selected from one or more of the following structures:

[0027]

[0028] in,

[0029] n is an integer between 0 and 6;

[0030] m is an integer between 0 and 8;

[0031] a is an integer between 0 and 5.

[0032] Preferably, the compound is selected from one or more of the following compounds:

[0033] 3-(Acetylamino)-N-(benzo[d][1,3]thiazolin-2-yl)benzamide;

[0034] 3-(Acetylamino)-N-(5-methylbenzo[d][1,3]thiazolin-2-yl)benzamide;

[0035] 3-(Acetylamino)-N-(5-methoxybenzo[d][1,3]thiazolin-2-yl)benzamide;

[0036] 3-(Acetylamino)-N-(5-fluorobenzo[d][1,3]thiazolin-2-yl)benzamide;

[0037] 3-(Acetylamino)-N-(7-fluorobenzo[d][1,3]thiazolin-2-yl)benzamide;

[0038] 3-(Acetylamino)-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)benzamide;

[0039] 3-(Acetylamino)-N-[6-(trifluoromethyl)benzo[d][1,3]thiazolin-2-yl]benzamide;

[0040] 3-(Acetylamino)-N-(5-chlorobenzo[d][1,3]thiazolin-2-yl)benzamide;

[0041] 3-(Acetylamino)-N-(6-chlorobenzo[d][1,3]thiazolin-2-yl)benzamide;

[0042] 3-(Acetylamino)-N-(4-chlorobenzo[d][1,3]thiazolin-2-yl)benzamide;

[0043] 3-(Acetylamino)-N-(5-bromobenzo[d][1,3]thiazolin-2-yl)benzamide;

[0044] 5-(Acetylamino)-2-chloro-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)benzamide;

[0045] 5-(Acetylamino)-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-2-methylbenzamide;

[0046] 3-(Acetylamino)-N-[6-(1,4-oxazepan-4-yl)benzo[d][1,3]thiazolin-2-yl]benzamide;

[0047] 3-(Acetylamino)-N-(5,6-dimethoxybenzo[d][1,3]thiazolin-2-yl)benzamide;

[0048] 3-(Acetylamino)-N-(6-ethoxybenzo[d][1,3]thiazolin-2-yl)benzamide;

[0049] 3-(Acetylamino)-N-(4-methoxy-7-methylbenzo[d][1,3]thiazolin-2-yl)benzamide;

[0050] 3-(Acetylamino)-N-[6-(propan-2-yloxy)benzo[d][1,3]thiazolin-2-yl]benzamide;

[0051] N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(4-{[2-(2,6-dioxaylidenehexahydropyridin-3-yl)-1,3-dioxaylidene-2,3-dihydro-1H-isoindol-5-yl]amino}-1-oxaylidenebutyl)amino]benzamide;

[0052] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(6-{[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-1-oxyylidenehexyl)amino]benzamide;

[0053] N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-({3-[(2-{[2-(2,6-dioxadienylhexahydropyridin-3-yl)-1,3-dioxadienyl-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoyl}amino)benzamide;

[0054] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide;

[0055] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxanylidene-3,6,9-trioxadodec-12-yl)amino]benzamide;

[0056] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxaylidenehexahydropyridin-3-yl)-1,3-dioxaylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-15-oxaylidene-3,6,9,12-tetraoxapentadecan-15-yl)amino]benzamide;

[0057] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxaylidenehexahydropyridin-3-yl)-1,3-dioxaylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-18-oxaylidene-3,6,9,12,15-pentaoxaoctadecan-18-yl)amino]benzamide;

[0058] N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-5-yl]amino}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide;

[0059] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1-oxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide;

[0060] N-(5,6-dimethylbenzo[d][1,3]thiazeol-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-4-yl]oxy}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide;

[0061] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(2-{4-[2-(2,6-dioxadienylhexahydropyridin-3-yl)-1,3-dioxadienyl-2,3-dihydro-1H-isoindol-4-yl]piperazin-1-yl}acetyl)amino]benzamide;

[0062] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1-oxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxanylidene-3,6,9-trioxadodec-12-yl)amino]benzamide;

[0063] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-5-yl]amino}-12-oxanylidene-3,6,9-trioxadodec-12-yl)amino]benzamide;

[0064] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(1-methyl-2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxanylidene-3,6,9-trioxadodec-12-yl)amino]benzamide;

[0065] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(3-{4-[2-(2,6-dioxaylidenehexahydropyridin-3-yl)-1,3-dioxaylidene-2,3-dihydro-1H-isoindol-4-yl]piperazin-1-yl}propanoyl)amino]benzamide;

[0066] N-(5,6-dimethoxybenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxanylidene-3,6,9-trioxadodec-12-yl)amino]benzamide;

[0067] N-(5,6-dimethylbenzo[d][1,3]thiazeol-2-yl)-3-{[2-(4-{1-[2-(2,6-dioxoylidenehexahydropyridin-3-yl)-1,3-dioxoylidene-2,3-dihydro-1H-isoindol-4-yl]hexahydropyridin-4-yl}hexahydropyridin-1-yl)acetyl]amino}benzamide.

[0068] As a further preference, the compound is selected from one or more of the following compounds; the preferred compound can more effectively and directly target the KLF5 protein and induce the degradation of the KLF5 protein, and the preferred compound can more effectively degrade the KLF5 protein in tumor cells and can significantly inhibit the proliferation of various tumor cells such as breast cancer.

[0069] 3-(Acetylamino)-N-(5-fluorobenzo[d][1,3]thiazolin-2-yl)benzamide;

[0070] 3-(Acetylamino)-N-(7-fluorobenzo[d][1,3]thiazolin-2-yl)benzamide;

[0071] 3-(Acetylamino)-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)benzamide;

[0072] 3-(Acetylamino)-N-[6-(trifluoromethyl)benzo[d][1,3]thiazolin-2-yl]benzamide;

[0073] 3-(Acetylamino)-N-(4-chlorobenzo[d][1,3]thiazolin-2-yl)benzamide;

[0074] N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-({3-[(2-{[2-(2,6-dioxadienylhexahydropyridin-3-yl)-1,3-dioxadienyl-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoyl}amino)benzamide;

[0075] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide;

[0076] N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxanylidene-3,6,9-trioxadodec-12-yl)amino]benzamide;

[0077] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxaylidenehexahydropyridin-3-yl)-1,3-dioxaylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-15-oxaylidene-3,6,9,12-tetraoxapentadecan-15-yl)amino]benzamide;

[0078] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxaylidenehexahydropyridin-3-yl)-1,3-dioxaylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-18-oxaylidene-3,6,9,12,15-pentaoxaoctadecan-18-yl)amino]benzamide;

[0079] N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-5-yl]amino}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide;

[0080] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-5-yl]amino}-12-oxanylidene-3,6,9-trioxadodec-12-yl)amino]benzamide;

[0081] N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1-oxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide;

[0082] N-(5,6-dimethylbenzo[d][1,3]thiazeol-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-4-yl]oxy}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide.

[0083] The present invention also provides a pharmaceutical composition comprising the aforementioned compound for targeting degradation of KLF5, and a pharmaceutically acceptable carrier;

[0084] and / or the pharmaceutical composition further comprises a second agent for preventing and / or treating cancer;

[0085] And / or the pharmaceutical composition further comprises an excipient, a diluent, an adjuvant, a vehicle or a combination thereof.

[0086] And / or the pharmaceutical composition is formulated into an injectable fluid, aerosol, cream, gel, pill, capsule, syrup, transdermal patch or excipient.

[0087] The present invention also provides use of the compound or the pharmaceutical composition in preparing a KLF5 regulator or an anti-tumor drug.

[0088] Preferably, the tumor comprises one or more of breast cancer, liver cancer, lung cancer, colorectal cancer, leukemia, gastric cancer, glioma, and prostate cancer. That is, the cancer cells are at least one of human breast cancer cells, human liver cancer cells, human leukemia cells, human cervical cancer cells, human lung cancer cells, human skin cancer cells, human colorectal cancer cells, human kidney cancer cells, human ovarian cancer cells, human glioma cells, human pancreatic cancer cells, human osteosarcoma cells, and human gastric cancer cells. Further preferably, the tumor is breast cancer.

[0089] The compounds and degraders for targeting KLF5 degradation described in the present invention and their related analogs or pharmaceutically acceptable salts, metabolites or prodrugs are used to inhibit the proliferation, growth, migration, infiltration, cloning and metastasis of cancer cells, promote the apoptosis of cancer cells, promote the autophagy of tumor cells, and / or prolong the survival of tumor patients.

[0090] The present invention also provides a method for preparing a drug for preventing and / or treating diseases related to KLF5 protein, wherein an effective amount of the compound targeting degradation of KLF5 and its related analogs or pharmaceutically acceptable salts, metabolites or prodrugs is administered to an individual in need.

[0091] The term "C1-C4 alkyl" used in the present invention refers to a straight-chain or branched alkane group having 1 to 4 carbon atoms, including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

[0092] The term "C1-C4 alkoxy" refers to a straight-chain or branched alkoxy group having 1 to 4 carbon atoms and containing at least one oxygen atom, including but not limited to methoxy, ethoxy, propoxy, ethoxymethoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and the like.

[0093] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0094] Preferably, the C1-C4 alkyl group is a methyl group, the C1-C4 alkoxy group is a methoxy group or a propoxy group, and the halogen group is a fluorine group, a chlorine group or a bromine group.

[0095] The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound of the invention having a specific substituent group and a relatively nontoxic acid or base. When the compound of the invention contains relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compound with a sufficient amount of base in neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, amine, organic amine, or magnesium salts, or similar salts. When the compound of the invention contains relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compound with a sufficient amount of acid in neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as 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, and methanesulfonic acid; and salts of amino acids such as arginine, and organic acids such as glucuronic acid. Certain specific compounds of the present invention contain acidic and basic functional groups and can be converted into either base or acid addition salts.

[0096] In addition to salt forms, the compounds provided herein also exist in prodrug form. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions, thereby converting to the compounds described herein. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound conforming to Formulas (I) to (III)) is a prodrug within the scope and spirit of the present invention. For example, a compound containing a carboxyl group can form a physiologically hydrolyzable ester, which, upon hydrolysis in vivo, can provide the compound conforming to Formulas (I) to (III) itself, thereby acting as a prodrug.

[0097] Certain compounds of the present invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers and individual isomers are included within the scope of the present invention, including cis and trans isomers, (-)- and (+)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, all of which are included within the scope of the present invention, as are mixtures thereof.

[0098] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with isotopes, such as deuterium ( 2 H), tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0099] The term "excipient" generally refers to a carrier, diluent and / or vehicle required to formulate an effective pharmaceutical composition.

[0100] With respect to a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a non-toxic amount of the drug or agent sufficient to achieve the intended effect. For the oral dosage forms of the present invention, an "effective amount" of one active substance in the composition refers to the amount required to achieve the intended effect when used in combination with another active substance in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active substance. The appropriate effective amount in each individual case can be determined by those skilled in the art through routine experimentation.

[0101] As used herein, the terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

[0102] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be substituted with up to two Rs, and each occurrence of R is an independent option. Furthermore, combinations of substituents and / or their variants are permitted only if such combinations result in stable compounds.

[0103] The term "comprising" in the present invention is an open expression, that is, including the contents specified in the present invention, but not excluding other contents.

[0104] Compared with the prior art, the present invention has the following beneficial effects:

[0105] 1. The compounds provided by the present invention can directly target KLF5 protein and induce the degradation of KLF5 protein. The preferred representative compound LS-23 binds to KLF5 protein. d It is 9.5 micromol / L.

[0106] 2. The compounds provided by the present invention can effectively degrade KLF5 protein in tumor cells. The representative compound LS-23 has a maximum degradation efficiency of 93% for KLF5.

[0107] 3. The compounds of the present invention can significantly inhibit the proliferation of various tumor cells such as triple-negative breast cancer. The representative compound LS-23 has an IC of 1.57 in triple-negative breast cancer cells SUM149PT with high expression of KLF5. 50 1.19 μmol / L,

[0108] 4. The compounds of the present invention have a significant inhibitory effect on the growth of subcutaneous transplanted tumors in mice with triple-negative breast cancer.

[0109] 5. It has excellent inhibitory activity against various types of human cancer cells that highly express KLF5, providing an alternative for broad-spectrum anti-cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 Activity screening of LS-23 and other compounds in breast cancer cell lines. A represents the degradation activity of representative compounds, B represents the cell inhibition activity of representative compounds, and C represents the strong inhibitory activity of LS-23 against breast cancer cells of different subtypes (TNBC: triple-negative breast cancer, HER2+: HER2-positive breast cancer, Luminal: Luminal breast cancer).

[0111] Figure 2 DC for LS-23 and LS-30 50 and IC 50Comparison, AB are the differences in cell viability and KLF5 protein levels after treatment with LS-23 and LS-30 in SUM149PT and HCC1806;

[0112] Figure 3 The results of the LS-23 cell scratch test, A is the migration picture, B is the statistical graph of the migration rate, NC is the control group;

[0113] Figure 4 Results of the LS-23 clone sphere formation experiment; A is a picture of clone formation, and B is a statistical chart of clone formation;

[0114] Figure 5 Figure 1 is the result of flow cytometry detection of LS-23 cell apoptosis; Figure A is the flow cytometry analysis image of apoptosis, and Figure B is the statistical graph of apoptosis;

[0115] Figure 6 Figure 5 is the experimental result of mouse xenograft tumor of LS-23; AB is the schematic diagram of the construction of mouse orthotopic breast cancer model and the administration time and frequency; after 13 consecutive days of administration, the mice were euthanized, the tumors were removed and photographed; C is the tumor volume measured every 2 days after the start of treatment, represented by a broken line graph; D is the change of mouse body weight after administration of compound LS-23; E is the serum alanine aminotransferase level after administration to evaluate the hepatotoxicity of compound LS-23 and the serum creatinine level to evaluate the nephrotoxicity of compound LS-23; F is the detection result of KLF5 level in tumor tissue of mice in the control group (DMSO) and experimental groups (20 mg / kg and 40 mg / kg);

[0116] Figure 7 This is the SPR (surface plasmon resonance) test result of LS-23. DETAILED DESCRIPTION

[0117] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The protection content of the present invention is not limited to the following embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

[0118] The low-temperature reaction apparatus used in the present invention was an EYELA (PSL-1810) magnetic stirring low-temperature constant-temperature water bath; an EYELA (N-1100) rotary evaporator; the purity results of the obtained compounds were obtained from an Agilent 1200 series LC system high-performance liquid chromatograph with UV detection wavelengths of 254 nm and 365 nm; a Bruker 500 nuclear magnetic resonance spectrometer was used, using DMSO-d6 as the solvent; and purification of reaction intermediates and final products was performed using chromatographic columns (200-300 mesh silica gel) purchased from Qingdao Ocean Chemical Plant. Unless otherwise specified, all reactions were monitored by TLC.

[0119] Example 1: Preparation of the compound provided by the present invention

[0120] Example 1-1. Preparation of 3-(acetylamino)-N-(benzo[d][1,3]thiazolin-2-yl)benzamide (LS-01):

[0121]

[0122] 2-Aminobenzothiazole (100 mg, 0.689 mmol) and 3-acetamidobenzoic acid (124 mg, 0.689 mmol) were dissolved in 2 mL of dimethyl sulfoxide (DMSO). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (393 mg, 1.033 mmol) and N,N-diisopropylethylamine (DIPEA) (267 mg, 2.067 mmol) were added to the mixture, followed by stirring at room temperature for 2 h. After completion of the reaction, the mixture was extracted with ethyl acetate and water. The organic phases were collected, washed with saturated sodium chloride solution, combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Compound LS-01 (68 mg, 40%) was obtained by column chromatography. 1 H NMR (500MHz, DMSO-d6) δ12.87(s,1H),10.18(s,1H),8.31(s,1H),8.03(d,J=7.9Hz,1H),7.84(t d,J=7.3,1.9Hz,2H),7.80(d,J=8.2Hz,1H),7.52–7.44(m,2H),7.38–7.32(m,1H),2.09(s,3H).

[0123] Example 1-2. Preparation of 3-(acetylamino)-N-(5-methylbenzo[d][1,3]thiazolin-2-yl)benzamide (LS-02):

[0124]

[0125] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-methylbenzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-02 (36 mg, 40%). 1 H NMR(500MHz,DMSO-d6)δ12.84(s,1H),10.18(s,1H),8.30(s,1H),7.89(d,J=8.1Hz,1H),7.87–7.80 (m,2H),7.61(s,1H),7.48(t,J=7.9Hz,1H),7.18(dd,J=8.1,1.6Hz,1H),2.45(s,3H),2.09(s,3H).

[0126] Example 1-3. Preparation of 3-(acetylamino)-N-(5-methoxybenzo[d][1,3]thiazolin-2-yl)benzamide (LS-03):

[0127]

[0128] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-methoxybenzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-03 (141 mg, 57%). 1 H NMR (500MHz, DMSO-d6) δ12.83(s,1H),10.18(s,1H),8.29(s,1H),7.89(d,J=8.7Hz,1H),7.83(dd,J=7.6 ,1.8Hz,2H),7.48(t,J=7.9Hz,1H),7.31(s,1H),6.99(dd,J=8.7,2.5Hz,1H),3.85(s,3H),2.09(s,3H).

[0129] Example 1-4. Preparation of 3-(acetylamino)-N-(5-fluorobenzo[d][1,3]thiazolin-2-yl)benzamide (LS-04):

[0130]

[0131] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-fluorobenzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-04 (207 mg, 64%). 1H NMR (500MHz, DMSO-d6) δ12.97(s,1H),10.19(s,1H),8.30(s,1H),8.07(dd,J=8.8,5.4Hz,1H),7.84(dd,J =8.7, 3.5Hz, 2H), 7.62 (d, J = 10.7Hz, 1H), 7.49 (t, J = 7.9Hz, 1H), 7.24 (td, J = 9.0, 2.5Hz, 1H), 2.09 (s, 3H).

[0132] Example 1-5. Preparation of 3-(acetylamino)-N-(7-fluorobenzo[d][1,3]thiazolin-2-yl)benzamide (LS-05):

[0133]

[0134] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced with 2-amino-7-fluoro-1,3-benzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-05 (113 mg, 58%). 1 H NMR (500MHz, DMSO-d6) δ13.13(s,1H),10.20(s,1H),8.32(d,J=2.4Hz,1H),7.85(ddd,J=7.5 ,5.2,1.9Hz,2H),7.68(d,J=8.0Hz,1H),7.56-7.47(m,2H),7.28-7.21(m,1H),2.09(s,3H).

[0135] Example 1-6. Preparation of 3-(acetylamino)-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)benzamide (LS-06):

[0136]

[0137] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced with 2-amino-5,6-dimethylbenzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-06 (75 mg, 26%). 1 H NMR (500MHz, DMSO-d6) δ12.76(s,1H),10.17(s,1H),8.28(t,J=1.9Hz,1H),7.83(dq,J=8.1,2.7,1 .9Hz,2H),7.76(s,1H),7.58(s,1H),7.47(t,J=7.9Hz,1H),2.35(s,3H),2.34(s,3H),2.09(s,3H).

[0138] Example 1-7. Preparation of 3-(acetylamino)-N-[6-(trifluoromethyl)benzo[d][1,3]thiazolyl-2-yl]benzamide (LS-07):

[0139]

[0140] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 2-amino-6-(trifluoromethyl)benzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-07 (108 mg, 34%). 1 H NMR (500MHz, DMSO-d6) δ13.12(s,1H),10.20(s,1H),8.56(s,1H),8.32(t,J=2.0Hz,1H),7.96(d, J=8.5Hz,1H),7.89–7.82(m,2H),7.79(dd,J=8.5,1.9Hz,1H),7.50(t,J=7.9Hz,1H),2.09(s,3H).

[0141] Example 1-8. Preparation of 3-(acetylamino)-N-(5-chlorobenzo[d][1,3]thiazolin-2-yl)benzamide (LS-08):

[0142]

[0143] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-chlorobenzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-08 (87 mg, 36%). 1 H NMR (500MHz, DMSO-d6) δ13.02(s,1H),10.19(s,1H),8.31(d,J=2.2Hz,1H),8.08(d,J=8.5H z,1H),7.87–7.81(m,3H),7.49(t,J=7.9Hz,1H),7.40(dd,J=8.4,2.0Hz,1H),2.09(s,3H).

[0144] Example 1-9. Preparation of 3-(acetylamino)-N-(4-chlorobenzo[d][1,3]thiazolin-2-yl)benzamide (LS-09):

[0145]

[0146] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 2-amino-4-chlorobenzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-09 (96 mg, 39%). 1 H NMR (500MHz, DMSO-d6) δ13.20(s,1H),10.18(s,1H),8.33(t,J=2.0Hz,1H),8.02(d,J=7.9Hz,1H),7.92–7.87(m,1 H), 7.83 (dd, J=8.2, 2.1Hz, 1H), 7.57 (d, J=7.8Hz, 1H), 7.48 (t, J=8.0Hz, 1H), 7.34 (t, J=7.8Hz, 1H), 2.09 (s, 3H).

[0147] Example 1-10. Preparation of 3-(acetylamino)-N-(6-chlorobenzo[d][1,3]thiazolin-2-yl)benzamide (LS-10):

[0148]

[0149] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 2-amino-6-chlorobenzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-10 (115 mg, 66%). 1 H NMR (500 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.19 (s, 1H), 8.30 (s, 1H), 8.19 (d, J = 2.2 Hz, 1H), 7.87–7.82 (m, 2H), 7.79 (d, J = 8.6 Hz, 1H), 7.53–7.45 (m, 2H), 2.09 (s, 3H). Example 1-11. Preparation of 3-(acetylamino)-N-(5-bromobenzo[d][1,3]thiazolin-2-yl)benzamide (LS-11):

[0150]

[0151] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-bromobenzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-11 (91 mg, 44%). 1H NMR(500MHz,DMSO-d6)δ12.97(s,1H),10.19(s,1H),8.33–8.28(m,2H),7.84(dd,J=7.7,1.8H z, 2H), 7.73 (d, J = 8.6Hz, 1H), 7.62 (dd, J = 8.6, 2.1Hz, 1H), 7.49 (t, J = 7.9Hz, 1H), 2.09 (s, 3H).

[0152] Example 1-12. Preparation of 5-(acetylamino)-2-chloro-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)benzamide (LS-12):

[0153]

[0154] Similar to the synthesis scheme of Example 1-1, 3-acetylaminobenzoic acid was replaced by 5-(acetylamino)-2-chlorobenzoic acid, and 2-aminobenzothiazole was replaced by 2-amino-5,6-dimethylbenzothiazole. The crude product obtained was purified by column chromatography to obtain compound LS-12 (55 mg, 16%). 1 H NMR (500MHz, DMSO-d6) δ12.85(s,1H),10.25(s,1H),7.91(d,J=2.6Hz,1H),7.77(s,1H),7.69( dd,J=8.8,2.6Hz,1H),7.59(s,1H),7.51(d,J=8.8Hz,1H),2.34(d,J=1.8Hz,6H),2.08(s,3H).

[0155] Example 1-13. Preparation of 5-(acetylamino)-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-2-methylbenzamide (LS-13):

[0156]

[0157] Similar to the synthesis scheme of Example 1-1, 3-acetylaminobenzoic acid was replaced by 5-acetylamino-2-methylbenzoic acid, and 2-aminobenzothiazole was replaced by 2-amino-5,6-dimethylbenzothiazole. The crude product obtained was purified by column chromatography to obtain compound LS-13 (77 mg, 39%). 1H NMR(500MHz,DMSO-d6)δ12.50(s,1H),10.12(s,1H),7.74(s,1H),7.61(d,J=8.5Hz,1H ), 7.55 (d, J = 8.4Hz, 2H), 7.51 (s, 1H), 2.43 (s, 3H), 2.34 (d, J = 2.8Hz, 6H), 2.08 (s, 3H).

[0158] Example 1-14. Preparation of 3-(acetylamino)-N-[6-(1,4-oxazepan-4-yl)benzo[d][1,3]thiazolin-2-yl]benzamide (LS-14):

[0159]

[0160] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 6-morpholinobenzo[D]thiazol-2-amine, and the crude product obtained was purified by column chromatography to obtain compound LS-14 (88 mg, 40%). 1 H NMR (500MHz, DMSO-d6) δ12.68(s,1H),10.18(s,1H),8.29(t,J=2.0Hz,1H),7.82(dt,J=8.0,1.8Hz,2H),7.64(d,J=8.9Hz,1H) ,7.53(d,J=2.5Hz,1H),7.47(t,J=8.0Hz,1H),7.17(dd,J=5.7,3.3Hz,1H),3.79-3.77(m,4H),3.18-3.15(m,4H),2.09(s,3H).

[0161] Example 1-15. Preparation of 3-(acetylamino)-N-(5,6-dimethoxybenzo[d][1,3]thiazolin-2-yl)benzamide (LS-15):

[0162]

[0163] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced with 5,6-dimethoxy-1,3-benzothiazol-2-amine, and the crude product obtained was purified by column chromatography to obtain compound LS-15 (68 mg, 26%). 1H NMR(500MHz,DMSO-d6)δ12.71(s,1H),10.17(s,1H),8.28(s,1H),7.82(dd,J=7.9,1.9Hz, 2H), 7.60 (s, 1H), 7.47 (t, J = 7.9Hz, 1H), 7.32 (s, 1H), 3.84 (d, J = 7.9Hz, 6H), 2.09 (s, 3H).

[0164] Example 1-16. Preparation of 3-(acetylamino)-N-(6-ethoxybenzo[d][1,3]thiazolin-2-yl)benzamide (LS-16):

[0165]

[0166] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 2-amino-6-ethoxybenzothiazole, and the crude product obtained was purified by column chromatography to obtain compound LS-16 (79 mg, 31%). 1 H NMR (500MHz, DMSO-d6) δ12.73(s,1H),10.17(s,1H),8.29(s,1H),7.82(dt,J=8.1,2.0Hz,2H),7.67(d,J=8.8Hz,1H),7.60(d ,J=2.6Hz,1H),7.47(t,J=7.9Hz,1H),7.06(dd,J=8.8,2.6Hz,1H),4.10(q,J=6.9Hz,2H),2.09(s,3H),1.37(t,J=6.9Hz,3H).

[0167] Example 1-17. Preparation of 3-(acetylamino)-N-(4-methoxy-7-methylbenzo[d][1,3]thiazolin-2-yl)benzamide (LS-17):

[0168]

[0169] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 4-methoxy-7-methylbenzo[D]thiazol-2-amine, and the crude product obtained was purified by column chromatography to obtain compound LS-17 (56 mg, 27%). 1H NMR (500MHz, DMSO-d6) δ13.00(s,1H),10.19(s,1H),8.30(t,J=1.9Hz,1H),7.90–7.81(m,2H),7.48(t,J=7 .9Hz,1H),7.11(dd,J=8.0,1.0Hz,1H),6.95(d,J=8.1Hz,1H),3.91(s,3H),2.48-2.44(m,3H),2.09(s,3H).

[0170] Example 1-18. Preparation of 3-(acetylamino)-N-[6-(propan-2-yloxy)benzo[d][1,3]thiazolin-2-yl]benzamide (LS-18):

[0171]

[0172] Similar to the synthesis scheme of Example 1-1, 2-aminobenzothiazole was replaced by 6-isopropoxy-1,3-benzothiazol-2-amine, and the crude product obtained was purified by column chromatography to obtain compound LS-18 (56 mg, 23%). 1 H NMR (500MHz, DMSO-d6) δ12.73(s,1H),10.18(s,1H),8.29(t,J=2.0Hz,1H),7.82(dt,J=8.0,2.0Hz,2H),7.66(d,J=8.8Hz,1H),7.60 (d,J=2.5Hz,1H),7.47(t,J=7.9Hz,1H),7.04(dd,J=8.8,2.5Hz,1H),4.66(hept,J=6.1Hz,1H),2.09(s,3H),1.30(d,J=6.0Hz,6H).

[0173] Preparation of the key intermediate 3-amino-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)benzamide (L1):

[0174]

[0175] 3-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)benzoic acid (1 g, 4.215 mmol) and 5,6-dimethylbenzo[d][1,3]thiazolin-2-amine (751 mg, 4.215 mmol) were dissolved in 2 mL of dimethyl sulfoxide (DMSO). 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (2.424 g, 6.377 mmol) and N,N-diisopropylethylamine (DIPEA) (1.634 g, 12.645 mmol) were added to the mixture in sequence. The mixture was stirred at room temperature for 2 h. After completion of the reaction, the mixture was extracted with ethyl acetate and water. The organic phase was collected, washed with saturated sodium chloride solution, combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Column chromatography separation and purification gave 2-methylpropan-2-yl [(3-{[(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)amino]carbonyl}phenyl)amino]methane (636 mg, 38% yield).

[0176] 2-Methylpropane-2-[(3-{[(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)amino]carbonyl}phenyl)amino]methane (636 mg, 1.602 mg) was dissolved in DCM (10 mL), trifluoroacetic acid (1.5 mL) was added, and the reaction was carried out for 5 h. DCM and TFA were evaporated to dryness, and the crude product obtained was purified by column chromatography to give the key intermediate 3-amino-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)benzamide (L1) (300 mg, yield 63%).

[0177] Example 1-19. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(4-{[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-5-yl]amino}-1-oxyylidenebutyl)amino]benzamide (LS-19).

[0178]

[0179] 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (300 mg, 1.086 mmol) and 4-aminobutyric acid (168 mg, 1.629 mmol) were dissolved in 2 mL of dimethyl sulfoxide (DMSO). N,N-diisopropylethylamine (DIPEA) (280 mg, 2.172 mmol) was added to the mixture. The mixture was reacted at 90°C for 10 h under nitrogen. After completion of the reaction, the mixture was extracted with ethyl acetate and water. The organic phases were collected, washed with saturated sodium chloride solution, combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. 4-{[2-(2,6-dioxoylidenehexahydropyridin-3-yl)-1,3-dioxoylidene-2,3-dihydro-1H-isoindol-4-yl]amino}butyric acid (150 mg) was obtained by column chromatography.

[0180] 4-{[2-(2,6-dioxyhexahydropyridin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}butyric acid and intermediate L1 were dissolved in DMSO. HATU and DIPEA were added sequentially to the reaction mixture and stirred at room temperature for 2 hours. After completion, the reaction was extracted with EA and water. The organic phase was collected, dried, and concentrated. The crude product obtained was purified by column chromatography to obtain the target product, N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(4-{[2-(2,6-dioxyhexahydropyridin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-5-yl]amino}-1-oxyylidenebutyl)amino]benzamide (LS-19). 1 H NMR(500MHz,DMSO-d6)δ12.76(s,1H),11.10(s,1H),10.18(s,1H),8.30(s,1H),7.83(dd,J=7.9,1.9Hz,2H ),7.76(s,1H),7.64–7.57(m,2H),7.47(t,J=7.9Hz,1H),7.17(d,J=8.6Hz,1H),7.04(d,J=7.0Hz,1H),6.70 (t,J=6.2Hz,1H),5.06(dd,J=12.8,5.4Hz,1H),2.89(ddd,J=16.9,13.8,5.4Hz,1H),2.65–2.55(m,2H),2.4 7(t,J=7.3Hz,2H),2.35(d,J=5.0Hz,6H),2.02(dp,J=10.7,3.5Hz,1H),1.93(p,J=7.2Hz,2H),1.24(s,2H).

[0181] Example 1-20. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(6-{[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-1-oxyylidenehexyl)amino]benzamide (LS-20).

[0182]

[0183] Similar to the synthesis scheme of Example 1-19, 4-aminobutyric acid was replaced by 6-aminohexanoic acid, and the crude product obtained was purified by column chromatography to obtain compound LS-20. 1 H NMR(500MHz,DMSO-d6)δ12.76(s,1H),11.09(s,1H),10.11(s,1H),8.32–8.28(m,1H),7.85–7.80(m,2H),7.76(s,1 H),7.61–7.55(m,2H),7.46(t,J=7.9Hz,1H),7.11(d,J=8.6Hz,1H),7.02(d,J=7.0Hz,1H),6.56(t,J=6.0Hz,1H),5 .05(dd,J=12.8,5.4Hz,1H),2.96–2.83(m,1H),2.65–2.55(m,1H),2.54–2.52(m,1H),2.38(d,J=7.5Hz,2H),2.34( d,J=5.3Hz,7H),2.02(dtd,J=11.3,5.4,2.7Hz,1H),1.65(dp,J=22.1,7.3Hz,4H),1.47–1.37(m,2H),1.24(s,1H).

[0184] Example 1-21. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-({3-[(2-{[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoyl}amino)benzamide (LS-21).

[0185]

[0186] 2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindole-1,3-dione (200 mg, 0.724 mmol) and 2-methylprop-2-yl 3-[(2-aminoethyl)oxy]propanoate (206 mg, 1.806 mmol) were dissolved in 2 mL of dimethyl sulfoxide (DMSO). N,N-diisopropylethylamine (DIPEA or DIEA) (187 mg, 1.448 mmol) was added to the mixed solution. The mixture was reacted at 90 ° C under nitrogen protection for 10 h. After completion of the reaction, the mixture was extracted with ethyl acetate and water, the organic phase was collected, washed with saturated sodium chloride solution, the organic phases were combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Column chromatography separation and purification gave 2-methylprop-2-yl 3-[(2-{[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoate (120 mg).

[0187] 2-Methylpropane-2-yl 3-[(2-{[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoate (120 mg, 0.269 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.5 mL) was added, and the mixture was stirred at room temperature for 3 h. After the reaction was completed, most of the solvent was dried and directly dissolved in DMSO (2 mL). Subsequently, intermediate L1 (70 mg), HATU (133 mg, 0.351 mmol) and DIPEA (90 mg, 0.702 mmol) were added to the reaction solution and stirred at room temperature for 3 h. After completion of the reaction, the mixture was extracted with EA and water, and the crude product obtained was subjected to silica gel column chromatography to obtain the target product, N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-({3-[(2-{[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propionyl}amino)benzamide (LS-21). 1H NMR(500MHz,DMSO-d6)δ12.76(s,1H),11.09(s,1H),10.18(s,1H),8.29(s,1H),7.86–7.80(m ,2H),7.76(s,1H),7.60–7.51(m,2H),7.46(t,J=7.9Hz,1H),7.14(d,J=8.6Hz,1H),7.01(d,J= 7.0Hz,1H),6.61(t,J=5.8Hz,1H),5.02(dd,J=12.9,5.4Hz,1H),3.78(t,J=6.3Hz,2H),3.64( t,J=5.5Hz,2H),3.48(q,J=5.6Hz,2H),2.64–2.58(m,2H),2.35(d,J=5.1Hz,6H),1.24(s,4H).

[0188] Example 1-22. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-4-yl]amino}-9-oxypyridin-3,6-dioxanonan-9-yl)amino]benzamide (LS-22).

[0189]

[0190] Similar to the synthesis scheme of Example 19, 2-methylprop-2-yl 3-[(2-aminoethyl)oxy]propanoate was replaced with 2-methylprop-2-yl 3-({2-[(2-aminoethyl)oxy]ethyl}oxy)propanoate, and the crude product obtained was purified by column chromatography to obtain compound LS-22. 1H NMR(500MHz,DMSO-d6)δ12.76(s,1H),11.09(s,1H),10.16(s,1H),8.31(s,1H),7.83(d,J=7.9Hz,2H) ,7.76(s,1H),7.56(dd,J=15.2,7.4Hz,2H),7.46(t,J=8.0Hz,1H),7.10(d,J=8.6Hz,1H),7.02(d,J=7. 1Hz,1H),6.59(s,1H),5.05(dd,J=12.5,5.4Hz,1H),3.74(t,J=6.1Hz,2H),3.62(t,J=5.5Hz,2H),3.59 –3.55(m,4H),3.43(d,J=5.5Hz,1H),2.85(d,J=17.2Hz,3H),2.65–2.54(m,4H),2.35(d,J=5.0Hz,6H).

[0191] Example 1-23. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxyhexahydropyridin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxy-3,6,9-trioxadodec-12-yl)amino]benzamide (LS-23):

[0192]

[0193] Similar to the synthesis scheme of Example 1-19, 3-[(2-aminoethyl)oxy]propanoic acid-2-methylpropan-2-yl ester was replaced with 3-[(8-amino-3,6-dioxaoctane-1-yl)oxy]propanoic acid-2-methylpropan-2-yl ester, and the crude product obtained was purified by column chromatography to obtain compound LS-23. 1H NMR (500MHz, DMSO-d6) δ12.72(s,1H),11.06(s,1H),10.18(s,1H),8.32(s,1H),7.84(dd,J=7.9,1.9Hz,2H),7.75(d, J=4.3Hz,1H),7.59–7.52(m,2H),7.52–7.44(m,1H),7.14(t,J=5.6Hz,1H),7.00(d,J=2.1Hz,1H),6.87(dd,J=8.4,2. 1Hz,1H),5.03(dd,J=12.8,5.4Hz,1H),4.10(q,J=5.2Hz,1H),3.72(q,J=6.2Hz,3H),3.57(t,J=5.4Hz,2H),3.52(d,J =3.4Hz, 8H), 2.59 (t, J = 6.2Hz, 3H), 2.55 (d, J = 2.3Hz, 1H), 2.34 (d, J = 5.3Hz, 7H), 1.99 (ddd, J = 10.5, 5.4, 3.0Hz, 1H).

[0194] Example 1-24. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxyhexahydropyridin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}-15-oxy-3,6,9,12-tetraoxapentadecan-15-yl)amino]benzamide (LS-24):

[0195]

[0196] Similar to the synthesis scheme of Example 1-19, 3-[(2-aminoethyl)oxy]propanoic acid-2-methylpropan-2-yl ester was replaced with 3-[(11-amino-3,6,9-trioxaundec-1-yl)oxy]propanoic acid-2-methylpropan-2-yl ester, and the crude product obtained was purified by column chromatography to obtain compound LS-24. 1H NMR(500MHz,DMSO-d6)δ12.78(s,1H),11.09(s,1H),10.17(s,1H),8.31(s,1H),7.83(dd,J=8.1,1.8Hz,2H),7.75(s,1H),7.60–7 .53(m,2H),7.47(t,J=7.9Hz,1H),7.12(d,J=8.6Hz,1H),7.03(d,J=7.0Hz,1H),6.58(t,J=5.8Hz,1H),5.05(dd,J=12.7,5.4Hz,1H ),4.10(q,J=5.2Hz,2H),3.72(t,J=6.2Hz,2H),3.60(t,J=5.5Hz,2H),3.54(dd,J=6.2,3.6Hz,2H),3.51(d,J=2.2Hz,4H),3.48(s ,4H),3.17(d,J=5.2Hz,4H),2.88(ddd,J=16.8,13.7,5.4Hz,1H),2.59(t,J=6.3Hz,2H),2.34(d,J=5.3Hz,6H),2.06–1.99(m,1H).

[0197] Example 1-25. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxyhexahydropyridin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}-18-oxy-3,6,9,12,15-pentaoxaoctadecan-18-yl)amino]benzamide (LS-25):

[0198]

[0199] Similar to the synthesis scheme of Example 1-19, 3-[(2-aminoethyl)oxy]propanoic acid-2-methylpropan-2-yl ester was replaced with 3-[(14-amino-3,6,9,12-tetraoxatetradec-1-yl)oxy]propanoic acid-2-methylpropan-2-yl ester, and the crude product obtained was purified by column chromatography to obtain compound LS-25. 1H NMR(500MHz,DMSO-d6)δ12.77(s,1H),11.09(s,1H),10.17(s,1H),8.31(s,1H),7.84(dd,J=8.0,1.6Hz,2H),7.75(s,1H),7 .57(dd,J=8.6,7.0Hz,2H),7.47(t,J=7.9Hz,1H),7.12(d,J=8.6Hz,1H),7.03(d,J=7.0Hz,1H),6.59(t,J=5.8Hz,1H),5.05( dd,J=12.7,5.4Hz,1H),4.23(t,J=6.6Hz,1H),3.72(t,J=6.2Hz,2H),3.60(t,J=5.4Hz,2H),3.57–3.40(m,16H),2.97–2.83 (m,1H),2.61–2.54(m,3H),2.34(d,J=5.4Hz,6H),2.02(ddt,J=11.2,6.5,3.6Hz,1H),1.69–1.60(m,1H),1.47–1.32(m,1H).

[0200] Example 1-26. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-5-yl]amino}-9-oxypyridin-3,6-dioxanonan-9-yl)amino]benzamide (LS-26).

[0201]

[0202] Similar to the synthesis scheme of Example 1-19, 2-(2,6-dioxypyridin-3-yl)-4-fluoroisoindole-1,3-dione was replaced with 2-(2,6-dioxypyridin-3-yl)-5-fluoroisoindole-1,3-dione, and 2-methylprop-2-yl 3-[(2-aminoethyl)oxy]propanoate was replaced with 2-methylprop-2-yl 3-({2-[(2-aminoethyl)oxy]ethyl}oxy)propanoate. The crude product thus obtained was purified by column chromatography to obtain compound LS-26. 1H NMR(500MHz,DMSO-d6)δ12.77(s,1H),11.05(s,1H),10.18(s,1H),8.31(s,1H),7.86–7.81(m,2H),7.78–7.74(m,1H ),7.60-7.52(m,2H),7.47(t,J=8.0Hz,1H),7.13(t,J=5.6Hz,1H),6.99(d,J=2.2Hz,1H),6.87(dd,J=8.5,2.1Hz,1H) ,5.02(dd,J=12.7,5.4Hz,1H),3.73(t,J=6.2Hz,2H),3.59(t,J=5.4Hz,2H),3.57(s,4H),2.87(ddd,J=16.6,13.6,5 .2Hz,1H),2.59(t,J=6.2Hz,2H),2.37(d,J=1.9Hz,1H),2.34(d,J=5.1Hz,6H),2.01–1.95(m,2H),0.98-0.78(m,2H).

[0203] Example 1-27. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxyhexahydropyridin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-5-yl]amino}-12-oxy-3,6,9-trioxadodec-12-yl)amino]benzamide (LS-27):

[0204]

[0205] Similar to the synthesis scheme of Example 1-19, 2-(2,6-dioxyhexahydropyridine-3-yl)-4-fluoroisoindole-1,3-dione was replaced with 2-(2,6-dioxyhexahydropyridine-3-yl)-5-fluoroisoindole-1,3-dione, and 3-[(2-aminoethyl)oxy]propionic acid-2-methylprop-2-yl ester was replaced with 3-[(8-amino-3,6-dioxaoct-1-yl)oxy]propionic acid-2-methylprop-2-yl ester. The crude product thus obtained was purified by column chromatography to obtain compound LS-27. 1H NMR (500MHz, DMSO-d6) δ12.72(s,1H),11.06(s,1H),10.18(s,1H),8.32(s,1H),7.84(dd,J=7.9,1.9Hz,2H),7.75(d, J=4.3Hz,1H),7.59–7.52(m,2H),7.52–7.44(m,1H),7.14(t,J=5.6Hz,1H),7.00(d,J=2.1Hz,1H),6.87(dd,J=8.4,2. 1Hz,1H),5.03(dd,J=12.8,5.4Hz,1H),4.10(q,J=5.2Hz,1H),3.72(q,J=6.2Hz,3H),3.57(t,J=5.4Hz,2H),3.52(d,J =3.4Hz, 8H), 2.59 (t, J = 6.2Hz, 3H), 2.55 (d, J = 2.3Hz, 1H), 2.34 (d, J = 5.3Hz, 7H), 1.99 (ddd, J = 10.5, 5.4, 3.0Hz, 1H).

[0206] Example 1-28. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxypyridin-3-yl)-1-oxyylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-9-oxyylidene-3,6-dioxanonan-9-yl)amino]benzamide (LS-28):

[0207]

[0208] Similar to the synthesis scheme of Example 1-19, 2-(2,6-dioxyylidenehexahydropyridine-3-yl)-4-fluoroisoindole-1,3-dione was replaced with 3-(4-amino-1-oxyylidene-2,3-dihydro-1H-isoindole-2-yl)hexahydropyridine-2,6-dione, and 3-[(2-aminoethyl)oxy]propionic acid-2-methylprop-2-yl ester was replaced with 3-({2-[(2-bromoethyl)oxy]ethyl}oxy)propionic acid-2-methylprop-2-yl ester. The crude product thus obtained was purified by column chromatography to obtain compound LS-28. 1H NMR (500MHz, DMSO-d6) δ12.77(s,1H),11.00(s,1H),10.18(s,1H),8.31(s,1H),7.84(d,J=7.1Hz,2H),7.76(s,1H),7.59(s,1H ),7.47(t,J=7.9Hz,1H),7.27(t,J=7.7Hz,1H),6.93(d,J=7.5Hz,1H),6.77(d,J=8.0Hz,1H),5.56(t,J=5.8Hz,1H),5.10(dd,J= 13.3,5.1Hz,1H),4.22(d,J=17.1Hz,1H),4.15–4.07(m,1H),3.74(t,J=6.2Hz,2H),3.58(d,J=15.2Hz,6H),3.29(d,J=6.4Hz,1H ), 2.91 (ddd, J = 18.1, 13.5, 5.4Hz, 1H), 2.59 (t, J = 6.1Hz, 2H), 2.35 (d, J = 4.9Hz, 6H), 2.30 (dd, J = 13.6, 4.3Hz, 1H), 1.24 (s, 3H). Example 1-29. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxyhexahydropyridin-3-yl)-1-oxyylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxyylidene-3,6,9-trioxadodec-12-yl)amino]benzamide (LS-29):

[0209]

[0210] Similar to the synthesis scheme of Example 1-19, 2-(2,6-dioxypyridin-3-yl)-4-fluoroisoindole-1,3-dione was replaced with 3-(4-amino-1-oxypyridin-2,3-dihydro-1H-isoindole-2-yl)pyridin-2,6-dione, and 3-[(2-aminoethyl)oxy]propionic acid-2-methylprop-2-yl ester was replaced with 3-[(8-bromo-3,6-dioxaoct-1-yl)oxy]propionic acid-2-methylprop-2-yl ester. The crude product thus obtained was purified by column chromatography to obtain compound LS-29. 1H NMR (500MHz, DMSO-d6) δ12.77(s,1H),11.00(s,1H),10.17(s,1H),8.31(s,1H),7.84(dd,J=7.9,1.8Hz,2H),7.75(s,1H),7.58(s,1H ),7.47(t,J=8.0Hz,1H),7.27(t,J=7.7Hz,1H),6.94(d,J=7.2Hz,1H),6.78(d,J=8.0Hz,1H),5.56(t,J=5.8Hz,1H),5.11(dd,J=13.2, 5.1Hz,1H),4.22(d,J=17.1Hz,1H),4.12(d,J=17.1Hz,1H),3.72(t,J=6.2Hz,2H),3.56(q,J=8.1,7.0Hz,2H),3.52(s,8H),3.29(t,J =5.8Hz,1H),2.92(ddd,J=17.3,13.6,5.4Hz,1H),2.59(t,J=6.1Hz,3H),2.34(d,J=5.3Hz,6H),2.31–2.24(m,2H),2.06–1.99(m,1H).

[0211] Example 1-30. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(1-methyl-2,6-dioxoylidenehexahydropyridin-3-yl)-1,3-dioxoylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxoylidene-3,6,9-trioxadodec-12-yl)amino]benzamide (LS-30):

[0212]

[0213] Similar to the synthesis scheme of Example 1-19, 2-(2,6-dioxypyridin-3-yl)-4-fluoroisoindole-1,3-dione was replaced with 4-fluoro-2-(1-methyl-2,6-dioxypyridin-3-yl)isoindole-1,3-dione, and 3-[(2-aminoethyl)oxy]propanoic acid-2-methylprop-2-yl ester was replaced with 3-[(8-amino-3,6-dioxaoct-1-yl)oxy]propanoic acid-2-methylprop-2-yl ester, and the reaction conditions were changed to the crude product obtained by column chromatography to obtain compound LS-30. 1H NMR (500MHz, DMSO-d6) δ12.77(s,1H),10.16(s,1H),8.31(s,1H),7.83(dd,J=7.9,1.9Hz,2H),7.73(d,J=9.4Hz,1H),7.60–7 .53(m,2H),7.46(t,J=7.9Hz,1H),7.12(d,J=8.6Hz,1H),7.03(d,J=7.0Hz,1H),6.58(t,J=5.8Hz,1H),5.12(dd,J=13.0,5.4 Hz,1H),3.71(t,J=6.2Hz,2H),3.59(t,J=5.5Hz,2H),3.53(d,J=4.6Hz,8H),3.44(q,J=5.6Hz,2H),3.01(s,3H),2.75(ddd,J =17.1,4.4,2.6Hz,1H),2.58(t,J=6.2Hz,2H),2.34(d,J=5.2Hz,6H),2.04(ddq,J=10.7,5.3,2.6Hz,1H),0.88–0.80(m,2H).

[0214] Example 1-31. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-4-yl]oxy}-9-oxypyridin-3,6-dioxanonan-9-yl)amino]benzamide (LS-31).

[0215]

[0216] 2-(2,6-Dioxyylidenehexahydropyridin-3-yl)-4-hydroxyisoindole-1,3-dione and 2-methylpropan-2-yl 3-({2-[(2-bromoethyl)oxy]ethyl}oxy)propanoate were dissolved in 2 mL of DMF. Sodium bicarbonate was added to the mixture, and the mixture was reacted at 60°C for 10 h under nitrogen. After completion of the reaction, the mixture was extracted with ethyl acetate and water. The organic phase was collected, washed with saturated sodium chloride solution, combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The product was separated and purified by column chromatography to obtain 2-methylpropan-2-yl 3-({2-[(2-{[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]oxy}ethyl)oxy]ethyl}oxy)propanoate.

[0217] 2-Methylpropane-2-yl 3-({2-[(2-{[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]oxy}ethyl)oxy]ethyl}oxy)propanoate was dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.5 mL) was added, and the mixture was stirred at room temperature for 3 h. After the reaction was completed, most of the solvent was dried and directly dissolved in DMSO (2 mL). Intermediate L1, HATU, and DIPEA were then added to the reaction solution and stirred at room temperature for 3 h. After completion of the reaction, the mixture was extracted with EA and water, and the crude product obtained was subjected to silica gel column chromatography to obtain the target product, N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-4-yl]oxy}-9-oxypyridin-3,6-dioxanonan-9-yl)amino]benzamide (LS-31). 1 H NMR(500MHz,DMSO-d6)δ12.76(s,1H),11.10(s,1H),10.17(s,1H),8.31(s,1H),7.85–7.74( m,5H),7.51–7.39(m,3H),5.08(dd,J=12.8,5.4Hz,1H),4.31(t,J=4.6Hz,2H),3.83–3.77(m, 2H),3.74(t,J=6.2Hz,3H),3.66(dd,J=5.9,3.5Hz,2H),3.57(dd,J=5.9,3.6Hz,2H),2.88(dd d,J=16.9,13.9,5.5Hz,1H),2.59(t,J=6.2Hz,2H),2.35(d,J=4.9Hz,7H),2.05–1.98(m,1H).

[0218] Example 1-32. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(2-{4-[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-4-yl]piperazin-1-yl}acetyl)amino]benzamide (LS-32).

[0219]

[0220] 2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (500 mg, 1.810 mmol) and 2-methylprop-2-piperazine-1-carboxylate (506 mg, 2.715 mmol) were dissolved in 2 mL of dimethyl sulfoxide (DMSO). N,N-diisopropylethylamine (DIPEA) (468 mg, 3.620 mmol) was added to the mixed solution. The mixture was reacted at 90 ° C under nitrogen protection for 10 h. After completion of the reaction, the mixture was extracted with ethyl acetate and water, the organic phase was collected, washed with saturated sodium chloride solution, the organic phases were combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Column chromatography separation and purification gave 2-methylprop-2-yl 4-[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]piperazine-1-carboxylate (795 mg).

[0221] Dissolve 2-methylpropane-2-yl-4-[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-4-yl]piperazine-1-carboxylate (795 mg, 1.799 mmol) in dichloromethane (5 mL). Add trifluoroacetic acid (2.5 mL) and stir at room temperature for 3 hours. After the reaction, most of the solvent was evaporated to dryness to obtain 2-(2,6-dioxypyridin-3-yl)-4-(piperazin-1-yl)isoindole-1,3-dione (755 mg).

[0222] 2-(2,6-Dioxyylidenehexahydropyridin-3-yl)-4-(piperazin-1-yl)isoindole-1,3-dione (755 mg, 2.207 mmol) was dissolved in DMF (2 mL). 2-Methylpropan-2-yl bromoacetate (516 mg, 2.649 mmol) and DIPEA (571 mg, 4.414 mmol) were added to the reaction mixture. The reaction was allowed to proceed at room temperature for 10 h. After completion, the mixture was extracted with ethyl acetate and water. The organic phases were collected, washed with saturated sodium chloride solution, combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The product was separated and purified by column chromatography to obtain 2-methylpropan-2-yl {4-[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]piperazin-1-yl}acetate.

[0223] 2-Methylpropan-2-yl {4-[2-(2,6-dioxyylidenehexahydropyridin-3-yl)-1,3-dioxyylidene-2,3-dihydro-1H-isoindol-4-yl]piperazin-1-yl}acetate was dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.5 mL) was added, and the mixture was stirred at room temperature for 3 h. After the reaction, most of the solvent was dried, and 2-methylpropane-2-yl {4-[2-(2,6-dioxypyridin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]piperazin-1-yl}acetate (100 mg, 0.250 mmol) was directly dissolved in DMSO (2 mL). Subsequently, intermediate L1 (74 mg, 0.250 mmol), HATU (143 mg, 0.375 mmol) and DIPEA (97 mg, 0.750 mmol) were added to the reaction solution and stirred at room temperature for 3 h. After completion of the reaction, the mixture was extracted with EA and water, and the crude product obtained was subjected to silica gel column chromatography to obtain the target product, N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(2-{4-[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-4-yl]piperazin-1-yl}acetyl)amino]benzamide (LS-32). 1 H NMR(500MHz,DMSO-d6)δ12.77(s,1H),11.09(s,1H),10.02(s,1H),8.36(s,1H),7.99–7.84(m,2H),7.78–7.70(m,2H),7.61–7.49(m, 2H), 7.39 (d, J = 9.0Hz, 2H), 5.11 (dd, J = 12.8, 5.4Hz, 1H), 3.42 (s, 4H), 3.28 (s, 3H), 2.77 (s, 3H), 2.35 (d, J = 5.1Hz, 6H), 1.24 (s, 4H).

[0224] Example 1-33. Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(3-{4-[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-4-yl]piperazin-1-yl}propanoyl)amino]benzamide (LS-33).

[0225]

[0226] Similar to the synthesis scheme of Example 1-32, 2-methylprop-2-yl bromoacetate was replaced with tert-butyl 3-bromopropionate, and the crude product obtained was purified by column chromatography to obtain compound LS-33. 1H NMR(500MHz,DMSO-d6)δ12.78(s,1H),11.09(s,1H),10.28(s,1H),8.31(s,1H),7.84(t, J=6.7Hz,2H),7.76(s,1H),7.70(q,J=6.9,5.9Hz,1H),7.58(s,1H),7.48(t,J=7.9Hz,1H) ,7.36(dd,J=7.9,4.6Hz,2H),5.10(dd,J=12.8,5.4Hz,1H),3.31(s,6H),2.75(t,J=7.2Hz ,2H),2.67–2.60(m,5H),2.58(d,J=6.7Hz,1H),2.35(d,J=5.2Hz,7H),2.05–2.00(m,1H).

[0227] Example 1-34. Preparation of N-(5,6-dimethoxybenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxyhexahydropyridin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxyylidene-3,6,9-trioxadodec-12-yl)amino]benzamide (LS-34).

[0228]

[0229] Similar to the synthesis scheme of Example 1-23, 5,6-dimethylbenzo[d][1,3]thiazolin-2-amine was replaced with 5,6-dimethoxy-1,3-benzothiazol-2-amine, and the crude product obtained was purified by column chromatography to obtain compound LS-34. 1H NMR (500MHz, DMSO-d6) δ12.71(s,1H),11.09(s,1H),10.16(s,1H),8.31(s,1H),7.82(t,J=6.3Hz,2H),7.59(d,J=1.7Hz, 1H),7.59–7.49(m,1H),7.47(t,J=7.9Hz,1H),7.31(s,1H),7.14–6.96(m,2H),6.57(dt,J=12.3,6.0Hz,1H),5.05(dd,J=1 2.7,5.4Hz,1H),4.09(q,J=5.2Hz,2H),3.84(d,J=7.8Hz,6H),3.72(t,J=6.2Hz,2H),3.61–3.54(m,2H),3.51–3.38(m,2H) ,3.17(d,J=5.3Hz,5H),2.88(ddd,J=16.7,13.6,5.3Hz,1H),2.62–2.56(m,2H),2.02(tt,J=7.6,4.7Hz,1H),1.24(s,3H).

[0230] Example 1-35 Preparation of N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-{[2-(4-{1-[2-(2,6-dioxypyridin-3-yl)-1,3-dioxypyridin-2,3-dihydro-1H-isoindol-4-yl]pyridin-4-yl}pyridin-1-yl)acetyl]amino}benzamide (LS-35):

[0231]

[0232] Similar to the synthesis scheme of Example 1-32, 2-methylpropane-2-ylpiperazine-1-carboxylate was replaced by 2-methylpropane-2-yl [4-(hexahydropyridin-4-yl)hexahydropyridin-1-yl]acetate, and the crude product obtained was purified by column chromatography to obtain compound LS-35. 1H NMR (500MHz, DMSO-d6) δ12.78(s,1H),11.09(s,1H),9.89(s,1H),8.34(s,1H),7.93(d,J=8.3Hz,1H),7.85(d,J=7.5Hz,1H),7.76(s,1H),7 .68(dd,J=8.4,7.1Hz,1H),7.58(s,1H),7.50(t,J=7.9Hz,1H),7.33(t,J=7.4Hz,2H),5.09(dd,J=12.7,5.5Hz,1H),4.46-4.42(m,1H),4.1 3-4.00(m,1H),3.74(s,1H),3.42-3.37(m,1H),3.17(d,J=5.1Hz,1H),2.97–2.85(m,3H),2.82(d,J=12.0Hz,1H),2.56(d,J=13.4Hz,1H),2 .35(d,J=5.0Hz,6H),2.29(t,J=7.4Hz,1H),2.16(d,J=23.8Hz,2H),2.06-2.00(m,1H),1.83(s,1H),1.73(s,1H),1.40(s,3H),1.24(s,5H).

[0233] Example 2 Evaluation of the Effect of the Compounds of the Present Invention on Inhibiting KLF5 Expression in Tumor Cells

[0234] Example 2-1 Cell culture

[0235] The triple-negative breast cancer cells HCC1806, HCC1937, and SUM149PT used in the present invention were obtained from the Kunming Cell Bank of the Chinese Academy of Sciences. HCC1806, SUM149PT, and HCC1937 were cultured in RPMI 1640 medium plus 10% FBS in a 37°C, 5% CO2 constant temperature cell incubator.

[0236] Example 2-2 Evaluation of the KLF5 degradation-inducing activity of the compounds of the present invention

[0237] A series of KLF5-targeting compounds (LS-01 to LS-48) synthesized in Example 1 were dissolved in DMSO to final concentrations of 1 μmol / L and 10 μmol / L, respectively. An equal amount of DMSO was added to the control group and treated with breast cancer cells SUM149PT and HCC1806, respectively, for 24 hours. The cells were harvested by centrifugation and denatured for lysis to collect total cell protein. The compounds were then analyzed for KLF5 inhibition and degradation using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0238] The Western Blot protocol is as follows: After cell culture under experimental conditions, cells were washed with cold phosphate-buffered saline (PBS) and collected. The cell pellet was solubilized with 2× protein sample buffer (1 M Tris-HCl, pH 6.8, 50% glycerol, 10% SDS, 2-mercaptoethanol, 1% bromophenol blue) and boiled at 100°C for 10 min. The prepared protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Millipore). The membrane was blocked with 5% skim milk solution to facilitate the specific coupling reaction between the protein and the antibody on the PVDF membrane. The skim milk solution was then discarded, and the membrane was washed with PBST (PBS supplemented with 0.5% Tween 20). The coupling reaction between the protein and the antibody on the membrane was carried out overnight at 4°C, followed by another wash with PBST. Finally, a secondary antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology) was diluted in PBST and reacted at room temperature for 2 hours. The protein content on the membrane was determined using Luminata Forte HRP substrate (Millipore).

[0239] Image J software was used to analyze the grayscale values ​​of KLF5 and Vinculin in different lanes. The relative grayscale ratio of KLF5 / Vinculin corresponding to each compound and the relative grayscale ratio of KLF5 / Vinculin in the control group were calculated in Excel. The KLF5 protein degradation rate (%) was calculated according to the following formula. Vinculin was used as the internal reference protein.

[0240]

[0241] The ability of compounds LS-01 to LS-32 of the present invention to degrade KLF5 in tumor cells is expressed as the KLF5 protein degradation rate (%) of the compounds at concentrations of 1 μmol / L and 10 μmol / L. "a," "b," "c," and "d" indicate that the degradation rate of KLF5 in tumor cells by the compounds of the present invention at these concentrations is less than 25%, between 25% and 50%, between 50% and 75%, and greater than 75%, respectively. "NT" indicates that the ability of the compound to induce KLF5 degradation at the specified concentration was not evaluated. The results of the evaluation of the KLF5 degradation efficiency of the compounds of the present invention in breast cancer cells SUM149PT and HCC1806 are shown in Table 1.

[0242] Table 1 Evaluation results of compounds LS-01 to LS-35 of the present invention inducing KLF5 degradation in different tumor cells

[0243]

[0244]

[0245] The results in Table 1 show that the compounds of the present invention exhibited significant KLF5 protein degradation in at least one breast cancer cell line at a concentration of 10 μmol / L. Some compounds, such as LS-04, LS-05, LS-06, LS-07, LS-09, LS-24, LS-26, and LS-31, were able to induce greater than 50% KLF5 protein degradation in triple-negative breast cancer HCC1937 and HCC1806 cells at a concentration of 10 μmol / L. LS-23 and LS-24 were able to induce greater than 50% KLF5 protein degradation in breast cancer SUM149PT cells at a concentration of 1 μmol / L.

[0246] In order to further illustrate that the compounds of the present invention have the ability to induce KLF5 protein degradation in tumor cells, the present invention selected some compounds for Western blot (WB) experiments for confirmation. Figure 1 The western blot results of some representative compounds of the present invention that induced KLF5 degradation in breast cancer cells HCC1806, HCC1937, and SUM149PT were shown. The results showed that LS-22, LS-23, NTZ, etc. had obvious ability to degrade KLF5 protein.

[0247] Example 2-3 DC activity of representative compound LS-23 in HCC1806 and SUM149PT cells 50 and Dmax

[0248] To evaluate the ability of the compounds described herein to degrade KLF5 in different breast cancer cell lines, HCC1806 and SUM149PT cells were treated with LS-23, a representative compound from the examples described herein, for 24 hours. The cells were then harvested and lysed with an appropriate amount of SDS protein lysis buffer. The cells and lysate were thoroughly mixed by pipetting. After mixing, the cells were denatured in a metal bath at 98°C for 10 minutes. The denatured proteins were separated by 10% SDS-PAGE electrophoresis (stacking gel 80 V, 40 min; separating gel 120 V, 90 min), and then transferred to a PVDF membrane using the wet transfer method (250 mA, 120 min), blocked with 5% skim milk for 1 hour, incubated with primary antibodies (KLF5 (CST, #5650S), Vinculin (MCE, HY-P80372)) at 4°C overnight, washed three times with TBST buffer (10 min / time), incubated with secondary antibodies (Millipore, 401315) at room temperature for 1 hour, washed three times with TBST buffer (10 min / time), and developed after incubation with enhanced chemiluminescence horseradish peroxidase substrate (ThermoFisher Scientific, 32106).

[0249] Image J software was used to analyze the gray values ​​of KLF5 and Vinculin in different lanes. The relative gray ratio of KLF5 / Vinculin corresponding to each compound and the relative gray ratio of KLF5 / Vinculin in the control group were calculated in Excel. The KLF5 protein degradation rate (%) was calculated according to the following formula.

[0250]

[0251] DC was calculated using Graphpad Prism software 50 and D max , and draw a compound concentration-KLF5 protein degradation curve, see Figure 2 , Figure 2 DCs that degrade KLF5 protein in HCC1806 and SUM149PT cells by the representative compound LS-23 in this example 50 and D max .like Figure 2 As shown, LS-23 in HCC1806 and SUM149PT cells 50 0.926 μmol / L and 1.384 μmol / L respectively, D max The results showed that the representative compound LS-23 in the examples of the present invention can significantly degrade KLF5 protein in triple-negative breast cancer cell lines, further demonstrating the effect of the compounds of the present invention in degrading KLF5 protein.

[0252] Degradation activity of representative compounds such as Figure 1 As shown in A, the effect of LS-23 in degrading KLF5 protein is stronger than that of NTZ and other compounds, and has a certain concentration gradient dependence.

[0253] Example 3: Evaluation of the antiproliferative activity of the compounds of the present invention in tumor cells

[0254] CCK8 assay to determine the inhibitory activity of compounds on breast cancer cell proliferation

[0255] The CCK8 method was used to detect cell growth inhibition. The test compound was dissolved in DMSO (dimethyl sulfoxide), and then prepared using cell culture medium to prepare compound concentrations of 10 μM in 0.1% DMSO solution, 1 μM in 0.1% DMSO solution, and 0.1 μM in 0.1% DMSO solution.

[0256] Proliferation is measured using the CCK8 cell proliferation kit: cells in the logarithmic growth phase are digested and prepared into a single-cell suspension. These cells are seeded at a specific density into a 96-well plate or other culture plate. Experimental and control groups are then set up and placed in an incubator for a specified period of time to allow the cells to adhere and grow. Depending on the experimental requirements, 1 μL of each compound dissolved in DMSO is added to each well of the cell culture, resulting in a final concentration of one percent of the original concentration. For the control group, the same volume of DMSO alone is added to the cell culture. The culture is continued for an appropriate duration. After 72 hours, 10 μL of fresh culture medium containing CCK8 is added to each well and incubated for 1 hour to allow the reagent to fully react with the viable cells. The absorbance of each well is measured using a microplate reader at a specific wavelength (around 450 nm). By comparing the absorbance data of different groups, cell proliferation is analyzed. Higher absorbance values ​​generally indicate a greater number of viable cells and better proliferation. The percentage of the absorbance value of the experimental group relative to the absorbance value of the control group represents the cell survival rate or cell proliferation level, with the control group defaulted to 100%.

[0257] Statistical analysis was continued on the effects of other compounds of the present invention at different concentrations on breast cancer cell proliferation. GraphPadPrism software was used for the IC values ​​of different compounds. 50 The calculation and test results are shown in Table 2.

[0258] The inhibitory effects of compounds LS-01 to LS-32 on tumor cell proliferation were analyzed by IC 50 Indicated by “+”, “++” and “+++” respectively represent the IC 50The IC values ​​of the compounds of the present invention on breast cancer cells HCC1937, HCC1806 and breast cancer cells SUM149PT were greater than 10 μmol / L, between 1.0 μmol / L and 10 μmol / L and less than 1.0 μmol / L. "NT" indicates that the anti-proliferative effect of the compound on these tumor cells has not been evaluated. 50 As shown in Table 2.

[0259] Table 2 Evaluation results of the antiproliferative activity of compounds LS-01 to LS-35 in different breast cancer tumor cells

[0260]

[0261]

[0262] The results showed that compounds LS-01 to LS-18 of the present invention had certain proliferation inhibitory activity against breast cancer cells HCC1937, HCC1806, and SUM149PT, while compounds LS-19 to LS-32 had significant proliferation inhibitory activity against breast cancer cells HCC1937, HCC1806, and SUM149PT. Compounds LS-22, LS-23, LS-24, LS-25, LS-26, and LS-31 had IC values ​​of 0.05 for breast cancer cells HCC1806 and SUM149PT. 50 The range is between 1.0 μmol / L and 10 μmol / L. The cell inhibitory activity of representative compounds is as follows Figure 1 As shown in Figure 3B, NTZ, LS-21, LS-28, LS-22, LS-26, LS-31, LS-24, LS-25, LS-27, and LS-23 all had strong inhibitory effects on HCC1806 and SUM149PT cells, and the cell survival rate was less than 50% at the concentration of LS-23 at 1 μM.

[0263] In order to further test the proliferation inhibitory activity of the compounds of the present invention in breast cancer, representative compounds from the examples of the present invention were selected to further determine the proliferation inhibitory activity of the compounds on various breast cancer cells using the SRB method. Figure 1 As shown in Figure C, LS-23 has strong inhibitory activity against breast cancer cells of different subtypes (TNBC: triple-negative breast cancer, HER2+: HER2-positive breast cancer, Luminal: Luminal breast cancer).

[0264] To further evaluate the effects of the compounds of the present invention on the proliferation of other tumor cells, the present invention selected a representative compound, LS-23, and tested its anti-proliferative activity in various other cancer cells. The results are shown in Table 3. The test results show that the compound LS-23 has strong cytotoxic activity against lung cancer cells, liver cancer cells, leukemia cells, colorectal cancer cells, prostate cancer cells, human glioblastoma cells, and human gastric cancer cell lines, demonstrating that the compounds of the present invention have good therapeutic potential against various tumors.

[0265] Table 3 Evaluation results of the antiproliferative activity of the compound LS-23 of the present invention in different tumor cells

[0266] cell lines <![CDATA[IC 50 (μM)]]> cell lines <![CDATA[IC 50 (μM)]]> Human lung cancer A549 cells 11.19 Human colorectal cancer cell line HCT116 18.24 Human hepatocellular carcinoma cell line SMMC-7721 7.124 Human prostate cancer cell PC3 4.733 Human hepatocellular carcinoma cell line LM3 8.29 Human glioblastoma cells T98G 0.655 Human leukemia cells Jurkat 3.156 Human gastric cancer cell line MKN45 20.61

[0267] Example 4: Detection of LS-30 and LS-23 DCs using cell viability assays and Western Blot assays 50 (compound treatment concentration at which half of the protein is degraded) and IC 50 (Compound treatment concentration at which cell proliferation is inhibited by half)

[0268] The CCK8 and Western Blot assay protocols were as described above.

[0269] Experimental results: After adding LS-23 and LS-30 to SUM149PT and HCC1806, the differences in cell activity and KLF5 protein levels were compared. Figure 2 As shown in A, it can be seen that LS-23 can significantly degrade KLF5, while LS-30 has a poor effect in degrading KLF5. Figure 2 As shown in Figure B, LS-23 has good anti-tumor activity in SUM149PT and HCC1806 cells, and its anti-tumor activity is stronger than that of LS-30.

[0270] Example 5: Cell scratch assay

[0271] The method of selecting compounds with better activity and using the scratch healing experiment to detect the effect of the compounds on cell migration is as follows: the cells are plated in a 6-well plate. After the cells grow into a dense layer, a 200μL pipette tip is used to scratch the middle of the cell layer. Use 1×PBS to wash away the cell debris, then add complete culture medium containing low concentration of serum and different concentrations of compounds and place them in the incubator for continued cultivation. Immediately, three scratches are randomly selected for image acquisition. Signals are collected at the same position every 4 hours. The experiment is terminated when one group of scratches is close to healing. The migration ability of the cells is analyzed by analyzing the degree of healing of the planned scratch area. Cell migration rate = (initial scratch width - final scratch width) / initial scratch width × 100%.

[0272] Test results: Figure 3As shown in Figures AB, wound healing experiments were performed on SUM149PT and HCC1806 cell lines, using different concentrations of LS-23 (0.5, 1, and 2 μM). Images were then taken at 24 and 48 hours. It was found that increasing LS-23 concentration significantly inhibited cancer cell migration. Figure 3 A is the migration picture, Figure 3 B is a statistical graph of migration rate. *p<0.05, **p<0.01, ***p<0.001. NC is the control group.

[0273] Example 6: Clonal sphere formation experiment

[0274] After digestion, count the cultured cells, take an appropriate amount of cell suspension and inoculate it into a 6-well plate (800 cells per well), add different concentrations of drugs for culture, and use an equal amount of DMSO as a control. Culture for 7-14 days until clones are visible to the naked eye. When clones are formed, remove the culture medium and wash the cells 1-2 times with PBS. Add an appropriate amount of paraformaldehyde solution to fix the cells for 10-15 minutes to fix the cells on the bottom of the culture plate. Remove the fixative and wash the cells again with PBS. Add crystal violet staining solution and stain for 10-30 minutes to dye the clones purple or blue for easy observation and counting. Wash off the excess staining solution with PBS and dry the culture plate or dish. Observe and count the clones under a microscope. The size and number of clones can be defined according to experimental requirements. It is generally considered that a cell cluster containing more than 50 cells is a clone. Use automatic cell counting software or manually count the number of clones.

[0275] Test results: Figure 4 Figures AB show colony formation experiments conducted in SUM149PT and HCC1806 cell lines. Different concentrations of LS-23 were administered and cultured for 14 days before staining and photographing to quantify the results. It was found that increasing LS-23 concentrations inhibited the number of cancer cell colonies. Figure 4 A is a picture of clone formation, Figure 4 B is a statistical graph of colony formation. **p<0.01, ***p<0.001. DMSO served as the control group.

[0276] Example 7: Cell apoptosis experiment

[0277] For cell apoptosis detection, cells were stained with FITC Annexin V and PI dyes, and the percentage of apoptotic cells was analyzed by flow cytometry according to the kit instructions.

[0278] Test results: Figure 5As shown in Figures A and B, SUM149PT cells were treated with LS-2 for 48 hours, and the percentage of apoptotic cells was analyzed by staining. Quantitative results show that the number of apoptotic cells increased significantly with increasing LS-23 concentration. Data were analyzed using a Student's T-test. ns, no significant difference; ***, p < 0.001. DMSO served as the control group. Figure 5 A is the flow cytometry analysis picture of apoptosis. Figure 5 B is a statistical diagram of apoptosis.

[0279] Example 8: Evaluation of the inhibitory activity of LS-23, a representative compound of the present invention, on the growth of breast cancer xenografts in nude mice

[0280] To evaluate the potential therapeutic effect of the compounds of the present invention on tumors in vivo, we took HCC1806 cells in the logarithmic growth phase, digested them, collected them, and performed cell counting. 6 After centrifugation, the cells were resuspended in pre-cooled 1× PBS and then Matrigel was added. After thorough mixing with a pre-cooled pipette tip, 75 μL of each injection volume was injected into the fourth pair of mammary fat pads of the mice using an insulin injection needle. One week after inoculation, the mice were observed for in situ tumor formation. When the tumor volume grew to 50-60 mm 3 The mice were randomly divided into groups of 5 each, and different drug treatment concentration groups were set up. The drug injection solution was prepared according to the formula of 100 μL per mouse, 10% (DMSO or drug) drug mixture + 30% PEG300 + 5% Towen80 + 30% normal saline. Intraperitoneal administration was continued every 3 days, and one day was stopped. The weight of the mice was weighed and recorded. At the same time, the mouse tumor was measured with a vernier caliper, and the tumor volume was calculated by the formula V = 0.5 × length × width 2. After the drug treatment, the mice were killed by cervical dislocation. The tumor tissue was separated using surgical instruments, weighed, photographed, and the mouse serum was taken for subsequent liver and kidney function tests.

[0281] Figure 6 The results show the in vivo inhibition of compound LS-23 on human breast cancer xenograft tumors in mice; Figure 6 AB are schematic diagrams of the establishment of an orthotopic breast cancer model in mice and the dosing schedule and frequency. After 13 consecutive days of dosing, the mice were euthanized, and the tumors were removed and photographed. Figure 6 C is the tumor volume measured every 2 days after the start of treatment, which is represented by a line graph. Figure 6 C shows that compared with the control group, after administration of compound LS-23, the tumor volume of mice was lower than that of the control group; the tumor weight of mice in the control group (DMSO) and experimental groups (20 mg / kg and 40 mg / kg) was lower than that of the control group; Figure 6D shows the changes in the body weight of mice after administration of compound LS-23. There is basically no difference in the body weight of mice in the experimental group and the control group; Figure 6 E represents the completion of drug administration. After the mice were anesthetized, orbital blood was collected and the level of aspartate aminotransferase in the serum was measured to evaluate the hepatotoxicity of compound LS-23; the level of alanine aminotransferase in the serum was measured to evaluate the hepatotoxicity of compound LS-23; and the level of creatinine in the serum was measured to evaluate the nephrotoxicity of compound LS-23; **P<0.01, ***P<0.001, ns represents no difference; Figure 6 As can be seen from E, compound LS-23 significantly inhibited the growth of tumor tissue in mice without causing liver and kidney toxicity. Figure 6 As can be seen in Figure F, in the tumor tissues of mice in the control group (DMSO) and the experimental group (20 mg / kg and 40 mg / kg), the KLF5 level in the tumor tissues of the experimental group was significantly reduced, proving that LS-23 has the effect of degrading KLF5 in vivo.

[0282] The use and welfare of experimental animals were carried out in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals were monitored daily. Routine examinations included observing the effects of the test substances and drugs on the animals' daily behaviors, such as activity, weight changes, and physical appearance.

[0283] The results showed that the representative compound LS-23 in the examples of the present invention has a good in vivo anti-cancer effect, can effectively inhibit the growth of breast cancer cells in mice, and does not significantly affect the weight and liver and kidney function of the mice.

[0284] Example 9: Evaluation of the binding activity of LS-23, a representative compound of the present invention, to KLF5 protein

[0285] The binding and binding force of LS-23 and KLF5 were determined using SPR (surface plasmon resonance) experiments: the KLF5 protein was coupled to a CM5 chip using a Biacore S200 instrument, coupling buffer: sodium acetate buffer pH = 4.5; regeneration solution: Glyline pH = 2.5; running buffer: PBSP (PBS + 0.005% P20 + 5% DMSO); LS-23 concentrations of 0 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 20 μM, and 30 μM were used for binding tests with the coupled KLF5 protein.

[0286] Test results: Figure 7As shown, 0.5 μM of compound LS-23 begins to bind to the KLF5 protein (RU>0, the vertical axis represents the binding ability of compound LS-23 to the KLF5 protein; larger values ​​indicate stronger binding ability, and the horizontal axis represents the reaction time). The binding signal increases with increasing molar concentration of compound LS-23. The results show that the maximum dissociation constant for the binding of compound LS-23 to the deubiquitinating enzyme KLF5 is KD = 9.507 μM. These results indicate that compound LS-23 can directly bind to the KLF5 protein.

[0287] In summary, the compounds of the present invention not only exhibit KLF5 protein degradation induction activity, but also exhibit significant anti-proliferative effects and exhibit significant dose-dependent inhibition of tumor growth in vivo, and are a class of effective compounds with potential for the treatment of triple-negative breast cancer.

[0288] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A compound targeting the degradation of transcription factor KLF5, characterized in that: The compound is selected from one or more of the following compounds: 3-(Acetylamino)-N-(5-fluorobenzo[d][1,3]thiazolin-2-yl)benzamide (LS-04); 3-(Acetylamino)-N-(7-fluorobenzo[d][1,3]thiazolin-2-yl)benzamide (LS-05); 3-(Acetylamino)-N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)benzamide (LS-06); 3-(Acetylamino)-N-[6-(trifluoromethyl)benzo[d][1,3]thiazolin-2-yl]benzamide (LS-07); 3-(Acetylamino)-N-(6-chlorobenzo[d][1,3]thiazolin-2-yl)benzamide (LS-09); N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-4-yl]amino}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide (LS-22); N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxadienylhexahydropyridin-3-yl)-1,3-dioxadienyl-2,3-dihydro-1H-isoindol-4-yl]amino}-12-oxadienyl-3,6,9-trioxadodec-12-yl)amino]benzamide (LS-23); N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxadienylhexahydropyridin-3-yl)-1,3-dioxadienyl-2,3-dihydro-1H-isoindol-4-yl]amino}-15-oxadienyl-3,6,9,12-tetraoxapentadecan-15-yl)amino]benzamide (LS-24); N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxadienylhexahydropyridin-3-yl)-1,3-dioxadienyl-2,3-dihydro-1H-isoindol-4-yl]amino}-18-oxadienyl-3,6,9,12,15-pentaoxaoctadec-18-yl)amino]benzamide (LS-25); N-(5,6-dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-5-yl]amino}-9-oxanylidene-3,6-dioxanonan-9-yl)amino]benzamide (LS-26); N-(5,6-Dimethylbenzo[d][1,3]thiazolin-2-yl)-3-[(1-{[2-(2,6-dioxanylidenehexahydropyridin-3-yl)-1,3-dioxanylidene-2,3-dihydro-1H-isoindol-5-yl]amino}-12-oxanylidene-3,6,9-trioxadodec-12-yl)amino]benzamide (LS-31).

2. A pharmaceutical composition, characterized in that The invention comprises the compound for targeting and degrading the transcription factor KLF5 as claimed in claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition includes a second agent for preventing and / or treating cancer.

4. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition includes an excipient, a diluent, an adjuvant, a vehicle or a combination thereof.

5. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition is formulated as an injectable fluid, aerosol, cream, gel, pill, capsule, syrup or transdermal patch.

6. Use of the compound according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a KLF5 regulator or an anti-tumor drug.

7. The use according to claim 6, characterized in that The tumor includes one or more of breast cancer, liver cancer, lung cancer, colorectal cancer, leukemia, gastric cancer, glioma and prostate cancer.

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