CN120136812B

CN120136812B - Compound for targeted degradation of transcription factor KLF5 and application thereof

Info

Publication number: CN120136812B
Application number: CN202510307009.5A
Authority: CN
Inventors: 陈策实; 陈益华; 张龙龙; 李申; 杨书
Original assignee: Kunming Medical University
Current assignee: Kunming Medical University
Priority date: 2025-03-15
Filing date: 2025-03-15
Publication date: 2025-09-30
Anticipated expiration: 2045-03-15
Also published as: CN120136812A

Abstract

The present invention discloses a class of compounds and applications thereof that target the degradation of transcription factor KLF5. 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

Compound for targeted degradation of transcription factor KLF5 and application thereof

Technical Field

The invention belongs to the technical field of antitumor drugs, and particularly relates to a compound for targeted degradation of transcription factor KLF5 and application thereof.

Background

Cancer is increasingly becoming a global problem, breast cancer remains the most common female cancer, accounting for 23% of the total number of cancer cases. Although mortality from breast cancer has been decreasing in many developed countries, with continued improvements in diagnostic techniques and treatments, the incidence of breast cancer has increased worldwide, being a major cause of cancer death in women, causing serious social health problems and economic hazards (Arch Gynecol obset.2016; 293 (2): 247-69.). According to whether the estrogen receptor (ERalpha) and the Progestogen Receptor (PR) are expressed or not, whether the human epidermal growth factor receptor 2 (HER 2) is amplified or not, breast cancers are classified into LuminalA type, luminalB type, HER2 type and triniyin Type (TNBC), and TNBC has high chemosensitivity, high invasiveness, high transfer probability and high recurrence rate, and the survival time after transfer is shorter, so that the method is important and difficult for treating the breast cancers. (Breast CANCER RES Treat.2018;167 (2): 459-468.) there is currently no therapeutic regimen targeting TNBC, so finding molecular targets for TNBC and developing new drugs remain the current focus of research.

Transcription factors are key factors for transcriptional regulation and play an important role in physiological and pathological processes. Transcription factors are widely studied in various diseases, especially cancers, and become an important novel drug target. KLF5 is a member of the Kruppel-like transcription factor family and is widely expressed in a variety of tissues including the digestive tract, pancreas, placenta, testis, prostate, skeletal muscle, lung, bladder and uterus, and the like. As a transcription factor of zinc finger structure, KLF5 is capable of regulating the expression of a variety of genes. The study shows 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 suggests that KLF5 promotes stem cell, proliferation, survival, adhesion and migration of breast cancer cells. KLF5 inhibits transcription of p27 and p21 by inducing transcription of FGF-BP1, mPGES1, TNFAIP2 and Cyclin D14 in breast cancer, promoting cancer cell proliferation and cell cycle. KLF5 maintains the stem cellularity of cells by inducing the transcription of Slug and Nanog. The mammary gland specific KLF5 gene knockout mice can obviously reduce proliferation, survival and dryness of mammary gland epithelial cells and inhibit PyMT induced tumorigenesis. KLF5 can promote the proliferation of bladder Cancer cells, and KLF5 can combine with a Cyclin E1 gene enhancer to activate transcription of the bladder Cancer cells, so that the susceptibility of bladder Cancer (Mol Cancer Res.2016; 14:1078-1086.) is increased, and besides, KLF5 is closely related to the occurrence and development of various cancers such as prostate Cancer, hepatocellular carcinoma, intestinal Cancer, esophageal squamous cell carcinoma and the like. In addition, high-expression KLF5 was found to be associated not only with early recurrence but also with early death, and KLF5 expression was a prognostic factor for disease-free survival and overall survival of breast cancer patients. The prognostic value of KLF5 may be related to its effect in promoting cell proliferation. (CLIN CANCER Res.2006;12 (8): 2442-8.) in conclusion, KLF5 plays a broad role in cancer of the breast and other diverse cancers, and therefore targeting KLF5 would be of great therapeutic benefit.

Current research on KLF5 inhibitors has focused mainly on the regulation of expression of genes downstream thereof, and reported inhibitors such as mifepristone inhibit KLF5 expression in TNBC by inducing miR-153 and inhibit 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 ubiquitination of KLF5 (Cell discovery.2017; 3:17010.), and mithramycin a inhibits TNBC by inhibiting Sp1 down-regulation of KLF5 (Sci rep.2018;8 (1): 1138.). RSK inhibitor JH685 inhibits YB-1-KLF5 axis and significantly inhibits basal breast cancer cell graft tumor growth (CELL DEATH differ.2022;29 (6): 1283-1295.). JQ1 derivative compounds 870 and PROTAC inhibit basal breast cancer growth by targeted inhibition of BRD4 down-regulating KLF5 expression (Int J Biol Sci.2019;15 (8): 1733-1742.). CID5951923 and its two structural derivatives inhibit proliferation of colorectal Cancer epithelial cells by inhibiting the down-regulation of KLF5 expression by EGR1 (Mol Cancer Ther.2011;10 (11): 2043-51). ML-264, SR18662 induce apoptosis of colorectal Cancer epithelial cells and inhibit tumor growth in vivo by inhibiting down-regulated KLF5 expression by EGR1 (Mol Cancer Ther.2016;15 (1): 72-83;Mol Cancer Ther.2019;18 (11): 1973-1984.). However, the current inhibitors are all indirect inhibitors to KLF5, and there is no effective way to directly target KLF5 to interfere with its function.

Proteolytic targeting chimera (PROTAC) is a promising therapeutic strategy, utilizing endogenous proteasomes to degrade disease-related proteins. PROTAC use of a linker to link the ligand of the E3 ubiquitin ligase with the ligand of the target Protein (POI) (Science 2017;355 (6330): 1163-1167.) and facilitate subsequent degradation of the POI by recruiting the E3 ubiquitin ligase in the vicinity of the POI. In recent years, PROTACs has made remarkable progress in clinical studies (Nat Rev Drug discovery.2022; 21 (3): 181-200.), which has potential therapeutic implications and market development prospects for cancer patients. Compared with the traditional small molecule inhibitor, PROTAC molecules play a role in degrading proteins, have higher selectivity and effectiveness, and show greater advantages than the traditional small molecule drugs in preclinical researches of tumor treatment.

In general, due to the current lack of therapeutic regimens for targeting TNBC, various targeted therapeutic approaches are becoming increasingly interesting. Through continuous research and development, the invention aims to provide a small molecular compound and a degradation agent which can target transcription factor KLF5, and the small molecular organic compound and the degradation agent have potential therapeutic significance and market development prospect for various cancer patients.

Disclosure of Invention

The invention discovers that the insect repellent NTZ (nitazoxanide) is an effective invasion inhibitor in the early screening and experiments. In KLF5 ^k369Q -induced bone metastasis, NTZ exerts potent inhibitory effects in both prophylactic and therapeutic modes. NTZ also inhibits osteoclast differentiation, a cellular process of KLF5 ^K369Q -induced bone metastasis. Based on the research result, the invention uses NTZ as a lead compound and uses a plurality of technologies such as structure-based drug design (SBDD) and the like to greatly change the parent nucleus structure, reduce the rigidity of a structural framework, improve the patentability of the compound and creatively design and synthesize the small molecular compound and the degradation agent of the targeted KLF5 protein by combining PROTACs design strategies. The compound and the degradation agent provided by the invention have obvious inhibition effect on proliferation of various tumor cells, and the representative compound also has obvious anti-tumor effect in tumor-bearing mice. The related results also show that the compound of the invention can directly target KLF5 protein, thereby providing a new idea for TNBC tumor treatment. Compared with the existing KLF5 inhibitor, the compound can directly target KLF5 and can induce KLF5 protein degradation and obviously inhibit proliferation of various tumor cells such as triple negative breast cancer and the like.

The invention provides a small molecule compound for targeted degradation of transcription factor KLF5, which comprises a compound shown in a formula (I), related analogues or pharmaceutically acceptable salts, metabolites or prodrugs of the compound shown in the formula (I), wherein the analogues comprise stereoisomers, geometric isomers, tautomers, nitrogen oxides or solvates;

R ₁ in the formula I is selected from any one or more of halogen, C ₁～C₄ alkyl, hydroxyl, amino, ester, trifluoromethyl, cyano and C ₁～C₄ alkoxy;

R ₂ in the formula I is selected from any one or more of hydrogen, halogen, hydroxyl, cyano, trifluoromethyl, C ₁～C₄ alkyl, C ₁～C₄ alkoxy, morpholine, piperidine and piperazine;

the invention also provides PROTACs compounds of a targeted degradation transcription factor KLF5, which comprise compounds shown in a formula (II), related analogues or pharmaceutically acceptable salts, metabolites or prodrugs of the compounds shown in the formula (II);

Wherein X represents a ligand binding to KLF5 protein, a small molecule compound selected from the group consisting of the targeted degradation transcription factor KLF5, Z represents a ligand of E3 ubiquitin ligase, and Y represents a linking group linking X and Z.

As a further description of the above schemes, X is selected from the group consisting of compounds represented by the following structural formulas;

as a further description of the above schemes, Z is selected from compounds of the structure shown in formula (III):

wherein:

A is selected from any one of CH ₂, C (O), SO or SO ₂;

B, E and W are respectively and independently selected from any one of CH or CF;

M is selected from any one of the following groups:

as a further description of the above schemes, Z is selected from one or more of the following structures:

as a further description of the above schemes, Y is selected from one or more of the following structures:

Wherein, the

N is an integer between 0 and 6;

m is an integer between 0 and 8;

a is an integer between 0 and 5.

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

3- (acetylamino) -N- (benzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide;

3- (acetylamino) -N- (5-methylbenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (5-methoxybenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (5-fluorobenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (7-fluorobenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (5, 6-dimethylbenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- [6- (trifluoromethyl) benzo [ d ] [1,3] thiazacyclopent-2-yl ] benzamide;

3- (acetylamino) -N- (5-chlorobenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (6-chlorobenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (4-chlorobenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (5-bromobenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

5- (acetylamino) -2-chloro-N- (5, 6-dimethylpheno [ d ] [1,3] thiazacyclopentan-2-yl) benzamide;

5- (acetylamino) -N- (5, 6-dimethylbenzo [ d ] [1,3] thiazepin-2-yl) -2-methylbenzamide;

3- (acetylamino) -N- [6- (1, 4-oxazepin-4-yl) benzo [ d ] [1,3] thiazepin-2-yl ] benzamide;

3- (acetylamino) -N- (5, 6-dimethoxy benzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide;

3- (acetylamino) -N- (6-ethoxybenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (4-methoxy-7-methylbenzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide;

3- (acetylamino) -N- [6- (prop-2-yloxy) benzo [ d ] [1,3] thiazepin-2-yl ] benzamide;

n- (5, 6-dimethylbenzo [ d ] [1,3] thiazapenta-2-yl) -3- [ (4- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-5-yl ] amino } -1-oxon-butyl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapenta-2-yl) -3- [ (6- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -1-oxon-exyl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- ({ 3- [ (2- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } ethyl) oxy ] propionyl } amino) benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -9-oxo-3, 6-dioxanon-9-yl) amino ] benzamide;

n- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -12-oxo-3, 6, 9-trioxadode-12-yl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiapentadecan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -15-oxo-3, 6,9, 12-tetraoxapentadecan-15-yl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapenta-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -18-oxo-3, 6,9,12, 15-pentoxaoctadeca-18-yl) amino ] benzamide;

n- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-5-yl ] amino } -9-oxo-3, 6-dioxanon-9-yl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1-oxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -9-oxo-3, 6-dioxanon-9-yl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] oxy } -9-oxo-3, 6-dioxanon-9-yl) amino ] benzamide;

n- (5, 6-dimethylbenzo [ d ] [1,3] thiazapenta-2-yl) -3- [ (2- {4- [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] piperazin-1-yl } acetyl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1-oxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -12-oxo-3, 6, 9-trioxadoden-12-yl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-5-yl ] amino } -12-oxo-3, 6, 9-trioxadode-12-yl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (1-methyl-2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -12-oxo-3, 6, 9-trioxadoden-12-yl) amino ] benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (3- {4- [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] piperazin-1-yl } propionyl) amino ] benzamide;

n- (5, 6-dimethoxybenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -12-oxo-3, 6, 9-trioxadode-12-yl) amino ] benzamide;

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

Further preferably, the compound is selected from one or more of the following compounds, the preferred compound can more effectively and directly target KLF5 protein and induce the degradation of KLF5 protein, and the preferred compound can more effectively degrade KLF5 protein in tumor cells and can remarkably inhibit proliferation of various tumor cells such as breast cancer.

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

3- (acetylamino) -N- (7-fluorobenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (5, 6-dimethylbenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

3- (acetylamino) -N- (4-chlorobenzo [ d ] [1,3] thiazepin-2-yl) benzamide;

N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] oxy } -9-oxo-3, 6-dioxanon-9-yl) amino ] benzamide.

The invention also provides a pharmaceutical composition comprising a compound which targets and degrades KLF5 as described above, and a pharmaceutically acceptable carrier;

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

and/or the pharmaceutical composition further comprises an excipient, diluent, adjuvant, vehicle, or combination thereof.

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

The invention also provides application of the compound or the pharmaceutical composition in preparing KLF5 modulator or antitumor drugs.

Preferably, the tumor comprises one or more of breast cancer, liver cancer, lung cancer, colorectal cancer, leukemia, gastric cancer, glioma, prostate cancer. 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. As a further preference, the tumor is breast cancer.

The compound for targeted degradation of KLF5, a degradation agent and related analogues or pharmaceutically acceptable salts, metabolites or prodrugs thereof are used for inhibiting proliferation, growth, migration, infiltration, clone formation and metastasis of cancer cells, promoting apoptosis of the cancer cells, promoting autophagy of tumor cells and/or prolonging the survival period of tumor patients.

The invention also provides a method for preparing a medicament for preventing and/or treating diseases related to KLF5 protein, and applying an effective amount of the compound for targeted degradation of KLF5 and related analogues or pharmaceutically acceptable salts, metabolites or prodrugs thereof to individuals in need.

The term "C ₁～C₄ alkyl" as used herein refers to straight or branched chain alkyl groups having 1 to 4 carbon atoms and includes, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and the like.

The term "C ₁～C₄ alkoxy" refers to a straight or branched chain alkoxy group containing at least one oxygen atom having 1 to 4 carbon atoms, including without limitation methoxy, ethoxy, propoxy, ethoxymethoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like.

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

Preferably, the C1-C4 alkyl is methyl, the C1-C4 alkoxy is methoxy or propoxy, and the halogen is fluorine, chlorine or bromine.

The term "pharmaceutically acceptable salt" refers to a compound found in the present invention to have a specific substituent group prepared from the compound and a relatively non-toxic acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting neutral forms of such compounds with a sufficient amount of a base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, amine, organic amine or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like, and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like, and salts of amino acids (e.g., arginine, and the like), and salts of organic acids such as glucuronic acid, and the like. Certain specific compounds of the invention contain both acidic and basic functionalities and can be converted to either base or acid addition salts.

In addition to salt forms, the compounds provided herein exist in prodrug forms. Prodrugs of the compounds of the present invention are susceptible to chemical changes under physiological conditions that convert to the compounds of the present invention. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound according to formulas (I) - (III)) is a prodrug within the scope and spirit of the invention. For example, compounds containing carboxyl groups can form physiologically hydrolyzable esters that act as prodrugs by hydrolyzing in vivo to give the compounds themselves according to formulas (I) - (III).

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 and other mixtures thereof, and all such isomers and mixtures thereof are included within the scope of the present invention.

The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more of the atoms comprising the compounds. For example, 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 intended to be encompassed within the scope of the present invention.

The term "excipient" generally refers to the carrier, diluent, and/or medium required to formulate an effective pharmaceutical composition.

For a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect. For the purposes of the oral dosage form of the present invention, an "effective amount" of one active agent in a composition refers to that amount of the other active agent in the composition which is required to achieve the desired effect when combined. Determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, a suitable effective amount in an individual case can be determined by one skilled in the art according to routine experimentation.

The term "active ingredient", "therapeutic agent", "active substance" or "active agent" as used herein refers to a chemical entity that is effective in treating a disorder, disease or condition of interest.

When any variable (e.g., R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0 to 2R, the group may optionally be substituted with up to two R's, and R's in each case are independent options. Furthermore, combinations of substituents and/or variants thereof are only permissible if such combinations result in stable compounds.

The term "comprising" in this application is an open-ended expression, i.e., including what is indicated in the present application, but not excluding other aspects.

Compared with the prior art, the invention has the beneficial effects that:

1. The compounds provided by the invention are capable of directly targeting KLF5 protein and inducing degradation of KLF5 protein, preferably a representative compound LS-23 binding K _d to KLF5 protein of 9.5 micromoles/liter.

2. The compound provided by the invention can effectively degrade KLF5 protein in tumor cells, and the maximum degradation efficiency of the representative compound LS-23 on KLF5 is 93%.

3. The compound of the invention can obviously inhibit proliferation of various tumor cells such as triple negative breast cancer and the like, the IC ₅₀ of the representative compound LS-23 in the triple negative breast cancer cell SUM149PT with high KLF5 expression is 1.19 micromole/liter,

4. The compound has remarkable inhibition effect on the growth of subcutaneous transplantation tumor of triple negative breast cancer mice.

5. Has excellent inhibitory activity on various different types of human cancer cells highly expressing KLF5, and provides a selectable scheme for broad-spectrum anticancer drugs.

Drawings

FIG. 1 shows the activity screening of LS-23 and other compounds in breast cancer cell lines, wherein A is representative compound degradation activity, B is representative compound cell inhibition activity, and C is LS-23 and has strong inhibition activity on breast cancer cells of different subtypes (TNBC: triple negative breast cancer, HER2+: HER2 positive breast cancer, luminal: luminal type breast cancer);

FIG. 2 shows the comparison of LS-23 with LS-30 DC ₅₀ and IC ₅₀, and A-B shows the difference in cellular activity and KLF5 protein levels between SUM149PT and HCC1806 after treatment with LS-23 and LS-30;

FIG. 3 shows the results of LS-23 cell scratch experiments, A is a migration picture, B is a statistical graph of mobility, and NC is a control group;

FIG. 4 shows the results of LS-23 clone ball formation experiments, A is a clone formation picture, and B is a statistical plot of clone formation;

FIG. 5 shows the result of detecting apoptosis by LS-23 flow cytometer, wherein A is the analysis picture of apoptosis by flow cytometer, and B is the statistical picture of apoptosis;

FIG. 6 shows the results of LS-23 mice xenograft tumor experiments, wherein A-B is the in-situ breast cancer model construction and administration time and frequency of the mice, the mice are euthanized after 13 days of continuous administration, tumors are excised and photographed, C is the tumor volume measured every 2 days after the beginning of the administration treatment and represented by a line graph, D is the weight change of the mice after the administration of compound LS-23, E is the results of measuring the level of glutamic pyruvic transaminase in serum to evaluate the hepatotoxicity of compound LS-23 and the level of creatinine in serum to evaluate the nephrotoxicity of compound LS-23 after the completion of the administration, F is the results of measuring the level of KLF5 in tumor tissues of mice in control group (DMSO) and experimental group (20 mg/kg and 40 mg/kg);

FIG. 7 shows the SPR (surface plasmon resonance) detection results of LS-23.

Detailed Description

The technical scheme of the present invention will be described in further detail below with reference to specific embodiments and the accompanying drawings, and the protection content of the present invention is not limited to the following embodiments. Variations and advantages that would occur to one skilled in the art are included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

The low-temperature reaction device used in the invention is EYELA (PSL-1810) magnetic stirring low-temperature and constant-temperature water tank, EYELA (N-1100) rotary evaporator, the purity results of the obtained compounds are all obtained from Agilent 1200series LC system high-performance liquid chromatography analyzer, the ultraviolet detection wavelength is 254nm and 365nm, the nuclear magnetic resonance spectrometer is Bruker 500 type, the solvent is DMSO-d ₆, the purification of the reaction intermediate and the final product is chromatographic column (200-300 meshes of silica gel used), and the silica gel used is purchased from Qingdao ocean chemical plant. Unless otherwise specified, all reactions were followed by TLC.

EXAMPLE 1 preparation of Compounds provided by the invention

Example 1-1.3 preparation of- (acetylamino) -N- (benzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide (LS-01):

2-aminobenzothiazole (100 mg,0.689 mmol) and 3-acetamidobenzoic acid (124 mg,0.689 mmol) were dissolved in 2mL of dimethyl sulfoxide (DMSO), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) (393 mg,1.033 mmol) and N, N-Diisopropylethylamine (DIPEA) (267 mg,2.067 mmol) were added sequentially to the mixed solution and stirred at room temperature for 2h. After the reaction was completed, the mixture was extracted with ethyl acetate and water, and the organic phases were collected, washed with a saturated sodium chloride solution, combined, dried over anhydrous sodium sulfate, and distilled under reduced pressure. Separating and purifying by column chromatography to obtain compound LS-01(68mg,40％).¹H 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(td,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).

Example 1-2.3 preparation of N- (5-methylbenzo [ d ] [1,3] thiazepin-2-yl) benzamide (LS-02):

in analogy to the synthesis of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-methylbenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-02(36mg,40％).¹H 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).

Examples 1-3.3 preparation of N- (5-methoxybenzo [ d ] [1,3] thiazepin-2-yl) benzamide (LS-03):

in analogy to the synthesis of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-methoxybenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-03(141mg,57％).¹H 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).

Examples 1-4.3 preparation of- (acetylamino) -N- (5-fluorobenzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide (LS-04):

In analogy to the synthetic scheme of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-fluorobenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-04(207mg,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).

Examples 1-5.3 preparation of- (acetylamino) -N- (7-fluorobenzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide (LS-05):

In analogy to the synthetic scheme of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-7-fluoro-1, 3-benzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-05(113mg,58％).¹H 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).

Examples 1-6.3 preparation of- (acetylamino) -N- (5, 6-dimethylpheno [ d ] [1,3] thiazacyclopent-2-yl) benzamide (LS-06):

in analogy to the synthesis of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5, 6-dimethylbenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-06(75mg,26％).¹H 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).

Examples 1-7.3 preparation of- (acetylamino) -N- [6- (trifluoromethyl) benzo [ d ] [1,3] thiazepin-2-yl ] benzamide (LS-07):

In analogy to the synthesis of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-6- (trifluoromethyl) benzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-07(108mg,34％).¹H 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).

Examples 1-8.3 preparation of- (acetylamino) -N- (5-chlorobenzo [ d ] [1,3] thiazafen-2-yl) benzamide (LS-08):

In analogy to the synthesis of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-chlorobenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-08(87mg,36％).¹H NMR(500MHz,DMSO-d₆)δ13.02(s,1H),10.19(s,1H),8.31(d,J＝2.2Hz,1H),8.08(d,J＝8.5Hz,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).

Examples 1-9.3 preparation of (acetylamino) -N- (4-chlorobenzo [ d ] [1,3] thiazepin-2-yl) benzamide (LS-09):

In analogy to the synthesis of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-4-chlorobenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-09(96mg,39％).¹H 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,1H),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).

Examples 1-10.3 preparation of- (acetylamino) -N- (6-chlorobenzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide (LS-10):

In analogy to the synthetic scheme of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-6-chlorobenzothiazole and the crude product thus obtained was purified by column chromatography to give compound LS-10(115mg,66％).¹H NMR(500MHz,DMSO-d₆)δ12.97(s,1H),10.19(s,1H),8.30(s,1H),8.19(d,J＝2.2Hz,1H),7.87–7.82(m,2H),7.79(d,J＝8.6Hz,1H),7.53–7.45(m,2H),2.09(s,3H)., example 1-11.3- (acetylamino) -N- (5-bromobenzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide (LS-11):

In analogy to the synthetic scheme of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-5-bromobenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-11(91mg,44％).¹H NMR(500MHz,DMSO-d₆)δ12.97(s,1H),10.19(s,1H),8.33–8.28(m,2H),7.84(dd,J＝7.7,1.8Hz,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).

Examples 1-12.5- (acetylamino) -2-chloro-N- (5, 6-dimethylbenzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide (LS-12) preparation:

In analogy to the synthesis of example 1-1, 3-acetamidobenzoic acid was replaced with 5- (acetylamino) -2-chlorobenzoic acid and 2-aminobenzothiazole was replaced with 2-amino-5, 6-dimethylbenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-12(55mg,16％).¹H 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).

Examples 1-13.5 preparation of- (acetylamino) -N- (5, 6-dimethylpheno [ d ] [1,3] thiazacyclopent-2-yl) -2-methylbenzamide (LS-13):

In analogy to the synthesis of example 1-1, 3-acetamidobenzoic acid was replaced with 5-acetamido-2-methylbenzoic acid and 2-aminobenzothiazole was replaced with 2-amino-5, 6-dimethylbenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-13(77mg,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).

Examples 1-14.3 preparation of- (acetylamino) -N- [6- (1, 4-oxazepin-4-yl) benzo [ d ] [1,3] thiazepin-2-yl ] benzamide (LS-14):

In analogy to the synthetic scheme of example 1-1, 2-aminobenzothiazole was replaced by 6-morpholinobenzo [ D ] thiazol-2-amine and the crude product thus obtained was purified by column chromatography to give the compound LS-14(88mg,40％).¹H 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).

Examples 1-15.3 preparation of N- (5, 6-dimethoxybenzo [ d ] [1,3] thiazacyclopent-2-yl) benzamide (LS-15):

In analogy to the synthetic scheme of example 1-1, 2-aminobenzothiazole was replaced by 5, 6-dimethoxy-1, 3-benzothiazol-2-amine and the crude product thus obtained was purified by column chromatography to give the compound LS-15(68mg,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).

Examples 1-16.3 preparation of- (acetylamino) -N- (6-ethoxybenzo [ d ] [1,3] thiazepin-2-yl) benzamide (LS-16):

In analogy to the synthesis of example 1-1, 2-aminobenzothiazole was replaced by 2-amino-6-ethoxybenzothiazole and the crude product thus obtained was purified by column chromatography to give the compound LS-16(79mg,31％).¹H 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).

Examples 1-17.3 preparation of N- (4-methoxy-7-methylbenzo [ d ] [1,3] thiazafen-2-yl) benzamide (LS-17):

in analogy to the synthetic scheme of example 1-1, 2-aminobenzothiazole was replaced by 4-methoxy-7-methylbenzo [ D ] thiazol-2-amine and the crude product thus obtained was purified by column chromatography to give the compound LS-17(56mg,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).

Examples 1-18.3 preparation of- (acetylamino) -N- [6- (prop-2-yloxy) benzo [ d ] [1,3] thiazepin-2-yl ] benzamide (LS-18):

In analogy to the synthetic scheme of example 1-1, 2-aminobenzothiazole was replaced by 6-isopropoxy-1, 3-benzothiazol-2-amine and the crude product thus obtained was purified by column chromatography to give the compound LS-18(56mg,23％).¹H 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).

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

3- ({ [ (2-methylpropan-2-yl) oxy ] carbonyl } amino) benzoic acid (1 g,4.215 mmol) and 5, 6-dimethyl benzo [ d ] [1,3] thiazapentan-2-amine (751mg, 4.215 mmol) were dissolved in 2mL of dimethyl sulfoxide (DMSO), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) (2.284 g,6.377 mmol) and N, N-Diisopropylethylamine (DIPEA) (1.244 g,12.645 mmol) were added sequentially to the mixed solution, and stirred at room temperature for 2h. After the reaction was completed, the mixture was extracted with ethyl acetate and water, and the organic phases were collected, washed with a saturated sodium chloride solution, combined, dried over anhydrous sodium sulfate, and distilled under reduced pressure. Column chromatography separation and purification gave [ (3- { [ (5, 6-dimethylpheno [ d ] [1,3] thiazacyclopent-2-yl) amino ] carbonyl } phenyl) amino ] propan-2-yl ester of methane acid (636 mg, 38% yield).

2-Methylpropan-2-yl [ (3- { [ (5, 6-dimethylpheno [ d ] [1,3] thiazacyclopent-2-yl) amino ] carbonyl } phenyl) amino ] methanoate (636 mg,1.602 mg) was dissolved in DCM (10 mL), trifluoroacetic acid (1.5 mL) was added, reacted for 5h, the DCM and TFA were evaporated, and the crude product thus obtained was purified by column chromatography to give the key intermediate 3-amino-N- (5, 6-dimethylpheno [ d ] [1,3] thiazacyclopent-2-yl) benzamide (L1) (300 mg, yield 63%).

Example 1-19 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazepin-2-yl) -3- [ (4- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-5-yl ] amino } -1-oxosubunit butyl) amino ] benzamide (LS-19).

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 2mL of dimethyl sulfoxide (DMSO), N-Diisopropylethylamine (DIPEA) (280 mg,2.172 mmol) was added to the mixed solution, and reacted under nitrogen protection at 90℃for 10 hours, after the completion of the reaction, extracted with ethyl acetate and water, the organic phase was collected, washed with a saturated sodium chloride solution, the organic phase was combined, dried over anhydrous sodium sulfate, and distilled under reduced pressure. The 4- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } butyric acid (150 mg) was isolated and purified by column chromatography.

4- { [2- (2, 6-Dioxohexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } butyric acid and intermediate L1 were dissolved in DMSO, HATU and DIPEA were added in sequence to the reaction solution and stirred at room temperature for 2H. After the completion of the reaction, EA and water were extracted, the organic phase was collected, dried and concentrated, and the crude product thus obtained was purified by column chromatography to give the target product N- (5, 6-dimethylbenzo [ d ] [1,3] thiaazacyclopent-2-yl) -3- [ (4- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-5-yl ] amino } -1-oxobutylen-amino ] benzamide (LS-19).¹H 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),2.47(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).

Example 1-20 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazepin-2-yl) -3- [ (6- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -1-oxoalkenylhexyl) amino ] benzamide (LS-20).

In analogy to the synthetic schemes of examples 1-19, 4-aminobutyric acid was replaced by 6-aminocaproic acid and the crude product thus obtained was purified by column chromatography to give the compound LS-20.¹H 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,1H),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).

Example 1-21 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazafen-2-yl) -3- ({ 3- [ (2- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } ethyl) oxy ] propionyl } amino) benzamide (LS-21).

2- (2, 6-Dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (200 mg,0.724 mmol) and 3- [ (2-aminoethyl) oxy ] propionic acid-2-methylpropan-2-yl ester (206 mg,1.806 mmol) were dissolved in 2mL of dimethyl sulfoxide (DMSO), N-diisopropylethylamine (DIPEA or DIEA) (87 mg, 1.4478 mmol) was added to the mixed solution, the mixture was reacted under nitrogen protection for 10 hours at 90℃and, after the completion of the reaction, the organic phase was collected by extraction with ethyl acetate and water, washed with saturated sodium chloride solution, the organic phase was combined, dried over anhydrous sodium sulfate and distilled under reduced pressure. Column chromatography was used to separate and purify 3- [ (2- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } ethyl) oxy ] prop-2-methylpropan-2-yl ester (120 mg).

2-Methylpropan-2-yl 3- [ (2- { [2- (2, 6-dioxohexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } ethyl) oxy ] propanoate (120 mg, 0.399 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.5 mL) was added and stirred at room temperature for 3H. After the reaction, most of the solvent was spin-dried and directly dissolved in DMSO (2 mL). Intermediate L1 (70 mg), HATU (133 mg,0.351 mmol) and DIPEA (90 mg,0.702 mmol) were then added to the reaction solution and stirred at room temperature for 3h. After the reaction, EA and water are extracted, and the crude product obtained is subjected to silica gel column chromatography to obtain the target product N- (5, 6-dimethylbenzo [ d ] [1,3] thiazapentan-2-yl) -3- ({ 3- [ (2- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-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).

Example 1-22 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazepin-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -9-oxo-3, 6-dioxanon-9-yl) amino ] benzamide (LS-22).

In analogy to the synthesis of example-19, 3- [ (2-aminoethyl) oxy ] prop-2-yl-3- ({ 2- [ (2-aminoethyl) oxy ] ethyl } oxy) prop-2-yl-3-ate was replaced and the crude product thus obtained was purified by column chromatography to give the 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).

Examples 1-23 preparation of N- (5, 6-dimethyl benzo [ d ] [1,3] thiazafen-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -12-oxo-3, 6, 9-trioxadode-12-yl) amino ] benzamide (LS-23):

In analogy to the synthesis of examples 1-19, 3- [ (2-aminoethyl) oxy ] prop-2-yl-3- [ (8-amino-3, 6-dioxaoct-1-yl) oxy ] prop-2-yl-3-methylpropan-2-yl-ate was replaced by the crude product obtained therefrom which was purified by column chromatography to give the 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).

Example 1-24 preparation of N- (5, 6-dimethyl benzo [ d ] [1,3] thiapentadec-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -15-oxo-3, 6,9, 12-tetraoxapentadec-15-yl) amino ] benzamide (LS-24):

In analogy to the synthesis of examples 1-19, 3- [ (2-aminoethyl) oxy ] prop-2-yl-2-ate was replaced by 3- [ (11-amino-3, 6, 9-trioxaundec-1-yl) oxy ] prop-2-yl-2-ate and the crude product thus obtained was purified by column chromatography to give the 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).

Example 1-25 preparation of N- (5, 6-dimethyl benzo [ d ] [1,3] thiazapenn-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -18-oxo-3, 6,9,12, 15-pentoxaoctadeca-18-yl) amino ] benzamide (LS-25):

In analogy to the synthesis of examples 1-19, 3- [ (2-aminoethyl) oxy ] prop-2-yl-3- [ (14-amino-3, 6,9, 12-tetraoxatetradecan-1-yl) oxy ] prop-2-yl-3- [ (2-aminoethyl) oxy ] prop-2-yl-3-ate was replaced and the crude product thus obtained was purified by column chromatography to give the 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).

Example 1-26 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazepin-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-5-yl ] amino } -9-oxo-3, 6-dioxanon-9-yl) amino ] benzamide (LS-26).

In analogy to the synthesis of examples 1-19, 2- (2, 6-dioxo-hexahydropyridin-3-yl) -4-fluoroisoindol-1, 3-dione was replaced with 2- (2, 6-dioxo-hexahydropyridin-3-yl) -5-fluoroisoindol-1, 3-dione and 3- [ (2-aminoethyl) oxy ] propionic acid-2-methylpropan-2-yl ester was replaced with 3- ({ 2- [ (2-aminoethyl) oxy ] ethyl } oxy) propionic acid-2-methylpropan-2-yl ester and the crude product thus obtained was purified by column chromatography to give the 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).

Example 1-27 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazafen-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-5-yl ] amino } -12-oxo-3, 6, 9-trioxadode-12-yl) amino ] benzamide (LS-27):

In analogy to the synthesis of examples 1-19, 2- (2, 6-dioxo-hexahydropyridin-3-yl) -4-fluoroisoindol-1, 3-dione was replaced with 2- (2, 6-dioxo-hexahydropyridin-3-yl) -5-fluoroisoindol-1, 3-dione and 3- [ (2-aminoethyl) oxy ] propionic acid-2-methylpropan-2-yl ester was replaced with 3- [ (8-amino-3, 6-dioxaoct-1-yl) oxy ] propionic acid-2-methylpropan-2-yl ester and the crude product thus obtained was purified by column chromatography to give the 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).

Example 1-28 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazepin-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1-oxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -9-oxo-3, 6-dioxan-9-yl) amino ] benzamide (LS-28):

In analogy to the synthesis of examples 1-19, 2- (2, 6-dioxo-hexahydropyridin-3-yl) -4-fluoroisoindol-1, 3-dione was replaced with 3- (4-amino-1-oxo-2, 3-dihydro-1H-isoindol-2-yl) hexahydropyridine-2, 6-dione and 3- [ (2-aminoethyl) oxy ] propionic acid-2-methylpropan-2-yl ester was replaced with 3- ({ 2- [ (2-bromoethyl) oxy ] ethyl } oxy) propionic acid-2-methylpropan-2-yl ester and the crude product thus obtained was purified by column chromatography to give 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.N- (5, 6-dimethylbenzo [ d ] [1,3] thiaazacyclopentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1-oxo-2, 3-dihydro-1H-4-amino } -indol-2-yl ] indol-2-yl } -3-oxo-12-amino-dodecanamide (29-3-dodecyl).

In analogy to the synthesis of examples 1-19, 2- (2, 6-dioxo-hexahydropyridin-3-yl) -4-fluoroisoindol-1, 3-dione was replaced with 3- (4-amino-1-oxo-2, 3-dihydro-1H-isoindol-2-yl) hexahydropyridine-2, 6-dione and 3- [ (2-aminoethyl) oxy ] prop-2-yl-3- [ (8-bromo-3, 6-dioxaoct-1-yl) oxy) prop-2-yl-ate and the crude product thus obtained was purified by column chromatography to give the 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).

Example 1-30 preparation of N- (5, 6-dimethyl benzo [ d ] [1,3] thiazacyclopentan-2-yl) -3- [ (1- { [2- (1-methyl-2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -12-oxo-3, 6, 9-trioxadode-12-yl) amino ] benzamide (LS-30):

In analogy to the synthesis of examples 1-19, 2- (2, 6-dioxo-hexahydropyridin-3-yl) -4-fluoroisoindol-1, 3-dione was replaced with 4-fluoro-2- (1-methyl-2, 6-dioxo-hexahydropyridin-3-yl) isoindol-1, 3-dione and 3- [ (2-aminoethyl) oxy ] propionic acid-2-methylpropan-2-yl ester was replaced with 3- [ (8-amino-3, 6-dioxaoct-1-yl) oxy ] propionic acid-2-methylpropan-2-yl ester and the crude product thus obtained was purified by column chromatography to give the 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＝13.0,5.4Hz,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).

Example 1-31 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazepin-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] oxy } -9-oxo-3, 6-dioxanon-9-yl) amino ] benzamide (LS-31).

2- (2, 6-Dioxyylidene hexahydropyridin-3-yl) -4-hydroxyisoindole-1, 3-dione and 3- ({ 2- [ (2-bromoethyl) oxy ] ethyl } oxy) propanoic acid-2-methylpropan-2-yl ester were dissolved in 2mL of DMF, sodium hydrogencarbonate was added to the mixed solution, and reacted under the condition of nitrogen protection for 10 hours at 60℃and, after the reaction was completed, extracted with ethyl acetate and water, the organic phase was collected, washed with saturated sodium chloride solution, and the organic phase was combined, dried over anhydrous sodium sulfate and distilled under reduced pressure. Separating and purifying by column chromatography to obtain 2-methylpropan-2-yl 3- ({ 2- [ (2- { [2- (2, 6-dioxy-hexahydro-pyridin-3-yl) -1, 3-dioxy-2, 3-dihydro-1H-isoindol-4-yl ] oxy } ethyl) oxy ] ethyl } oxy) propanoate.

2-Methylpropan-2-yl 3- ({ 2- [ (2- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-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 stirred at room temperature for 3H. After the reaction, most of the solvent was spin-dried and directly dissolved in DMSO (2 mL). Then, the intermediates L1, HATU and DIPEA were added to the reaction solution and stirred at room temperature for 3 hours. After the reaction, EA and water are extracted, and the crude product obtained by the extraction is subjected to silica gel column chromatography to obtain a target product N- (5, 6-dimethylbenzo [ d ] [1,3] thiaazacyclopent-2-yl) -3- [ (1- { [2- (2, 6-dioxy hexahydropyridin-3-yl) -1, 3-dioxy-2, 3-dihydro-1H-isoindol-4-yl ] oxy } -9-oxo-3, 6-dioxanon-9-yl) amino ] benzamide (LS-31).¹H 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(ddd,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).

Example 1-32 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazepin-2-yl) -3- [ (2- {4- [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] piperazin-1-yl } acetyl) amino ] benzamide (LS-32).

2- (2, 6-Dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (500 mg, 1.81mmol) and piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (506 mg, 2.015 mmol) were dissolved in 2mL of dimethyl sulfoxide (DMSO), N-Diisopropylethylamine (DIPEA) (4638 mg,3.620 mmol) was added to the mixed solution, and the mixture was reacted under nitrogen protection for 10 hours at 90℃and, after the reaction was completed, the organic phase was collected by extraction with ethyl acetate and water, washed with saturated sodium chloride solution, and the organic phase was combined, dried over anhydrous sodium sulfate and distilled under reduced pressure. Column chromatography separation and purification are carried out to obtain 4- [2- (2, 6-dioxy hexahydropyridine-3-yl) -1, 3-dioxy-2, 3-dihydro-1H-isoindol-4-yl ] piperazine-1-formic acid-2-methylpropan-2-yl ester (795 mg).

4- [2- (2, 6-Dioxypyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] piperazine-1-carboxylic acid 2-methylpropan-2-yl ester (795 mg,1.799 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (2.5 mL) was added, and the mixture was stirred at room temperature for 3H. After the completion of the reaction, most of the solvent was spin-dried to give 2- (2, 6-dioxo-hexahydropyridin-3-yl) -4- (piperazin-1-yl) isoindole-1, 3-dione (755 mg).

2- (2, 6-Dioxypyridin-3-yl) -4- (piperazin-1-yl) isoindole-1, 3-dione (755 mg,2.207 mmol) was dissolved in DMF (2 mL), and 2-methylpropan-2-yl bromoacetate (516 mg,2.649 mmol) and DIPEA (571 mg,4.414 mmol) were added to the reaction. After the reaction was completed, the mixture was extracted with ethyl acetate and water, and the organic phase was collected, washed with a saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Separating and purifying by column chromatography to obtain {4- [2- (2, 6-dioxy-hexahydropyridine-3-yl) -1, 3-dioxy-2, 3-dihydro-1H-isoindol-4-yl ] piperazine-1-yl } acetic acid-2-methylpropan-2-yl ester.

{4- [2- (2, 6-Dioxypyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] piperazin-1-yl } acetic acid-2-methylpropan-2-yl ester was dissolved in methylene chloride (2 mL), trifluoroacetic acid (0.5 mL) was added, and stirred at room temperature for 3H. After completion of the reaction, most of the solvent was dried by spinning, {4- [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] piperazin-1-yl } acetic acid-2-methylpropan-2-yl ester (100 mg,0.250 mmol) was directly dissolved in DMSO (2 mL), and then 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 3H. After the reaction, EA and water are extracted, and the crude product obtained by the extraction is subjected to silica gel column chromatography to obtain the target product N- (5, 6-dimethylbenzo [ d ] [1,3] thiazacyclopent-2-yl) -3- [ (2- {4- [2- (2, 6-dioxy hexahydropyridin-3-yl) -1, 3-dioxy-2, 3-dihydro-1H-isoindol-4-yl ] piperazin-1-yl } acetyl) amino ] benzamide (LS-32).¹H 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).

Example 1-33 preparation of N- (5, 6-dimethyl-benzo [ d ] [1,3] thiazepin-2-yl) -3- [ (3- {4- [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] piperazin-1-yl } propionyl) amino ] benzamide (LS-33).

In analogy to the synthetic schemes of examples 1-32, bromoacetic acid-2-methylpropan-2-yl ester was replaced by tert-butyl 3-bromopropionate and the crude product thus obtained was purified by column chromatography to give the 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).

Example 1-34 preparation of N- (5, 6-Dimethoxybenzo [ d ] [1,3] thiazacyclopentan-2-yl) -3- [ (1- { [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] amino } -12-oxo-3, 6, 9-trioxadode-12-yl) amino ] benzamide (LS-34).

In analogy to the schemes of examples 1-23, 5, 6-dimethylpheno [ d ] [1,3] thiazapentan-2-amine was replaced by 5, 6-dimethoxy-1, 3-benzothiazol-2-amine and the crude product thus obtained was purified by column chromatography to give the 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＝12.7,5.4Hz,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).

Example 1-35 preparation of N- (5, 6-dimethylpheno [ d ] [1,3] thiazafen-2-yl) -3- { [2- (4- {1- [2- (2, 6-dioxo-hexahydropyridin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-yl ] hexahydropyridin-4-yl } hexahydropyridin-1-yl) acetyl ] amino } benzamide (LS-35):

In analogy to the schemes of examples 1-32, piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (replaced by [4- (hexahydropyridin-4-yl) hexahydropyridin-1-yl ] acetic acid-2-methylpropan-2-yl ester) and the crude product thus obtained was purified by column chromatography to give the 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),4.13-4.00(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).

EXAMPLE 2 evaluation of the inhibitory Effect of the Compounds of the invention on KLF5 expression in tumor cells

Example 2-1 cell culture

Triple negative breast cancer cells HCC1806 and HCC1937, SUM149PT used in the present invention were from the kunming cell bank of the national academy of sciences. HCC1806, SUM149PT and HCC1937 were cultured in RPMI 1640 medium+10% fbs,37 ℃ 5% co ₂ thermostated cell culture incubator.

EXAMPLES 2-2 evaluation of the Activity of Compounds of the invention to induce degradation of KLF5

A series of compounds (LS-01 to LS-48) synthesized in example 1, which target degradation of KLF5, were dissolved in DMSO to a final concentration of 1. Mu. Mol/liter and 10. Mu. Mol/liter, respectively, and the control group was added with an equivalent amount of DMSO to treat breast cancer cells SUM149PT and HCC1806, respectively, and incubated for 24 hours. Cells were collected by centrifugation and total cell proteins were collected by denaturing lysis. The compounds were then analyzed for inhibition and degradation of KLF5 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Western Blot protocol following cell culture under experimental conditions, cells were collected after washing with cold Phosphate Buffered Saline (PBS). Cell pellet was solubilized with 2 x protein sample buffer (1M Tris-hclph= 6.8,50% glycerol, 10% sds, 2-mercaptoethanol, 1% bromophenol blue) and boiled at 100 ℃ for 10min. The prepared protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto PVDF membranes (Millipore). Blocking was performed in 5% skim milk solution for specific coupling reaction between protein and antibody on PVDF membrane. The skim milk solution was then discarded and the membrane was washed with PBST (0.5% tween 20 in PBS). The coupling reaction between the protein and antibody on the membrane was carried out overnight at 4 ℃ and then washed again with PBST. Finally, horseradish peroxidase (HRP) -conjugated secondary antibodies (CELL SIGNALING Technology) were diluted in PBST and reacted at room temperature for 2 hours. The protein content on the membrane was determined using Luminata Forte HRP substrates (Millipore).

The grey values of KLF5 and Vinculein in different lanes were analyzed by Image J software, the relative grey ratio of KLF 5/Vinculein for each compound was calculated in Excel and the relative grey ratio of KLF 5/Vinculein in the control group, and KLF5 protein degradation (%) was calculated according to the following formula. Vingulin is an internal reference protein.

The ability of the compounds LS-01 to LS-32 of the present invention to degrade KLF5 in tumor cells is expressed as KLF5 protein degradation (%) of the compounds at concentrations of 1. Mu. Mol/liter and 10. Mu. Mol/liter. Wherein, "a", "b", "c", "d" respectively indicate that the compound of the present invention has a degradation rate of less than 25%, between 25% and 50%, between 50% and 75% and greater than 75% of KLF5 in tumor cells at the concentration, and "NT" indicates that the compound has no evaluation of the ability to induce KLF5 degradation at the indicated concentration. The evaluation results of KLF5 degradation efficiency of the compound of the present invention on breast cancer cells SUM149PT, HCC1806 are shown in table 1.

TABLE 1 evaluation of the results of the induction of KLF5 degradation by the compounds LS-01 to LS-35 according to the invention in different tumor cells

From the results shown in Table 1, the compounds of the present invention showed a remarkable effect of degrading KLF5 protein in at least one strain of breast cancer cells at a concentration of 10. Mu. Mol/liter, and some of the compounds such as LS-04, LS-05, LS-06, LS-07, LS-09, LS-24, LS-26 and LS-31 were capable of inducing degradation of 50% or more of KLF5 protein in triple negative breast cancer HCC1937 and HCC1806 cells at a concentration of 10. Mu. Mol/liter, and LS-23 and LS-24 were capable of inducing degradation of 50% or more of KLF5 protein in breast cancer SUM149PT cells at a concentration of 1. Mu. Mol/liter.

To further illustrate the ability of the compounds of the present invention to induce KLF5 protein degradation in tumor cells, the present invention has been confirmed by selecting a portion of the compounds for Westernblot (WB) experiments, and fig. 1 shows the western blot results of the present invention for a portion of the representative compounds to induce KLF5 degradation in breast cancer cells HCC1806, HCC1937, SUM149PT, and the results show that LS-22, LS-23, NTZ, etc. have significant ability to degrade KLF5 protein.

EXAMPLES 2-3 DC ₅₀ and Dmax in HCC1806 and SUM149PT cells of representative Compounds LS-23 in examples of the invention

In order to evaluate the ability of the compound of the invention to degrade KLF5 in different breast cancer cells, the representative compound LS-23 in the embodiment of the invention is selected to treat HCC1806 and SUM149PT cells for 24 hours respectively, the cells are collected and added with an appropriate amount of SDS protein lysate to lyse the cells, and the cells and the lysate are fully blown down to mix uniformly. After mixing, denaturation was carried out for 10 min at 98 ℃ using a metal bath. The denatured proteins were separated by 10% SDS-PAGE electrophoresis (80V for 40 min; 120V for 90 min for separation), followed by transfer of the proteins to PVDF membrane using wet transfer (250 mA for 120 min), blocking with 5% skim milk for 1 hour, incubating overnight with primary antibody (KLF 5 (CST, # 5650S), vinculin (MCE, HY-P80372) at 4 ℃, washing the membrane three times (10 min/time) with TBST buffer, incubating the secondary antibody (Millipore, 401315) for 1 hour at room temperature, washing the membrane three times (10 min/time) with TBST buffer, incubating with enhanced chemiluminescent horseradish peroxidase substrate (ThermoFisher Scientific, 32106), and developing.

The grey values of KLF5 and Vinculein in different lanes were analyzed by Image J software, the relative grey ratio of KLF 5/Vinculein for each compound was calculated in Excel and the relative grey ratio of KLF 5/Vinculein in the control group, and KLF5 protein degradation (%) was calculated according to the following formula.

DC ₅₀ and D _max were calculated using GRAPHPAD PRISM software, and compound concentration-KLF 5 protein degradation graphs were plotted, see FIG. 2, which shows that the representative compound LS-23 in this example degraded KLF5 protein DC ₅₀ and D _max in HCC1806, SUM149PT cells. As shown in FIG. 2, LS-23 had DC ₅₀ of 0.926 and 1.384 micromoles/liter, respectively, and D _max of 93% and 91% in HCC1806 and SUM149PT cells, respectively. The results show that the representative compounds LS-23 and the like in the examples of the invention can significantly degrade KLF5 protein in triple negative breast cancer cell lines, and further illustrate the effect of the compounds of the invention on degradation of KLF5 protein.

Representative compounds have degradation activity as shown in figure 1A, LS-23 has a stronger effect on degrading KLF5 protein than NTZ and other compounds, and has a certain concentration gradient dependence.

EXAMPLE 3 evaluation of the antiproliferative Activity of the Compounds of the invention in tumor cells

Determination of proliferation inhibitory Activity of Compounds on breast cancer cells by CCK8 method

Cell growth inhibition was examined using the CCK8 method. Test compounds were dissolved in DMSO (dimethyl sulfoxide), and then prepared into 0.1% DMSO solutions, 1 μm 0.1% DMSO solutions, 0.1 μm 0.1% DMSO solutions with respective compound concentrations of 10 μm using cell culture media.

The CCK8 cell proliferation kit is used for detecting proliferation, namely, cells in logarithmic phase are digested, then single cell suspension is prepared, the single cell suspension is inoculated into culture plates such as 96-well plates according to a certain density, each experimental group and each control group are arranged, and the culture plates are placed into an incubator for culturing for a certain time to enable the cells to grow on the wall. According to the experimental requirements, 1 μl of each of the compounds at different concentrations dissolved in DMSO was added to each well of cell culture, so that the final concentration of each compound was one percent of the previous, and the control group was to add only the same volume of DMSO to the cell culture. The culture was continued for a suitable period of time. After 72h, 10. Mu.L of fresh medium containing CCK8 was added to each well and incubated for 1 hour to allow the reagent to react well with the living cells. The absorbance value of each well is detected at a specific wavelength (about 450 nm) by using an enzyme-labeled instrument, and the proliferation condition of the cells is analyzed by comparing absorbance data of different groups, wherein the higher the absorbance is, the more the number of living cells is, and the better the proliferation condition is. The percentage of absorbance values of the experimental group relative to absorbance values of the control group represents the survival rate of the cells or the proliferation level of the cells, i.e., the control group defaults to 100%.

The effect of other different concentrations of the compounds of the invention on breast cancer cell proliferation was continued to be statistically analyzed, graphPadPrism software was used for IC ₅₀ calculations for the different compounds, and the test results are shown in table 2.

The proliferation-inhibiting ability of the compounds LS-01 to LS-32 of the present invention on tumor cells is represented by IC ₅₀. Wherein "+", "++", and "++", respectively, indicate that the compound has an IC ₅₀ of greater than 10 micromoles per liter, between 1.0 micromoles per liter and 10 micromoles per liter, and less than 1.0 micromoles per liter. "NT" means that the antiproliferative effect of this compound on such tumor cells was not evaluated. The IC ₅₀ of the compounds of the invention against breast cancer cells HCC1937, HCC1806 and breast cancer cell SUM149PT are shown in table 2.

TABLE 2 evaluation of the antiproliferative Activity of the Compounds LS-01 to LS-35 of the invention in different breast cancer tumor cells

The results show that the compounds LS-01 to LS-18 have certain proliferation inhibition activity on breast cancer cells HCC1937, HCC1806 and SUM149PT, while the compounds LS-19 to LS-32 have obvious proliferation inhibition activity on breast cancer cells HCC1937, HCC1806 and SUM149PT, and the compounds LS-22, LS-23, LS-24, LS-25, LS-26 and LS-31 have the IC ₅₀ of 1.0 micromole/liter to 10 micromole/liter on breast cancer cells HCC1806 and SUM149 PT. The cell inhibitory activities of representative compounds are shown in FIG. 1B, and NTZ, LS-21, LS-28, LS-22, LS-26, LS-31, LS-24, LS-25, LS-27, LS-23 have strong inhibitory effects on HCC1806 and SUM149PT cells, and the cell viability of LS-23 at 1 μM is lower than 50%.

To further test the proliferation inhibitory activity of the compounds of the present invention in breast cancer, representative compounds in the examples of the present invention were selected to continue to determine the proliferation inhibitory activity of the compounds on various breast cancer cells using the SRB method. As shown in FIG. 1C, 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 type breast cancer).

To further evaluate the effect of the compounds of the present invention on proliferation of other tumor cells, a representative compound LS-23 was selected and tested for antiproliferative activity in a variety of other cancer cells, and the results are shown in Table 3. The test result shows that the compound LS-23 has stronger cancer cell toxicity activity on lung cancer cells, liver cancer cells, leukemia cells, colorectal cancer cells, prostate cancer cells, human glioma cells and human gastric cancer cell lines, and has better treatment potential on various tumors.

TABLE 3 evaluation of the antiproliferative Activity of the inventive Compound LS-23 in different tumor cells

Cell lines	IC₅₀(μM)	Cell lines	IC₅₀(μM)
				Human lung cancer cell A549	11.19	Human colorectal cancer cell HCT116	18.24
Human liver cancer cell SMMC-7721	7.124	Human prostate cancer cells PC3	4.733
				Human liver cancer cell LM3	8.29	Human glioblastoma cell T98G	0.655
Human leukemia cell Jurkat	3.156	Human gastric cancer cell MKN45	20.61

Example 4 detection of DC ₅₀ (Compound treatment concentration at half of protein degradation) and LS-30 and LS-23 by cell viability assay and Western Blot experiment and IC ₅₀ (Compound treatment concentration at half of cell proliferation inhibition)

CCK8 and Western Blot protocols are described above

Test results comparing the difference in cell activity and KLF5 protein levels between SUM149PT and HCC1806 after treatment with LS-23 and LS-30, it was seen that LS-23 was able to significantly degrade KLF5 while LS-30 degraded KLF5 poorly, as shown in FIG. 2A. As shown in FIG. 2B, LS-23 had good anti-tumor activity in SUM149PT and HCC1806 cells, which was stronger than LS-30.

Example 5 cell scratch test

The method for detecting the migration of the compound to the cells by adopting a scratch healing experiment to select the compound with better activity comprises the steps of paving the cells on a 6-hole plate, and scratching the cells in the middle of the cell layer by using a 200 mu L gun head after the cells grow into compact sheets. Cell debris was washed off using 1 XPBS, and then complete medium with low concentration serum was added and the culture was continued in an incubator with different concentrations of compound. Immediately randomly selecting 3 positions of scratches for image acquisition, acquiring signals at the same position every 4 hours, ending the experiment when a certain group of scratches are close to healing, and analyzing the migration capacity of cells by counting the healing degree of the scratch areas. Cell mobility= (initial scratch width-final scratch width)/initial scratch width x 100%.

Test results As shown in FIGS. 3A-B, scratch experiments were performed in SUM149PT and HCC1806 cell lines, treated with different concentrations of LS-23 (0.5, 1, 2. Mu.M), and then recorded at 24h, 48 h. LS-23 was found to significantly inhibit cancer cell migration rate with increasing concentration. Fig. 3A is a migration picture, and fig. 3B is a statistical diagram of mobility. * p <0.05, < p <0.01, < p <0.001.NC is control group.

Example 6 clone sphere formation experiments

The cultured cells were counted after digestion, and appropriate amount of cell suspension was inoculated into 6-well plates (800 cells per well), and cultured with different concentrations of drug, with the same amount of DMSO as a control. Culturing for 7-14 days until clone formation is visible to naked eyes. After the clone formation, the medium was removed and the cells were washed 1-2 times with PBS. Cells were fixed in a proper amount of paraformaldehyde solution for 10-15 minutes to fix the cells at the bottom of the plate. The fixative was removed and the cells were washed again with PBS. And adding crystal violet staining solution, and staining for 10-30 minutes to dye the clone into purple or blue, so that the clone is convenient to observe and count. Excess staining solution was washed off with PBS and the plates or dishes were air dried. Clones were observed under a microscope and counted, and the size and number of clones were defined according to experimental requirements, and a cell mass containing 50 or more cells was generally considered as one clone. The number of clones was counted using automatic cell counting software or manually.

Test results As shown in FIGS. 4A-B, cloning experiments were performed in SUM149PT and HCC1806 cell lines, and staining photographs were taken after 14 days of drug administration culture with different concentrations of LS-23, and the results were quantified. LS-23 was found to inhibit the number of cancer cell clonogenic events with increasing concentration. FIG. 4A is a picture of clone formation, and FIG. 4B is a statistical picture of clone formation. * P <0.01, p <0.001.DMSO was control.

EXAMPLE 7 apoptosis experiments

Apoptosis detection cells were stained with FITC Annexin V and PI dye, and flow cytometry was used to analyze the percentage of apoptotic cells according to the kit instructions.

As a result of the experiment, SUM149PT cells were treated with LS-2 for 48 hours, respectively, and their apoptosis ratio was analyzed by staining, as shown in FIGS. 5A-B. From the quantitative results, it can be seen that the number of apoptotic cells increases significantly with increasing LS-23 concentration. Data statistics were analyzed using the T-test method. ns, show no significant difference; p <0.001.DMSO was control. Fig. 5A is a flow cytometer analysis picture of apoptosis, and fig. 5B is a statistical diagram of apoptosis.

EXAMPLE 8 evaluation of the growth inhibitory Activity of the representative Compound LS-23 of the present invention on nude mice with Breast cancer

To assess the potential therapeutic effect of the compounds of the invention on tumors in vivo, we took HCC1806 cells in log phase, digested, collected and subjected to cell counting procedures. After centrifugation of 1×10 ⁶ cells per spot, the cells were resuspended in pre-chilled 1×pbs, matrigel was added, and after thorough mixing with pre-chilled gun tips, insulin injection needles were used to inject into the 4 th pair of mammary fat pads of mice at a volume of 75 μl per spot. After one week of inoculation, mice were observed for in situ neoplasia, and when tumor volumes were as long as 50-60mm ³, mice were randomly grouped, 5 each, with different drug treatment concentration groups set. A drug injection was prepared according to a formulation of 10% (DMSO or drug) drug cocktail+30% PEG300+5% Towen80+30% physiological saline at a volume of 100. Mu.L each. The intraperitoneal administration was continued every 3 days, stopped for one day, and the body weight of the mice was weighed and recorded, while the tumor of the mice 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, mice were sacrificed by cervical dislocation. Tumor tissue was isolated using surgical instruments, weighed, photographed, and mouse serum was taken for subsequent liver and kidney function detection.

Fig. 6 shows the in vivo inhibition results of compound LS-23 on xenograft tumors of human breast cancer mice, fig. 6A-B shows the in situ breast cancer model construction and administration time and frequency of mice, mice are euthanized after 13 days of continuous administration, tumors are resected and photographed, fig. 6C shows the measurement of tumor volume every 2 days after the start of administration treatment, as shown by a line graph, compared with a control group, the tumor volume of mice is lower than that of the control group after administration of compound LS-23, the tumor weights of mice in the control group (DMSO) and experimental group (20 mg/kg and 40 mg/kg) are lower than that of the control group, the tumor weights of mice in the experimental group are lower than that of the control group, fig. 6D shows the change of body weight of mice after administration of compound LS-23, the mice in the experimental group and the control group are basically not different, fig. 6E shows the liver toxicity of compound LS-23 by measuring the level of glutamic-oxaloacetic transaminase in serum after the anesthesia of mice, the liver toxicity of compound LS-23 is evaluated by detecting the level of C in serum, and the liver toxicity of compound LS-23 is not significantly lower than that the liver toxicity of mouse P is evaluated by detecting the level of serum, and the liver toxicity of the compound is not significantly lower than that of P-23 is measured, and the toxicity of the compound is not significantly lower than that is evaluated by the contrast of P-23. As can be seen from FIG. 6F, in tumor tissues of control (DMSO) and experimental (20 mg/kg and 40 mg/kg) mice, KLF5 level in tumor tissues of the experimental was significantly reduced, demonstrating the effect of LS-23 on degradation of KLF5 in vivo.

The use and welfare of the experimental animals were carried out in compliance with the regulations of the international commission on assessment and approval of experimental animals (AAALAC). The health condition and death of animals are monitored daily, and routine examination includes observing the effects of the test substances and drugs on the daily performance of the animals, such as behavioral activity, weight change, and appearance signs.

The results show that the representative compound LS-23 in the embodiment of the invention has better in-vivo anticancer effect, can effectively inhibit the growth of breast cancer cells in mice, and can not obviously influence the weight and liver and kidney functions of the mice.

EXAMPLE 9 evaluation of binding Activity of representative Compound LS-23 of the present invention on KLF5 protein

Binding and force of LS-23 to KLF5 was determined using SPR (surface plasmon resonance assay) experiments by coupling KLF5 protein to CM5 chips using Biacore S200 instrument assay, coupling buffer sodium acetate buffer PH=4.5, regeneration buffer GLYLINE PH =2.5, running buffer PBSP (PBS+0.005% P20+5% DMSO) binding assays were performed with LS-23 concentrations of 0. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 4. Mu.M, 6. Mu.M, 8. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, respectively, to coupled KLF5 protein.

As a result of the test, as shown in FIG. 7, 0.5. Mu.M of compound LS-23 started to have binding ability to KLF5 protein (RU >0, the larger the ordinate in the figure represents the binding ability of compound LS-23 to KLF5 protein, the stronger the binding ability, and the abscissa represents the reaction time), and the binding signal increased with the increase in the molar concentration of compound LS-23, showing that the maximum dissociation constant of compound LS-23 binding to deubiquitinase KLF5 was KD= 9.507. Mu.M according to the result. The results demonstrate that compound LS-23 can bind directly to KLF5 protein.

In conclusion, the compound of the invention not only shows induced KLF5 protein degradation activity, but also has obvious antiproliferative effect, and also has obvious dose-dependent inhibition of tumor growth in vivo, and is an effective compound with potential for treating triple negative breast cancer.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined 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 it comprises the compound for targeting and degrading the transcription factor KLF5 according to claim 1.

3 . The pharmaceutical composition according to claim 2 , wherein the pharmaceutical composition comprises a second agent, and the second agent is used to prevent and/or treat cancer.

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

5. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is formulated into an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup or a 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 comprises one or more of breast cancer, liver cancer, lung cancer, colorectal cancer, leukemia, gastric cancer, glioma and prostate cancer.