WO2023109004A1 - 一类萘基脲-哌嗪类化合物及其制备方法和应用 - Google Patents

一类萘基脲-哌嗪类化合物及其制备方法和应用 Download PDF

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WO2023109004A1
WO2023109004A1 PCT/CN2022/092953 CN2022092953W WO2023109004A1 WO 2023109004 A1 WO2023109004 A1 WO 2023109004A1 CN 2022092953 W CN2022092953 W CN 2022092953W WO 2023109004 A1 WO2023109004 A1 WO 2023109004A1
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add
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徐学军
杨玉坡
段超群
杨争艳
徐红运
裴梦富
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河南省锐达医药科技有限公司
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D213/40—Acylated substituent nitrogen atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/26—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
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    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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Definitions

  • the invention belongs to the field of tumor targeting therapy, and specifically relates to a class of naphthyl urea-piperazine compounds and a preparation method and application thereof.
  • STATs Signal transducer and activators of transcriptions
  • Overexpression and constitutive activation of STAT3 are common in a variety of solid and hematological cancers.
  • STAT3 is a member of the STATs family and a substrate protein of JAK2 (Janus kinase 2), which has been proven to be closely related to the occurrence, development and malignant transformation of cancer. Under normal circumstances, STAT3 exists in the cytoplasm as an inactive monomer and has a strict negative feedback regulation mechanism.
  • the present invention aims to provide the anti-tumor effect and potential pharmacological mechanism of a new class of naphthyl urea-piperazine compounds and their derivatives, as well as the potential of these compounds in the clinical treatment of breast cancer, liver cancer, lung cancer, colon cancer and leukemia. application.
  • the object of the present invention is to provide a class of naphthyl urea-piperazine compounds and their preparation method and application.
  • R 1 is selected from R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 , R 50 , Each R 51 is independently selected from H, F, Cl, Br, -CN, -CH 3 , -CF 3 , -OCH 3 , -OCF 3 ;
  • R2 is selected from
  • R3 is selected from H, CH3 , CF3 ;
  • n represents the number of CH 2 substituents, m is 1, 2, 3, 4...10;
  • k, z represent the number of CH 2 substituents, k, z are 0, 1, 2, 3, 4, 5, 6, wherein, k, z are not 0 at the same time.
  • naphthyl urea-piperazine compounds are specifically compounds of the following structure:
  • the preparation method of above-mentioned naphthyl urea-piperazine compound comprises the following steps:
  • the preparation process is as follows:
  • step (1) The molar ratio to potassium tert-butoxide is 1:1.2:1.2;
  • step (2) The molar ratio to iron powder is 1:8, and the volume ratio of tetrahydrofuran, ethanol and saturated ammonium chloride aqueous solution is 1:2.4:2.4;
  • step (3) in step (3), triphosgene, R 1 NH 2 ,
  • the molar ratio of N,N-diisopropylethylamine and N,N-diisopropylethylamine is 1:2.3:3:11, and the molar ratio of N,N-diisopropylethylamine twice is 4:7.
  • step (a) The molar ratio of triphenylphosphine and diisopropyl azodicarboxylate is 1:1:1.2:1.2;
  • step (b) The molar ratio of sodium borohydride and sodium borohydride is 1:4.
  • the antineoplastic drugs refer to drugs for treating breast cancer, liver cancer, lung cancer, colon cancer or leukemia.
  • Another object of the present invention is to provide a class of small molecule compounds with targeted anti-tumor activity.
  • the tumor can specifically be a tumor with high expression or constitutive activation of STAT3, including but not limited to liver cancer, breast cancer, lung cancer, colon cancer and leukemia.
  • the present invention synthesized a class of naphthyl urea-piperazine compounds ID210916B-1, ID210917B-1, ID210918B-1, ID210919B-1, ID211130C-1, ID211203B-1, IY211214C-1, IY211228B-1, IY220302B-1, IY220209A-1, IY220313A-1, IY220219A-1, ID210928B-1, ID210929B-1, IY220319A-1, ID211008B-1, ID211009B-1, ID21101 0B-1, ID211116B-1 and ID211012B- 1 etc.
  • the inhibitory effect of these compounds on tumor cell proliferation was detected by MTT assay, the effect of compounds on the cell cycle and apoptosis of tumor cells was detected by flow cytometry, and the inhibitory effect on JAK2/STAT3 signaling was confirmed by immunoblotting and other methods .
  • the present invention provides the application and potential molecular mechanism of a new naphthylurea-piperazine compound and its derivatives in tumor treatment.
  • Figure 1 is ID210916B-1, ID210917B-1, ID210918B-1, ID210919B-1, D211130C-1, ID211203B-1, IY211214C-1, IY211228B-1, IY220302B-1, IY220209A-1, IY220 313A-1, IY220219A-1 , ID210928B-1, ID210929B-1, IY220319A-1, ID211008B-1, ID211009B-1, ID211010B-1, ID211116B-1 and ID211012B-1 etc.
  • tumor cells such as afatinib-resistant lung cancer cell PC9-AR, colon cancer cell HT29 and leukemia cell Jurkat;
  • Figure 2 is the effect of ID210916B-1 on inducing TNBC cell cycle arrest
  • Figure 3 is the inhibitory effect of ID210916B-1 on TNBC cell scratch repair
  • Figure 4 is the inhibitory effect of ID210916B-1 on TNBC cell migration
  • Figure 5 shows the inhibitory effect of ID210916B-1 on the nuclear entry of p-STAT3 in TNBC cells
  • Figure 6 is the inhibitory effect of ID210916B-1 on STAT3 transcriptional activity in TNBC cells
  • Figure 7 shows the inhibitory effect of ID210916B-1 on the phosphorylation of STAT3 and the expression of Cyclin D1 in TNBC cells.
  • the left side of the figure is MDA-MB-468 cells, and the right side is 4T1 cells.
  • the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature; more specifically, the room temperature refers to 20-30°C;
  • the organic solvent is The drying method is drying, and the evaporation of the solvent is evaporated under reduced pressure using a rotary evaporator, and the bath temperature is not higher than 50 ° C; the developing solvent and the eluent are volume ratios;
  • the reaction process is followed by thin-layer chromatography (TLC);
  • TLC thin-layer chromatography
  • final product has satisfactory proton nuclear magnetic resonance ( 1 H-NMR).
  • Embodiment 1 the synthesis of compound
  • compound ID210916B-1 is tert-butyl 4-(2-(4-(((4-(3-(pyridin-4-ylmethyl)ureido)naphthalen-1-yl)oxy)methyl)phenoxy)ethyl)piperazine -1-carboxylate,
  • Step 4 tert-butyl 4-(2-(4-(((4-aminonaphthalen-1-yl)oxy)methyl)phenoxy)ethyl)piperazine-1-carboxylate(int 4)
  • reaction time was 18 hours, and then TLC was monitored, and the results showed that the raw materials were reacted and new products were generated, so the reaction liquid was processed later;
  • step 1 the raw material 1 is replaced with the corresponding aldehyde, or 1a is replaced with the piperazine of the corresponding substituent, or in step 5, the raw material 4-picolylamine is replaced with into the corresponding amine.
  • Example 2 ID210916B-1, ID210917B-1, ID210918B-1, ID210919B-1, D211130C-1, ID211203B-1, IY211214C-1, IY211228B-1, IY220302B-1, IY220209A-1, IY2 20313A-1, IY220219A- 1. Inhibitory effect of ID210928B-1, ID210929B-1, IY220319A-1, ID211008B-1, ID211009B-1, ID211010B-1, ID211116B-1 and ID211012B-1 on the proliferation of breast cancer and liver cancer cells
  • the tumor cells in the logarithmic growth phase were collected separately, the concentration of the cell suspension was adjusted to 5 ⁇ 10 4 cells/mL, and added to a 96-well cell culture plate with a volume of 100 ul per well.
  • WP1066 Choinese name: (2E)-3-(6-bromo-2-pyridyl)-2-cyano-N-[(1S)-1-phenylethyl]-2- Acrylamide, CAS: 857064-38-1, the structure is ) as a positive control, the novel naphthylurea-piperazine compounds ID210916B-1, ID210917B-1, ID210918B-1 etc.
  • the statistical results of the half inhibition rate (IC50 value) of ID210928B-1, ID210929B-1, IY220319A-1, ID211008B-1, ID211009B-1, ID211010B-1, ID211116B-1 and ID211012B-1 on tumor cells are shown in Figure 1 shown.
  • MDA-MB-468 or 4T1 cells in the logarithmic growth phase were taken, digested, centrifuged and the cells were made into a single cell suspension. After counting, the cells were plated into a 12-well plate, and two kinds of cells were inoculated at 2 ⁇ 10 5 cells per well, and 3 wells were plated for parallel control. 16h after plating, the cells were treated with compounds. Using DMSO as the compound solvent, the final concentrations of compound ID210916B-1 in 4T1 cell suspension were 0, 2.5, 5 and 10 ⁇ M, and the final concentrations of compound ID210916B-1 in MDA-MB-468 cell suspension were 0, 0.025 , 0.05 and 0.1 ⁇ M.
  • each empty cell was digested with trypsin, counted after resuspension, and the cell concentration in each well was adjusted to 5 ⁇ 10 5 .
  • the digestion was completed, discard the supernatant, then wash the cells twice with PBS (2000rpm, centrifuge for 5min), then discard the supernatant, add 980 ⁇ l of 70% cold ethanol and 20 ⁇ l of 5% BSA to each tube (adding a small amount of BSA can reduce the number of operations). Cell loss during the process) fixed overnight at 4°C. Discard the fixative, wash with PBS 3 times to remove residual fixative (1000rpm, centrifuge for 3min).
  • the ratio of cells in the G1 phase of the compound ID210916B-1 treatment groups at higher concentrations (5 and 10 ⁇ M) was significantly increased in a dose-dependent manner.
  • the ratio of cells in G2 phase was significantly reduced. It shows that compound ID210916B-1 can induce G1 phase arrest in 4T1 cells and inhibit cell cycle progression.
  • MDA-MB-468 or 4T1 cells in the logarithmic growth phase were seeded in 6-well plates with 5 ⁇ 10 5 cells per well. After about 24 hours, the cells evenly covered the bottom of the well in a single layer. Use a 200 ⁇ L gun tip to scratch vertically and evenly. After washing twice with PBS, add different doses of ID210916B-1 (0, 0.015, 0.03 and 0.06 ⁇ M for MDA-MB-468 cells, 0.01 ⁇ M for 4T1 cells, , 0.25, 0.5 and 1 ⁇ M), observed and photographed under a microscope at 0, 24 and 36h (MDA-MB-468 cells) and 0, 24 and 48h (4T1 cells) respectively.
  • the cells were treated with different concentrations of ID210916B-1 (MDA-MB-468 cells were treated with concentrations of 0, 0.015, and 0.03 ⁇ M, and 4T1 cells were treated with concentrations of 0, 2, and 4 ⁇ M), and cultured in an incubator. . Stained with crystal violet after 48h.
  • Crystal violet staining steps 1) Discard the culture medium and wash the chamber 3 times with 1 ⁇ PBS; 2) Discard the PBS and fix the chamber with 75% ethanol for 15 minutes; 3) Discard the ethanol after fixation and wash the chamber again with 1 ⁇ PBS 3 times; 4) Discard PBS, and stain the chamber with 0.1% crystal violet for 30 minutes; 5) Rinse the chamber with tap water until the water flow is clear and no purple can be seen; Migratable cells, then place the upper chamber back into the 24-well plate. Photographs were taken under a microscope to record cell migration and invasion.
  • Example 6 Inhibitory effect of ID210916B-1 on p-STAT3 nuclear entry in TNBC cells
  • MDA-MB-468 and 4T1 cells are spread in 35mm small dishes respectively, and the number of cells in each dish is 1 ⁇ 10 5 , and incubated at 37°C
  • MDA-MB-468 or 4T1 cells were treated with ID210916B-1, the final concentration of ID210916B-1 for MDA-MB-468 was 0.04 ⁇ M and ID210916B-1 for 4T1 was 6 ⁇ M.
  • Fig. 5 show that, compared with the cells in the control well, the expression of p-STAT3 in MDA-MB-468 cells, especially the nucleus, was significantly reduced after 24 hours of treatment with 0.04 ⁇ M ID210916B-1. After 24 hours of treatment with 6 ⁇ M ID210916B-1, the expression of p-STAT3 in the cytoplasm and nucleus of 4T1 decreased significantly. It indicated that ID210916B-1 could interfere with the nuclear entry of p-STAT3 in TNBC cells.
  • Example 7 Inhibitory effect of ID210916B-1 on STAT3 transcriptional activity in TNBC cells
  • STAT3-luciferase reporter plasmid purchased from Shanghai Quanyang Biotechnology Co., Ltd., TSB10127-1
  • MDA-MB-468 cells (0, 0.015, 0.03, 0.06 and 0.12 ⁇ M) and 4T1 cells (0, 3, 6 and 12 ⁇ M) were pretreated with gradient doses of compound ID210916B-1 for 2 h each. Cells were then stimulated with IL-6 (50 ng/ml) for 1 h. Discard the supernatant and wash the cells once with PBS. Lyse with 200 ⁇ L of 5 ⁇ PLB Lysis Solution for 20 min at room temperature. Transfer the cell lysate into a 1.5ml Ep tube.
  • the relative luciferase activity ratio without IL-6 treatment was about 10, indicating that the background STAT3 transcriptional activity of the cells was strong.
  • the relative luciferase activity ratio was increased by about 5 times, indicating that the cells and the detection system such as the reporter carrier had sensitivity and specificity to the STAT3 signal response.
  • the ratio of luciferase activity decreased continuously. Under the action of 0.03 ⁇ M ID210916B-1, the transcriptional activity of STAT3 was reduced by about 50%.
  • ID210916B-1 when the concentration of ID210916B-1 was 0.12 ⁇ M, IL-6-induced STAT3 transcriptional activation was inhibited to the background level. Similar results were obtained in 4T1 cells. It indicated that ID210916B-1 could dose-dependently inhibit the gene transcription regulation function of STAT3 in TNBC cells.
  • Example 8 Inhibitory effect of ID210916B-1 on phosphorylation of STAT3 and expression of Cyclin D1 in TNBC cells
  • One, cell culture and dosing a. get the MDA-MB-468 or 4T1 cell of logarithmic growth phase, after digesting with trypsin, make density with the L-15 or DMEM medium that contains 10% fetal bovine serum 4 ⁇ 10 5 cells/mL of single cell suspension were inoculated into 6-well plates with 2 mL of cell suspension per well. b. Incubate in a 37°C, 5% CO 2 incubator (5% CO 2 is not required for MDA-MB-468 cell culture). After the cells adhere to the wall, add different concentrations of ID210916B-1. c. After continuing to culture for 48 hours, the cells were lysed with RIPA lysate and the protein was collected.
  • Cell collection and lysis a. Discard the upper medium, and wash the cells twice with pre-cooled PBS. Add 100 ⁇ L of pre-cooled RIPA cell lysate (protease inhibitors, PMSF and lysate are mixed in advance at a ratio of 1:100) to each well. b. Lyse on ice for 3 minutes, scrape off the cells with a cell scraper, and collect them in a 1.5mL EP tube; place on ice for 30 minutes and vortex every 6 minutes. c. Centrifuge at 12000g for 10min at 4°C. d. Transfer the cell supernatant to a new EP tube.
  • Tris-HCL 100ml: Dissolve 18.15gtris in 80ml water, adjust to 8.8 with 4N Hcl, and set the volume to 100ml.
  • Tris-HCL 1000ml: Weigh 60.5g of tris base, add water to 850ml, add concentrated hydrochloric acid and stir until completely dissolved, adjust pH to 6.8, add water to 1L.
  • TBS buffer Weigh 8.8g of NaCl in 800mL of distilled water, dissolve it, add 10mL of 1mol/L Tris HCl (pH7.5), dilute to 1L, and store at room temperature.
  • TBST buffer Add 500 ⁇ L of 20% Tween20 to 1L of TBS buffer, so that the final concentration of Tween20 is 0.1%.
  • ID210916B-1 In order to further clarify the inhibitory effect of ID210916B-1 on the phosphorylation of STAT3 in TNBC cells.
  • the results of Western blot showed that 0.06 ⁇ M and 4 ⁇ M ID210916B-1 could effectively down-regulate the key markers of STAT3 activation p-STAT3 (Y705), total STAT3 protein (T-STAT3) and the downstream target protein Cyclin of STAT3 in the two types of cells, respectively.
  • Expression levels of D1 FIG. 7 ).
  • the above data fully demonstrate that the compound ID210916B-1 can effectively inhibit the growth and migration of TNBC cells, and can inhibit the phosphorylation and activation of STAT3. It is a STAT3 targeting inhibitor and a potential therapeutic drug for TNBC.
  • the above results show that the piperazine compounds represented by ID210916B-1 can significantly inhibit the proliferation and metastasis of breast cancer and liver cancer cells, induce tumor cell cycle arrest, and have obvious inhibitory effects on STAT3 and its related proteins , showing a good anticancer effect.
  • the compound of the present invention can be applied to cancer treatment drugs related to abnormal cell proliferation, and can be used in combination with human acceptable ingredients. salt or mixed with pharmaceutical carriers to prepare antitumor drugs.

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Abstract

一类萘基脲-哌嗪化合物及其制备方法和应用，该类化合物在低剂量下可显著抑制JAKs/STATs 信号的活化，细胞增殖实验、免疫印迹实验、细胞周期实验、蛋白传膜转运实验以及细胞侵袭和迁移实验等实验结果显示，该类化合物可以特异抑制JAK2 信号活化及下游STAT3、CyclinD1、CyclinB1 和MMP9 等靶基因的表达，诱导细胞周期G1 期阻滞和细胞凋亡，该类化合物能显著抑制乳腺癌、肝癌、肺癌、结肠癌、白血病、淋巴瘤、多发性骨髓瘤以及视网膜母细胞瘤等多种肿瘤细胞株的增殖。

Description

一类萘基脲-哌嗪类化合物及其制备方法和应用 技术领域
本发明属于肿瘤靶向治疗领域，具体涉及一类萘基脲-哌嗪类化合物及其制备方法和应用。
背景技术
已有大量研究证明STATs(Signal transducer and activators of transcriptions)信号的异常活化与许多疾病相关，包括癌症及免疫相关疾病。STAT3的过表达和组成型活化在多种实体瘤和血液系统癌症中常见。STAT3是STATs家族的成员之一，是JAK2(Janus kinase2)底物蛋白，已被证实与癌症的发生、发展和恶性转化密切相关。在正常情况下，STAT3以无活性的单体形式存在于胞浆中，并存在严格的负反馈调控机制。当JAK2或STAT3的负反馈调控机制异常或基因突变，可以导致STAT3磷酸化水平持续升高和内源性亢奋，与另外一个STAT3蛋白的SH2结构域形成同源二聚体或异源二聚体进入细胞核，通过DNA结合域结合到特定的基因启动子序列上，启动下游基因的转录和蛋白表达，其中包括与线粒体凋亡相关的BCL-2蛋白家族的BCL-2和BCL-XL，以及与细胞周期调控相关的Cyclin-D1等一系列抗凋亡因子的表达。
由于Cyclin-D1、BCL-XL、MMP9、VEGF和c-Myc等众多促增殖、侵袭和抗凋亡的基因都是STAT3信号的靶基因，在STAT3持续活化的动物肿瘤模型或体外培养的肿瘤细胞中，抑制STAT3蛋白可以有效的抑制肿瘤细胞的生长或诱导肿瘤细胞调亡，并减少肿瘤细胞的转移。STAT3已成为肿瘤治疗热门靶点。尽管目前国外已有三款JAK抑制剂在免疫性疾病中获批上市，多个JAKs抑制剂针对肿瘤治疗的研究在临床后期，针对STAT3的一些靶向抑制剂也已进入临床研究阶段，JAKs/STAT3抑制剂在肿瘤市场的需求还远未被满足。
为了开发STAT3靶向抗肿瘤药物，我们近期合成了一类具有全新结构式的萘基脲-哌嗪类化合物。通过一些生物学技术分析，发现该类化合物能够显著抑制STAT3信号的活化，抑制乳腺癌和肝癌细胞株的细胞增殖，诱导细胞发生G1/S或G2/M期阻滞，并促进肿瘤细胞凋亡，显示了极强的抑瘤活性。
本发明旨在提供一类新型萘基脲-哌嗪类化合物及其衍生物的抗肿瘤作用及其潜在药理机制，以及此类化合物在乳腺癌、肝癌、肺癌、结肠癌和白血病临床治疗的潜在应用。
发明内容
本发明的目的是提供一类萘基脲-哌嗪类化合物及其制备方法和应用。
基于上述目的，本发明采取如下技术方案：
一类萘基脲-哌嗪类化合物，结构式如通式I所示：
Figure PCTCN2022092953-appb-000001
其中，R 1选自
Figure PCTCN2022092953-appb-000002
Figure PCTCN2022092953-appb-000003
Figure PCTCN2022092953-appb-000004
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11、R 12、R 13、R 14、R 15、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39、R 40、R 41、R 42、R 43、R 44、R 45、R 46、R 47、R 48、R 49、R 50、R 51各自独立地选自H、F、Cl、Br、-CN、-CH 3、-CF 3、-OCH 3、-OCF 3；
R 2选自
Figure PCTCN2022092953-appb-000005
Figure PCTCN2022092953-appb-000006
R 3选自
Figure PCTCN2022092953-appb-000007
Figure PCTCN2022092953-appb-000008
H、CH 3、CF 3；
m代表CH 2取代基的个数，m为1、2、3、4...10；
k,z代表CH 2取代基的个数，k,z为0、1、2、3、4、5、6，其中，k、z不同时为0。
上述萘基脲-哌嗪类化合物，具体为如下结构的化合物：
Figure PCTCN2022092953-appb-000009
Figure PCTCN2022092953-appb-000010
上述萘基脲-哌嗪类化合物与乙酸、二氢叶酸、苯甲酸、柠檬酸、山梨酸、丙酸、草酸、富马酸、马来酸、盐酸、苹果酸、磷酸、亚硫酸、硫酸、香草酸、酒石酸、抗坏血酸、硼酸、乳酸和乙二胺四乙酸中的至少一种形成的生物学可接受的盐。
上述萘基脲-哌嗪类化合物的制备方法，包括以下步骤：
(1)将
Figure PCTCN2022092953-appb-000011
溶于1,4-二氧六环中，室温下加入叔丁醇钾，再加入
Figure PCTCN2022092953-appb-000012
80～85℃搅拌反应至完全，后处理得到
Figure PCTCN2022092953-appb-000013
(2)将
Figure PCTCN2022092953-appb-000014
溶于四氢呋喃、乙醇和饱和氯化铵水溶液的混合溶液中，45～55℃下加入铁粉，45～55℃搅拌反应至完全，经后处理得到
Figure PCTCN2022092953-appb-000015
(3)将三光气溶于二氯甲烷中并用冰浴降温至0±5℃，将化合物
Figure PCTCN2022092953-appb-000016
和N,N-二异丙基乙胺溶于二氯甲烷中，滴加入三光气的二氯甲烷溶液中，0±5℃保温反应1.5h～2.5h，再次加入剩余N,N-二异丙基乙胺、R 1NH 2,并补加 甲苯，110℃～120℃反应完全，柱层析得到
Figure PCTCN2022092953-appb-000017
其中，所述
Figure PCTCN2022092953-appb-000018
的制备过程如下：
(a)将
Figure PCTCN2022092953-appb-000019
和三苯基膦溶于四氢呋喃中，-5℃～5℃保护气氛下加入偶氮二甲酸二异丙酯，室温搅拌反应至完全，经后处理得到
Figure PCTCN2022092953-appb-000020
(b)将化合物
Figure PCTCN2022092953-appb-000021
溶于四氢呋喃中，-5℃～5℃分批加入硼氢化钠，室温搅拌至反应完全，经后处理得到
Figure PCTCN2022092953-appb-000022
进一步地，所述步骤(1)中
Figure PCTCN2022092953-appb-000023
与叔丁醇钾的摩尔比为1:1.2:1.2；
步骤(2)中
Figure PCTCN2022092953-appb-000024
与铁粉的摩尔比为1:8，四氢呋喃、乙醇以及饱和氯化铵水溶液的体积比为1:2.4:2.4；
步骤(3)中，步骤(3)中，三光气、R 1NH 2、
Figure PCTCN2022092953-appb-000025
和N,N- 二异丙基乙胺的摩尔比为1:2.3:3:11，两次N,N-二异丙基乙胺的摩尔比为4:7。
进一步地，步骤(a)中，
Figure PCTCN2022092953-appb-000026
三苯基膦、偶氮二甲酸二异丙酯的摩尔比为1:1:1.2:1.2；
步骤(b)中，
Figure PCTCN2022092953-appb-000027
和硼氢化钠的摩尔比为1:4。
上述的萘基脲-哌嗪类化合物及其生物学可接受的盐在制备抗肿瘤药物中的用途，其中所述抗肿瘤药物为治疗与STAT3信号传导相关的肿瘤的药物。
优选地，所述的抗肿瘤药物是指治疗乳腺癌、肝癌、肺癌、结肠癌或白血病的药物。
本发明的另一目的是提供一类具有靶向抗肿瘤活性的小分子化合物。
所述肿瘤具体可为STAT3高表达或组成型活化的肿瘤，包括但不限于肝癌、乳腺癌、肺癌、结肠癌和白血病等。
具体的说，本发明合成了一类具有全新结构的萘基脲-哌嗪类化合物ID210916B-1，ID210917B-1，ID210918B-1，ID210919B-1，ID211130C-1，ID211203B-1，IY211214C-1，IY211228B-1，IY220302B-1，IY220209A-1，IY220313A-1，IY220219A-1，ID210928B-1，ID210929B-1，IY220319A-1，ID211008B-1，ID211009B-1，ID211010B-1，ID211116B-1和ID211012B-1等。通过MTT法检测此类化合物对肿瘤细胞的增殖抑制作用，通过流式细胞术检测化合物对肿瘤细胞的细胞周期和凋亡的影响，并通过免疫印迹等方法明确其对JAK2/STAT3信号的抑制作用。
结果表明，本发明的化合物ID210916B-1，ID210917B-1，ID210918B-1，ID210919B-1，D211130C-1，ID211203B-1，IY211214C-1，IY211228B-1，IY220302B-1，IY220209A-1，IY220313A-1，IY220219A-1，ID210928B-1，ID210929B-1，IY220319A-1，ID211008B-1，ID211009B-1，ID211010B-1，ID211116B-1和ID211012B-1等，可以有效抑制乳腺癌和肝癌细胞的增殖，诱导癌细胞G1/S或G2/M期阻滞和细胞凋亡。
总之，本发明提供了一种新的萘基脲-哌嗪类化合物以及它的衍生物在肿瘤治疗上的用途和潜在分子机制。
附图说明
图1是ID210916B-1，ID210917B-1，ID210918B-1，ID210919B-1，D211130C-1，ID211203B-1，IY211214C-1，IY211228B-1，IY220302B-1，IY220209A-1，IY220313A-1，IY220219A-1，ID210928B-1，ID210929B-1，IY220319A-1，ID211008B-1，ID211009B-1， ID211010B-1，ID211116B-1和ID211012B-1等对乳腺癌细胞MDA-MB-468，肝癌细胞HepG2，肺癌细胞PC9，阿法替尼耐药的肺癌细胞PC9-AR，结肠癌细胞HT29和白血病细胞Jurkat等肿瘤细胞的半数抑制率(IC50值)的检测结果；
图2是ID210916B-1诱导TNBC细胞周期阻滞的作用；
图3是ID210916B-1对TNBC细胞划痕修复的抑制作用；
图4是ID210916B-1对TNBC细胞迁移的抑制作用；
图5是ID210916B-1对TNBC细胞中p-STAT3入核的抑制作用；
图6是ID210916B-1对TNBC细胞中STAT3转录活性的抑制作用；
图7是ID210916B-1对TNBC细胞中STAT3的磷酸化和Cyclin D1的表达的抑制作用，图左为MDA-MB-468细胞，图右为4T1细胞。
具体实施方式
为了使本发明的技术目的、技术方案和有益效果更加清楚，下面结合附图和具体实施例对本发明的技术方案作出进一步的说明。
在本发明合成式I化合物的方法中，反应所用的各种原材料是本领域技术人员根据已有知识可以制备得到的，或者是可以通过文献公知的方法制得的，或者是可以通过商业购得的。以上反应方案中所用的中间体、原材料、试剂、反应条件等均可以根据本领域技术人员已有知识做适当改变的。
在本发明中，除非另外说明，其中：(i)温度以摄氏度(℃)表示，操作在室温环境下进行；更具体地，所述室温是指20-30℃；(ii)有机溶剂用常用干燥方法干燥，溶剂的蒸发使用旋转蒸发仪减压蒸发，浴温不高于50℃；展开剂和洗脱剂均为体积比；(iii)反应过程用薄层色谱(TLC)跟踪；(iv)终产物具有满意的质子核磁共振( 1H-NMR)。
实施例1:化合物的合成
Figure PCTCN2022092953-appb-000028
ID210916B-1：
Figure PCTCN2022092953-appb-000029
ID210917B-1：
Figure PCTCN2022092953-appb-000030
ID210918B-1：
Figure PCTCN2022092953-appb-000031
ID210919B-1：
Figure PCTCN2022092953-appb-000032
ID211130C-1：
Figure PCTCN2022092953-appb-000033
ID211203B-1：
Figure PCTCN2022092953-appb-000034
IY211214C-1：
Figure PCTCN2022092953-appb-000035
IY211228B-1：
Figure PCTCN2022092953-appb-000036
IY220302B-1：
Figure PCTCN2022092953-appb-000037
IY220209A-1：
Figure PCTCN2022092953-appb-000038
IY220313A-1：
Figure PCTCN2022092953-appb-000039
IY220219A-1：
Figure PCTCN2022092953-appb-000040
ID210928B-1：
Figure PCTCN2022092953-appb-000041
ID210929B-1：
Figure PCTCN2022092953-appb-000042
IY220319A-1：
Figure PCTCN2022092953-appb-000043
ID211008B-1：
Figure PCTCN2022092953-appb-000044
ID211009B-1：
Figure PCTCN2022092953-appb-000045
R 3＝H；
ID211010B-1：
Figure PCTCN2022092953-appb-000046
R 3＝CH 3；
ID211116B-1：
Figure PCTCN2022092953-appb-000047
ID211012B-1：
Figure PCTCN2022092953-appb-000048
具体合成方法，以化合物ID210916B-1为例，结构式分别如下：
Figure PCTCN2022092953-appb-000049
化合物ID210916B-1的名称为tert-butyl 4-(2-(4-(((4-(3-(pyridin-4-ylmethyl)ureido)naphthalen-1-yl)oxy)methyl)phenoxy)ethyl)piperazine-1-carboxylate，
其合成路线如下：
Figure PCTCN2022092953-appb-000050
步骤1.tert-butyl 4-(2-(4-formylphenoxy)ethyl)piperazine-1-carboxylate(int 1)
①称取对羟基苯甲醛(化合物1)520.00g(相当于4.26mol)，置于50L双层玻璃反应釜中，用20L的四氢呋喃进行溶解，搅拌使其完全溶解；
②称取叔丁基-4-(2-羟乙基)哌嗪-1-羧酸酯(化合物1a)980.65g(相当于4.26mol)和三苯基膦1.34kg(相当于5.11mol)，并加入①中，搅拌使其完全溶解，然后搅拌降温至0℃；
③称取偶氮二甲酸二异丙酯(DIAD)1.03kg(相当于5.11mol)，然后用恒压滴液漏斗缓慢滴加到溶液②中，使体系温度维持在0±4℃；
④滴加完毕后0℃保温搅拌0.50小时，然后将体系升温至20℃反应16小时；
⑤采用旋转蒸发仪进行反应溶液旋转蒸馏，蒸至无馏分；
⑥将得到的液体用10L乙酸乙酯进行稀释，然后将其倒入50L双层玻璃反应釜中，然后加入10L水，然后用5.9L稀盐酸(1M)将溶液pH调至2-4之间，然后分出水相和有机相，然后再用乙酸乙酯对水相进行两次萃取，每次用量8L，然后将三次有机相合并废弃，水 相进行后续处理；
⑦将水相加入到50L双层玻璃反应釜中，然后用碳酸氢钠将其pH调至8，然后用乙酸乙酯萃取两次，每次用量8L，合并有机相，水相废弃(TLC监测无产品残留)，然后用无水硫酸钠干燥有机相，抽滤除去硫酸钠，滤液用旋转蒸发仪进行进行旋转蒸发，蒸至无馏分流出，得到黏稠状棕红色液体717.67g即为int 1；
1H NMR(CDCl 3,300MHz)δ:9.93(s,1H),7.87(d,J＝9.0Hz,2H),7.04(d,J＝9.0Hz,2H),4.23(t,J＝6.0Hz,2H),3.50(t,J＝6.0Hz,4H),2.89(t,J＝6.0Hz,2H),2.57(t,J＝6.0Hz,4H),,1.50(s,9H)
步骤2.tert-butyl 4-(2-(4-(hydroxymethyl)phenoxy)ethyl)piperazine-1-carboxylate(int 2)
①称取int 1 707.34g(相当于2.12mol)，置于50L双层玻璃反应釜中，用4.8L的四氢呋喃进行溶解，搅拌使其完全溶解；
②称取硼氢化钠320.08g(相当于8.46mol)，用甲醇(1.2L)将其配成浑浊液(防止加料过程中硼氢化钠吸潮)缓慢加到溶液①中；溶液颜色由棕红色变为浅黄色；
③置于室温反应；
④反应时长14小时，然后进行TLC监测，结果显示原料反应完毕且生成新产物，于是停止反应，开始后续处理；
⑤将反应液用旋转蒸发仪进行旋转蒸发，除去大部分溶剂，剩余1.20L液体后停止旋蒸，向剩余液体中加入5L二氯甲烷进行稀释(用二氯甲烷的目的是为了萃取时将产物全部萃取到有机相中，提高萃取效率)，然后加入5L水进行萃取，搅拌15分钟后静置分层，分出水相和有机相，水相再用5L二氯甲烷进行萃取，搅拌15分钟后静置分层，分出水相和有机相，水相TLC监测已无产品残留废弃，将两次有机相合并，用无水硫酸钠进行干燥；
⑥然后抽滤除去硫酸钠，滤液用旋转蒸发仪进行旋转蒸发，旋至无馏分蒸出，得666.74g浅黄色粘稠状液体，放置后固化成浅黄色固体即为int 2。
1H NMR(CDCl 3,300MHz)δ:7.33(d,J＝9.0Hz,2H),6.92(d,J＝9.0Hz,2H),4.66(s,2H),4.14(t,J＝6.0Hz,2H),3.49(t,J＝6.0Hz,4H),2.85(t,J＝6.0Hz,2H),2.54(t,J＝6.0Hz,4H),,1.50(s,9H)
步骤3.tert-butyl 4-(2-(4-(((4-nitronaphthalen-1-yl)oxy)methyl)phenoxy)ethyl)piperazine-1-carboxylate(int 3)
①称取int 2 512.14g(相当于1.52mol)，置于50L双层玻璃反应釜中，然后用8L的1,4-二氧六环进行溶解，搅拌使其全部溶解；
②称取叔丁醇钾170.82g(相当于1.52mol)缓慢加入到溶液①中，室温搅拌15分钟；
③称取1-氟-4-硝基萘242.50g(化合物2，相当于1.27mol)缓慢加入到溶液②中，然后使反应体系升温至80℃，保温反应；
④反应2小时后，TLC进行监测，检测结果显示，原料反应完毕有新产物生成，于是停止反应，开始后续处理；
⑤将反应液进行抽滤，除去其中的不溶物，硅藻土助虑，但仍然不好抽滤，抽滤速率很慢，抽滤完毕后滤液放置待明天浓缩拌样；
⑥用旋转蒸发仪将昨天抽滤得到的滤液进行旋转蒸发，并加硅胶进行拌样，旋干后进行装柱，待进行柱层析纯化产品；
⑦开始柱层析纯化产品，用甲醇/二氯甲烷＝1/100(V/V)体系的淋洗剂淋洗玻璃柱，最终得到产品323.64g黏稠状棕色液体即为int 3。但核磁检测和TLC检测均含有杂质点。
步骤4.tert-butyl 4-(2-(4-(((4-aminonaphthalen-1-yl)oxy)methyl)phenoxy)ethyl)piperazine-1-carboxylate(int 4)
①称取int 3 320.66g(相当于631.73mmol)，置于5L双口圆底烧瓶中，用四氢呋喃(500mL)将其溶解，然后继续加入95％乙醇(1.2L)使其完全溶解，溶液变为深棕色；
②然后向溶液①中加入饱和氯化铵溶液(1.2L)，此时溶液中会有固体析出，溶液颜色变为棕黄色，然后使溶液温度升高到50℃，固体会随之溶解；
③称取铁粉282.23g(相当于5.05mol)缓慢加入到溶液②中，保持50℃反应2小时；
④反应2小时后，TLC监测，结果显示原料反应完毕，开始后续处理；
⑤向反应液中加入200.00g硅藻土进行助虑，然后抽滤除去反应液中的铁粉和硅藻土，滤饼用二氯甲烷泡洗，滤液倒入50L双层玻璃反应釜中；
⑥然后向50L反应釜中加入8.00L水和10L二氯甲烷，搅拌25分钟，静置分层，分出水相和有机相后，水相TLC检测无产品残留，废弃；有机相用无水硫酸钠干燥；
⑦抽滤除去硫酸钠，然后滤液用旋转蒸发仪进行旋转蒸发，蒸至无馏分后称量有340.12g粗品放置于冰箱低温保存，待明天拌样进行柱层析纯化；
⑧将昨天得到的粗品用二氯甲烷(200mL)溶解，然后加入1.2倍粗品质量的硅胶400.00g左右进行拌样，然后采用旋转蒸发仪进行旋转蒸发，除去溶剂，旋至粉末状后进行拌样装柱(先在玻璃柱中加入5倍粗品质量的硅胶1.80kg左右，再将拌好的硅胶样倒入上面装柱)，准备进行柱层析纯化；
⑨开始用纯二氯甲烷进行淋洗硅胶柱，将硅胶柱润湿，并将比产品极性小的杂质除去，然后用甲醇：二氯甲烷＝1:100(v/v)淋洗剂进行淋洗，但冲出的淋洗剂中仍有一部分既含有 产品也含有杂质，将其收集在一起待处理，继续淋洗，得到纯的产品，用旋转蒸发仪旋干淋洗剂后得到119.37g棕色黏稠状液体即为int 4.
步骤5.tert-butyl 4-(2-(4-(((4-(3-(pyridin-4-ylmethyl)ureido)naphthalen-1-yl)oxy)methyl)phenoxy)ethyl)piperazine-1-carboxylate(ID210916B-1)
①称取三光气33.15g(相当于111.72mmol)置于1L单口圆底烧瓶中，然后用二氯甲烷(300mL)溶解，搅拌使其完全溶解，然后用冰水浴将体系里的温度降至0℃
②然后称取int 4 160.07g(相当于335.15mmol)和N,N-二异丙基乙胺57.76g(相当于448.87mmol)混合在一起，加入二氯甲烷(350mL)溶解后用恒压第一漏斗缓慢加入到溶液①中，然后0℃保温反应2小时；(滴加过程中有白色的烟生成，滴加完毕后白烟消失)
③反应2小时后，向反应液中继续滴加N,N-二异丙基乙胺99.63g(相当于770.85mmol)；
④滴加完毕后，称取4-吡啶甲胺27.79g(相当于256.95mmol)缓慢加入到反应液中，然后向反应体系中补加600mL甲苯，然后升温至110℃，保温反应过夜，待明天TLC监测；
⑤反应时长18小时，然后TLC进行监测，结果显示原料反应完，且生成新产物，于是将反应液做后续处理；
⑥向反应液中加入1.2倍粗品(投料总量大约300g)质量的硅胶450g左右进行拌样，然后用旋转蒸发仪将溶液中的溶剂旋干，旋至粉末状后进行装柱(玻璃柱中先加入5倍粗品质量的硅胶1.50kg，然后将拌好的硅胶样倒入硅胶上方)，待用柱层析进行产品的纯化；
⑦开始用柱层析对产品进行纯化，用MeOH/DCM＝1/20(V/V)体系的淋洗剂进行淋洗，然后将得到的含有纯品的淋洗剂进行旋转蒸发，浓缩至剩余12.5L后将其转移至50L双层玻璃反应釜中，然后再向其中加入8L水进行洗涤，搅拌15分钟后静置分层，分出有机相和水相，水相废弃，有机相再次转移至50L双层玻璃反应釜中，再用8L水洗涤一次，搅拌15分钟后静置分层，分出有机相和水相，水相废弃，有机相用无水硫酸钠进行干燥，抽滤除去硫酸钠，滤液用旋转蒸发仪进行旋转蒸发，蒸干得到101.94g浅棕色(略微发白)固体即为ID210916B-1。
1H NMR(CDCl 3,300MHz)δ:8.52(d,J＝6.0Hz,2H),8.44(s,1H),8.19(d,J＝6.0Hz,1H),8.01(d,J＝6.0Hz,1H),7.62-7.45(m,5H),7.32(d,J＝6.0Hz,2H),7.06-6.92(m,4H),5.20(s,2H),4.36(d,J＝6.0Hz,2H),4.10(t,J＝6.0Hz,2H),3.31(t,J＝6.0Hz,4H),2.72(t,J＝6.0Hz,2H),2.44(t,J＝6.0Hz,4H),1.40(s,9H)
其他化合物的合成方法参照实施例1，区别在于，在步骤1中原料1换成相应的醛，或 者将1a替换成对应取代基的哌嗪，或者在步骤5中将原料4-吡啶甲胺换成对应的胺即可。
实施例2：ID210916B-1，ID210917B-1，ID210918B-1，ID210919B-1，D211130C-1，ID211203B-1，IY211214C-1，IY211228B-1，IY220302B-1，IY220209A-1，IY220313A-1，IY220219A-1，ID210928B-1，ID210929B-1，IY220319A-1，ID211008B-1，ID211009B-1，ID211010B-1，ID211116B-1和ID211012B-1等对乳腺癌和肝癌等细胞的增殖抑制作用
分别收集对数生长期的肿瘤细胞，调整细胞悬液浓度为5×10 4个/mL，加入96孔细胞培养板，每孔体积100ul。以DMSO为溶剂对照，WP1066(中文名称：(2E)-3-(6-溴-2-吡啶基)-2-氰基-N-[(1S)-1-苯基乙基]-2-丙烯酰胺,CAS:857064-38-1，结构为
Figure PCTCN2022092953-appb-000051
)为阳性对照，将本发明所述的新型萘脲-哌嗪类化合物ID210916B-1,ID210917B-1,ID210918B-1等用DMSO稀释后加入培养孔，使体系中化合物的终浓度分别为0.1、0.3、1、3、10、30、100和300(μmol/L)。继续培养48h后，每孔加入MTT溶剂(5mg/ml)10μL，37℃孵育4h，吸弃培养上清，每孔加入150μl DMSO，摇床震荡脱色10min，酶标仪读值，测定在吸收波长为490nm下的OD值，记录结果，以化合物的剂量为横坐标，吸光值为纵坐标绘制细胞生长曲线。所述的ID210916B-1,ID210917B-1,ID210918B-1,ID210919B-1,D211130C-1,ID211203B-1,IY211214C-1,IY211228B-1,IY220302B-1,IY220209A-1,IY220313A-1,IY220219A-1,ID210928B-1，ID210929B-1，IY220319A-1，ID211008B-1，ID211009B-1，ID211010B-1，ID211116B-1和ID211012B-1等对肿瘤细胞的半数抑制率(IC50值)的统计结果如图1所示。
图1的结果表明：与阳性对照药物WP1066相比，ID210916B-1，IY211214C-1和ID211130C-1等对乳腺癌和肝癌等肿瘤细胞均有良好的增殖抑制作用，特别是在乳腺癌和肝癌细胞的抑瘤活性更强，本申请重点对这3种化合物在乳腺癌和肝癌的抗肿瘤效应进行了进一步研究。
实施例3：ID210916B-1诱导TNBC细胞周期阻滞的作用
取对数生长期的MDA-MB-468或4T1细胞，消化后离心并将细胞制成单细胞悬液。计数后将细胞铺入1个12孔板，两种细胞均每孔接种2×10 5个细胞，铺3个孔做平行对照。铺板16h后，加化合物处理细胞。以DMSO为化合物的溶剂，化合物ID210916B-1在4T1细胞悬液的终浓度分别为0、2.5、5和10μM，化合物ID210916B-1在MDA-MB-468细胞悬液的终浓度分别为0、0.025、0.05和0.1μM。加药48h后，用胰酶分别消化各空细胞，重悬 之后计数，将各孔细胞浓度调整为5×10 5个。消化完成后离心弃上清，再用PBS洗细胞两遍(2000rpm，离心5min)，之后弃尽上清，每管加入980μl的70％冷乙醇和20μl的5％BSA(添加少量BSA可以减少操作过程中的细胞损失)在4℃条件下固定过夜。弃固定液，用PBS洗3遍以去除残余的固定液(1000rpm，离心3min)。细胞洗涤完成后，按DNA含量检测试剂盒(北京索莱宝公司产品)的说明书的要求进行后续操作。每个样品分别用100μl Rnase A于37度孵育30min，然后每个样品中加入500μl已经配制好的PI(碘化丙啶)工作液，室温避光孵育30min。最后，通过流式细胞仪测定细胞周期。采用ModFit软件对实验结果进行分析，通过Graphpad prism 6.0进一步分析得到两种细胞各自的细胞周期比例。
如图2，在MDA-MB-468细胞中，与溶剂(DMSO)对照组相比，化合物ID210916B-1较高浓度(0.05和0.1μM)处理组的G2期的细胞所占比率呈升高趋势，分别为19.67％、25.63％和36.04％。在0.1μM剂量处理下，S期细胞比率也明显增加(25.67％：41.66％)，G1期比率相应降低(54.65％:22.3％)。因此，化合物ID210916B-1可以诱导MDA-MB-468细胞发生S/G2期阻滞。在4T1细胞中，化合物ID210916B-1较高浓度(5和10μM)处理组的G1期细胞的比率显著增加，并呈剂量依赖性。相应地，G2期细胞的比率显著降低。表明化合物ID210916B-1能够诱导4T1细胞发生G1期阻滞，抑制细胞周期进程。
实施例4：ID210916B-1对TNBC细胞划痕修复的抑制作用
取对数生长期的MDA-MB-468或4T1细胞，接种于6孔板，每孔5×10 5个细胞。约24h后细胞呈单层均匀铺满孔底。以200μL枪尖垂直均匀划痕。PBS洗2次后，用含0.5％胎牛血清的DMEM培养液，加入不同剂量的ID210916B-1(MDA-MB-468细胞处理浓度为0、0.015、0.03和0.06μM，4T1细胞处理浓度为0、0.25、0.5和1μM)，分别于0、24和36h(MDA-MB-468细胞)和0、24和48h(4T1细胞)在显微镜下观察拍照。
图3的结果显示，在MDA-MB-468细胞和4T1细胞中，未加ID210916B-1处理的对照组在24时的弥合面积接近50％，在36h接近完全弥合。而应用0.015μM和0.03μM的ID210916B-1处理后，细胞的愈合速度显著滞后。用0.06μM的ID210916B-1几乎可以完全抑制细胞的划痕修复。在4T1细胞中可以观察到相似的现象，表明该化合物可以抑制TNBC细胞的横向迁移能力。
实施例5：ID210916B-1对TNBC细胞迁移的抑制作用
取对数生长期的MDA-MB-468或4T1细胞，常规消化离心，PBS洗细胞1次，500g离心5min后，用基础培养基重悬计数。Transwell上层小室(8μm pore sizes,Corning,US)加30μL预冷的Matrigel，下室加600μL含20％血清的培养基做诱饵。上层小室每孔接种1×105个细胞。以DMSO为溶剂对照孔，不同浓度的ID210916B-1分别处理细胞(MDA-MB-468 细胞处理浓度为0、0.015、0.03μM，4T1细胞处理浓度为0、2、4μM)，置孵箱继续培养。48h后用结晶紫染色。
结晶紫染色步骤：1)弃培养液，用1×PBS洗小室3遍；2)弃PBS，用75％的乙醇固定小室15min；3)固定完成后弃尽乙醇，用1×PBS再洗小室3遍；4)弃PBS，用0.1％的结晶紫对小室进行染色，染色时间为30min；5)自来水冲洗小室至水流清澈看不出紫色；6)用棉棒沾水小心擦去上室未能迁移的细胞，然后将上室放回24孔板中。显微镜下拍照记录细胞迁移和侵袭情况。
从Transwell(图4)的结果可以观察到，对照孔有大量的紫红色颗粒，表明不加ID210916B-1处理的MDA-MB-468和4T1细胞均具有很强的细胞纵向迁移能力，可以穿过插入物上室的底膜。而ID210916B-1处理后各孔中染色阳性的细胞明显减少，表明TNBC细胞的纵向迁移能力被抑制。
实施例6：ID210916B-1对TNBC细胞中p-STAT3入核的抑制作用
按照实施例2的常规细胞培养法进行传代培养，接下来进行如下操作：(1)MDA-MB-468和4T1细胞分别铺35mm小皿，每皿细胞数量为1×10 5个，置37℃孵箱培养过夜；(2)用ID210916B-1处理MDA-MB-468或4T1细胞，ID210916B-1处理MDA-MB-468的终浓度为0.04μM和ID210916B-1处理4T1的终浓度为6μM，以每毫升培养液加入1μl的DMSO的细胞作为溶剂对照(Control)，继续培养48小时；(3)用吸头尽弃培养基；(4)每孔加免疫固定液1ml，室温固定15min(注意：在皿外围加，迅速晃一下，不要对细胞冲击，不要干板)；(5)弃固定液，1×PBS洗3次，每次5min，摇床最慢速摇；(6)用预冷的甲醇1ml/孔，-20℃透化10min或4℃透化15min；(7)弃甲醇，1×PBS洗3次，每次5min；(8)加封闭液，150-200μl/孔，湿盒中室温封闭60min；(9)用抗体稀释液1:100稀释一抗，弃封闭液，加一抗150μl/孔，湿盒中孵育过夜；(10)弃一抗，1×PBS洗3次，每次5min；(11)加用抗体稀释液配好的二抗，室温避光反应30min；(12)弃二抗，1×PBS洗3次，每次5min；(13)DAPI染核，加入少量DAPI，覆盖住样品即可，室温孵育5min；(14)去除DAPI，以1×PBS洗3次，每次5min；(15)每皿加入1ml的1×PBS，避光观察或固定。
图5的结果显示，与对照孔的细胞相比，0.04μM的ID210916B-1处理24h后，MDA-MB-468细胞特别是细胞核的p-STAT3表达明显减少。应用6μM的ID210916B-1处理后24h后，4T1细胞浆和细胞核中的p-STAT3表达均明显减少。表明ID210916B-1可以干扰TNBC细胞中p-STAT3的入核。
实施例7：ID210916B-1对TNBC细胞中STAT3转录活性的抑制作用
按照实施例2的常规细胞培养法进行传代培养，接下来进行如下操作：(1)以MDA-MB-468细胞为例：将MDA-MB-468细胞接种于12孔板，每孔细胞数量1.5×10 5个，置37℃培养箱孵育24h；(2)取出12孔板，弃去培养液，并用1mL PBS缓冲液清洗细胞一遍，以500μL无血清的培养液再清洗一遍，备用；(3)用无血清的培养基稀释STAT3-荧光素酶报告质粒(购自上海权阳生物科技有限公司，TSB10127-1)和pRL-TK海肾质粒(购自Promega,US)质粒与脂质体(Lipofectamine TM 2000,CAT.NO.11668-027)。每孔取250μL无血清培养基稀释4μg质粒，轻轻吹打混匀。每孔取250μL无血清培养基稀释10μg脂质体，轻轻吹打混匀，室温静置5min。将稀释好的质粒加入到稀释好的脂质体中，轻轻吹打，室温静置20min。将混合物慢慢滴加到细胞中，轻轻混匀，37℃孵育4h。每孔更换1mL含10％FBS的新鲜培养基，37℃培养箱继续培养60h。(4)用梯度剂量的化合物ID210916B-1分别预处理MDA-MB-468细胞(0、0.015、0.03、0.06和0.12μM)及4T1细胞(0、3、6和12μM)各2h。然后以IL-6(50ng/ml)刺激细胞1h。弃去上清，PBS清洗细胞一次。用5×PLB裂解液200μL室温裂解20min。将细胞裂解液转入1.5ml Ep管中。按1：50的比例加入双荧光素酶报告基因检测试剂盒(Promega,US，E1910)检测缓冲液，于多功能酶标仪(Varioskan Flash,Thermo-Fisher,US)分别检测萤火虫和海参荧光素酶的活性。(5)计算相对荧光强度(RF＝萤火虫荧光值/海肾荧光值)，并与空白对照组的荧光强度比较。
如图6，在MDA-MB-468细胞中，未采用IL-6处理时相对荧光素酶的活性比值约为10，表明该细胞的本底STAT3转录活性较强。用50ng/ml的IL-6刺激细胞1h后，相对荧光素酶活性比约提高5倍，表明该细胞和报告载体等检测系统对STAT3信号的响应具有灵敏性和特异性。而用0、0.015、0.03、0.06和0.12μM的ID210916B-1预处理后，荧光素酶活性比值不断降低。在0.03μM的ID210916B-1作用下，STAT3的转录活性约降低50％。而当ID210916B-1的浓度为0.12μM时，IL-6诱导的STAT3转录激活被抑制到本底水平。在4T1细胞中可以得到相似的结果。表明ID210916B-1可剂量依赖性地抑制TNBC细胞中STAT3的基因转录调控功能。
实施例8：ID210916B-1对TNBC细胞中STAT3的磷酸化和Cyclin D1的表达的抑制作用
一、细胞培养及加药:a.取对数生长期的MDA-MB-468或4T1细胞，以胰酶消化后，用含10％胎牛血清的L-15或DMEM培养基制成密度为4×10 5个/mL的单细胞悬液，以每孔2mL细胞悬液接种至6孔板中。b.37℃、5％CO 2培养箱孵育(MDA-MB-468细胞培养不需5％CO 2)，待细胞贴壁后，加入不同浓度的ID210916B-1。c.继续培养48h后，用RIPA裂解液裂解细胞并收集蛋白。
二、细胞收集和裂解：a.弃上层培养基，用预冷的PBS洗细胞两次。每孔加入预冷的RIPA细胞裂解液(蛋白酶抑制剂和PMSF与裂解液均以1:100的比例提前加入混匀)100μL。b.在冰上裂解3min，用细胞刮子将细胞刮下来，收集到1.5mL EP管中；放在冰上裂解30min，每隔6min涡旋一次。c.4℃，12000g离心10min。d.将细胞上清转移到新的EP管中。细胞上清分两部分：取5μL加入到1.5mL的EP管中用于BCA测蛋白含量，再加入45μL的1×PBS混匀备用；剩余的细胞上清定量都取80μL，加入5×SDS Loading Buffer 20μL，混匀后在沸水中煮沸10min，离心后上样或置于-20℃冰箱中保存。e.蛋白浓度测定步骤：(1)BCA工作液配制：根据标准品和待测样品的个数，计算出总共所需A与B混合工作液的量。按BCA实剂A与B体积比50：1的比例，配制好工作液，涡旋振荡混匀备用。(2)1×PBS稀释蛋白标准品：
7管号 1×PBS(μl) BSA标准品用量 BSA标准品(μg/ml)
A 0 100 2000
B 200 200 1000
C 200 200(从B管中取) 500
D 200 200(从C管中取) 250
E 200 200(从D管中取) 125
F 400 100(从E管中取) 25
G 200 0 0(空白)
(3)将蛋白标准液和用PBS稀释好的样品上清液(10倍稀释)各取25μL加入到新的96孔板中。然后再分别加入200μL提前配好的BCA工作液充分混匀。切记不要吹打产生气泡，盖紧96孔板板盖，在37℃恒温箱中反应30min。(4)取出96孔板恢复至室温3～5min，在酶标仪上测定A562的吸光度值，拷出获得的数值保存于Excel表中。作出标准曲线并计算出每个样品1μL的蛋白含量以备蛋白上样。
三、SDS-PAGE：(1)固定制胶板，配制10％的SDS-PAGE分离胶。按照下表配制分离胶：10mL
去离子水 4.0mL
30％(m/v)Acrylamide 3.3mL
1.5M Tris-HCl(PH 8.8)缓冲液 2.5mL
10％(m/v)SDS 0.1mL
10％(m/v)APS 0.1mL
TEMED 4μL
Total 10mL
(2)将混好的分离胶分别加入到2块胶板中，加到离顶部1.0cm的位置，用无水乙醇填满胶板，静置30～45min。(3)分离胶凝好后，倒出剩余的无水乙醇，并用滤纸将剩余无水乙醇吸干净。(4)按照下表配制5％浓缩胶5mL
去离子水 2.77mL
30％(m/v)Acrylamide 830μL
0.5M Tris-HCl(PH 6.8)缓冲液 1.26mL
10％(m/v)SDS 50μL
10％(m/v)APS 50μL
TEMED 5μL
Total 5mL
(5)将配制好的浓缩胶缓慢地加入胶板中，避免产生气泡，插入梳子，静置30～45min。
(6)取出蛋白样品，100℃水浴加热5min，10000rpm，离心5min。(7)将胶板固定到电泳槽中，加入SDS-PAGE电泳缓冲液，拔出梳子，按照顺序将处理好的蛋白样品加入到样品槽中，每孔蛋白50μg。(8)80V电泳40min。(9)换电压120V电泳大约1.5h直至溴酚蓝跑出胶体；
四、Western-blot：(1)将电泳完毕的SDS-PAGE胶放入TBST缓冲液中漂洗一次，蛋白胶置于转膜缓冲液中浸泡。(2)将一层棉垫在膜转移缓冲液中浸湿，用镊子夹到转膜仪上，按照黑板、棉垫、滤纸、蛋白胶、PVDF膜、滤纸、棉垫和白板的顺序放好，夹紧，放入转膜仪。每层之间若有气泡应用玻璃实管轻轻滚动将其赶出，。(3)打开转膜仪，以300mA恒流转印80min。(4)将膜放入TBST缓冲液中，漂洗3次，每次8min。(5)用5％BSA-TBST封闭液20mL，室温封闭2h。(6)加入一抗，4℃60rpm过夜孵育。(7)室温，摇床60rpm，以TBST洗膜三次，每次10min。(8)加入二抗，室温孵育1h。(9)室温，摇床60rpm，以TBST洗膜三次，每次10min。(10)取化学发光底物实剂溶液A和溶液B各1ml，室温显色2min。(11)用滤纸将膜上的液体吸干，上机曝片。
五、试剂配制：
(1)10％SDS：称量1g高纯度(电泳级)的SDS置于10mL离心管中，加入约8mL去离子水，加热溶解，定容至10mL，室温保存。
(2)10％过硫酸铵(Ammonium persulfate，AP)：称量1g过硫酸铵，加入约10mL去离子水后搅拌溶解，储存于4℃。
(3)5×电泳缓冲液：称取Tris 15.1g、Glycine 94g、SDS 5.0g于烧杯中，加入1L双蒸水溶解，室温保存，用时稀释5倍。
(4)转膜缓冲液：称取Tris 5.8g，甘氨酸11.6g、SDS 0.75g于烧杯中，加入700mL双蒸水，溶解后定容至800mL，最后加入甲醇200mL。
(5)1.5mol/L的Tris-HCL，100ml:：则用18.15gtris溶于80ml水中用4N Hcl调至8.8，定容100ml即可。
(6)0.5mol/L的Tris-HCL，1000ml：称60.5克tris碱，加水到850ml，加浓盐酸搅拌到全部溶解后调pH到6.8，加水到1L。
(7)TBS缓冲液：称取NaCl 8.8g于800mL蒸馏水中，溶解，加入10mL 1mol/L Tris HCl(pH7.5)，定容至1L，室温保存。
(8)TBST缓冲液：1L TBS缓冲液中加入20％Tween20 500μL，使Tween20的终浓度为0.1％，现用现配。
(9)封闭液，抗体稀释液：TBST缓冲液中加入5％脱脂奶粉或BSA，现用现配。
为了进一步明确ID210916B-1对TNBC细胞中STAT3的磷酸化的抑制作用。我们用不同浓度的ID210916B-1分别处理MDA-MB-468和4T1细胞各48h。Western blot的结果显示，0.06μM和4μM的ID210916B-1可以分别有效下调两种细胞中STAT3活化的关键标志物p-STAT3(Y705)、STAT3总蛋白(T-STAT3)和STAT3的下游靶蛋白Cyclin D1的表达水平(图7)。
综上数据充分说明，化合物ID210916B-1可以有效抑制TNBC细胞的生长和迁移，可以抑制STAT3的磷酸化和活化，是STAT3靶向抑制剂和TNBC的潜在治疗药物。
综上结果表明，以ID210916B-1为代表的此种哌嗪类化合物可以显著抑制乳腺癌和肝癌细胞增殖和转移，诱导肿瘤细胞发生周期阻滞，并且对STAT3及其相关蛋白具有明显的抑制作用，显示了良好的抗癌作用。按照药物开发的一般途径(先进行常规的抗肿瘤体外筛选，然后进行针对性的研究)，本发明的化合物可以应用到与细胞增殖异常相关的癌症治疗药物中，可通过与人体可接受的成盐或与药用载体混合制备抗肿瘤药物。

Claims (9)

  1. 一类萘基脲-哌嗪类化合物，其特征在于，结构式如通式I所示：
    Figure PCTCN2022092953-appb-100001
    其中，R 1选自
    Figure PCTCN2022092953-appb-100002
    Figure PCTCN2022092953-appb-100003
    Figure PCTCN2022092953-appb-100004
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11、R 12、R 13、R 14、R 15、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39、R 40、R 41、R 42、R 43、R 44、R 45、R 46、R 47、R 48、R 49、R 50、R 51各自独立地选自H、F、Cl、Br、-CN、-CH 3、-CF 3、-OCH 3、-OCF 3；
    R 2选自
    Figure PCTCN2022092953-appb-100005
    Figure PCTCN2022092953-appb-100006
    R 3选自
    Figure PCTCN2022092953-appb-100007
    Figure PCTCN2022092953-appb-100008
    H、CH 3、CF 3；
    m代表CH 2取代基的个数，m为1、2、3、4...10；
    k,z代表CH 2取代基的个数，k,z为0、1、2、3、4、5、6，其中，k、z不同时为0。
  2. 根据权利要求1所述的萘基脲-哌嗪类化合物，其特征在于，具体为如下结构的化合物：
    Figure PCTCN2022092953-appb-100009
  3. 权利要求1或2所述的萘基脲-哌嗪类化合物与乙酸、二氢叶酸、苯甲酸、柠檬酸、山梨酸、丙酸、草酸、富马酸、马来酸、盐酸、苹果酸、磷酸、亚硫酸、硫酸、香草酸、酒石酸、抗坏血酸、硼酸、乳酸和乙二胺四乙酸中的至少一种形成的生物学可接受的盐。
  4. 权利要求1或2所述的萘基脲-哌嗪类化合物的制备方法，其特征在于，包括以下步骤：
    (1)将
    Figure PCTCN2022092953-appb-100010
    溶于1,4-二氧六环中，室温下加入叔丁醇钾，再加入
    Figure PCTCN2022092953-appb-100011
    80～85℃搅拌反应至完全，后处理得到
    Figure PCTCN2022092953-appb-100012
    (2)将
    Figure PCTCN2022092953-appb-100013
    溶于四氢呋喃、乙醇和饱和氯化铵水溶液的混合溶液中，45～55℃下加入铁粉，45～55℃搅拌反应至完全，经后处理得到
    Figure PCTCN2022092953-appb-100014
    (3)将三光气溶于二氯甲烷中并用冰浴降温至0±5℃，将化合物
    Figure PCTCN2022092953-appb-100015
    和N,N-二异丙基乙胺溶于二氯甲烷中，滴加入三光气的二氯甲烷溶液中，0±5℃保温反应1.5h～2.5h，再次加入剩余N,N-二异丙基乙胺、R 1NH 2,并补加甲苯，110℃～120℃反应完全，柱层析得到
    Figure PCTCN2022092953-appb-100016
  5. 根据权利要求4所述的萘基脲-哌嗪类化合物的制备方法，其特征在于，所述
    Figure PCTCN2022092953-appb-100017
    的制备过程如下：
    (a)将
    Figure PCTCN2022092953-appb-100018
    和三苯基膦溶于四氢呋喃中，-5℃～5℃下加入偶氮二甲酸二异丙酯，室温搅拌反应至完全，经后处理得到
    Figure PCTCN2022092953-appb-100019
    (b)将化合物
    Figure PCTCN2022092953-appb-100020
    溶于四氢呋喃中，-5℃～5℃分批加入硼氢化钠，室温搅拌至反应完全，经后处理得到
    Figure PCTCN2022092953-appb-100021
  6. 根据权利要求4所述的萘基脲-哌嗪类化合物的制备方法，其特征在于，所述步骤(1)中
    Figure PCTCN2022092953-appb-100022
    与叔丁醇钾的摩尔比为1:1.2:1.2；
    步骤(2)中
    Figure PCTCN2022092953-appb-100023
    与铁粉的摩尔比为1:8，四氢呋喃、乙醇以及饱和氯化铵水溶液的体积比为1:2.4:2.4；
    步骤(3)中，三光气、R 1NH 2、
    Figure PCTCN2022092953-appb-100024
    和N,N-二异丙基乙胺的摩尔比为1:2.3:3:11，两次N,N-二异丙基乙胺的摩尔比为4:7。
  7. 根据权利要求5所述的萘基脲-哌嗪类化合物的制备方法，其特征在于，步骤(a)中，
    Figure PCTCN2022092953-appb-100025
    三苯基膦、偶氮二甲酸二异丙酯的摩尔比为1:1:1.2:1.2；
    步骤(b)中，
    Figure PCTCN2022092953-appb-100026
    和硼氢化钠的摩尔比为1:4。
  8. 权利要求1至3任一项所述的萘基脲-哌嗪类化合物及其生物学可接受的盐在制备抗肿瘤药物中的用途，其特征在于，所述抗肿瘤药物为治疗与STAT3细胞信号传导异常相关疾病的药物。
  9. 根据权利要求8所述的用途，其特征在于，所述抗肿瘤药物为治疗乳腺癌、肝癌、肺癌、结肠癌或白血病的药物。
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