Targeted TEA domain transcription factor 1 compound and application of antitumor activity thereof

Abstract

The invention relates to a compound targeting a TEA domain transcription factor 1 and application of antitumor activity of the compound, the structural general formula of the compound is as follows: # imgabs0 #, and R is independently selected from one of the following groups: # imgabs1 #. The compound provided by the invention can be effectively covalently combined with CYS359 or CYS327 of the TEA domain transcription factor 1, and can inhibit the growth of tumor cells; good application prospects are realized.

Description

Technical Field

[0001] The present invention relates to a class of compounds targeting TEA domain transcription factor 1 and their related analogs, and the compound or a pharmaceutical composition containing this compound can be used as a drug for preparing drugs targeting TEAD for treating various malignant tumors and related diseases of tumor metastasis, with particular attention paid to its application in the treatment of pancreatic cancer. Background Art

[0002] The Hippo signaling pathway plays a central role in controlling organ size, tissue homeostasis, stem cell renewal, cell proliferation, angiogenesis, and tumorigenesis. The activation of YAP / TAZ in the Hippo signaling pathway is closely associated with the malignant development of various tumors, such as mesothelioma, lung cancer, liver cancer, and meningioma (Miyanaga, A.; Masuda, M.; Tsuta, K.; et al J. Thorac. Oncol. 2015, 10, 844 - 851. Moroishi, T.; Hansen, C.G.; Guan, K.-L.; et al. Nat. Rev. Cancer 2015, 15, 73 - 79.). Targeting the interface of TEAD - YAP protein interaction has been proven to be a feasible strategy for inhibiting the Hippo - YAP signaling pathway, and there have been multiple reports in the literature (Zhou, Z.; Hu, T.; Xu, Z.; et al. FASEB J 2015, 29, 724 - 732. Jiao, S.; Wang, H.; Shi, Z.; Cancer Cell 2014, 25, 166 - 180. Smith, S.A.; Sessions, R.B.; Shoemark, D.K.; et al. J. Med. Chem. 2019, 62, 1291 - 1305.). The TEAD (transcriptional enhancer factor domain) family has been identified to complete the stability and normal function of TEAD proteins through post - transcriptional palmitoylation modification (Chan, P.; Han, X.; Zheng, B.; et al. Nat. Chem. Biol. 2016, 12, 282 - 289.), which is very important for the pro - oncogenic effect of the TEAD - YAP / TAZ signaling pathway (Chan, P.; Han, X.; Zheng, B.; et al. Nat. Chem. Biol. 2016, 12, 282 - 289). Some TEAD inhibitors have entered clinical research for solid tumors (NCT05228015, NCT04665206, NCT04857372). However, the application of TEAD in pancreatic cancer is rarely reported, and it has been reported that the recurrence of pancreatic cancer may be related to the activation of TEAD / YAP (Kapoor, A.; Yao, W.; Ying, H.; et al. Cell, 2014, 158, 185 - 197). Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a class of compounds targeting transcriptional enhancer factor domain 1 and their application in anti - tumor activity. Such compounds can effectively inhibit the growth of tumor cells, especially pancreatic cancer cell lines, and have good application prospects.

[0004] The present invention provides a class of compounds targeting TEA domain transcription factor 1, and the general structural formula of the compounds is shown as follows:

[0005] Wherein, R independently selects one from the following groups:

[0006] Preferably, the structural formula of the compound 2-chloro-1-(4-(2,3-dihydrobenzo[b][1,4]dioxin-2-carbonyl)piperazin-1-yl)ethan-1-one is shown as follows:

[0007] Denoted as YL10.

[0008] The present invention also provides a compound 2-((1-(2-chlorobenzyl)-4-formyl-3-methyl-1H-pyrazol-5-yl)thio)acetic acid targeting TEA domain transcription factor 1, and the structural formula of the compound is shown as follows:

[0009] Denoted as YL4.

[0010] The present invention also provides a compound 8-(benzyloxy)-5-(oxiran-2-yl)quinolin-2(1H)-one targeting TEA domain transcription factor 1, and the structural formula of the compound is shown as follows:

[0011] Denoted as YL16.

[0012] The present invention also provides a compound 2-(tert-butyl)-6-fluoro-1-(2-oxo-2-(4-phenylpiperazin-1-yl)ethyl)-1,2-dihydro-3H-indazol-3-one targeting TEA domain transcription factor 1, and the structural formula of the compound is shown as follows:

[0013] Denoted as YQ8.

[0014] The present invention also provides a compound (1-((5-cyclopropyl-1,2,4-oxadiazol-3-yl)methyl)-1H-pyrrol-2-yl)(4-phenylpiperazin-1-yl)methanone targeting TEA domain transcription factor 1, and the structural formula of the compound is shown as follows:

[0015] Denoted as YQ11.

[0016] The present invention also provides a compound 2-((5-(4-(tert-butyl)phenyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)-1-morpholinoethan-1-one targeting TEA domain transcription factor 1, and the structural formula of the compound is as follows:

[0017] Denoted as YQ14.

[0018] The present invention also provides a pharmaceutical composition, which contains the compound or a pharmaceutically acceptable salt thereof, a radioactive group, a fluorescent group or a biotin to form a marker, and a pharmaceutically acceptable carrier.

[0019] The present invention also provides the use of the compound or the pharmaceutically acceptable salt thereof in the preparation of a tool compound targeting the TEAD protein family.

[0020] The present invention also provides the use of the compound or the pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting the proliferation, growth, migration and invasion of tumor cells; wherein, the tumor cells are selected from one or more of pancreatic cancer cells, breast cancer cells, melanoma cells, liver cancer cells, lung cancer cells, prostate cancer cells, skin cancer cells, colon cancer cells, leukemia cells, lymphoma cells, ovarian cancer cells, gastric cancer cells, bladder cancer cells, renal cancer cells and oral cancer cells.

[0021] The present invention also provides the use of the compound or the pharmaceutically acceptable salt thereof in the preparation of a drug for treating malignant tumors; wherein, the malignant tumors are selected from one or more of pancreatic cancer, breast cancer, osteosarcoma, melanoma, liver cancer, lung cancer, prostate cancer, skin cancer, colon cancer, leukemia, ovarian cancer, gastric cancer, bladder cancer, renal cancer and oral cancer.

[0022] The present invention also provides the use of the compound or the pharmaceutically acceptable salt thereof in the preparation of a drug for treating the metastasis and recurrence of malignant tumors; wherein, the malignant tumors are selected from one or more of pancreatic cancer, breast cancer, osteosarcoma, melanoma, liver cancer, lung cancer, prostate cancer, skin cancer, colon cancer, leukemia, ovarian cancer, gastric cancer, bladder cancer, renal cancer and oral cancer.

[0023] The present invention also provides the application of the compound or the pharmaceutically acceptable salt thereof in the study of the TEAD1 protein signaling pathway.

[0024] The present invention also provides the use of the compound or the pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting the proliferation and growth of tumor cells; wherein, the tumor cells are selected from pancreatic cancer cells.

[0025] The present invention also provides the use of the described compound or the pharmaceutically acceptable salt thereof in the preparation of a drug for treating malignant tumors; wherein, the malignant tumor is selected from pancreatic cancer.

[0026] The present invention also provides the use of the described compound or the pharmaceutically acceptable salt thereof in the preparation of a drug for treating the metastasis and recurrence of malignant tumors; wherein, the malignant tumor is selected from pancreatic cancer.

[0027] Beneficial effects

[0028] The compound of the present invention can effectively covalently bind to CYS359 or CYS327 of the TEA domain transcription factor 1, and can inhibit the growth of tumor cells, especially inhibit the growth of pancreatic cancer cell lines, and the compound can be used in combination with other clinical drugs to enhance the killing effect of chemotherapeutic drugs, so it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the characterization results of the high-resolution mass spectrum and 1H NMR spectrum of compound YL10.

[0030] Figure 2 It is the high-resolution mass spectrum structure characterization results of compounds YL16, YQ8, YQ11, and YQ14.

[0031] Figure 3 It shows that compound YL10 inhibits the growth of breast cancer cell line MCF7 in a gradient-dependent manner, IC50 = 9.01 μM.

[0032] Figure 4 It shows that compound YL10 inhibits the growth of osteosarcoma cell line 143B in a concentration gradient-dependent manner.

[0033] Figure 5 It shows that compound YL10 inhibits the growth of pancreatic cancer cell lines Aspc-1 (A), PANC-1 (B), Patu8988t (C), and CFPAC-1 (D) in a concentration gradient-dependent manner.

[0034] Figure 6 It is the intact protein mass spectrometry analysis results of compound YL10; wherein, A: TEAD1 is co-incubated with DMSO; B: TEAD1 is co-incubated with YL10.

[0035] Figure 7 A - C show that compound YL10 binds to specific cysteine sites of TEAD1.

[0036] Figure 8 It shows that compound YL10 can inhibit the proliferation of Aspc-1 cells in a concentration gradient-dependent manner.

[0037] Figure 9 It is shown that compound YL10 can promote the apoptosis of Aspc-1 cells in a concentration gradient-dependent manner. Detailed implementation manners

[0038] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0039] The compounds selected from the covalent compound library in Table 1 and their codes are as follows:

[0040]

[0041]

[0042] The compounds selected from the diverse scaffold library in Table 2:

[0043]

[0044] Example 1

[0045] Virtual screening

[0046] Download the crystal structure file 7ZJP of TEAD1 from the Protein Data Bank, and use the Schrödinger virtual screening software to prepare the protein; select PHE239 as the center to generate a grid file, and perform virtual screening on the cysteine-targeted covalent compound library of MedChemExpress (HY-L153, containing 4,812 compounds targeting cysteine covalent warheads) and the drug-like diversity library (HY-L901, a total of 50,000 compounds) in the standard precision docking mode. After purchasing 17 covalent small molecule compounds with high scoring rankings and covalent warheads close to CYS in the protein and 17 non-covalent compounds from MCE, subsequent experimental screening was carried out.

[0047] Example 2

[0048] The breast cancer cell line MCF-7 cells (from ATCC) were cultured in DMEM high-glucose basal medium supplemented with 10% FBS and 1% double antibody. After washing with PBS, they were digested with trypsin, the supernatant was removed after centrifugation, and they were resuspended with fresh complete medium and seeded in 96-well plates, with 3,000 - 4,000 cells per well and a medium volume of 200 μl. The cells were cultured at 37 °C, 5% CO 2Overnight in an incubator. The next day, the supernatant of cell culture was removed, washed with PBS, and then complete cell culture medium containing the drug was added. The concentration of the drug in each well was 20 μM, and the concentration of DMSO was 0.2%, with three replicate wells. After culturing for 48 hours in a cell incubator at 37 °C and 5% CO 2 Under the conditions of, the cell viability was detected using the CellTiter-Glo Luminescent assay kit (Promega, G7573), and the readings were taken with a microplate reader. The DMSO well was used as the negative control, and the well without cells was used as the blank control. The cell survival rate was reflected by the value obtained by subtracting the mean of the blank control from the mean of the drug-treated wells and then dividing by the value obtained by subtracting the mean of the blank control from the mean of the negative control group. The experimental results showed that some compounds such as YL4, YL10, YL12, YL16, YQ8, YQ11, and YQ14 had good effects on inhibiting the growth of cancer cells, and among them, YL10 was superior to the reported TEAD inhibitor YL12 (.Xu, xiaofeng, et al. Bicyclic compounds, compositions and use thereof. WO2022037568A1). Therefore, subsequent multi-concentration tests of the compound and tests on multiple cell lines were carried out on YL10. The test conditions were similar, and the concentrations of the compound were 30 μM, 10 μM, 3.333 μΜ, 1.111 μΜ, 0.3704 μΜ, 0.1234 μΜ, 0.04115 μΜ, with two replicate wells. The experimental data processing software was Graphpad Prism 8.3.0. The experimental results showed that the compound YL10 could inhibit the proliferation of MCF-7 cells in a concentration-gradient-dependent manner ( Figure 3 ).

[0049] Table 3 Single-concentration cell survival experiment for primary screening of compounds.

[0050]

[0051]

[0052] Example 3

[0053] Compound structure characterization

[0054] High-resolution mass spectrometry was performed using a Bruker MicroTOF-Q II liquid chromatography-mass spectrometry instrument, and the detection method was ESI high-resolution mass spectrometry. 1 1H-NMR was measured using a Bruker Avance NEO 700 MHz instrument, and the solvent was deuterated chloroform.

[0055] As Figure 1As shown, for the compound 2-chloro-1-(4-(2,3-dihydrobenzo[b][1,4]dioxin-2-carbonyl)piperazin-1-yl)ethan-1-one (YL10) in [C 15 H 17 ClN 2 O 4 +Na], the theoretical m / z value is 347.0769 and the target peak 347.0766 was found; 1 H NMR (700 MHz, CDCl 3 ) δ 6.97–6.83 (m, 4H), 4.88–4.79 (m, 1H), 4.54–4.47 (m, 1H), 4.40–4.32 (m, 1H), 4.11 (d, J = 3.4 Hz, 2H), 4.01–3.83 (m, 3H), 3.74–3.42 (m, 5H).

[0056] As Figure 2 shown, for the compound 8-(benzyloxy)-5-(oxiran-2-yl)quinolin-2(1H)-one (YL16) in [C 18 H 15 NO 3 +Na], the theoretical m / z value is 316.0944 and the target peak 316.0940 was found;

[0057] For the compound 2-(tert-butyl)-6-fluoro-1-(2-oxo-2-(4-phenylpiperazin-1-yl)ethyl)-1,2-dihydro-3H-indazol-3-one (YQ8) in [C 23 H 27 FN 4 O 2 +Na], the theoretical m / z value is 433.2010 and the target peak 433.2016 was found;

[0058] For the compound (1-((5-cyclopropyl-1,2,4-oxadiazol-3-yl)methyl)-1H-pyrrol-2-yl)(4-phenylpiperazin-1-yl)methanone (YQ11) in [C 21 H 23 N 5 O 2 +Na], the theoretical m / z value is 400.1744 and the target peak 400.1752 was found;

[0059] For the compound 2-((5-(4-(tert-butyl)phenyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)-1-morpholinoethan-1-one (YQ14) in [C 24 H 28 N 4 O 2The theoretical m / z value of [S+Na] was 459.1825, and the target peak 459.1840 was found.

[0060] Example 4

[0061] Other cancer cell lines such as osteosarcoma cell line 143B (from ATCC) were also tested for activity inhibition with compound YL10. The culture conditions for the 143B cell line were DMEM high-glucose medium supplemented with 10% FBS. After washing with PBS, the cells were digested with trypsin, the supernatant was removed after centrifugation, and the cells were resuspended in fresh complete medium and seeded in 96-well plates at 4000 cells per well with a medium volume of 200 μl. The cells were incubated overnight in an incubator at 37 °C, 5% CO 2 The next day, the supernatant of the cell culture was removed, the cells were washed with PBS, and then the cell complete medium containing the drug was added. The concentrations of the drug were 30 μM, 10 μM, 3.333 μM, and the concentration of DMSO was 0.3%, with three replicates. Incubated in a cell incubator at 37 °C, 5% CO 2 After culturing for 48 hours under the conditions of, the cell viability was tested using a CCK8 assay kit (abcam, ab155902), and the absorbance value was read using a microplate reader. The data processing software was Graphpad Prism 8.3.0. The processing results were as Figure 4 , and the results showed that compound YL10 could inhibit the growth of osteosarcoma cell line 143B in a concentration-gradient-dependent manner. At a concentration of 10 μM, the cell survival rate was 25%.

[0062] Example 5

[0063] Pancreatic cancer cell line Aspc-1 cells (from ATCC) were cultured in RPMI-1640 basal medium supplemented with 10% FBS and 1% penicillin-streptomycin. After washing with PBS, the cells were digested with trypsin, the supernatant was removed after centrifugation, and the cells were resuspended in fresh complete medium and seeded in 96-well plates at 4000 cells per well with a medium volume of 200 μl. The cells were incubated overnight in an incubator at 37 °C, 5% CO 2 The next day, the supernatant of the cell culture was removed, the cells were washed with PBS, and then the cell complete medium containing the compound was added. The concentrations of the compound were 30 μM, 10 μM, 3.333 μΜ, 1.111 μΜ, 0.3704 μΜ, 0.1234 μΜ, 0.04115 μΜ, with two replicates. The concentration of DMSO was 0.3%. Incubated in a cell incubator at 37 °C, 5% CO 2After culturing for 48 hours under the given conditions, the cell viability was detected using the CellTiter-Glo Luminescent assay kit (Promega, G7573), and the readings were taken with a microplate reader. The DMSO well was used as the negative control, and the well without cells was used as the blank control. The cell survival rate was reflected by the value obtained by subtracting the mean of the blank control from the mean of the wells with the drug and then dividing by the value obtained by subtracting the mean of the blank control from the mean of the negative control group. The experimental data processing software was Graphpad Prism 8.3.0. The experimental results showed that compound YL10 could inhibit the proliferation of Aspc-1 cells in a concentration-gradient-dependent manner ( Figure 5 A).

[0064] Example 6

[0065] PANC-1 cells (derived from ATCC), a pancreatic cancer cell line, were cultured in DMEM high-glucose basal medium supplemented with 10% FBS and 1% penicillin-streptomycin. After washing with PBS, the cells were digested with trypsin, the supernatant was removed after centrifugation, and the cells were resuspended in fresh complete medium and seeded in a 96-well plate at 4000 cells per well with a medium volume of 200 μl. The cells were incubated overnight in an incubator at 37 °C and 5% CO 2 The next day, the supernatant of the cell culture was removed, the cells were washed with PBS, and then the cell complete medium containing the drug was added. The concentrations of the compound were 30 μM, 10 μM, 3.333 μM, 1.111 μM, 0.3704 μM, 0.1234 μM, 0.04115 μM, with two replicates. The concentration of DMSO was 0.3%. After culturing for 48 hours under the conditions of 37 °C and 5% CO 2 in the cell incubator, the cell viability was detected using the CellTiter-Glo Luminescent assay kit (Promega, G7573), and the readings were taken with a microplate reader. The DMSO well was used as the negative control, and the well without cells was used as the blank control. The cell survival rate was reflected by the value obtained by subtracting the mean of the blank control from the mean of the wells with the drug and then dividing by the value obtained by subtracting the mean of the blank control from the mean of the negative control group. The experimental data processing software was Graphpad Prism 8.3.0. The experimental results showed that compound YL10 could inhibit the proliferation of PANC-1 cells in a concentration-gradient-dependent manner ( Figure 5 B).

[0066] Example 7

[0067] Patu8988t cells (derived from ATCC), a pancreatic cancer cell line, were cultured in DMEM high-glucose basal medium supplemented with 10% FBS and 1% penicillin-streptomycin. After washing with PBS, the cells were digested with trypsin, the supernatant was removed after centrifugation, and the cells were resuspended in fresh complete medium and seeded in a 96-well plate at 4000 cells per well with a medium volume of 200 μl. The cells were incubated overnight in an incubator at 37 °C and 5% CO 2Overnight in an incubator. The next day, the supernatant of cell culture was removed, washed with PBS, and then complete cell culture medium containing the drug was added. The concentrations of the compound were 30 μM, 10 μM, 3.333 μM, 1.111 μM, 0.3704 μM, 0.1234 μM, 0.04115 μM, with duplicate wells. The concentration of DMSO was 0.3%. After culturing for 48 hours under the conditions of 37 °C and 5% CO 2 in a cell incubator, the cell viability was detected using the CellTiter-Glo Luminescent assay kit (Promega, G7573), and the readings were taken with a microplate reader. The DMSO well was used as the negative control, and the cell-free well was used as the blank control. The cell survival rate was reflected by the value obtained by subtracting the mean of the blank control from the mean of the wells with the added drug and then dividing by the value obtained by subtracting the mean of the blank control from the mean of the negative control group. The experimental data processing software was Graphpad Prism 8.3.0. The experimental results showed that compound YL10 could inhibit the proliferation of Patu8988t cells in a concentration-gradient-dependent manner ( Figure 5 C).

[0068] Example 8

[0069] The pancreatic cancer cell line CFPAC-1 cells (obtained from ATCC) were cultured in IMDM basal medium supplemented with 10% FBS and 1% penicillin-streptomycin. After washing with PBS, they were digested with trypsin, the supernatant was removed by centrifugation, and then resuspended in fresh complete medium and seeded onto a 96-well plate at 4000 cells per well with a medium volume of 200 μl. The cells were cultured overnight in an incubator at 37 °C and 5% CO 2 The next day, the supernatant of cell culture was removed, washed with PBS, and then complete cell culture medium containing the drug was added. The concentrations of the compound were 30 μM, 10 μM, 3.333 μM, 1.111 μM, 0.3704 μM, 0.1234 μM, 0.04115 μM, with duplicate wells. The concentration of DMSO was 0.3%. After culturing for 48 hours under the conditions of 37 °C and 5% CO 2 in a cell incubator, the cell viability was detected using the CellTiter-Glo Luminescent assay kit (Promega, G7573), and the readings were taken with a microplate reader. The DMSO well was used as the negative control, and the cell-free well was used as the blank control. The cell survival rate was reflected by the value obtained by subtracting the mean of the blank control from the mean of the wells with the added drug and then dividing by the value obtained by subtracting the mean of the blank control from the mean of the negative control group. The experimental data processing software was Graphpad Prism 8.3.0. The experimental results showed that compound YL10 could inhibit the proliferation of CFPAC-1 cells in a concentration-gradient-dependent manner ( Figure 5 D).

[0070] Example 9

[0071] TEAD1 protein expression

[0072] The TEAD1 - YBD domain fragment (amino acids 206 - 426) was inserted into an expression plasmid, and a 6XHis tag was added at the N - terminus for subsequent purification steps. Induction was carried out in Rosetta Escherichia coli (DE3) using 0.4 mM isopropyl - 1 - thio - D - galactopyranoside (IPTG). After culturing the bacteria to an appropriate density, they were treated at 18 °C for 16 hours to induce protein expression. Next, the induced cells were collected and suspended in lysis buffer (containing 50 mM Tris pH 8.5, 300 mM NaCl, 10 mM imidazole pH 8.0, and 5% glycerol). Sonication was performed on ice to break the cells, and the lysate was recovered by centrifugation at 20000 x g for 30 minutes. The lysate was purified by nickel column, and gradient elution was carried out using imidazole buffer at different concentrations. The eluted protein sample was further purified by gel filtration using a buffer solution of 20 mM Tris - HCl pH 8, 150 mM NaCl, and 1 mM TCEP. Finally, for long - term storage, 10% glycerol was added to the buffer solution, and the protein sample was frozen at - 80 °C.

[0073] Example 10

[0074] Full - length protein mass spectrometry analysis.

[0075] After replacing the buffer solution of TEAD1 protein with PBS, compound YL10 or DMSO was incubated with the protein at a molar ratio of 10:1 at 37 °C for 3 hours. The protein mass spectrometry conditions were as follows: liquid phase (H CLASS, Waters), Q - TOF mass spectrometer (Xevo G2, Waters), C4 column (100 mM, Waters), gradient elution (A = water containing 1‰ formic acid, B = acetonitrile containing 1‰ formic acid, the proportion of B increased from 0 to 45% within 15 minutes), and after electrospray ionization (spray voltage = 40 kV), it entered the mass spectrometry detection. The software for demodulating the mass spectrum was Waters UNIFI, Version: 3.1.0.16.

[0076] The experimental result figures are as Figure 6 , where Figure 6 B is the co - incubation of compound YL10 with TEAD1, and 6A is the DMSO control. The results show that the compound binds to the TEAD1 protein in a 1:1 ratio, and almost 100% of the m / z peak of TEAD1 (26589) is converted to the target m / z peak of TEAD1 + 288 (26886).

[0077] Example 11

[0078] Experiment on confirming the binding rate and binding site of covalent compounds by protein mass spectrometry.

[0079] After replacing the buffer solution of TEAD1 protein with PBS, the compound YL10 or DMSO was incubated with the protein at a molar ratio of 10:1 at 37 °C for 3 hours. 5x pre-cooled acetone was added to the protein sample. After the sample was placed at -20 °C for 1 hour, it was centrifuged at 12,000 g for 7 minutes. The sample was washed twice with 5x pre-cooled acetone and dried in the fume hood. It was resuspended with 8M GHCl and 50 mM ammonium bicarbonate solution, and then reduced (5 mM DTT, 37 °C, treated for 0.5 h) and alkylated (20 mM IAA, 25 °C, treated for 0.5 h). Trypsin with a molar ratio of 1:50 was added to the mixture, and the target protein was enzymatically digested overnight at 37 °C. The next day, 0.2% formic acid was added to quench the digestion reaction, and then desalted with C18 Ziptip. Data acquisition was performed by using Orbitrap Exploris 240 in conjunction with nano-liquid phase, and data analysis was performed by PEAKS software. The experimental results are as Figure 7 shown. As Figure 7 can be seen, the compound covalently binds to the amino acid residues CYS359 or CYS327 of TEAD1 protein.

[0080] Example 13

[0081] Pancreatic cancer cell line Aspc-1 cells (from ATCC) were cultured in RPMI-1640 basal medium supplemented with 10% FBS and 1% penicillin-streptomycin. After washing with PBS, they were digested with trypsin. After centrifugation, the supernatant was removed, and they were resuspended with fresh complete medium and seeded on a six-well plate, with 800 cells per well and a medium volume of 2 ml. The cells were incubated overnight in an incubator at 37 °C, 5% CO 2 . The next day, the supernatant of cell culture was removed. After washing with PBS, the cell complete medium containing the drug was added. The concentrations of the compound were 1 μM and 0.1 μM, with two replicates. The concentration of DMSO was 0.3%. After culturing for 7 days under the conditions of 37 °C, 5% CO 2 in the cell incubator, the supernatant of cell culture was removed. After washing with PBS, it was fixed with 4% paraformaldehyde, stained with crystal violet, and then photographed and counted. The experimental data processing software was Image J. The experimental results showed that the compound YL10 could inhibit the proliferation of Aspc-1 cells in a concentration gradient-dependent manner ( Figure 8 ).

[0082] Example 14

[0083] The pancreatic cancer cell line, Aspc-1 cells (obtained from ATCC), were cultured in RPMI-1640 basal medium supplemented with 10% FBS and 1% penicillin-streptomycin. After washing with PBS, the cells were digested with trypsin, the supernatant was removed after centrifugation, and the cells were resuspended in fresh complete medium and seeded in a six-well plate at a density of 100,000 cells per well with a medium volume of 2 ml. The cells were incubated overnight in an incubator at 37°C and 5% CO 2 . The next day, the supernatant of the cell culture was removed, the cells were washed with PBS, and then complete cell medium containing the drug was added. The concentrations of the compound were 10 μM, 3.333 μM, 1.111 μM, 0.3704 μM, and 0.1234 μM. The concentration of DMSO was 0.3%. After culturing for 2 days in an incubator at 37°C and 5% CO 2 , the supernatant was collected, the cells were digested with trypsin, and the cells were collected by centrifugation together with the supernatant. After centrifugation, the supernatant was removed, and the cells were washed twice with PBS. After staining with Annexin V-FITC / PI Apoptosis Detection Kit (Vazyme, A211-01), flow cytometry was performed. The experimental results showed that compound YL10 could promote the apoptosis of Aspc-1 cells in a concentration gradient-dependent manner ( Figure 9 ).

Claims

1. A compound targeting TEA domain transcription factor 1, characterized in that: The general structural formula of the compound is shown below: Wherein, R is independently selected from one of the following groups:

2. The compound according to claim 1, characterized in that: The structural formula of the compound is shown below:

3. A compound targeting TEA domain transcription factor 1, characterized in that: The structural formula of the compound is shown below:

4. A compound targeting TEA domain transcription factor 1, characterized in that: The structural formula of the compound is shown below:

5. A compound targeting TEA domain transcription factor 1, characterized in that: The structural formula of the compound is shown below:

6. A compound targeting TEA domain transcription factor 1, characterized in that: The structural formula of the compound is shown below:

7. A compound targeting TEA domain transcription factor 1, characterized in that: The structural formula of the compound is shown below:

8. A pharmaceutical composition, characterized in that It contains the compound or pharmaceutically acceptable salt according to any one of claims 1 to 7, a radioactive group, a fluorescent group or a biotin-bound marker, and a pharmaceutically acceptable carrier.

9. Use of the compound according to any one of claims 1 to 7 or the pharmaceutically acceptable salt according to claim 8 in the preparation of a tool compound targeting the TEAD protein family.

10. Use of the compound according to any one of claims 1 to 7 or the pharmaceutically acceptable salt according to claim 8 in the preparation of a drug for inhibiting tumor cell proliferation, growth, migration and infiltration; wherein, The tumor cells are selected from one or more of pancreatic cancer cells, breast cancer cells, melanoma cells, liver cancer cells, lung cancer cells, prostate cancer cells, skin cancer cells, colon cancer cells, leukemia cells, lymphoma cells, ovarian cancer cells, gastric cancer cells, bladder cancer cells, kidney cancer cells and oral cancer cells.

11. Use of the compound according to any one of claims 1 to 7 or the pharmaceutically acceptable salt according to claim 8 in the preparation of a drug for treating malignant tumors; wherein, The malignant tumor is selected from one or more of pancreatic cancer, breast cancer, osteosarcoma, melanoma, liver cancer, lung cancer, prostate cancer, skin cancer, colon cancer, leukemia, ovarian cancer, gastric cancer, bladder cancer, kidney cancer and oral cancer.

12. Use of the compound according to any one of claims 1 to 7 or the pharmaceutically acceptable salt according to claim 8 in the preparation of a drug for treating metastasis and recurrence of malignant tumors; wherein, The malignant tumor is selected from one or more of pancreatic cancer, breast cancer, osteosarcoma, melanoma, liver cancer, lung cancer, prostate cancer, skin cancer, colon cancer, leukemia, ovarian cancer, gastric cancer, bladder cancer, kidney cancer and oral cancer.

13. Use of the compound according to any one of claims 1 to 7 or the pharmaceutically acceptable salt according to claim 8 in studying the targeting of TEAD1 protein signaling pathway.

14. Use of the compound according to any one of claims 1 to 7 or the pharmaceutically acceptable salt according to claim 8 in the preparation of a drug for inhibiting the proliferation and growth of tumor cells; wherein, The tumor cells are selected from pancreatic cancer cells.

15. Use of the compound according to any one of claims 1 to 7 or the pharmaceutically acceptable salt according to claim 8 in the preparation of a drug for treating malignant tumors; wherein, The malignant tumor is selected from pancreatic cancer.

16. Use of the compound according to any one of claims 1 to 7 or the pharmaceutically acceptable salt according to claim 8 in the preparation of a drug for treating metastasis and recurrence of malignant tumors; wherein, The malignant tumor is selected from pancreatic cancer.

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