WO2023168851A1 - 一种硼酸化合物、制备方法及用途 - Google Patents

一种硼酸化合物、制备方法及用途 Download PDF

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WO2023168851A1
WO2023168851A1 PCT/CN2022/099680 CN2022099680W WO2023168851A1 WO 2023168851 A1 WO2023168851 A1 WO 2023168851A1 CN 2022099680 W CN2022099680 W CN 2022099680W WO 2023168851 A1 WO2023168851 A1 WO 2023168851A1
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boric acid
compound
acid compound
formula
stat3
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PCT/CN2022/099680
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English (en)
French (fr)
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王元相
张小雷
邓琳
莫建珊
刘培庆
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中山大学
广州中大南沙科技创新产业园有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the fields of medicinal chemistry and drug therapy, in particular to a boric acid compound, its preparation method and its use.
  • STAT3 Signal transducer and activator of transcription 3
  • STAT3 is a type of bifunctional protein with signal transduction and transcription activation, which is responsible for regulating a series of important physiological processes such as cell growth, proliferation, differentiation, and apoptosis.
  • the SH2 domain plays a crucial role in the activation of STAT3. It can specifically recognize phosphorylated tyrosine residues and thereby be phosphorylated and activated. After phosphorylation, STAT3 quickly enters the nucleus and forms homodimers or heterodimers from monomers, thereby acting as a transcription factor, binding to the promoter of target genes and activating transcription. Since the SH2 domain selectively binds to phosphorylated tyrosine (PTyr) peptides and mediates cell signaling pathways related to cancer, inflammation, allergies and other diseases, it is desirable to develop specific STAT3 inhibitors to disrupt the pTyr-SH2 interaction. A research hotspot in the field of tumor treatment.
  • PTyr phosphorylated tyrosine
  • the purpose of the present invention is to overcome the defects or deficiencies of existing STAT3-targeting drug candidates and provide a boronic acid compound.
  • the boric acid compound provided by the invention has high activity on STAT3 protein, and can significantly inhibit the cellular activity of STAT3 overexpression by inhibiting STAT3 phosphorylation, the binding of STAT3 dimers to DNA and other mechanisms, and the compound has high binding force to the STAT3 target, and can Selectively inhibits STAT3.
  • Another object of the present invention is to provide a method for preparing the above-mentioned boric acid compound.
  • Another object of the present invention is to provide the use of the above-mentioned boric acid compound in preparing drugs that inhibit STAT3 activity.
  • Another object of the present invention is to provide the use of the above-mentioned boric acid compound in preparing drugs for preventing and/or treating tumors or cancer.
  • the present invention provides the following technical solutions:
  • n is an integer from 0 to 2;
  • t an integer from 0 to 1;
  • X and Y are independently selected from CH or N;
  • R 1 is selected from one or more hydrogen, halogen, hydroxyl, amino, nitro, cyano, trifluoromethyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, -C(O)R a , -OC(O)R a , -NR a R b , -CO 2 R a or -CONHR a ;
  • R 2 is selected from hydrogen, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 3 -C 6 heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted 5-10 membered heterocycle Aryl;
  • R 3 is selected from aryl, 5-10 membered heterocyclic aryl, C 5 -C 10 bridged ring, C 5 -C 10 fused ring, C 1 -C 6 alkyl or C 3 -C 6 heterocycloalkyl base;
  • R a , R b are independently selected from hydrogen or C 1 -C 6 alkyl, or R a , R b form C 3 -C 6 cycloalkyl.
  • the present invention uses phenylboronic acid as a drug backbone, which can simulate phosphorylation and has druggability and other characteristics.
  • the boric acid compound obtained after specific substitution has high activity on STAT3 protein by inhibiting STAT3 phosphorylation and the binding of STAT3 dimers to DNA. Mechanisms such as this can significantly inhibit the activity of cells overexpressing STAT3, and the compound has high binding capacity to the STAT3 target and can selectively inhibit STAT3.
  • n is 0 or 1.
  • t is 1.
  • X is CH.
  • Y is CH.
  • R1 is hydrogen
  • the substituents in R 2 are halogen, hydroxyl, amino, nitro, cyano, trifluoromethyl, aryl, heterocyclic aryl, C 1 -C 6 alkyl, C 2 -C 6 alkene base, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 3 -C 7 cycloalkenyl, C 3 -C 6 Heterocycloalkyl, C 3 -C 7 saturated heterocycle, -COOR a or -CONHR a ; or the aryl group in R 2 or the 5-10-membered heterocyclic aryl group and the substituent form naphthyl, indolyl, benzo Furan or benzothienyl.
  • the substituent in R 2 is an aryl group (such as phenyl, naphthyl, indolyl, benzofuran or benzothienyl, the aryl group in the present invention can be understood in this way), substituted
  • the number of bases is 1.
  • R2 is hydrogen, aryl (eg phenyl, naphthyl, indolyl, benzofuran or benzothienyl) or 5-6 membered heterocyclic aryl.
  • the substituents in R 3 are halogen, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy, aryloxy, arylmercapto, arylamine, aryl , heteroaryl, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 1 -C 6 alkoxy, C 1 - C 6 alkylamino, C 3 -C 7 cycloalkenyl, C 3 -C 6 heterocycloalkyl, C 3 -C 7 saturated heterocycle, C 5 -C 10 bridged ring group, -CO 2 R a , -CONHR a or -R a NR b SO 2 R c , R c is trifluoromethyl, substituted or unsubstituted aryl, or substituted or unsubstituted heterocyclic ary
  • the number of substituents in R 3 is 1 to 5.
  • R 3 is -R a NR b SO 2 R c .
  • the substituents in the substituted aryl group and the substituted heterocyclic aryl group in R c are independently selected from C 1 -C 6 alkyl, halogen, hydroxyl, cyano or trifluoromethyl, and the number of substituents is 1 to 5.
  • the boric acid compound has the structure shown in the following numbering:
  • the preparation method of the above-mentioned boric acid compound includes the following steps:
  • the reaction process is as follows:
  • the solvent in S1 is one or more of N,N-dimethylformamide, acetone, tetrahydrofuran, dichloromethane, acetonitrile or water.
  • the reaction temperature of the condensation reaction and hydrolysis reaction in S1 is room temperature; the reaction temperature of the substitution reaction is 0-25°C.
  • the molar ratio of the corresponding amine represented by formula (1) and the corresponding acid represented by formula (2) in the condensation reaction described in S1 is 1: (1.2-1.5).
  • the base in S2 is one or more of N,N-diisopropylethylamine, cesium carbonate, sodium hydride or triethylamine.
  • the molar ratio of the intermediate represented by formula (3) and the substance represented by formula (4) in the substitution reaction described in S2 is 1: (1.0-1.5).
  • the acid described in S3 is hydrochloric acid.
  • the boric acid compound is prepared by the following process: dissolving the corresponding amine shown in formula (1) and the corresponding acid shown in formula (2) in a solvent, and After the condensation reaction, the intermediate shown in formula (6) is generated; the intermediate shown in formula (6) is hydrolyzed under acidic conditions to generate the boric acid compound shown in formula (I).
  • the reaction process is as follows:
  • the boric acid compound is prepared by the following process:
  • the intermediate represented by formula (9) removes the protecting group under acidic conditions, and then undergoes an acylation reaction with the corresponding acid chloride under alkaline conditions to obtain the intermediate represented by formula (10);
  • the intermediate of formula (10) is hydrolyzed under acidic conditions to generate the boric acid compound represented by formula (I).
  • the reaction process is as follows:
  • the reaction temperature of the acylation reaction and hydrolysis reaction is room temperature.
  • the molar ratio of the substance represented by formula (9) and the corresponding acid chloride in the acylation reaction is 1: (1.0-1.1).
  • the pharmaceutically acceptable salt is a pharmaceutically acceptable acid addition salt, ie a pharmaceutically acceptable salt formed by acid treatment.
  • the acid is a pharmaceutically acceptable inorganic acid and a pharmaceutically acceptable organic acid;
  • the acid addition salt is hydrochloride, hydrobromide, nitrate, methyl nitrate, sulfate, hydrogen sulfate Salt, aminosulfate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleic acid Salt, acrylate, fumarate, malate, tartrate, citrate, salicylate, para-aminosalicylate, glycolate, lactate, enanthate, phthalate, Oxalates, succinates, benzoates, o-acetoxybenzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, parabens, methoxybenzoates Benzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pas
  • the boric acid compound and its pharmaceutically acceptable salts, metabolites or prodrugs are used in the preparation of treatments for diseases related to abnormal cell proliferation, morphological changes, hypermotility, etc. with high expression of STAT3.
  • boric acid compounds and their pharmaceutically acceptable salts, metabolites or prodrugs in the preparation of drugs for preventing and/or treating tumors.
  • boric acid compound can inhibit the proliferation, survival, migration and invasion of cancer cells and promote their apoptosis, and is useful in preparing drugs for preventing and/or treating tumors or cancer.
  • the boric acid compound and its pharmaceutically acceptable salts, metabolites or prodrugs are used in the preparation of medicaments for preventing and/or treating cancer.
  • the cancer is lung cancer, bone cancer, gastric cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, brain cancer, etc.
  • Carcinoma pituitary adenoma, epidermoid carcinoma, T-cell lymphoma, chronic and acute leukemia, colorectal cancer, kidney cancer, esophageal cancer, breast cancer, cervical cancer, bladder cancer, fibrosarcoma, breast cancer, esophageal cancer, bladder cancer, Hematopoietic system cancer, lymphoma, medulloblastoma, medulloblastoma, rectal adenocarcinoma, colon cancer, liver cancer, adenoid cystic carcinoma, prostate cancer, head and neck squamous cell carcinoma, brain cancer, hepatocellular carcinoma , melanoma, oligodendroglioma, glioblastoma, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma, thyroid cancer, multiple myeloma (AML), mantle cell lymphoma, triple-negative breast cancer, Non-small cell lung cancer, etc.
  • AML multiple
  • the present invention has the following advantages and effects:
  • the boric acid compound provided by the invention has high activity on STAT3 protein, and can significantly inhibit the cellular activity of STAT3 overexpression by inhibiting STAT3 phosphorylation, the binding of STAT3 dimers to DNA and other mechanisms, and the compound has high binding force to the STAT3 target, and can Selectively inhibits STAT3.
  • Figure 1 is a graph showing the effect of compound 1 on the clonogenesis of gastric cancer cells.
  • Figure 1(A) is a graph of compound 1 inhibiting the clonogenesis of AGS cells and inhibiting the growth of MGC803 cells.
  • Figure 1(B) is a graph of Figure 1(A). statistical results;
  • Figure 2 is a graph showing the results of Compound 1 inhibiting the migration of gastric cancer cells.
  • Figure 2(A) is a graph showing the effect of compound 1 on the migration of gastric cancer cells in a scratch test;
  • Figure 2(B) is the statistical result of Figure 2(A);
  • Figure 3 is a graph of Compound 1 invading cells at different concentrations.
  • Figure 3(A) is a graph of Compound 1 invading cells at different concentrations under a microscope.
  • Figure 3(B) is a statistical result graph of Figure 3(A).
  • Figure 4 is a graph showing the apoptosis results of compound 1 in gastric cancer cells.
  • Figure 4(A) is a graph showing the detection of gastric cancer cell apoptosis using flow cytometry.
  • Figure 4(B) is the statistical result of Figure 4(A);
  • Figure 5 is a graph showing the binding results of Compound 1 and STAT3 protein, Figure 5(A) is the SPR result, and Figure 5(B) is the CESTA result;
  • Figure 6 shows the results of Compound 1 inhibiting the phosphorylation of pY705-STAT3 and the expression of downstream genes
  • Figure 7 is a graph showing the results of compound 1 inhibiting the transcriptional activity of STAT3;
  • Figure 8 shows the results of compound 1 inhibiting STAT3 dimerization
  • Figure 9 is a graph showing the results of compound 1 inhibiting tumor growth;
  • Figure (9A) is a graph of mouse tumors after 21 days of treatment with compound 1 and control compound SH-4-54;
  • Figure (9B) is a tumor growth curve graph;
  • Figure (9C) is a statistical chart of the weight of key tumors;
  • Figure (9D) is a Western Blot test result chart;
  • Figure (9E) is a statistical chart of mouse weight growth;
  • Figure (9F) is the effect of Compound 1 and SH-454 on the morphology of major organs picture.
  • the present invention will be further explained below with reference to the examples and drawings, but the examples do not limit the invention in any way.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.
  • Step 1 Preparation of tert-butylmethyl (2-oxo-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxobenzofuran-2-yl))phenyl )Amino)ethyl)carbamate (1a)
  • Step 2 Preparation of tert-butyl (2-((4-cyclohexylbenzyl))(4-(4,4,5,5-tetramethyl-1,3,2-dioxobenzaldehyde-2-yl) Phenyl)amino)-2-oxoethyl(methyl)carbamate (1b)
  • Step 3 Preparation of N-(4-cyclohexylbenzyl)-2-((2,3,4,5,6-pentafluoro-N-methylphenyl)sulfonamide)-N-(4-(4 ,4,5,5-Tetramethyl-1,3,2-dioxobenzaldehyde-2-yl)phenyl)acetamide (1c)
  • Step 4 Preparation of (4-(N-(4-cyclohexylbenzyl)-2-((2,3,4,5,6-pentafluoro-N-methylphenyl)sulfonylamino)acetamido) phenyl)boric acid
  • Steps Replace tert-butoxycarbonylsarcosine with (R)-N-BOC-azetidine-2-carboxylic acid.
  • the remaining required raw materials, reagents and preparation methods are the same as in Example 1 to obtain a white solid.
  • Steps Replace pentafluorobenzenesulfonyl chloride with p-toluenesulfonyl chloride.
  • the remaining required raw materials, reagents and preparation methods are the same as in Example 1 to obtain a white solid.
  • Steps Replace pentafluorobenzenesulfonyl chloride with p-toluenesulfonyl chloride.
  • the remaining required raw materials, reagents and preparation methods are the same as steps 1, 3 and 4 in Example 1 to obtain a white solid.
  • Step 1 Preparation of 3-(4-cyanophenyl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxobenzaldehyde-2-yl)phenyl )propylamine(8a)
  • Step 2 Preparation of 3-(4-cyanophenyl)-N-(4-cyclohexylbenzyl)-N-(4-(4,4,5,5-tetramethyl-1,3,2- Dioxybenzaldehyde-2-yl)phenyl)propylamine (8b)
  • Step 3 Preparation of (4-(3-(4-cyanophenyl)-N-(4-cyclohexylbenzyl)propionamido)phenyl)boronic acid
  • the boric acid compounds prepared in Examples 1 to 26 are respectively designated as compounds 1 to 26.
  • the boric acid compound can significantly and effectively inhibit the expression level of STAT3, thereby inhibiting the proliferation, survival, migration and invasion of cancer cells and promoting their apoptosis.
  • CCK8 method was used to detect cell viability to detect the effect of compounds on the proliferation of gastric cancer cells.
  • the experimental method is as follows: Take cells in the logarithmic growth phase, inoculate them into a 96-well plate at a cell density of (1.5 ⁇ 3) ⁇ 10 3 cells/well and 100 ⁇ L/well. After culturing for 12 to 24 hours, set up a control group without compound. , blank group and compound group. The cells in the control group without adding any drug were added with only an equal volume of culture medium; the cells in the blank group were not added with either cells or compounds, only culture medium was added; the cells in the compound group were added with different concentrations of small molecule compounds and continued to be cultured for 72 hours, and then placed in a 96-well plate.
  • Figure 1(A) show that compound 1 can significantly inhibit the colony formation of AGS cells at 0.5 ⁇ M and 1 ⁇ M, and can significantly inhibit the growth of MGC803 cells at 1 ⁇ M and 3 ⁇ M, and in two gastric cancer cell lines The effect of inhibiting colony formation was stronger than that of the carboxylic acid positive control compound SH-4-54.
  • Figure 1(B) is a statistical result graph of Figure 1(A), showing that compound 1 can significantly inhibit the clonogenesis of gastric cancer cells, and there is a statistical difference compared with the control drug SH-4-54.
  • Compound 1 can significantly inhibit the migration of gastric cancer cells
  • Figure 2(A) shows the scratch test to detect the effect of compounds on the migration of gastric cancer cells.
  • Compound 1 can significantly inhibit the migration of gastric cancer cells, and is compared with the positive control drug SH-4.
  • Compared with -54, boric acid compound 1 has a more significant effect in inhibiting the migration of gastric cancer cells.
  • Figure 2(B) is a statistical result graph of Figure 2(A), showing that the superior effect of Compound 1 is statistically different from SH-4-54 in inhibiting the migration of gastric cancer cells.
  • Compound 1 can significantly inhibit the invasion of gastric cancer cells
  • the bottom of the chamber was replaced with a medium containing 20% FBS, and the upper layer of the nest was replaced with a medium containing 2% FBS containing or not containing compounds, and the culture was continued in the incubator for 24 hours. After 24 hours, discard the culture medium, wash once with PBS, and discard the PBS.
  • Add crystal violet dyeing solution to both the upper and lower layers of the nest, and dye at room temperature for 30 minutes in the dark. Discard the crystal violet dyeing solution. Add PBS and wash until the background becomes clean, and let the chamber dry naturally at room temperature. Then take pictures and observe and count the number of cells passing through the Transwell nested membrane at each concentration. The experiment was repeated three times independently.
  • Figure 3(A) is a picture of the invading cells under each compound concentration taken under a microscope. The results show that after treatment with Compound 1 and SH-4-54, there are fewer invading cells, indicating that Compound 1 and SH -4-54 can significantly inhibit the invasion of gastric cancer cells, but compared with SH-4-54, boric acid compound 1 has a more obvious inhibitory effect on gastric cancer cell invasion, indicating that boric acid compounds are more effective than carboxylic acid compounds in inhibiting the invasion of gastric cancer cells.
  • Figure 3(B) is the statistical result graph of Figure 3(A). The results show that compound 1 can inhibit the invasion of gastric cancer cells better than SH-4-54, with a statistical difference.
  • Compound 1 can promote gastric cancer cell apoptosis
  • Annexin V-FITC/PI double staining method was used to detect the ability of compound 1 to promote apoptosis of gastric cancer cells. Take gastric cancer cells in the logarithmic growth phase, inoculate them into a 6-well plate, and culture them in an incubator for 24 hours until the cells adhere to the wall. Set up a control group and a compound group. No drug is added to the control group, and different concentrations of compound 1 or SH are added to the compound group. -4-54, and after continuing to culture for 72 hours, use Bebo's Annexin V-FITC/PI double-stained apoptosis detection kit (BB-4101-1) for detection.
  • BB-4101-1 Bebo's Annexin V-FITC/PI double-stained apoptosis detection kit
  • Figure 4(A) shows the detection of apoptosis of gastric cancer cells using flow cytometry.
  • compound 1 was treated for 72 hours. Afterwards, it can significantly promote the apoptosis of gastric cancer cells, and at the same concentration, the effect of Compound 1 on promoting the apoptosis of gastric cancer cells is significantly better than that of SH-4-54 compound;
  • Figure 4(B) is the statistical graph of Figure 4(A), The results showed that compound 1 was better than SH-4-54 in promoting the apoptosis of gastric cancer cells, with a statistical difference.
  • This application example takes Compound 1 as an example to illustrate the mechanism of the boric acid compound of the present invention inhibiting the growth of gastric cancer cells.
  • Compound 1 can bind to STAT3 protein
  • SPR verifies that the compound binds to the STAT3 protein: Thaw the STAT3 protein on ice and perform low-temperature ultrafiltration. Use PBS to prepare the protein to a concentration of 100 ⁇ g/mL, and anchor it to the CM5 chip through amino coupling. Compound 1 and compound SH-4 -54 was dissolved in PBS buffer containing 1% DMSO filtered through a 0.22 ⁇ m membrane, and then diluted half-fold to obtain different compound concentrations (containing 1% DMSO). Compounds of different concentrations are passed through the chip sequentially from low to high, and the response signal is obtained. The kinetics and affinity are calculated by software to determine the binding affinity (K D ).
  • the cell thermal transfer (CETSA) experiment verified that the compound binds to the intracellular STAT3 protein: Gastric cancer cells MGC803 in the logarithmic growth phase were inoculated into a 15cm large dish and cultured in an incubator for 24 hours until the cells grew to adhere to the wall and reached a confluence of 70% ⁇ At 80%, compound 1 and SH-4-54 were added respectively to a final concentration of 5 ⁇ M, and the plate was collected after continuing to culture for 3 hours. Aspirate off the medium, add PBS and wash once. After trypsin digestion, medium digestion and centrifugation, discard the supernatant, add PBS and wash twice, centrifuge and discard the PBS.
  • polyacrylamide gel SDS-PAGE electrophoresis was used to separate the protein samples, and the proteins were transferred to nitrocellulose membrane (PVDF membrane) by wet transfer.
  • PVDF membrane nitrocellulose membrane
  • the primary antibodies of STAT3, c-Myc, Bcl-XL and ⁇ -Actin were incubated overnight at 4°C, and then incubated with fluorescently labeled rabbit secondary antibodies and mouse secondary antibodies respectively at room temperature for 1 hour, and incubated with ECL chemiluminescence solution in the dark for 2 minutes. , and finally use Bio-RAD imaging instrument to detect the protein expression level.
  • FIG. 5(A) SPR results show that compound 1 can significantly bind to STAT3 protein in vitro, and compared with SH-4-54, compound 1 has higher binding activity.
  • Figure 5(B) CESTA results show that Compound 1 can enter cancer cells and bind to the STAT3 protein in the cells, and compared with the control drug SH-4-54, Compound 1 has a higher binding activity to the STAT3 protein in gastric cancer cells.
  • Compound 1 can inhibit the phosphorylation of pY705-STAT3 and the expression of downstream genes
  • Collect cells that have been treated with compound 1 or SH-4-54, discard the culture medium, wash twice with PBS, add lysis buffer (RIPA:PMSF:phosphatase A:phosphatase B 100:1:1:1), ice Shake and lyse for 15 minutes. Use a cell scraper to scrape off the cell lysates. Place the cell plate on ice and let it stand for 5 minutes. Transfer the cell lysates to an EP tube, then centrifuge at 4°C and 15,000 rpm for 15 minutes. Take the supernatant. For protein quantification, add 5 ⁇ Loading Buffer and boil at 100°C for 5 minutes. Then the protein immunoblotting (Western Blot) method was used for detection.
  • ice Shake and lyse for 15 minutes.
  • a cell scraper to scrape off the cell lysates. Place the cell plate on ice and let
  • polyacrylamide gel SDS-PAGE electrophoresis was used to separate the protein samples.
  • the protein was transferred to a nitrocellulose membrane (PVDF membrane) by wet transfer and passed through 5% BSA. After blocking for 1 hour, incubate overnight at 4°C with primary antibodies of pY705-STAT3, T-STAT3, c-Myc, Bcl-XL and ⁇ -Actin, and then use fluorescently labeled rabbit secondary antibodies and mouse secondary antibodies respectively. Incubate at room temperature for 1 hour, incubate in ECL chemiluminescence solution in the dark for 2 minutes, and finally use a Bio-RAD developer to detect the protein expression level.
  • PVDF membrane nitrocellulose membrane
  • 293T cells were cultured in DMEM containing 10% fetal bovine serum. Cells in the logarithmic growth phase were digested and counted. The cells were seeded into a 96-well plate at a density of 1 to 2 ⁇ 104 cells/100 ⁇ L. The cells were cultured in 5% CO2. , in a 37°C incubator, wait for the cells to adhere to the wall and reach a confluence of 70% to 80% before transfecting the cells.
  • a and B Prepare two mixed solutions A and B in sterile 1.5mL EP tubes (the following is the amount of each well: A: 0.25 ⁇ L Lipo 2000+OPTI-MEMI culture medium, the volume is 5 ⁇ L; B: 50ng pGL3-STAT3- promoter plasmid + 50ng STAT3C plasmid + 40ng TKRL (Renilla luciferin reporter gene plasmid) + OPTI-MEM culture medium, the volume is 5 ⁇ L), mix gently, let stand for 5 minutes, and then transfer solution A to tube B. Mix gently and let stand at room temperature for 15 minutes.
  • Renilla luciferase assay buffer To the Renilla luciferase assay buffer, add Renilla luciferase assay substrate (100 ⁇ ) at a ratio of 1:100 to prepare the Renilla luciferase assay working solution; take out the 96-well plate, discard the culture medium, and mix each Add 50 ⁇ L of reporter gene cell lysate to the well, vortex and mix for 10 minutes; take 25 ⁇ L of lysate into a 96-well white plate, add 25 ⁇ L of firefly luciferase detection reagent to each well, shake and mix for 5 minutes, and detect RLU1; after completing the above determination After the firefly luciferase step, add 25 ⁇ L of Renilla luciferase detection working solution to each well, shake and mix for 5 minutes, and detect RLU2; obtain the ratio of RLU1/RLU2.
  • the dimerization of STAT3 was detected using co-immunoprecipitation (Co-IP) method.
  • 293T cells were cultured in DMEM containing 10% fetal bovine serum.
  • 293T cells in the logarithmic growth phase were inoculated into a 6-well plate and cultured for 12 to 24 hours until the cells grew to adhere to the wall and reached a confluency of approximately 70% to 80%. Perform cell transfection.
  • the 6-well plate Take out the 6-well plate, suck off the old culture medium, add 800 ⁇ L OPTI-MEMI medium, use a pipette to drop 200 ⁇ L of the prepared transfection solution into the 6-well plate, place it in a cell culture incubator to continue culturing, and transfect After 6 hours of staining, the medium in the 6-well plate was replaced with DMEM medium containing 10% FBS, and the culture was continued. Different concentrations of compound 1 were added 24 hours after transfection, and culture was continued for 24 hours.
  • IL-6 at a final concentration of 100ng/mL for stimulation 1 hour before protein collection, then extract the total protein of the cells, collect cells that have been treated with Compound 1 or SH-4-54, and discard the culture medium.
  • This application example takes Compound 1 as an example to illustrate the pharmacological efficacy, pharmacology and toxicology research of the boric acid compound of the present invention on the whole animal model.
  • MGC803 cells in the logarithmic growth phase, digest and centrifuge them, count them, wash them twice with pre-cooled PBS, resuspend the cells in pre-cooled PBS and Matrigel at a ratio of 1:1, and prepare a concentration of 2 ⁇ 10 6 cells/100 ⁇ L. Place on ice. Subcutaneously inject 100 ⁇ L of cell suspension on both sides of the abdomen and back of nude mice. When the tumor volume reaches about 70 mm3 , drug administration in groups begins. The nude mice are randomly divided into 4 groups, and the solvent control group is intraperitoneally injected with an equal amount of solvent (containing 15% Trichosanthes).
  • the low-dose group was intraperitoneally injected with 5 mg/kg of compound 1
  • the high-dose group was intraperitoneally injected with 15 mg/kg of compound 1
  • the positive control group was intraperitoneally injected with 15 mg/kg of SH-4-54, administered continuously. 21 days. Tumor volume and mouse weight were measured using vernier calipers and electronic scales every two days. After 21 days of administration, the mice were sacrificed and dissected, and the tumors and organs such as heart, liver, spleen, lungs, and kidneys were removed for subsequent experiments.
  • the experimental results are shown in Figure 9.
  • the tumor map in Figure (9A) shows that after 21 days of treatment with compound 1 and control compound SH-4-54, the tumors of mice were significantly smaller than those without treatment, indicating that compound 1 and SH -4-54 can inhibit tumor growth in mice.
  • Compound 1 already has a significant inhibitory effect on tumor growth at a low dose of 5 mg/kg, and the inhibitory effect is more obvious than that of the control compound SH-4-54 at a high dose of 15 mg/kg, indicating that Compound 1 inhibits tumor growth in vivo.
  • the effect is better than SH-4-54.
  • Figures (9B) and (9C) are respectively the tumor growth curve and the key tumor weight statistics. The same verification can also be obtained.
  • Compound 1 is better than SH-4-54 in inhibiting tumor growth.

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  • Inorganic Compounds Of Heavy Metals (AREA)

Abstract

本发明公开了一种硼酸化合物、制备方法及用途。该硼酸化合物具有如式(I)所示结构：本发明提供的硼酸化合物对STAT3蛋白具有高活性，通过抑制STAT3磷酸化、STAT3二聚体与DNA的结合等机制显著抑制STAT3过表达的细胞活性，且化合物与STAT3靶点的结合力高，可以选择性的抑制STAT3。

Description

一种硼酸化合物、制备方法及用途 技术领域
本发明涉及药物化学和药物治疗学领域，特别涉及一种硼酸化合物、制备方法及用途。
背景技术
信号转导与转录激活因子3(STAT3)是一类具有信号转导和转录激活的双功能蛋白，负责调控细胞的生长、增值、分化以及凋亡等一系列重要生理过程。研究发现STAT3持续性激活及异常高表达，能够诱导细胞增殖、侵袭、迁移，抑制细胞凋亡，促进血管生成，在肿瘤的发生发展转移过程中发挥重要的作用。
其中，SH2结构域对STAT3的激活起着至关重要的作用，它能特异性识别磷酸化酪氨酸残基，从而被磷酸化激活。磷酸化后的STAT3迅速进入细胞核，由单体形成同源或异源二聚体从而作为转录因子，与靶基因的启动子结合，激活转录。由于SH2结构域选择性地与磷酸化酪氨酸(PTyr)肽结合，介导与癌症、炎症、过敏等疾病有关的细胞信号通路，因此开发特异性STAT3抑制剂来破坏pTyr-SH2相互作用是肿瘤治疗领域中的一个研究热点。
迄今为止，已有数个靶向STAT3候选药物进入I/II期临床试验(Eur J Med Chem.2020 Feb 1；187:111922.)，如：S31-201、SF-1-066、STA-21、LLL-3、BP-1-102和SH-4-54，但多数化合物的亲水能力强、膜通透性差，在较高浓度下(IC 50>10μmol·L -1)才能对STAT3高表达肿瘤细胞株的增殖产生抑制作用，因此需进一步的结构优化提高成药性。
综上所述，靶向STAT3的特异性抑制剂已经成为研发的热点，但现今为止已研发的药物，都存在膜通透性差、药效作用小、成药性差等缺点，限制了STAT3抑制剂的临床应用与后期开发。而STAT3作为一个具有前景的肿瘤治疗靶点，本领域迫切需要开发新的一类化合物，能特异性的抑制STAT3，具有选择性高、药效强、成药性好等特征。
发明内容
本发明的目的在于克服现有靶向STAT3候选药物存在的缺陷或不足，提供一种硼酸化合物。本发明提供的硼酸化合物对STAT3蛋白具有高活性，通过抑制STAT3磷酸化、STAT3二聚体与DNA的结合等机制显著抑制STAT3过表达的细胞活性，且化合物与STAT3靶点的结合力高，可以选择性的抑制STAT3。
本发明的另一目的在于提供上述硼酸化合物的制备方法。
本发明的另一目的在于提供上述硼酸化合物在制备抑制STAT3活性的药物中的用途。
本发明的另一目的在于提供上述硼酸化合物在制备预防和/或治疗肿瘤或癌症的药物中的用途。
为了实现本发明的上述目的，本发明提供了如下技术方案：
一种硼酸化合物，具有如式(Ⅰ)所示结构：
Figure PCTCN2022099680-appb-000001
其中，n为0～2的整数；
t＝0～1的整数；
X、Y独立地选自CH或N；
R 1选自一个或多个氢、卤素、羟基、氨基、硝基、氰基、三氟甲基、C 1-C 6烷基、C 1-C 6烷氧基、-C(O)R a、-OC(O)R a、-NR aR b、-CO 2R a或-CONHR a；
R 2选自氢、取代或非取代C 3-C 6环烷基、取代或非取代C 3-C 6杂环烷基、取代或非取代芳基、取代或非取代5-10元杂环芳基；
R 3选自芳基、5-10元杂环芳基、C 5-C 10桥环基、C 5-C 10稠环基、C 1-C 6烷基或C 3-C 6杂环 烷基；
R a、R b独立地选自氢或C 1-C 6烷基，或R a、R b形成C 3-C 6环烷基。
本发明以苯硼酸作为成药骨架，能够模拟磷酸化，具备成药性等特征，并进行特定取代后得到的硼酸化合物对STAT3蛋白具有高活性，通过抑制STAT3磷酸化、STAT3二聚体与DNA的结合等机制显著抑制STAT3过表达的细胞活性，且化合物与STAT3靶点的结合力高，可以选择性的抑制STAT3。
优选地，n为0或1。
优选地，t为1。
优选地，X为CH。
优选地，Y为CH。
优选地，R 1为氢。
优选地，所述R 2中取代基为卤素、羟基、氨基、硝基、氰基、三氟甲基、芳基、杂环芳基、C 1-C 6烷基、C 2-C 6烯基、C 2-C 6炔基、C 3-C 8环烷基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 3-C 7环烯基、C 3-C 6杂环烷基、C 3-C 7饱和杂环、-COOR a或-CONHR a；或R 2中芳基或5-10元杂环芳基与取代基形成萘基、吲哚基、苯并呋喃或苯并噻吩基。
更为优选地，所述R 2中取代基为芳基(例如苯基、萘基、吲哚基、苯并呋喃或苯并噻吩基，本发明中的芳基均可按此理解)，取代基的数量为1个。
优选地，R 2为氢、芳基(例如苯基、萘基、吲哚基、苯并呋喃或苯并噻吩基)或5-6元杂环芳基。
优选地，所述R 3中取代基为卤素、羟基、氨基、硝基、氰基、三氟甲基、三氟甲氧基、芳基氧基、芳基巯基、芳基胺基、芳基、杂芳基、C 1-C 6烷基、C 2-C 6烯基、C 2-C 6炔基、C 3-C 8环烷基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 3-C 7环烯基、C 3-C 6杂环烷基、C 3-C 7饱和杂环、C 5-C 10桥环基、-CO 2R a、-CONHR a或-R aNR bSO 2R c，R c为三氟甲基、取代或非取代芳基，或取代或非取代杂环芳基。
更为优选地，所述R 3中取代基的数量为1～5个。
更为优选地，所述R 3中取代基为-R aNR bSO 2R c。
更为优选地，R c中取代芳基、取代杂环芳基中的取代基独立地选自C 1-C 6烷基、卤素、羟基、氰基或三氟甲基，取代基的数量为1～5个。
优选地，所述硼酸化合物具有如下编号所示结构：
Figure PCTCN2022099680-appb-000002
Figure PCTCN2022099680-appb-000003
上述硼酸化合物的制备方法，包括如下步骤：
S1：将式(1)所示相应的胺和式(2)所示相应的酸溶于溶剂中，经缩合反应后生成式(3)所示中间体；
S2：式(3)所示中间体与式(4)所示物质在碱性条件下经取代反应得到式(5)所示中间体；
S3：式(5)所示中间体在酸性条件下水解，即得式(Ⅰ)所示硼酸化合物。
Figure PCTCN2022099680-appb-000004
反应过程如下：
Figure PCTCN2022099680-appb-000005
优选地，S1中所述溶剂为N,N-二甲基甲酰胺、丙酮、四氢呋喃、二氯甲烷、乙腈或水中的一种或几种。
优选地，S1中所述缩合反应和水解反应的反应温度为室温；所述取代反应的反应温度为0～25℃。
优选地，S1中所述缩合反应中式(1)所示相应的胺和式(2)所示相应的酸的摩尔比为1:(1.2～1.5)。
优选地，S2中所述碱为N,N-二异丙基乙胺、碳酸铯、氢化钠或三乙胺的一种或几种。
优选地，S2中所述取代反应中式(3)所示中间体和式(4)所示物质的摩尔比为1:(1.0～1.5)。
优选地，S3中所述酸为盐酸。
优选的，当R 2为氢，n为0时，所述硼酸化合物通过如下过程制备得到：将式(1)所示相应的胺和式(2)所示相应的酸溶于溶剂中，经缩合反应后生成式(6)所示中间体；式(6)所示中间体在酸性条件下水解生成式(Ⅰ)所示硼酸化合物。
Figure PCTCN2022099680-appb-000006
反应过程如下：
Figure PCTCN2022099680-appb-000007
优选地，当R 3为-R aNR bSO 2R c时，所述硼酸化合物通过如下过程制备得到：
将式(1)所示相应的胺和式(7)所示相应的酸溶于溶剂中，经缩合反应后生成式(8)所示中间体；
式(8)所示中间体与式(4)所示物质在碱性条件下经取代反应得到式(9)所示中间体；
式(9)所示中间体在酸性条件下去除保护基，然后在碱性条件下与相应的酰氯发生酰化反应得到式(10)所示中间体；
式(10)中间体在酸性条件下水解生成式(Ⅰ)所示硼酸化合物。
Figure PCTCN2022099680-appb-000008
反应过程如下：
Figure PCTCN2022099680-appb-000009
优选地，所述酰化反应和水解反应的反应温度为室温
优选地，所述酰化反应中式(9)所示物质和相应的酰氯的摩尔比为1:(1.0～1.1)。
上述硼酸化合物及其药学可接受的盐、代谢物或前药在制备抑制STAT3活性的药物中的用途也在本发明的保护范围内。
优选地，药学可接受的盐为药学上可接受的酸加成盐，即通过酸处理形成的药学上可接受的盐。
所述酸为药学上可接受的无机酸和药学上可接受的有机酸；所述的酸加成盐为盐酸盐、氢溴酸盐、硝酸盐、甲基硝酸盐、硫酸盐、硫酸氢盐、氨基硫酸盐、磷酸盐、乙酸盐、羟基 乙酸盐、苯基乙酸盐、丙酸盐、丁酸盐、异丁酸盐、戊酸盐、马来酸盐、羟基马来酸盐、丙烯酸盐、延胡索酸盐、苹果酸盐、酒石酸盐、柠檬酸盐、水杨酸盐、对氨基水杨酸盐、乙醇酸盐、乳酸盐、庚酸盐、邻苯二甲酸盐、草酸盐、琥珀酸盐、苯甲酸盐、邻乙酰氧基苯甲酸盐、氯苯甲酸盐、甲基苯甲酸盐、二硝基苯甲酸盐、羟苯酸盐、甲氧基苯甲酸盐、扁桃酸盐、丹宁酸盐、甲酸盐、硬脂酸盐、抗坏血酸盐、棕榈酸盐、油酸盐、丙酮酸盐、双羟奈酸盐、丙二酸盐、月桂酸盐、戊二酸盐、谷氨酸盐、丙酸酯月桂硫酸盐(estolate)、甲磺酸盐、乙磺酸盐、2-羟基乙磺酸盐、苯磺酸盐、对氨基苯磺酸盐、对甲苯磺酸盐(甲苯磺酸盐)和萘-2-磺酸盐等。
优选地，所述硼酸化合物及其药学可接受的盐、代谢物或前药在制备治疗STAT3高表达的细胞异常增殖、形态变化以及运动功能亢进等相关的疾病中的用途。
上述硼酸化合物及其药学可接受的盐、代谢物或前药在制备预防和/或治疗肿瘤的药物中的用途。
研究表明，该硼酸化合物可抑制癌细胞增殖、生存、迁移和侵袭并促进其凋亡，具有制备预防和/或治疗肿瘤或癌症的药物中的用途。
优选地，所述硼酸化合物及其药学可接受的盐、代谢物或前药在制备预防和/或治疗癌症的药物中的用途。
更为优选地，所述癌症为肺癌、骨癌、胃癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、脑癌、垂体腺瘤、表皮样癌、T细胞淋巴瘤、慢性和急性白血病、大肠癌、肾癌、食道癌、乳房癌、宫颈癌、膀胱癌、纤维肉瘤、乳腺癌、食道癌、膀胱癌、造血系统癌、淋巴瘤、髓母细胞瘤、成神经管细胞瘤、直肠腺癌、结肠癌、肝癌、腺样囊性癌、前列腺癌、头颈部鳞状细胞癌、脑癌、肝细胞癌、黑色素瘤、少突神经胶质瘤、胶质母细胞癌、卵巢透明细胞癌、卵巢浆液性囊腺癌、甲状腺癌、多发性骨髓瘤(AML)、套细胞淋巴瘤、三阴性乳腺癌、非小细胞肺癌等。
相对于现有技术，本发明具有如下的优点及效果：
本发明提供的硼酸化合物对STAT3蛋白具有高活性，通过抑制STAT3磷酸化、STAT3二聚体与DNA的结合等机制显著抑制STAT3过表达的细胞活性，且化合物与STAT3靶点的结合力高，可以选择性的抑制STAT3。
附图说明
图1为化合物1对胃癌细胞克隆形成的影响结果图，其中图1(A)为化合物1抑制AGS细胞的克隆形成及抑制MGC803细胞的生长图，图1(B)为图1(A)的统计学结果；
图2为化合物1抑制胃癌细胞的迁移结果图，其中图2(A)为划痕实验检测化合物对胃癌细胞迁移的影响结果图；图2(B)为图2(A)的统计学结果；
图3为化合物1在不同浓度下侵袭细胞图，图3(A)为显微镜拍照下化合物1不同浓度下侵袭细胞图；图3(B)为图3(A)的统计结果图；
图4为化合物1进胃癌细胞凋亡结果图，图4(A)为利用流式细胞仪检测胃癌细胞凋亡的情况图，图4(B)为图4(A)的统计学结果；
图5为化合物1与STAT3蛋白结合结果图，图5(A)为SPR结果，图5(B)为CESTA结果；
图6为化合物1抑制pY705-STAT3的磷酸化及下游基因的表达结果图；
图7为化合物1抑制STAT3的转录活性结果图；
图8为化合物1抑制STAT3二聚化结果图；
图9为化合物1抑制肿瘤生长结果图；其中，图(9A)为经化合物1和对照化合物SH-4-54治疗21天后的小鼠肿瘤图；图(9B)为肿瘤的生长曲线图；图(9C)为重点肿瘤重量统计图；图(9D)为Western Blot试验结果图；图(9E)为小鼠重量生长统计图；图(9F)为化合物1和SH-454对主要脏器形态影响图。
具体实施方式
以下结合实施例和附图进一步解释本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，本发明所用试剂和材料均为市购。
实施例1 (4-(N-(4-环己基苄基)-2-((2,3,4,5,6-五氟-N-甲基苯基)磺酰氨基)乙酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000010
具体制备过程为：
步骤1：制备叔丁基甲基(2-氧代-2-((4-(4,4,5,5-四甲基-1,3,2-二氧苯并呋喃-2-基)苯基)氨基)乙基)氨基甲酸酯(1a)
Figure PCTCN2022099680-appb-000011
将4-氨基苯硼酸频哪醇酯(4.0g,18.26mmol)、叔丁氧羰酰基肌氨酸(4.16g,21.91mmol)、HATU(8.33g,21.91mmol)和N,N-二异丙基乙胺(9.5ml,54.78mmol)溶于适量的N,N-二甲基甲酰胺中，室温搅拌过夜。反应完成，乙酸乙酯以及水萃取，无水硫酸钠干燥，旋干溶剂得粗产品。柱层析得白色固体4.27g，产率为60％。 1H NMR(400MHz,CDCl 3)δ7.77(d,J＝8.1Hz,2H),7.51(d,J＝8.2Hz,2H),3.96(s,2H),3.01(s,3H),1.49(s,9H),1.33(s,12H).LCMS:m/z(M+H +):390.23.
步骤2：制备叔丁基(2-((4-环己基苄基)(4-(4,4,5,5-四甲基-1,3,2-二氧苯甲醛-2-基)苯基)氨基)-2-氧乙基(甲基)氨基甲酸酯(1b)
Figure PCTCN2022099680-appb-000012
取氢化钠(1.23g,30.75mmol)，加入适量的超干四氢呋喃，冰浴条件下缓慢加入化合物1a(4.0g,10.25mmol)，搅拌1h后，加入4-环己基苄溴(2.6g,10.25mmol)，室温搅拌过夜。反应完成后，加水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，旋干溶剂得粗产品。柱层析得白色固体4.09g，产率为70％。 1H NMR(400MHz,CDCl 3)δ7.77(dd,J＝12.5,8.0Hz,2H),7.10(dd,J＝14.2,6.8Hz,4H),7.06–6.92(m,2H),4.82(d,J＝5.5Hz,2H),3.64(d,J＝52.1Hz,2H),2.88(d,J＝12.5Hz,3H),2.45(s,1H),1.78(dd,J＝41.3,12.0Hz,6H),1.44(s,4H),1.40(d,J＝18.5Hz,9H),1.33(t,J＝5.0Hz,12H).LCMS:m/z(M+H +):562.36.
步骤3：制备N-(4-环己基苄基)-2-((2,3,4,5,6-五氟-N-甲基苯基)磺酰胺)-N-(4-(4,4,5,5-四甲 基-1,3,2-二氧苯甲醛-2-基)苯基)乙酰胺(1c)
Figure PCTCN2022099680-appb-000013
取化合物1b(3.5g,6.22mmol)，加入适量的二氯甲烷溶解，冰浴条件下加入三氟乙酸(4.6ml,62.24mmol)，搅拌1小时。反应完全后，乙酸乙酯萃取，旋干，用适量乙腈溶解，加入N,N-二异丙基乙胺(2.3ml,31.12mmol)、五氟苯磺酰氯(1.02ml,6.85mmol)，室温搅拌。反应完成后，乙酸乙酯萃取，无水硫酸钠干燥，旋干溶剂得粗产品。柱层析得白色固体1.91g，产率为62％。 1H NMR(400MHz,DMSO)δ7.69(d,J＝7.7Hz,2H),7.19(d,J＝8.2Hz,2H),7.11(d,J＝7.9Hz,2H),7.01(d,J＝7.6Hz,2H),4.75(s,2H),3.98(s,2H),2.99(s,3H),2.44(s,1H),1.78–1.66(m,5H),1.35(dd,J＝21.2,9.7Hz,5H),1.28(s,12H).LCMS:m/z(M+H +):692.25.
步骤4：制备(4-(N-(4-环己基苄基)-2-((2,3,4,5,6-五氟-N-甲基苯基)磺酰氨基)乙酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000014
取化合物1c(1.9g,2.74mmol)，加入适量的四氢呋喃和水溶解，加入高碘酸钠(1.76g,8.23mmol)和2ml 2M稀盐酸溶液，室温搅拌。反应完成后，用乙酸乙酯萃取，无水硫酸钠干燥，旋干溶剂得粗产品。柱层析得白色固体1.59g，产率为95.5％。 1H NMR(400MHz,DMSO)δ7.85(dd,J＝25.2,7.6Hz,2H),7.17–7.06(m,4H),7.01(d,J＝7.3Hz,2H),4.74(s,2H),4.01(s,2H),3.02(s,3H),2.48–2.40(m,1H),1.71(dd,J＝29.9,10.5Hz,5H),1.38–1.19(m,5H).HRMS(ESI)calcd for C 28H 28N 2O 5BF 5S(M+H +):611.1810；found 611.1808.
实施例2 (R)-(4-(N-(4-环己基苄基)-1-((五氟苯基)磺酰基)吡咯烷-2-甲酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000015
步骤：将叔丁氧羰酰基肌氨酸换成Boc-D-脯氨酸，其余所需原料、试剂以及制备方法同实施例1，得白色固体。 1H NMR(400MHz,DMSO)δ8.16(s,2H),7.79(d,J＝7.9Hz,2H),7.11(d,J＝7.9Hz,4H),7.01(d,J＝7.9Hz,2H),4.89(d,J＝14.8Hz,1H),4.60(d,J＝14.9Hz,1H),4.33(dd,J＝7.2,4.9Hz,1H),3.57–3.51(m,1H),3.44(dd,J＝14.9,7.0Hz,1H),2.44(s,1H),1.94(dd,J＝11.8,4.1Hz,2H),1.72(dd,J＝33.4,8.9Hz,6H),1.36(dd,J＝20.5,9.4Hz,4H),1.30–1.14(m,2H).HRMS(ESI)calcd for C 30H 30N 2O 5BF 5S(M+H +):637.1904；found 637.1967.
实施例3 (R)-(4-(N-(4-环己基苄基)-2-((2,3,4,5,6-五氟-N-甲基苯基)磺酰氨基)丙酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000016
步骤：将叔丁氧羰酰基肌氨酸换成BOC-D-丙氨酸，其余所需原料、试剂以及制备方法同实施例1，得白色固体。 1H NMR(400MHz,DMSO)δ8.16(s,2H),7.81(d,J＝8.0Hz,2H),7.11(dd,J＝11.7,8.0Hz,4H),6.97(d,J＝7.8Hz,2H),4.93(d,J＝14.8Hz,1H),4.68(q,J＝6.8Hz,1H),4.56(d,J＝14.8Hz,1H),3.08(s,3H),2.47–2.38(m,1H),1.71(dd,J＝28.2,9.4Hz,5H),1.43–1.25(m,5H),1.16(d,J＝7.1Hz,3H).HRMS(ESI)calcd for C 29H 30N 2O 5BF 5S(M+H +):625.1967；found 625.1943.
实施例4 (S)-(4-(N-(4-环己基苄基)-2-((2,3,4,5,6-五氟-N-甲基苯基)磺酰氨基)丙酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000017
步骤：将叔丁氧羰酰基肌氨酸换成N-叔丁氧羰基-L-丙氨酸，其余所需原料、试剂以及制备方法同实施例1，得白色固体。 1H NMR(500MHz,DMSO)δ8.15(s,1H),7.81(d,J＝7.5Hz,1H),7.16–7.04(m,4H),6.98(d,J＝7.2Hz,2H),4.93(d,J＝14.5Hz,1H),4.68(d,J＝6.5Hz,1H),4.57(d,J＝14.7Hz,1H),3.08(s,3H),2.44(s,1H),1.72(dd,J＝34.1,8.8Hz,5H),1.35(t,J＝9.4Hz,4H),1.18(t,J＝11.3Hz,4H).HRMS(ESI)calcd for C 29H 30N 2O 5BF 5S(M+H +):625.1967；found 625.1969.
实施例5 (R)-(4-(N-(4-环己基苄基)-1-((五氟苯基)磺酰基)氮杂环丁烷-2-甲酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000018
步骤：将叔丁氧羰酰基肌氨酸换成(R)-N-BOC-氮杂环丁烷-2-羧酸，其余所需原料、试剂以及制备方法同实施例1，得白色固体。 1H NMR(500MHz,DMSO)δ8.13(s,2H),7.79(d,J＝7.9Hz,2H),7.11(d,J＝8.0Hz,2H),7.05(d,J＝7.4Hz,4H),4.89(d,J＝15.0Hz,1H),4.69(d,J＝14.4Hz,2H),3.88(d,J＝7.4Hz,2H),2.47–2.40(m,1H),2.32–2.22(m,1H),1.86(tt,J＝7.5,3.9Hz,1H),1.71(dd,J＝37.0,9.2Hz,5H),1.29(dt,J＝38.0,7.6Hz,5H).HRMS(ESI)calcd for C 29H 28N 2O 5BF 5S(M+H +):623.1812；found 623.1810.
实施例6 (4-(N-(4-环己基苄基)-2-((N,4-二甲基苯基)磺酰胺)乙酰胺)苯基)硼酸
Figure PCTCN2022099680-appb-000019
步骤：将五氟苯磺酰氯换成对甲苯磺酰氯，其余所需原料、试剂以及制备方法同实施例1，得白色固体。 1H NMR(400MHz,DMSO)δ8.15(s,1H),7.78(d,J＝8.0Hz,2H),7.52(d,J＝8.1Hz,2H),7.35(d,J＝8.1Hz,2H),7.14(dd,J＝11.6,8.2Hz,4H),7.05(d,J＝7.8Hz,2H),4.77(s,2H),3.72(s,2H),2.78(s,3H),2.45(s,1H),2.37(s,3H),1.75(d,J＝7.1Hz,4H),1.67(s,1H),1.35(t,J＝10.8Hz,4H),1.24(s,1H).HRMS(ESI)calcd for C 29H 35N 2O 5BS(M+H +):535.2438；found 535.2446.
实施例7 (4-(2-((N,4-二甲基苯基)磺酰胺)乙酰胺)苯基)硼酸
Figure PCTCN2022099680-appb-000020
步骤：将五氟苯磺酰氯换成对甲苯磺酰氯，其余所需原料、试剂以及制备方法同实施例1中的步骤1、3、4，得白色固体。 1H NMR(400MHz,DMSO)δ10.03(s,1H),7.91(s,2H),7.74(s,1H),7.72(d,J＝5.2Hz,2H),7.69(s,1H),7.51(d,J＝8.2Hz,2H),7.44(d,J＝8.0Hz,2H),3.90(s,2H),2.80(s,3H),2.41(s,3H).HRMS(ESI)calcd for C 26H 19N 2O 5BS(M+H +):363.1184；found 363.1184.
实施例8 (4-(3-(4-氰基苯基)-N-(4-环己基苄基)丙酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000021
具体制备过程为：
步骤1：制备3-(4-氰基苯基)-N-(4-(4,4,5,5-四甲基-1,3,2-二氧苯甲醛-2-基)苯基)丙胺(8a)
Figure PCTCN2022099680-appb-000022
将4-氨基苯硼酸频哪醇酯(1.0g,4.57mmol)、4-氰基-3-苯基丙酸(0.96g,5.48mmol)、HATU(2.08g,5.48mmol)和N,N-二异丙基乙胺(2.4ml,13.71mmol)溶于适量的N,N-二甲基甲酰胺中，室温搅拌过夜。反应完成，乙酸乙酯以及水萃取，无水硫酸钠干燥，旋干溶剂得粗产品。柱层析得白色固体1.49g，产率为86.5％。 1H NMR(400MHz,DMSO)δ10.06(s,1H),7.76(d,J＝8.2Hz,2H),7.59(s,4H),7.47(d,J＝8.2Hz,2H),3.00(t,J＝7.5Hz,2H),2.69(t,J＝7.6Hz,2H),1.28(s,12H).LCMS:m/z(M+H +):376.20.
步骤2：制备3-(4-氰基苯基)-N-(4-环己基苄基)-N-(4-(4,4,5,5-四甲基-1,3,2-二氧苯甲醛-2-基)苯基)丙胺(8b)
Figure PCTCN2022099680-appb-000023
取氢化钠(0.3g,3.99mmol)，加入适量的超干四氢呋喃，冰浴条件下缓慢加入化合物8a(0.5g,1.33mmol)，搅拌1h，加入4-环己基苄溴(0.33g,1.33mmol)，室温搅拌过夜。反应完成后，加水淬灭，乙酸乙酯萃取，无水硫酸钠干燥，旋干溶剂得粗产品。柱层析得白色固体0.57g，产率为78.1％。 1H NMR(500MHz,DMSO)δ7.69(d,J＝7.5Hz,2H),7.61(d,J＝7.4Hz,2H),7.31(d,J＝7.6Hz,2H),7.06(dd,J＝15.8,7.3Hz,4H),6.98(d,J＝7.6Hz,2H),4.79(s,2H),2.89(t,J＝7.2Hz,2H),2.41(d,J＝10.8Hz,3H),1.77–1.66(m,5H),1.44–1.30(m,5H),1.26(s,12H).LCMS:m/z(M+H +):548.32.
步骤3：制备(4-(3-(4-氰基苯基)-N-(4-环己基苄基)丙酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000024
取化合物8b(0.37g,0.67mmol)，加入适量的四氢呋喃和水溶解，加入高碘酸钠(0.43g,2.02mmol)和0.3ml 2M稀盐酸溶液，室温搅拌。反应完成后，用乙酸乙酯萃取，无水硫酸钠干燥，旋干溶剂得粗产品。柱层析得白色固体0.24g，产率为76.2％。 1H NMR(400MHz,DMSO)δ8.09(s,2H),7.70(dd,J＝13.1,8.1Hz,4H),7.30(d,J＝8.0Hz,2H),7.09(d,J＝8.0Hz,2H),6.99(d,J＝7.8Hz,4H),4.79(s,2H),2.89(t,J＝7.3Hz,2H),2.48–2.35(m,3H),1.71(dd,J＝26.1,10.9Hz,5H),1.41–1.21(m,5H).HRMS(ESI)calcd for C 29H 31N 2O 3B(M+H +):467.2506；found 467.2447.
实施例9 (4-(N-(4-环己基苄基)-3-苯基丙酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000025
步骤：将4-氰基-3-苯基丙酸换成苯丙酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(400MHz,DMSO)δ7.74(d,J＝7.9Hz,2H),7.21(t,J＝7.3Hz,2H),7.14(t,J＝7.1Hz,1H),7.08(t,J＝10.4Hz,5H),7.04–6.98(m,3H),4.82(s,2H),2.83(t,J＝7.5Hz,2H),2.40(d,J＝22.1Hz,3H),1.70(dd,J＝31.2,10.8Hz,5H),1.41–1.24(m,5H).HRMS(ESI)calcd for C 28H 32NO 3B(M+H +):442.2553；found 442.2552.
实施例10 (4-((3r,5r,7r)-N-(4-环己基苄基)金刚烷-1-甲酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000026
步骤：将4-氰基-3-苯基丙酸换成1-金刚烷甲酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(400MHz,DMSO)δ8.12(s,2H),7.74(d,J＝8.2Hz,2H),7.24–6.95(m,6H),4.69(s,2H),2.48–2.38(m,1H),1.90–1.57(m,15H),1.51(d,J＝12.0Hz,3H),1.45–1.29(m,7H).HRMS(ESI)calcd for C 30H 38NO 3B(M+H +):472.3023；found 472.3028.
实施例11 (4-(2-((3r,5r,7r)-金刚烷-1-基)-N-(4-环己基苄基)乙酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000027
步骤：将4-氰基-3-苯基丙酸换成1-金刚烷乙酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(500MHz,DMSO)δ7.78(dd,J＝23.4,7.7Hz,2H),7.07(dt,J＝8.3,6.3Hz,6H),4.81(s,2H),2.47–2.39(m,1H),1.92(s,2H),1.85(s,3H),1.76–1.65(m,5H),1.61(d,J＝11.8Hz,3H),1.53(d,J＝12.2Hz,9H),1.36–1.16(m,5H).HRMS(ESI)calcd for C 31H 40NO 3B(M+H +):486.3180；found 486.3183.
实施例12 (4-(N-(4-环己基苄基)-4-氟苯甲酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000028
步骤：将4-氰基-3-苯基丙酸换成对氟苯甲酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(400MHz,DMSO)δ7.60(t,J＝7.5Hz,2H),7.39–7.35(m,2H),7.21–7.17(m,2H),7.12(t,J＝7.0Hz,2H),7.02(dd,J＝12.9,4.9Hz,4H),5.06(d,J＝7.0Hz,2H),2.42(s,1H),1.75–1.64(m,5H),1.38–1.25(m,5H).HRMS(ESI)calcd for C 26H 27NO 3BF(M+H +):432.2145；found 432.2158.
实施例13 (4-(N-(4-环己基苄基)-4-甲氧基苯甲酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000029
步骤：将4-氰基-3-苯基丙酸换成对甲氧基苯甲酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(400MHz,DMSO)δ7.61(d,J＝8.3Hz,2H),7.30–7.25(m,2H),7.19(d,J＝8.2Hz,2H),7.13(d,J＝8.2Hz,2H),7.01(d,J＝8.3Hz,2H),6.78–6.73(m,2H),5.06(s,2H),3.69(s,3H),2.43(d,J＝2.5Hz,1H),1.76–1.65(m,5H),1.41–1.24(m,5H).HRMS(ESI)calcd for C 27H 30NO 4B(M+H +):444.2345；found 444.2348.
实施例14 (4-(4-氰基-N-(4-环己基苄基)苯甲酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000030
步骤：将4-氰基-3-苯基丙酸换成对氰基苯甲酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(500MHz,DMSO)δ8.04(s,2H),7.69(d,J＝8.0Hz,2H),7.62(d,J＝7.9Hz,2H),7.50(d,J＝7.9Hz,2H),7.21(d,J＝7.8Hz,2H),7.14(d,J＝7.8Hz,2H),7.06(d,J＝7.6Hz,2H),5.09(s,2H),2.43(t,J＝9.2Hz,1H),1.74–1.64(m,4H),1.38–1.17(m,6H).HRMS(ESI)calcd for C 27H 27N 2O 3B(M+H +):439.2192；found 439.2192.
实施例15 (4-(N-(4-环己基苄基)-2,4-二氟苯甲酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000031
步骤：将4-氰基-3-苯基丙酸换成2,4-二氟苯甲酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(400MHz,DMSO)δ7.57(dd,J＝13.5,7.7Hz,2H),7.52–7.42(m,1H),7.22–7.06(m,5H),6.98(d,J＝21.4Hz,3H),5.05(d,J＝7.4Hz,2H),2.43(s,1H),1.70(dd,J＝30.5,10.3Hz,5H),1.50–1.23(m,5H).HRMS(ESI)calcd for C 26H 26NO 3BF 2(M+H +):450.2051；found 450.2054.
实施例16 (4-(N-(4-环己基苄基)-2-苯基乙酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000032
步骤：将4-氰基-3-苯基丙酸换成苯乙酰氯，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(500MHz,DMSO)δ8.12(s,2H),7.79(d,J＝7.9Hz,2H),7.25(t,J＝7.3Hz,2H),7.19(t,J＝7.2Hz,1H),7.16–7.05(m,8H),4.84(s,2H),3.47(s,2H),2.46–2.39(m,1H),1.77–1.65(m,5H),1.39–1.22(m,5H).HRMS(ESI)calcd for C 27H 30NO 3B(M+H +):428.2396；found 428.2392.
实施例17 (4-(N-(4-环己基苄基)-2-苯氧基乙酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000033
步骤：将4-氰基-3-苯基丙酸换成苯氧乙酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(500MHz,DMSO)δ8.12(s,2H),7.80(d,J＝8.1Hz,2H),7.29–7.27(m,2H),7.26–7.22(m,2H),7.12(d,J＝2.5Hz,4H),6.92(t,J＝7.3Hz,1H),6.77(d,J＝8.0Hz,2H),4.86(s,2H),4.52(s,2H),2.44(dd,J＝11.0,8.1Hz,1H),1.75(d,J＝7.5Hz,3H),1.68(d,J＝12.4Hz,1H),1.35(dd,J＝12.7,8.9Hz,5H),1.25–1.19(m,1H).HRMS(ESI)calcd for C 27H 30NO 4B(M+H +):444.2345；found 444.2345.
实施例18 (4-(N-(4-环己基苄基)-3,4-二氟苯甲酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000034
步骤：将4-氰基-3-苯基丙酸换成3,4-二氟苯甲酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(500MHz,DMSO)δ8.04(s,2H),7.62(d,J＝8.3Hz,2H),7.44–7.37(m,1H),7.27(dt,J＝10.4,8.4Hz,1H),7.20(d,J＝8.1Hz,2H),7.13(d,J＝8.1Hz,3H),7.06(d,J＝8.2Hz,2H),5.06(s,2H),2.47–2.40(m,1H),1.76–1.65(m,5H),1.39–1.20(m,5H).HRMS(ESI)calcd for C 26H 26NO 3BF 2(M+H +):450.2051；found 450.2063.
实施例19 (4-(N-(4-环己基苄基)苯甲酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000035
步骤：将4-氰基-3-苯基丙酸换成苯甲酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(500MHz,DMSO)δ8.00(s,2H),7.56(d,J＝7.5Hz,2H),7.29(dd,J＝19.1,7.4Hz,3H),7.21(dd,J＝14.2,7.3Hz,4H),7.14(d,J＝7.7Hz,2H),7.01(d,J＝7.6Hz,2H),5.07(s,2H),2.44(s,1H),1.75(s,4H),1.68(d,J＝12.7Hz,1H),1.42–1.30(m,4H),1.24(s,1H).HRMS(ESI)calcd for C 26H 28NO 3B(M+H +):414.2240；found 414.2244.
实施例20 (4-(N-(4-环己基苄基)-2-(苯硫基)乙酰胺)苯基)硼酸
Figure PCTCN2022099680-appb-000036
步骤：将4-氰基-3-苯基丙酸换成苯硫基乙酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(500MHz,DMSO)δ8.11(s,1H),7.77(d,J＝8.0Hz,1H),7.31–7.22(m,4H),7.18(dd,J＝16.8,7.5Hz,3H),7.09(dd,J＝23.3,7.8Hz,4H),4.84(s,2H),3.68(s,2H),2.48–2.37(m,1H),1.82–1.63(m,5H),1.41–1.28(m,4H),1.21(dd,J＝15.0,6.0Hz,1H).HRMS(ESI)calcd for C 27H 30NO 3BS(M+H +):460.2117；found 460.2117.
实施例21 (4-(N-(4-环己基苄基)-4-苯氧基苯甲酰胺基)苯基)硼酸
Figure PCTCN2022099680-appb-000037
步骤：将4-氰基-3-苯基丙酸换成4-苯氧基苯甲酸，其余所需原料、试剂以及制备方法同实施例8，得白色固体。 1H NMR(400MHz,DMSO)δ8.01(s,2H),7.60(d,J＝8.2Hz,2H),7.41(d,J＝7.6Hz,2H),7.32(d,J＝8.7Hz,2H),7.19(d,J＝8.2Hz,3H),7.14(d,J＝8.1Hz,2H),7.00(dd,J＝13.4,8.0Hz,4H),6.80(d,J＝8.7Hz,2H),5.07(s,2H),2.45(s,1H),1.75(d,J＝9.0Hz,4H),1.67(s,1H),1.35(t,J＝10.0Hz,5H).HRMS(ESI)calcd for C 32H 32NO 4B(M+H +):506.2503；found 506.2502.
实施例22 (4-(4-苯氧基苯甲胺)苯基硼酸
Figure PCTCN2022099680-appb-000038
步骤：将叔丁氧羰酰基肌氨酸换成4-苯氧基苯甲酸，其余所需原料、试剂以及制备方法同实施例8中的步骤1和步骤3，得白色固体。 1H NMR(400MHz,DMSO)δ10.22(s,1H),8.01(d,J＝8.4Hz,2H),7.95(s,2H),7.76(q,J＝8.0Hz,4H),7.46(t,J＝7.6Hz,2H),7.23(t,J＝7.2Hz,1H),7.11(t,J＝7.8Hz,4H).HRMS(ESI)calcd for C 19H 16NO 4B(M+H +):334.1249；found334.1249.
实施例23 (4-(4-环己基苯甲酰胺)苯基硼酸
Figure PCTCN2022099680-appb-000039
步骤：将叔丁氧羰酰基肌氨酸换成4-环己基苯甲酸，其余所需原料、试剂以及制备方法同实施例8中的步骤1和步骤3，得白色固体。 1H NMR(400MHz,DMSO)δ10.18(s,1H),7.94(s,2H),7.88(d,J＝8.2Hz,2H),7.76(d,J＝3.4Hz,4H),7.37(d,J＝8.2Hz,2H),2.58(t,J＝10.9Hz,1H),1.80(d,J＝10.3Hz,4H),1.71(d,J＝12.5Hz,1H),1.41(dd,J＝25.7,11.9Hz,4H),1.25(d,J＝12.6Hz,1H).HRMS(ESI)calcd for C 19H 22NO 3B(M+H +):324.1780；found 324.1780.
实施例24 3-(N-(4-环己基苄基)-2-((N，4-二甲基苯基)磺酰胺基)乙酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000040
步骤：将4-氨基苯硼酸频哪醇酯换成3-氨基苯硼酸频哪醇酯，其余所需原料、试剂以及制备方法同实施例6，得白色固体。 1H NMR(500MHz,DMSO)δ8.19(s,2H),7.74(d,J＝6.7Hz,1H),7.61(s,1H),7.52(d,J＝7.7Hz,2H),7.36(dd,J＝17.2,7.9Hz,3H),7.20(d,J＝7.7Hz,1H),7.13(d,J＝7.6Hz,2H),7.05(d,J＝7.6Hz,2H),4.76(s,2H),3.68(s,2H),2.77(s,3H),2.45(s,1H),2.37(s,3H),1.79–1.74(m,4H),1.69(d,J＝12.6Hz,1H),1.36(t,J＝11.4Hz,4H),1.30(s,1H).HRMS(ESI)calcd for C 29H 35N 2O 5BS(M+H +):535.2438；found 535.2436.
实施例25 (3-(4-苯氧基苯甲胺)苯基硼酸
Figure PCTCN2022099680-appb-000041
步骤：将4-氨基苯硼酸频哪醇酯换成3-氨基苯硼酸频哪醇酯，其余所需原料、试剂以及制备方法同实施例22，得白色固体。 1H NMR(500MHz,DMSO)δ10.15(s,1H),8.04(t,J＝8.5Hz,5H),7.84(d,J＝8.0Hz,1H),7.55(d,J＝7.3Hz,1H),7.45(d,J＝7.8Hz,2H),7.32(t,J＝ 7.7Hz,1H),7.22(d,J＝7.4Hz,1H),7.11(dd,J＝13.8,8.3Hz,4H).HRMS(ESI)calcd for C 19H 16NO 4B(M+H +):334.1249；found 334.1249.
实施例26 (3-(N-(4-环己基苄基)-4-苯氧基苯甲酰氨基)苯基)硼酸
Figure PCTCN2022099680-appb-000042
步骤：将4-氨基苯硼酸频哪醇酯换成3-氨基苯硼酸频哪醇酯，其余所需原料、试剂以及制备方法同实施例21，得白色固体。 1H NMR(400MHz,DMSO)δ8.04(s,2H),7.53(d,J＝5.8Hz,2H),7.39(t,J＝7.8Hz,2H),7.30(d,J＝8.5Hz,2H),7.19(t,J＝7.4Hz,3H),7.15(d,J＝7.9Hz,3H),7.05(d,J＝7.8Hz,1H),6.95(d,J＝8.0Hz,2H),6.79(d,J＝8.6Hz,2H),5.06(s,2H),2.45(s,1H),1.76(s,4H),1.70(s,1H),1.35(t,J＝10.5Hz,4H),1.24(s,1H).HRMS(ESI)calcd for C 32H 32NO 4B(M+H +):506.2503；found 506.2503.
生物活性部分
实施例1～26制备得到的硼酸化合物分别记为化合物1～26。
研究发现，与阳性对照化合物SH-4-54相比，硼酸化合物能够显著有效抑制STAT3的表达水平，进而抑制癌细胞的增殖、生存、迁移和侵袭并促进其凋亡。
应用例1 硼酸化合物抑制胃癌细胞增殖、生存、迁移和侵袭并促进其凋亡
(1)抑制胃癌细胞增殖
采用CCK8法检测细胞活力来检测化合物对胃癌细胞增殖的影响。实验方法如下：取对数生长期的细胞，以(1.5～3)×10 3cells/孔，100μL/孔的细胞密度接种于96孔板中，培养12～24h后，设置不加化合物对照组、空白组和化合物组。不加药对照组细胞，只加等体积培养基；空白组不加细胞，也不加化合物，只加培养基；化合物组细胞加入不同浓度的小分子化合物，继续培养72h，然后往96孔板中避光加入10μL/孔的CCK8检测试剂，37℃避光孵育1～4h，用酶标仪检测450nm波长下的吸光度值(OD)。实验独立重复三次。细胞活力(％)＝[OD(加药)-OD(空白)]/[OD(对照)-OD(空白)]×100％。最后用GraphPad Prism 8软件进行非线性回归得到对应的半数抑制浓度(IC 50)，结果如表1所示。与羧酸类阳性对照化合物SH-4-54相比，化合物1-5抑制胃癌细胞增殖的能力有明显的优势。因此，根据活性结果，选取抑制效果最佳的化合物1进行后续的生物活性研究。
表1 化合物对胃癌细胞的抑制活性
Figure PCTCN2022099680-appb-000043
(2)化合物1对胃癌细胞克隆形成的影响
取对数生长期的细胞，以400～600cells/孔接种于6孔板中，置于37℃，5％CO 2细胞培养箱中，培养24h至细胞贴壁后，加入不同浓度的化合物1和阳性对照药物SH-4-54，继续培养两周，其间每2～3天换一次含有化合物的培养基。两周后，吸去培养基，加入PBS洗涤，重复洗涤两次，弃去PBS。加入4％多聚甲醛，室温固定15min，弃去固定液，加入PBS洗涤两次，弃去PBS。加入1mL结晶紫染色液，避光孵育30min，弃去结晶紫。加入PBS洗涤至背景变干净，室温晾干。然后拍照并统计每孔集落数，利用GraphPad Prism软件进行统计学分析，实验独立重复3次。
结果如图1所示，图1(A)结果表示化合物1在0.5μM和1μM时能够明显抑制AGS细胞的克隆形成，在1μM和3μM时能够明显抑制MGC803细胞的生长，且在两株胃癌细胞上抑制克隆形成的效果均比羧酸类阳性对照化合物SH-4-54强。图1(B)为图1(A)的统计结果图，表示化合物1能够显著抑制胃癌细胞的克隆形成，且与对照药SH-4-54相比，有统计学差异。
(3)化合物1能够明显抑制胃癌细胞的迁移
取对数生长期的细胞，接种于6孔板中，于培养箱中培养24h，待细胞贴壁并生长至汇合度约90％时，用枪头比着直尺划痕，弃去培养基，用PBS洗2次，加入新鲜培养基，显微镜下拍照，设置对照组和化合物组，对照组不加化合物，化合物组加入不同浓度化合物1和SH-4-54，置于37℃、5％CO 2培养箱培养24h。然后弃去培养基，加入PBS洗两次，弃去PBS，加入培养基，拍照并统计化合物处理24h后细胞迁移的程度，实验独立重复3次。
实验结果如图2所示，图2(A)表示划痕实验检测化合物对胃癌细胞迁移的影响，与对照组相比，化合物1能够显著抑制胃癌细胞的迁移，且与阳性对照药SH-4-54相比，硼酸类化合物1具有更显著的抑制胃癌细胞迁移的效果。图2(B)为图2(A)的统计结果图，显示在抑制胃癌细胞迁移方面，化合物1的优势效果与SH-4-54相比具有统计学差异。
(4)化合物1能够显著抑制胃癌细胞的侵袭
在Transwell小室中取出嵌套至另一新的24孔板，加入空白培养基浸泡，室温浸泡1h以上，进行嵌套活化。取对数生长期的胃癌细胞，用含10％FBS重悬细胞，在24孔板小室下层加入500μL含10％FBS的培养基，嵌套小室上层加入(1～2)×10 4cells/孔，300μL/孔细胞悬液，于培养箱中培养12～24h至细胞贴壁。然后小室底部换成含20％FBS的培养基，嵌套上层将培养基换成含有或不含有化合物的2％FBS的培养基，于培养箱中继续培养24h。24h后，弃去培养基，用PBS洗一次，弃去PBS。在嵌套上下层均加入500μL 4％多聚甲醛固定液，室温固定15min，弃去固定液。加入PBS洗一次，弃去PBS。在嵌套上下层均加入结晶紫染色液，室温避光染色30min，弃去结晶紫染色液。加入PBS洗至背景变干净，室温自然晾干小室。然后进行拍照观察并统计各浓度下穿过Transwell嵌套膜的细胞数，实验独立重复3次。
实验结果如图3所示，图3(A)为显微镜拍照下各化合物浓度下侵袭细胞图，结果显示，经化合物1和SH-4-54处理后，侵袭细胞变少，说明化合物1和SH-4-54均能够显著抑制胃癌细胞的侵袭，但是与SH-4-54相比，硼酸类化合物1具有更明显的抑制胃癌细胞侵袭的效果，说明硼酸类化合物比羧酸类化合物更能抑制胃癌细胞侵袭；图3(B)为图3(A)的统计结果图，结果显示化合物1比SH-4-54更能抑制胃癌细胞的侵袭，具有统计学差异。
(5)化合物1能够促进胃癌细胞凋亡
采用Annexin V-FITC/PI双染法检测化合物1促进胃癌细胞凋亡的能力。取对数生长期的胃癌细胞，接种于6孔板中，于培养箱中培养24h至细胞贴壁，设置对照组和化合物组，对照组不加药，化合物组加入不同浓度的化合物1或SH-4-54，继续培养72h后，利用贝博的Annexin V-FITC/PI双染细胞凋亡检测试剂盒(BB-4101-1)进行检测。弃去6孔板中的培养基，加入PBS洗2次，利用不含EDTA的胰酶消化，收集并离心，用预冷的PBS洗2次，加入400μL 1×Annexin V结合液重悬细胞，加入5μL Annexin V-FITC染色液，轻轻混匀，于冰上避光孵育15min。然后加入10μL PI染色液后轻轻混匀，于冰上避光孵育5min。然后立即用流式细胞仪进行检测。
实验结果如图4所示，图4(A)为利用流式细胞仪检测胃癌细胞凋亡的情况，如图所示，与对照组和化合物SH-4-54组相比，化合物1处理72h后能够明显促进胃癌细胞的凋亡，且在相同浓度下，化合物1促进胃癌细胞凋亡的效果明显比SH-4-54化合物好；图4(B)为图4(A)的统计图，结果显示，化合物1促进胃癌细胞凋亡的效果优于SH-4-54，具有统计学差异。
应用例2 硼酸类化合物抑制胃癌细胞生长的机制
本应用例以化合物1为例，说明本发明硼酸类化合物抑制胃癌细胞生长的机制
(1)化合物1能够与STAT3蛋白结合
SPR验证化合物与STAT3蛋白结合：将STAT3蛋白冰上融解后低温超滤，用PBS将蛋白配置成100μg/mL浓度，通过氨基耦联的方式锚定到CM5芯片上，化合物1和化合物SH-4-54分别溶于经0.22μm滤膜过滤后的含1％DMSO的PBS缓冲液中，然后倍半稀释得到不同化合物浓度(含有1％DMSO)。将不同浓度的化合物由低到高依次通过芯片，得到响应信号，通过软件计算动力学和亲和力，确定结合亲和度(K D)。
细胞热转移(CETSA)实验验证化合物与细胞内的STAT3蛋白结合：将对数生长期的胃癌细胞MGC803接种于15cm大皿中，于培养箱中培养24h，待细胞贴壁生长至汇合度70％～80％时，分别加入化合物1和SH-4-54，使其终浓度为5μM，继续培养3h后收板。吸去培养基，加入PBS洗涤一次，经过胰酶消化、培养基终止消化、离心后，弃去上清，加入PBS洗涤2次，离心弃去PBS。分别加入1mL PBS(含1％磷酸酶抑制剂和1％PMSF)重悬细胞，将细胞悬液分装于PCR管中，然后置于梯度PCR仪中，在不同温度下加热2min，室温静置3min，然后样品立即放入液氮中冷冻。为了使细胞溶解释放蛋白，需要将样品在液氮中冷冻，然后室温解冻，反复冻融3次。然后4℃，20000×g离心20min。取上清，加入5×Loading Buffer，100℃，煮沸5min。然后用聚丙烯酰胺凝胶SDS-PAGE电泳分离蛋白样品，通过湿转的方法将蛋白转移至硝酸纤维素薄膜(PVDF膜)上，经5％BSA封闭1h后，分别用pY705-STAT3、T-STAT3、c-Myc、Bcl-XL和β-Actin的一抗在4℃下孵育过夜，再分别用带有荧光标记的兔二抗、鼠二抗室温孵育1h，ECL化学发光液避光孵育2min，最后用Bio-RAD显影仪检测蛋白的表达水平。
实验结果如图5所示，图5(A)SPR结果显示，化合物1在体外能够与STAT3蛋白有明显的结合，而且与SH-4-54相比，化合物1具有更高的结合活性。图5(B)CESTA结果显示，化合物1能够进入癌细胞并与细胞内的STAT3蛋白结合，且与对照药SH-4-54相比，化合物1与胃癌细胞内的STAT3蛋白结合活性更高。这些写过说明本发明的硼酸类化合物可以与STAT3蛋白结合，并且结合活性比对照化合物SH-4-54更高。
(2)化合物1可以抑制pY705-STAT3的磷酸化及下游基因的表达
收集已经过化合物1或SH-4-54处理的细胞，弃去培养基，用PBS洗两次，加入裂解液(RIPA:PMSF:phosphatase A:phosphatase B＝100:1:1:1)，冰上振荡裂解15min，用细胞刮将细胞裂解物刮下，将细胞板斜躺于冰上，静置5min，将细胞裂解物转移到EP管中，然后4℃，15000rpm离心15min，取上清进行蛋白定量，加入5×Loading Buffer，100℃煮沸5min。然后蛋白质免疫印记(Western Blot)方法进行检测，首先用聚丙烯酰胺凝胶SDS-PAGE电泳分离蛋白样品，通过湿转的方法将蛋白转移至硝酸纤维素薄膜(PVDF膜)上，经5％BSA封闭1h后，分别用pY705-STAT3、T-STAT3、c-Myc、Bcl-XL和β-Actin的一抗在4℃下孵育过夜，再分别用带有荧光标记的兔二抗、鼠二抗室温孵育1h，ECL化学发光液避光孵育2min，最后用Bio-RAD显影仪检测蛋白的表达水平。
实验结果如图6所示，经化合物1处理后，胃癌细胞中pY705-STAT3的磷酸化水平和下游基因c-Myc、Bcl-XL的表达水平均明显下降，且与对照化合物SH-4-54相比，化合物1抑制STAT3信号通路的效果更明显。说明硼酸类化合物在抑制胃癌细胞内STAT3信号通路的效果优于SH-4-54。
(3)化合物1抑制STAT3的转录活性
293T细胞培养于含10％胎牛血清的DMEM中，取对数生长期的细胞消化，计数，将细胞以1～2×104cells/100μL密度接种于96孔板中，将细胞培养于5％CO2，37℃培养箱中，待细胞贴壁长到汇合度70％～80％时，进行细胞转染。分别在无菌1.5mL EP管中配制A、B两种混合溶液(以下为每一孔的用量：A:0.25μL Lipo 2000+OPTI-MEMI培养基，体积为5μL；B:50ng pGL3-STAT3-promoter质粒+50ng STAT3C质粒+40ng TKRL(海肾萤光素报告基因质粒)+OPTI-MEM培养基，体积为5μL)，轻轻混匀，静置5min，再将A溶液 转移到B管中，轻轻混匀，室温静置15min。用移液枪将10μL配制好的转染液滴加至96孔板中，置于细胞培养箱中继续培养。转染24h后，加入不同浓度的1或SH-4-54化合物继续处理24h。将萤火虫萤光素酶检测试剂和海肾萤光素酶检测缓冲液融解至室温，海肾萤光素酶检测底物(100×)置于冰浴上备用；按照每孔25μL用量，取适量海肾萤光素酶检测缓冲液，按照1:100加入海肾萤光素酶检测底物(100×)配制成海肾萤光素酶检测工作液；取出96孔板，弃去培养基，每孔加入50μL报告基因细胞裂解液，震荡混匀10min；取25μL裂解液至96孔白板中，每孔再加入25μL萤火虫萤光素酶检测试剂，震荡混匀5min，检测得到RLU1；在完成上述测定萤火虫萤光素酶步骤后，每孔加入25μL海肾萤光素酶检测工作液，震荡混匀5min，检测得到RLU2；得到RLU1/RLU2的比值。
实验结果如图7所示，经硼酸类化合物1处理后，STAT3的转录活性明显受到抑制，且与对照化合物SH-4-54对比，化合物1抑制STAT3转录活性的效果更明显，说明硼酸类化合物抑制STAT3信号转录活性优于SH-4-54。
(4)化合物1抑制STAT3二聚化
利用免疫共沉淀(Co-IP)的方法检测STAT3的二聚化。293T细胞培养于含10％胎牛血清的DMEM中，取对数生长期的293T细胞接种于6孔板中，培养12～24h待细胞贴壁生长至汇合度约为70％～80％时，进行细胞转染。分别在无菌1.5mL EP管中配制A、B两种混合溶液(以下为每一孔的用量：A:4μL Lipo 2000+OPTI-MEMI培养基，体积为100μL；B:2.5μg HA-STAT3质粒+2.5μg Flag-STAT3质粒+OPTI-MEM培养基，体积为100μL)，轻轻混匀，静置5min，再将A溶液转移到B管中，轻轻混匀，室温静置15min。取出6孔板，吸去旧的培养基，加入800μL OPTI-MEMI培养基，用移液枪将200μL配制好的转染液滴加至6孔板中，置于细胞培养箱中继续培养，转染6h后将6孔板中的培养基换成含10％FBS的DMEM培养基，继续培养。转染24h后加入不同浓度的化合物1，继续培养24h。
药物处理24h后，在收取蛋白的前1h加入终浓度为100ng/mL的IL-6刺激，然后提取细胞的总蛋白，收集已经过化合物1或SH-4-54处理的细胞，弃去培养基，用PBS洗两次，加入IP裂解液(RIPA:PMSF:phosphatase A:phosphatase B＝100:1:1:1)，冰上振荡裂解15min，用细胞刮将细胞裂解物刮下，将细胞板斜躺于冰上，静置5min，将细胞裂解物转移到EP管中，然后4℃，15000rpm离心15min，取上清进行蛋白定量，在不同EP管中分别吸取相同蛋白总量的不同处理的样品。取带有Flag抗体的免疫共沉淀的Beads，用PBST洗两遍，4℃，5000rpm离心30s，加入适量的PBS重悬。将Beads均分到装有相同蛋白量的EP管中，轻轻混匀，放入4℃冰箱旋转摇床中翻转过夜。过夜翻转后，5000rpm离心30s，弃上清，往沉淀中加入500μL PBST，旋转5min，5000rpm离心30s，重复洗2遍。往沉淀中加入2×Loading Buffer，煮沸5min。接下来用蛋白质免疫印记(Western Blot)检测STAT3的二聚化，首先用聚丙烯酰胺凝胶SDS-PAGE电泳分离蛋白样品，通过湿转的方法将蛋白转移至硝酸纤维素薄膜(PVDF膜)上，经5％BSA封闭1h后，分别用HA、Flag和β-Actin的一抗在4℃下孵育过夜，再分别用带有荧光标记的兔二抗、鼠二抗室温孵育1h，ECL化学发光液避光孵育2min，最后用Bio-RAD显影仪检测蛋白的表达水平。
实验结果如图8所示，经硼酸类化合物1处理后，细胞内HA-STAT3和Flag-STAT3蛋白的结合显著减少，且具有浓度依赖性。说明化合物1可以减少STAT3蛋白的二聚化。
应用例3 硼酸类化合物在动物整体模型上的药效、药理和毒理研究
本应用例以化合物1为例，说明本发明硼酸类化合物在动物整体模型上的药效、药理和毒理研究。
取对数生长期的MGC803细胞，消化离心后计数，用预冷的PBS洗2遍，将预冷PBS与Matrigel按照1:1的比例重悬细胞，配成2×10 6cells/100μL浓度，置于冰上。分别在裸鼠的腹背两侧皮下注射100μL细胞悬液，待肿瘤体积达到70mm 3左右时，开始分组给药；将裸鼠随机分成4组，溶剂对照组腹腔注射等量溶剂(含15％蓖麻油的灭菌PBS溶液)，低剂量组腹腔注射5mg/kg的化合物1，高剂量组腹腔注射15mg/kg的化合物1，阳性对照组腹腔 注射15mg/kg的SH-4-54，连续给药21天。每两天用游标卡尺和电子秤分别测量肿瘤体积大小和小鼠体重。给药21天后，处死小鼠进行解剖，取出肿瘤和心、肝、脾、肺、肾等脏器进行后续实验。
实验结果如图9所示，图(9A)肿瘤图显示，经化合物1和对照化合物SH-4-54治疗21天后，与未治疗相比，小鼠的肿瘤明显较小，说明化合物1和SH-4-54均能够抑制小鼠肿瘤生长。但是化合物1在低剂量5mg/kg时就已经有明显的抑制肿瘤生长的作用，并且比对照化合物SH-4-54在高剂量15mg/kg的抑制效果还明显，说明化合物1抑制体内肿瘤生长的效果优于SH-4-54。图(9B)图(9C)分别为肿瘤的生长曲线图和重点肿瘤重量统计图，也可以得到同样的验证，化合物1抑制肿瘤生长的效果优于SH-4-54。进一步在肿瘤组织中验证化合物1和SH-4-54抑制肿瘤生长的机制，图(9D)Western Blot结果显示，化合物1能够抑制肿瘤组织中pY705-STAT3的磷酸化水平和下游与肿瘤生长相关的c-Myc和Bcl-xL的表达，且抑制效果优于对照化合物SH-4-54。对化合物的毒性探究发现，从图(9E)小鼠重量生长统计图中可以看出，与溶剂组相比，化合物1和SH-4-54对小鼠的生长并没有明显影响；从图(9F)可以看出，化合物1和SH-454对主要脏器形态基本没有影响，说明化合物1抑制肿瘤生长的同时对小鼠的整体毒性较小。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (10)

  1. 一种硼酸化合物，其特征在于，具有如式(Ⅰ)所示结构：
    Figure PCTCN2022099680-appb-100001
    其中，n为0～2的整数；
    t＝0～1的整数；
    X、Y独立地选自CH或N；
    R 1选自一个或多个氢、卤素、羟基、氨基、硝基、氰基、三氟甲基、C 1-C 6烷基、C 1-C 6烷氧基、-C(O)R a、-OC(O)R a、-NR aR b、-CO 2R a或-CONHR a；
    R 2选自氢、取代或非取代C 3-C 6环烷基、取代或非取代C 3-C 6杂环烷基、取代或非取代芳基、取代或非取代5-10元杂环芳基；
    R 3选自取代或非取代的芳基、5-10元杂环芳基、C 5-C 10桥环基、C 5-C 10稠环基、C 1-C 6烷基或C 3-C 6杂环烷基；
    R a、R b独立地选自氢、C 1-C 6烷基，或R a、R b形成C 3-C 6环烷基。
  2. 根据权利要求1所述硼酸化合物，其特征在于，n为0或1；t为1。
  3. 根据权利要求1所述硼酸化合物，其特征在于，X为CH；Y为CH。
  4. 根据权利要求1所述硼酸化合物，其特征在于，所述R 2中取代基为卤素、羟基、氨基、硝基、氰基、三氟甲基、芳基、杂环芳基、C 1-C 6烷基、C 2-C 6烯基、C 2-C 6炔基、C 3-C 8环烷基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 3-C 7环烯基、C 3-C 6杂环烷基、C 3-C 7饱和杂环、-COOR a或-CONHR a。
  5. 根据权利要求1所述硼酸化合物，其特征在于，所述R 3中取代基为卤素、羟基、氨基、硝基、氰基、三氟甲基、三氟甲氧基、芳基氧基、芳基巯基、芳基胺基、芳基、杂芳基、C 1-C 6烷基、C 2-C 6烯基、C 2-C 6炔基、C 3-C 8环烷基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 3-C 7环烯基、C 3-C 6杂环烷基、C 3-C 7饱和杂环、C 5-C 10桥环基、-CO 2R a、-CONHR a或-R aNR bSO 2R c，R c为三氟甲基、取代或非取代芳基，或取代或非取代杂环芳基。
  6. 根据权利要求1所述硼酸化合物，其特征在于，R 1为氢；R 2为氢、芳基或5-6元杂环芳基。
  7. 根据权利要求1所述硼酸化合物，其特征在于，所述硼酸化合物具有如下编号所示结构：
    Figure PCTCN2022099680-appb-100002
    Figure PCTCN2022099680-appb-100003
  8. 权利要求1～7任一所述硼酸化合物的制备方法，其特征在于，包括如下步骤：
    S1：将式(1)所示相应的胺和式(2)所示相应的酸溶于溶剂中，经缩合反应后生成式(3)所示中间体；
    S2：式(3)所示中间体与式(4)所示物质在碱性条件下经取代反应得到式(5)所示中间体；
    S3：式(5)所示中间体在酸性条件下水解，即得式(Ⅰ)所示硼酸化合物。
    Figure PCTCN2022099680-appb-100004
  9. 权利要求1～7任一所述硼酸化合物及其药学可接受的盐、代谢物或前药在制备抑制STAT3活性的药物中的用途。
  10. 权利要求1～7任一所述硼酸化合物及其药学可接受的盐、代谢物或前药在制备预防和/或治疗肿瘤的药物中的用途。
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