WO2023077739A1 - Quinolone derivative, preparation method therefor, and use thereof - Google Patents

Quinolone derivative, preparation method therefor, and use thereof Download PDF

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WO2023077739A1
WO2023077739A1 PCT/CN2022/088657 CN2022088657W WO2023077739A1 WO 2023077739 A1 WO2023077739 A1 WO 2023077739A1 CN 2022088657 W CN2022088657 W CN 2022088657W WO 2023077739 A1 WO2023077739 A1 WO 2023077739A1
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compound
dmso
nmr
synthesis
stat3
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余文颖
查放
余大鑫
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中国药科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C07D215/54—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3
    • C07D215/56—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3 with oxygen atoms in position 4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur 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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  • the invention belongs to the field of medicinal chemistry and pharmacotherapeutics, and specifically relates to a quinolone derivative, a preparation method thereof, and an application for preparing a small molecule inhibitor of STAT3 and preparing a drug for treating tumors.
  • STAT The signal transducer and activator of transcription (STAT) protein family plays an important role in the occurrence of many human diseases.
  • STAT3 Signal Transducer and Activator of Transcription 3
  • Activated STAT3 protein The activated STAT3 protein will enter the nucleus and promote the expression of various anti-apoptotic protein genes, thereby supporting the occurrence and development of tumors. At present, this target is already an important anti-tumor target.
  • Binding of cytokines or growth factors to surface receptors promotes the STAT3 signaling cascade. Phosphorylation of pTyr705 residues activates cytoplasmic kinases, thereby promoting the phosphorylation of STAT3 monomers, and the two phosphorylated STAT3 monomers combine at the pTyr705-SH2 domain to form a dimerization complex of STAT3, and then the complex enters the nucleus In the process, the expression of related tumor genes is induced, so in the whole pathway, the pTyr705-SH2 domain plays a key role.
  • IL-6/GP130/STAT3 signaling pathway is related to tumorigenesis, survival and drug resistance.
  • the SH2 domain of STAT3 plays a key role in the activation of STAT3 recruitment and formation of STAT3 homodimers and formation of STAT3:STAT3/DNA complexes. It is therefore an object of the present invention to provide an effective STAT3 inhibitor against the SH2 domain.
  • ring A is selected from phenyl, five- and six-membered saturated or unsaturated heterocycles containing 1 to 2 heteroatoms, and the heteroatoms are selected from N, O, and S;
  • R1 is independently selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl, halogen-substituted C1-C3 alkoxy, nitro, amino, halogen; m is an integer from 1 to 5;
  • R is selected from hydrogen, methyl, ethyl, isopropyl
  • ring A is selected from phenyl, pyridine, pyrimidine, thiophene, pyrrole, furan;
  • R1 is independently selected from hydrogen, methyl, nitro, amino, trifluoromethyl, trifluoromethoxy, F, Br; m is an integer of 1 to 2;
  • R2 is selected from hydrogen.
  • quinolone derivatives shown in formula I are selected from:
  • compound 7d exhibited a strong growth inhibitory effect on breast cancer cell MDA-MB-231 and lung cancer cell A549. Further mechanism verification showed that compound 7d can selectively bind to the SH2 domain of STAT3 protein, inhibit the formation of STAT3 homodimers, inhibit the proliferation of breast cancer cells MDA-MB-231 and lung cancer cells A549, and induce breast cancer cells MDA- Apoptosis of MB-231 and lung cancer cell A549, etc.
  • the pharmaceutically acceptable salts of the quinolone derivatives described in the present invention are sodium salts and hydrochlorides.
  • Another object of the present invention is to provide the application of the quinolone derivatives or pharmaceutically acceptable salts or esters thereof in the preparation of STAT3 inhibitors.
  • Another object of the present invention is to provide the application of said quinolone derivatives or pharmaceutically acceptable salts or esters in the preparation of drugs for preventing and/or treating tumor-related diseases.
  • the tumor-related diseases are colon cancer, osteosarcoma, lung cancer, and breast cancer.
  • the lung cancer is non-small cell lung cancer.
  • Another object of the present invention is to provide a pharmaceutical composition, which contains a therapeutically effective amount of a quinolone derivative or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.
  • the quinolone derivatives can bind to the target protein STAT3 SH2 domain, thereby mediating the formation of STAT3 homodimers and selectively inhibiting the JAK-STAT3 cellular pathway. Specifically, it selectively inhibits the formation of p-STAT3, has little effect on the expression levels of other proteins of the same family, does not affect related kinases, inhibits tumor cell proliferation in vitro, and induces cell apoptosis.
  • Figure 1 shows the inhibitory rate of the test compound (concentration: 10 ⁇ M) on tumor cell A549.
  • Figure 2 shows the inhibitory rate of the test compound (concentration: 5 ⁇ M) on tumor cell A549.
  • Figure 3 is the level verification test that the test compound 7d does not affect related kinases
  • Fig. 4 is a validation test of the test compound 7d inducing tumor cell apoptosis in vitro.
  • Fig. 5 is a verification test for the test compound compound 7d to inhibit STAT3 pathway activation.
  • Figure 6 is a kinetic analysis of the test compound 7d on STAT3 protein, analyzing the binding force of the compound to the target protein.
  • Fig. 7 is a test of the effect of the test compound 7d on tumor growth in xenografted nude mice.
  • 3,5-bis(trifluoromethyl)benzoic acid (1.67g, 1mmol) was added to a solution of compound 9 (1.52g, 1.2mmol) in N,N-dimethylformamide (10mL), and then 1- Hydroxybenzotriazole (0.9g, 5.8mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1g, 7.4mmol), stirred at 80°C for 4h, added water Quenching and filtration afforded compound 13 as a yellow solid in 64% yield.
  • Dissolve compound 14 (0.2g, 423umol) in a mixed solvent of 5mL equal volume of absolute ethanol and 10% potassium hydroxide solution, heat at 90°C for 3 hours; cool to room temperature, add concentrated hydrochloric acid to adjust the pH to 2.0; filter and precipitate Washing with water and drying in vacuo afforded compound 15a as a tan solid in 84% yield.
  • Cancer cells (MDA-MB-231, MDA-MB-468, U2OS, A549, HT-29) were seeded in 96-well plates at a density of 4000-6000 cells per well at 37°C in a humidified 5% CO2 Incubate overnight in the incubator; remove the medium, use biological grade 99.9% DMSO to prepare different concentrations (5, 10 ⁇ M) of the compounds to be tested, and then add them in triplicate to 200 ⁇ L of fresh medium containing fetal bovine serum (HT- 29 cells use DMEM medium, other cell lines use RPMI-1640 medium), and incubate at 37°C for 72 hours. The percentage of DMSO in the medium does not exceed 0.1%.
  • Cell viability was assessed by addition of 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT). Absorbance was read by an ELISA reader (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA) at a test wavelength of 570 nm and a reference wavelength of 630 nm. Cell viability was calculated by the following formula:
  • Cell viability (At/As) ⁇ 100%; wherein, At and as represent the absorbance of the test compound and the solvent control, respectively.
  • Figure 1 is a preliminary screening of all synthetic compounds inhibiting the proliferation of A549 cells at (10 ⁇ M).
  • the positive control is BP-1-102, and most of the compounds have better inhibitory activity than BP-1-102.
  • Figure 2 is a re-screening of the activity of synthetic compounds with better primary screening activity in inhibiting the proliferation of A549 cells at (5 ⁇ M).
  • MTT experiment Cell culture was carried out according to Example 34 1.1, MDA-MB-231, MDA-MB-468, U2OS, A549, HT-29 cells were counted after passage, and cell suspension was prepared according to the density of 6000 cells/well. After mixing, pipette 100 ⁇ L of cell suspension into a 96-well plate, adhere to the wall overnight, and administer the cells the next day. Prepare 10 mMOL of the 7d compound stock solution with biological grade 99.9% DMSO, and dilute the compound with the corresponding medium. The final concentrations were 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 ⁇ M, a total of 10 concentration gradients.
  • the cell culture solution in the 96-well plate was aspirated, and 100 ⁇ L of compound 7d containing different concentrations were added to each well. Culture medium into 96-well plate and incubate for 24 hours. Then add 10 ⁇ L MTT to each well and incubate for 4 hours in the dark. After the end, take out the 96-well plate, carefully absorb the supernatant, add 150 ⁇ LDMSO to each well to redissolve, measure the absorbance value of each well with a fluorescent microplate reader, the detection wavelength is 570nm, and the reference wavelength is 630nm.
  • proliferation inhibition rate (%) (1—A experimental group /A control group ) ⁇ 100%.
  • Table 1 shows the results of the inhibitory activity of the compounds after re-screening on A549 cells, among which compounds 7a, 7d, 7h, 12d, and 12f have significant inhibitory activity, which is significantly better than the positive drug BP-1-102; and compound 7da shows the removal of quinolone core After the carboxyl group, the activity decreased significantly.
  • Compounds 7a, 7d, 7h, 12d, and 12f were further screened for activity using HT-29 cells, as shown in Table 2. Compound 7d had the best activity among the candidate compounds.
  • the positive drug BP-1-102 was used as a control to screen the inhibitory activity of compound 7d in different cell lines. The results are shown in Table 3, which shows that the activity of compound 7d is significantly better than that of the positive control BP-1-102.
  • a Data are mean values from at least three independent experiments.
  • a Data are mean values from at least three independent experiments.
  • a Data are mean values from at least three independent experiments.
  • Example 35 Compound 7d inhibits STAT3 protein activity
  • Example 34 for cell culture, use 99.9% biological grade DMSO to prepare 10mmol/L 7d compound mother solution, select RPMI-1640 medium to dilute compound 7d to 0, 2.5, 5, 10, 20 ⁇ M concentration, MDA-MB-231 and A549 cells in RPMI-1640 medium containing different concentrations of compound 7d (0, 2.5, 5, 10, 20 ⁇ M) and 0.1% DMSO (medium supplemented with 10% (v/v) FBS, 50 ⁇ g/mL penicillin and 50 ⁇ g/mL streptomycin) for 24 hours at 37°C in a humidified atmosphere containing 5% CO 2 .
  • DMSO medium supplemented with 10% (v/v) FBS, 50 ⁇ g/mL penicillin and 50 ⁇ g/mL streptomycin
  • Membranes were subsequently blocked with 5% bovine serum albumin (BSA) in TBST, and then incubated with primary antibodies overnight at 4°C, followed by secondary antibody incubation for 2 hours at room temperature. Bound immune complexes were detected using the ChemiDOC TM XRS+ system (BioRad Laboratories, Hercules, CA).
  • Figure 3A MDA-MB-231 cells
  • B A549 cells
  • Figure 4A MDA-MB-231 cells
  • B A549 cells
  • Figure 4A show that using GAPDH as a control, the expression of Cleaved Caspase-3, Cleaved PARP, and Bcl-2 in cell lines significantly increased under the action of compound 7d, indicating that compound 7d can Induces apoptosis.
  • Figure 5A MDA-MB-231 cells
  • B A549 cells
  • C MDA-MB-231 cells
  • MDA-MB-231 cells were seeded in six-well plates (RPMI-1640 medium), about 400,000 cells/well, and adhered overnight. On the next day, compound 7d was added to the final concentration of 0, 5, and 10 ⁇ M, and incubated for 24 hours; after the cell pellet was collected and fully resuspended in 200 ⁇ L buffer, the instructions of the Annexin V-FITC Apoptosis Kit (Bio-Vision) were followed. Add 5 ⁇ L Annexin V-FITC and 10 ⁇ L propidium iodide, and incubate at room temperature for 15 minutes in the dark; light sheet) to analyze the cells.
  • A549 cells were seeded in a six-well plate, about 400,000 cells/well, and adhered overnight. On the next day, compound 7d was added to the final concentration of 0, 1, 5, and 10 ⁇ M, and incubated for 24 hours; after the cell pellet was collected and fully resuspended in 200 ⁇ L buffer, the instructions of the Annexin V-FITC apoptosis kit (Bio-Vision) Add 5 ⁇ L Annexin V-FITC and 10 ⁇ L propidium iodide, and incubate for 15 minutes at room temperature in the dark; emission filter) to analyze the cells.
  • Annexin V-FITC apoptosis kit Bio-Vision
  • MDA-MB-231 cells were cultured as described in Example 34 1.1, and then seeded into 96-well plates so that the number of cells in each well was uniform and between 12,000-15,000. After inoculation, the 96-well plate was incubated overnight in a cell culture incubator to allow the cells to fully adhere to the wall. The culture medium prepared with 10 ⁇ M compound 7d was treated for 24 hours, and then the cells were fixed in 4% PFA/PBS for 15 minutes, and stored in PBS at 4°C. MDA-MB-231 cells were permeabilized with 0.2% TritonX-100 in PBS for 20 minutes and blocked with 5% milk, 2.5% BSA, 10% serum, 0.2% Triton X-100 in PBS for 30 minutes.
  • Electrophoretic mobility shift assay assay of DNA binding activity
  • MDA-MB-231 cells were cultured overnight in culture dishes and then treated with 10 ⁇ M compound 7d in RPMI-1640 medium for 0–24 hours. Cells were harvested and subjected to nuclear extract preparation and EMSA analysis as previously described. For specific steps, see Example 34. 1.1 Cell line cultivation and extraction, and other operations were completed according to the instructions of the EMSA/Gel-Shift kit (Beiyuntian). 32 P-labeled oligonucleotide hSIE probes were used to bind STAT3, and MGFe probes were used to bind STAT1 and STAT5.
  • Recombinant STAT3 was obtained by standard amine coupling procedures in Covalently immobilized on the CM5 sensor chip at pH5.0, the response value is about 20000RU.
  • compound 7d was injected into the fixed flow cell of STAT3 at a flow rate of 30 ⁇ L/min, and 10 mmol/L compound 7d stock solution was prepared with 99.9% biological grade DMSO, and the 7d stock solution was diluted with running buffer at a concentration of 0.078, 0.156, and 0.312 , 0.625, 1.25, 5, and 10 ⁇ M were injected respectively, the binding time was 120 s, and the dissociation time was 120 s.
  • Steady-state KD values were calculated using BIAcore T200 evaluation software. The results are shown in Figure 6, the K D value is 460nM, indicating that compound 7d has a strong affinity for STAT3 protein.
  • Example 37 Compound 7d tumor experiment in vivo
  • mice Female nude mice (4-6 weeks old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Mice were fed standard diet and water according to the standard and allowed to acclimatize for 5 days before dosing.
  • Athymic balb/c nude mice (15-18 g) were injected with 5 ⁇ 10 6 human lung cancer A549 cells suspended in 100 ⁇ L PBS buffer in the right abdominal region. Three days after tumor cell inoculation, the tumor volume reached about 70 mm 3 .
  • the mice were randomly divided into three groups (10 mg/kg compound 7d, 20 mg/kg compound 7d, normal saline), five in each group. The drug was dissolved in a mixed solvent of 40% polyethylene glycol and 60% physiological saline for in vivo administration.
  • Tumor-bearing mice received an intraperitoneal injection of 10 or 20 mg/kg compound 7d or normal saline (CT, control). The size of the tumor was measured with a caliper three times a week, the growth of the tumor was recorded, calculated according to the formula: length ⁇ width ⁇ width/2, and the body weight was measured and recorded. After 21 days of treatment, all mice were sacrificed, tumors were isolated, weighed and stored at -80°C for future use. The tumor tissue cells were cultured according to Example 34, and the expression levels of p-STAT1, p-STAT3, p-STAT5, Bcl-2 and cyclin D1 proteins in the tumor slices were evaluated by Western blotting.

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Abstract

The present invention belongs to the field of medicinal chemistry and pharmacotherapeutics. Disclosed are a quinolone derivative represented by formula I or a pharmaceutically acceptable salt or ester thereof. Further disclosed is a use of the quinolone derivative or a pharmaceutically acceptable salt or ester thereof in the preparation of an STAT3 inhibitor or in the preparation of a medicament for the prevention and/or treatment of tumor-related diseases. Pharmacological experiments prove that the quinolone derivative of the present invention or a pharmaceutically acceptable salt or ester thereof can achieve anti-tumor purposes by inhibiting the proliferation of tumor cells and inducing the apoptosis of tumor cells, thus having antitumor effects and low toxicity. The quinolone derivative can bind to a target protein STAT3 SH2 domain, thereby mediating the formation of STAT3 homodimers and selectively inhibiting the JAK-STAT3 cellular pathway.

Description

一种喹诺酮衍生物及其制备方法和用途A kind of quinolone derivative and its preparation method and application 技术领域technical field
本发明属于药物化学和药物治疗学领域，具体涉及一种喹诺酮衍生物及其制备方法和制备STAT3小分子抑制剂、制备治疗肿瘤药物的应用。The invention belongs to the field of medicinal chemistry and pharmacotherapeutics, and specifically relates to a quinolone derivative, a preparation method thereof, and an application for preparing a small molecule inhibitor of STAT3 and preparing a drug for treating tumors.
背景技术Background technique
信号转导及转录激活化因子(STAT)蛋白家族在人类众多疾病发生起到重要作用。STAT3(信号转导与转录激活因子3)是一种转录因子，它在包括乳腺癌在内的多种癌症的发生发展中起到重要的促进作用，且在多种肿瘤中都能检测到过度激活的STAT3蛋白。而活化的STAT3蛋白会进入细胞核，促进多种抗凋亡蛋白基因的表达，从而支持肿瘤的发生发展。目前该靶点已经是重要的抗肿瘤靶点。The signal transducer and activator of transcription (STAT) protein family plays an important role in the occurrence of many human diseases. STAT3 (Signal Transducer and Activator of Transcription 3) is a transcription factor that plays an important role in the development of various cancers, including breast cancer, and can be detected in a variety of tumors. Activated STAT3 protein. The activated STAT3 protein will enter the nucleus and promote the expression of various anti-apoptotic protein genes, thereby supporting the occurrence and development of tumors. At present, this target is already an important anti-tumor target.
细胞因子或者生长因子与表面受体结合后促进STAT3信号级联反应。pTyr705残基磷酸化后使细胞质激酶激活，从而促进STAT3单体磷酸化，两个磷酸化的STAT3单体在pTyr705-SH2结构域处结合并形成STAT3的二聚化复合物，随后复合物进入细胞核中，诱导相关肿瘤基因的表达，因此在整条通路中，pTyr705-SH2结构域起到关键作用。Binding of cytokines or growth factors to surface receptors promotes the STAT3 signaling cascade. Phosphorylation of pTyr705 residues activates cytoplasmic kinases, thereby promoting the phosphorylation of STAT3 monomers, and the two phosphorylated STAT3 monomers combine at the pTyr705-SH2 domain to form a dimerization complex of STAT3, and then the complex enters the nucleus In the process, the expression of related tumor genes is induced, so in the whole pathway, the pTyr705-SH2 domain plays a key role.
但是现有的小分子SH2结构域抑制剂都存在活性不足或者与阳性对照药相比无统计学差异的窘境。因此，设计合成新型小分子SH2结构域抑制剂对JAK-STAT3细胞通路的阻断有着重要作用。However, the existing small molecule SH2 domain inhibitors all have the dilemma of insufficient activity or no statistical difference compared with the positive control drug. Therefore, designing and synthesizing new small molecule SH2 domain inhibitors plays an important role in blocking the JAK-STAT3 cellular pathway.
发明内容Contents of the invention
研究表明，IL-6/GP130/STAT3信号通路与肿瘤发生、存活以及耐药性相关。STAT3的SH2结构域在激活STAT3的募集和形成STAT3同源二聚体以及形成STAT3：STAT3/DNA复合物的形成中起着关键作用。因此本发明的目的是针对SH2结构域提供一种有效的STAT3抑制剂。Studies have shown that IL-6/GP130/STAT3 signaling pathway is related to tumorigenesis, survival and drug resistance. The SH2 domain of STAT3 plays a key role in the activation of STAT3 recruitment and formation of STAT3 homodimers and formation of STAT3:STAT3/DNA complexes. It is therefore an object of the present invention to provide an effective STAT3 inhibitor against the SH2 domain.
本发明的目的是通过以下技术方案实现的：The purpose of the present invention is achieved through the following technical solutions:
式I所示的喹诺酮衍生物或其药学上可接受的盐或酯：The quinolone derivative shown in formula I or its pharmaceutically acceptable salt or ester:
Figure PCTCN2022088657-appb-000001
Figure PCTCN2022088657-appb-000001
其中，A环选自苯基、含1～2个杂原子的五、六元饱和或不饱和杂环，杂原子选自N、O、S；Among them, ring A is selected from phenyl, five- and six-membered saturated or unsaturated heterocycles containing 1 to 2 heteroatoms, and the heteroatoms are selected from N, O, and S;
R 1每次出现独立的选自氢、C1～C3烷基、C1～C3烷氧基、卤素取代的C1～C3烷基、卤素取代的C1～C3烷氧基、硝基、氨基、卤素；m为1～5的整数； Each occurrence of R1 is independently selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl, halogen-substituted C1-C3 alkoxy, nitro, amino, halogen; m is an integer from 1 to 5;
Y选自NH、O，X选自亚甲基(-CH 2-)、羰基
Figure PCTCN2022088657-appb-000002
n＝1～3的整数，结构单元
Figure PCTCN2022088657-appb-000003
可重复； Y is selected from NH, O, X is selected from methylene (-CH 2 -), carbonyl
Figure PCTCN2022088657-appb-000002
n=1～3 integer, structural unit
Figure PCTCN2022088657-appb-000003
repeatable;
R 2选自氢、甲基、乙基、异丙基、
Figure PCTCN2022088657-appb-000004
 R is selected from hydrogen, methyl, ethyl, isopropyl,
Figure PCTCN2022088657-appb-000004
优选的，A环选自苯基、吡啶、嘧啶、噻吩、吡咯、呋喃；Preferably, ring A is selected from phenyl, pyridine, pyrimidine, thiophene, pyrrole, furan;
R 1每次出现独立的选自氢、甲基、硝基、氨基、三氟甲基、三氟甲氧基、F、Br；m为1～2的整数； Each occurrence of R1 is independently selected from hydrogen, methyl, nitro, amino, trifluoromethyl, trifluoromethoxy, F, Br; m is an integer of 1 to 2;
Y选自NH、O，X选自亚甲基，n＝1；Y is selected from NH, O, X is selected from methylene, n=1;
R 2选自氢。 R2 is selected from hydrogen.
具体的，式I所示的喹诺酮衍生物选自：Specifically, the quinolone derivatives shown in formula I are selected from:
Figure PCTCN2022088657-appb-000005
Figure PCTCN2022088657-appb-000005
Figure PCTCN2022088657-appb-000006
Figure PCTCN2022088657-appb-000006
在上述优选的基础上，本发明所述的喹诺酮衍生物结构式如下：On the above-mentioned preferred basis, the quinolone derivative structural formula of the present invention is as follows:
Figure PCTCN2022088657-appb-000007
Figure PCTCN2022088657-appb-000007
化学名称：6-((3,5-双(三氟甲基)苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸。Chemical name: 6-((3,5-bis(trifluoromethyl)benzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid.
药理实验证明，化合物7d在乳腺癌细胞MDA-MB-231、肺癌细胞A549体现出强力的生长抑制作用。进一步的机制验证表明，化合物7d可以选择性结合STAT3蛋白SH2结构域，抑制STAT3同源二聚体的形成，抑制乳腺癌细胞MDA-MB-231、肺癌细胞A549的增殖，诱导乳腺癌细胞MDA-MB-231、肺癌细胞A549的凋亡等。Pharmacological experiments proved that compound 7d exhibited a strong growth inhibitory effect on breast cancer cell MDA-MB-231 and lung cancer cell A549. Further mechanism verification showed that compound 7d can selectively bind to the SH2 domain of STAT3 protein, inhibit the formation of STAT3 homodimers, inhibit the proliferation of breast cancer cells MDA-MB-231 and lung cancer cells A549, and induce breast cancer cells MDA- Apoptosis of MB-231 and lung cancer cell A549, etc.
本发明所述的喹诺酮衍生物药学上可接受的盐为钠盐、盐酸盐。The pharmaceutically acceptable salts of the quinolone derivatives described in the present invention are sodium salts and hydrochlorides.
本发明的再一目的是提供所述的喹诺酮衍生物或其药学上可接受的盐或酯在制备STAT3抑制剂中的应用。Another object of the present invention is to provide the application of the quinolone derivatives or pharmaceutically acceptable salts or esters thereof in the preparation of STAT3 inhibitors.
本发明的再一目的是提供所述的喹诺酮衍生物或其药学上可接受的盐或酯在制备预防和/或治疗与肿瘤有关疾病的药物中的应用。Another object of the present invention is to provide the application of said quinolone derivatives or pharmaceutically acceptable salts or esters in the preparation of drugs for preventing and/or treating tumor-related diseases.
所述的与肿瘤有关疾病为结肠癌、骨肉瘤、肺癌、乳腺癌。The tumor-related diseases are colon cancer, osteosarcoma, lung cancer, and breast cancer.
具体的，所述的肺癌为非小细胞肺癌。Specifically, the lung cancer is non-small cell lung cancer.
本发明的另一个目的在于提供一种药物组合物，含有治疗有效量的喹诺酮衍生物或其药学上可接受的盐或酯，可药用的载体、佐剂或媒剂。Another object of the present invention is to provide a pharmaceutical composition, which contains a therapeutically effective amount of a quinolone derivative or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.
本发明的有益效果：Beneficial effects of the present invention:
药理实验证明本发明所述的喹诺酮衍生物或其药学上可接受的盐或酯可以通过抑制肿瘤细胞的增殖、诱导肿瘤细胞的凋亡来达到抗肿瘤的目的，具有抗肿瘤效果，且毒性较低。Pharmacological experiments prove that the quinolone derivatives of the present invention or their pharmaceutically acceptable salts or esters can achieve the anti-tumor purpose by inhibiting the proliferation of tumor cells and inducing the apoptosis of tumor cells, with anti-tumor effect and relatively high toxicity. Low.
喹诺酮衍生物可以与靶蛋白STAT3SH2结构域结合，进而介导STAT3同源二聚体的形成，选择性抑制JAK-STAT3细胞通路。具体表现为选择性的抑制p-STAT3的形成，对其他同家族蛋白表达水平影响较小，不影响相关激酶，体外抑制肿瘤细胞增殖，诱导细胞凋亡。The quinolone derivatives can bind to the target protein STAT3 SH2 domain, thereby mediating the formation of STAT3 homodimers and selectively inhibiting the JAK-STAT3 cellular pathway. Specifically, it selectively inhibits the formation of p-STAT3, has little effect on the expression levels of other proteins of the same family, does not affect related kinases, inhibits tumor cell proliferation in vitro, and induces cell apoptosis.
附图说明Description of drawings
图1为待测化合物(浓度为10μM)对肿瘤细胞A549的抑制率。Figure 1 shows the inhibitory rate of the test compound (concentration: 10 μM) on tumor cell A549.
图2为待测化合物(浓度为5μM)对肿瘤细胞A549的抑制率。Figure 2 shows the inhibitory rate of the test compound (concentration: 5 μM) on tumor cell A549.
图3为待测化合物7d不影响相关激酶的水平验证试验Figure 3 is the level verification test that the test compound 7d does not affect related kinases
图4为待测化合物7d在体外诱导肿瘤细胞凋亡验证试验。Fig. 4 is a validation test of the test compound 7d inducing tumor cell apoptosis in vitro.
图5为待测化合物化合物7d抑制STAT3通路激活验证试验。Fig. 5 is a verification test for the test compound compound 7d to inhibit STAT3 pathway activation.
图6为待测化合物7d对STAT3蛋白的动力学分析，分析化合物与目标蛋白的结合力。Figure 6 is a kinetic analysis of the test compound 7d on STAT3 protein, analyzing the binding force of the compound to the target protein.
图7为待测化合物7d对异种移植裸鼠肿瘤生长的影响试验。Fig. 7 is a test of the effect of the test compound 7d on tumor growth in xenografted nude mice.
具体实施方式Detailed ways
为了进一步阐明本发明的技术方案，下面给出一系列实施例，这些实施例完全是例证性的，它们仅用来对本发明的技术方案具体描述，不应当理解为对本发明的限制。In order to further clarify the technical solution of the present invention, a series of examples are given below, these examples are completely illustrative, they are only used to specifically describe the technical solution of the present invention, and should not be construed as a limitation of the present invention.
化合物7a-7r的合成路线为：The synthetic route of compound 7a-7r is:
Figure PCTCN2022088657-appb-000008
Figure PCTCN2022088657-appb-000008
实施例1Example 1
2-(((4-羟苯基)氨基)亚甲基)丙二酸二乙酯(化合物3)的合成Synthesis of Diethyl 2-(((4-Hydroxyphenyl)amino)methylene)malonate (Compound 3)
将4-氨基苯酚(化合物1，0.5g，4.5mmol)加入到2.5mL乙醇中，再加入乙氧基亚甲基丙二酸二乙酯 (化合物2，925μL，4.5mmol)，室温反应2小时，待反应完全，低压旋去乙醇，并用甲基叔丁基醚进行冲洗，得到2-(((4-羟苯基)氨基)亚甲基)丙二酸二乙酯(化合物3)，白色固体(1.26g，产率98％)。Add 4-aminophenol (compound 1, 0.5 g, 4.5 mmol) to 2.5 mL of ethanol, then add ethoxymethylene malonate (compound 2, 925 μL, 4.5 mmol), and react at room temperature for 2 hours , until the reaction is complete, spin off ethanol under low pressure, and wash with methyl tert-butyl ether to obtain diethyl 2-(((4-hydroxyphenyl)amino)methylene)malonate (compound 3), white Solid (1.26 g, 98% yield).
1H NMR(500MHz,CDCl 3)δ10.95(d,J＝13.9Hz,1H,NH),8.41(d,J＝13.9Hz,1H,CH),7.07–6.95(m,2H,Ar-H),6.92–6.82(m,2H,Ar-H),6.62(s,1H,OH),4.3(q,J＝7.0,2H,CH 2),4.29(q,J＝7.0,2H,CH 2),1.35(t,J＝7.0Hz,3H,CH 3),1.32(t,J＝7.0Hz,3H,CH 3). 1 H NMR (500MHz, CDCl 3 ) δ10.95 (d, J = 13.9Hz, 1H, NH), 8.41 (d, J = 13.9Hz, 1H, CH), 7.07–6.95 (m, 2H, Ar-H ),6.92–6.82(m,2H,Ar-H),6.62(s,1H,OH),4.3(q,J=7.0,2H,CH 2 ),4.29(q,J=7.0,2H,CH 2 ), 1.35(t, J=7.0Hz, 3H, CH 3 ), 1.32(t, J=7.0Hz, 3H, CH 3 ).
2-[(4-乙酰氧基-苯基氨基)-亚甲基]-丙二酸二乙酯(化合物4)的合成Synthesis of 2-[(4-acetoxy-phenylamino)-methylene]-diethyl malonate (compound 4)
将2-(((4-羟苯基)氨基)亚甲基)丙二酸二乙酯(化合物3，0.5g，1.7mmol)与三乙胺(374μL，2.6mmol)加入到二氯甲烷(5mL)中，随后在氩气保护下加入乙酸酐(190μL，2.6mmol)，室温搅拌30分钟；加入水(5mL)萃取，取有机相，萃取3次后合并二氯甲烷层，用无水硫酸钠干燥并减压蒸发，得到化合物4，白色固体(0.56g，产率95％)。Diethyl 2-(((4-hydroxyphenyl)amino)methylene)malonate (compound 3, 0.5 g, 1.7 mmol) and triethylamine (374 μL, 2.6 mmol) were added to dichloromethane ( 5mL), then added acetic anhydride (190μL, 2.6mmol) under the protection of argon, stirred at room temperature for 30 minutes; added water (5mL) for extraction, took the organic phase, extracted 3 times and combined the dichloromethane layer, and washed with anhydrous sulfuric acid The sodium was dried and evaporated under reduced pressure to give compound 4 as a white solid (0.56 g, 95% yield).
1H NMR(500MHz,DMSO)δ10.70(d,J＝13.9Hz,1H,NH),8.36(d,J＝13.9Hz,1H,CH),7.41(d,J＝8.8Hz,2H,Ar-H),7.15(d,J＝8.8Hz,2H,Ar-H),4.21(q,J＝7.1Hz,2H,CH 2),4.12(q,J＝7.1Hz,2H,CH 2),2.26(s,3H,CH 3CO),1.26(t,J＝7.1Hz,3H,CH 3),1.24(t,J＝7.1Hz,3H,CH 3). 1 H NMR (500MHz, DMSO) δ10.70 (d, J = 13.9Hz, 1H, NH), 8.36 (d, J = 13.9Hz, 1H, CH), 7.41 (d, J = 8.8Hz, 2H, Ar -H), 7.15(d, J=8.8Hz, 2H, Ar-H), 4.21(q, J=7.1Hz, 2H, CH 2 ), 4.12(q, J=7.1Hz, 2H, CH 2 ), 2.26(s, 3H, CH 3 CO), 1.26(t, J=7.1Hz, 3H, CH 3 ), 1.24(t, J=7.1Hz, 3H, CH 3 ).
6-乙酰氧基-4-氧代-1,4-二氢喹啉-3-羧酸乙酯(化合物5)的合成Synthesis of ethyl 6-acetoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (compound 5)
将化合物4(0.8g，2.59mmol)加入到二苯醚(5mL)中，在氩气保护下回流2h；冷却到室温，加入石油醚(15mL)，析出固体，过滤收集固体，将固体加入五倍当量的DMF溶液中，加热至45℃使化合物完全溶解，随后加入十倍当量的水，沉淀迅速析出，过滤得到纯品，即为化合物5，白色固体(0.38g，产率42％)。Compound 4 (0.8g, 2.59mmol) was added to diphenyl ether (5mL), and refluxed for 2h under the protection of argon; cooled to room temperature, petroleum ether (15mL) was added, and a solid was precipitated, collected by filtration, and added to five In double equivalent of DMF solution, heated to 45°C to completely dissolve the compound, then added ten times equivalent of water, the precipitate precipitated out rapidly, and the pure product was obtained by filtration, namely compound 5, white solid (0.38g, yield 42%).
1H NMR(500MHz,DMSO)δ12.41(s,1H,NH),8.56(s,1H,CH),7.83(d,J＝2.6Hz,1H,Ar-H),7.67(d,J＝8.9Hz,1H,Ar-H),7.50(dd,J＝8.9,2.6Hz,1H,Ar-H),4.22(q,J＝7.1Hz,2H,CH 2),2.31(s,3H,CH 3CO),1.28(t,J＝7.1Hz,3H,CH 3). 1 H NMR (500MHz, DMSO) δ12.41(s, 1H, NH), 8.56(s, 1H, CH), 7.83(d, J=2.6Hz, 1H, Ar-H), 7.67(d, J= 8.9Hz, 1H, Ar-H), 7.50(dd, J=8.9, 2.6Hz, 1H, Ar-H), 4.22(q, J=7.1Hz, 2H, CH 2 ), 2.31(s, 3H, CH 3 CO), 1.28(t, J=7.1Hz, 3H, CH 3 ).
6-羟基-4-氧代-1,4-二氢喹啉-3-羧酸(化合物6)的合成Synthesis of 6-Hydroxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 6)
将化合物5(0.38g，1.38mmol)在10％氢氧化钾溶液(10mL)中回流3小时，在室温冷却后，过滤，用盐酸酸化调节滤液的pH至3，过滤，收集沉淀，用水和石油醚依次洗涤，然后用无水乙醇加热至60℃溶解，冷却至室温重结晶，过滤得到化合物6，白色固体(0.26g，产率92％)。Compound 5 (0.38g, 1.38mmol) was refluxed in 10% potassium hydroxide solution (10mL) for 3 hours, after cooling at room temperature, filtered, acidified with hydrochloric acid to adjust the pH of the filtrate to 3, filtered, and the precipitate was collected, water and petroleum The ether was washed sequentially, then heated to 60°C with absolute ethanol to dissolve, cooled to room temperature for recrystallization, and filtered to obtain compound 6 as a white solid (0.26 g, yield 92%).
1H NMR(500MHz,DMSO)δ15.64(s,1H,COOH),13.42(s,1H,OH),10.33(s,1H,NH),8.71(s,1H,CH),7.75(d,J＝9.0Hz,1H,Ar-H),7.57(d,J＝2.6Hz,1H,Ar-H),7.39(dd,J＝8.8,2.6Hz,1H,Ar-H). 1 H NMR (500MHz,DMSO)δ15.64(s,1H,COOH),13.42(s,1H,OH),10.33(s,1H,NH),8.71(s,1H,CH),7.75(d, J=9.0Hz, 1H, Ar-H), 7.57(d, J=2.6Hz, 1H, Ar-H), 7.39(dd, J=8.8, 2.6Hz, 1H, Ar-H).
6-((4-三氟甲氧基苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7a)的合成Synthesis of 6-((4-trifluoromethoxybenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7a)
将化合物6(1mmol)、氢氧化钾(0.3mmol，作为缚酸剂)和4-(三氟甲氧基)苄基溴(1.1mmol)在5mL乙醇和水(1:1V/V)中加热回流20小时；加入氢氧化钾水溶液(20％，10mL)，加热回流4小时；冷却，加入水，并用盐酸调节pH至2，待析出沉淀，过滤，收集固体，用无水乙醇加热至60℃溶解，冷却至室温重结晶，过滤，得到化合物7a，白色固体，产率80.3％。Compound 6 (1 mmol), potassium hydroxide (0.3 mmol, as an acid-binding agent) and 4-(trifluoromethoxy)benzyl bromide (1.1 mmol) were heated in 5 mL of ethanol and water (1:1 V/V) Reflux for 20 hours; add potassium hydroxide aqueous solution (20%, 10mL), heat and reflux for 4 hours; cool, add water, and adjust the pH to 2 with hydrochloric acid, wait for precipitation to precipitate, filter, collect the solid, and heat to 60°C with absolute ethanol Dissolved, cooled to room temperature for recrystallization, and filtered to obtain compound 7a as a white solid with a yield of 80.3%.
mp>250℃.IR(KBr):3427,2894,1693,1624,1488,1393cm -1. 1H NMR(500MHz,DMSO)δ15.47(s,1H),13.52(s,1H,COOH),8.81(d,J＝5.5Hz,1H,NH),7.82(d,J＝9.1Hz,1H,Ar-H),7.74(s,1H,Ar-H),7.64(d,J＝8.4Hz,2H,Ar-H),7.61(dd,J＝9.1,1.6Hz,1H,Ar-H),7.41(d,J＝8.2Hz,2H,Ar-H),5.31(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ177.43(s),166.44(s),156.15(s),143.56(s),141.33(s),134.32(s),128.00(s),125.60(s),125.62–125.11(m),124.72(s),121.55(s),106.95(s),105.60(s),68.83(s).MS(ESI)m/z 378.0[M-H] -；HRMS(ESI)calcd for C 18H 11F 3NO 5[M-H] -378.0595,found 378.0597. mp>250℃.IR(KBr):3427,2894,1693,1624,1488,1393cm -1 . 1 H NMR(500MHz,DMSO)δ15.47(s,1H),13.52(s,1H,COOH), 8.81(d, J=5.5Hz, 1H, NH), 7.82(d, J=9.1Hz, 1H, Ar-H), 7.74(s, 1H, Ar-H), 7.64(d, J=8.4Hz, 2H, Ar-H), 7.61(dd, J=9.1, 1.6Hz, 1H, Ar-H), 7.41(d, J=8.2Hz, 2H, Ar-H), 5.31(s, 2H, CH 2 ) .13 C NMR(126MHz,DMSO)δ177.43(s),166.44(s),156.15(s),143.56(s),141.33(s),134.32(s),128.00(s),125.60(s) ,125.62–125.11(m),124.72(s),121.55(s),106.95(s),105.60(s),68.83(s).MS(ESI) m/z 378.0[MH] - ;HRMS(ESI) calcd for C 18 H 11 F 3 NO 5 [MH] - 378.0595, found 378.0597.
实施例2Example 2
6-((4-甲基苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7b)的合成Synthesis of 6-((4-methylbenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7b)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为4-(甲基)苄基溴，得到化合物7b，白色固体，产率85.2％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced with 4-(methyl)benzyl bromide to obtain compound 7b as a white solid with a yield of 85.2%.
mp>250℃.IR(KBr):3451,2894,1685,1626,1490,1383cm -1. 1H NMR(500MHz,DMSO)δ15.47(s,1H,COOH),13.52(s,1H,NH),8.81(d,J＝5.5Hz,1H,CH),7.82(d,J＝9.1Hz,1H,Ar-H),7.74(s,1H,Ar-H),7.64(d,J＝8.4Hz,2H,Ar-H),7.61(dd,J＝9.1,1.6Hz,1H,Ar-H),7.41(d,J＝8.2Hz,2H,Ar-H),5.31(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ177.42(s),166.49(s),156.44(s),143.32(s),137.19(s),134.13(s),133.35(s),128.96(s),127.75(s),125.59(s),124.77(s),121.40(s),106.86(s),105.51(s),69.66(s),20.71(s).MS(ESI)m/z 308.1[M-H] -；HRMS(ESI)calcd for C 18H 14NO 4[M-H] -308.0928,found 308.0931. mp>250℃.IR(KBr):3451,2894,1685,1626,1490,1383cm -1 . 1 H NMR(500MHz,DMSO)δ15.47(s,1H,COOH),13.52(s,1H,NH ), 8.81(d, J=5.5Hz, 1H, CH), 7.82(d, J=9.1Hz, 1H, Ar-H), 7.74(s, 1H, Ar-H), 7.64(d, J=8.4 Hz, 2H, Ar-H), 7.61(dd, J=9.1, 1.6Hz, 1H, Ar-H), 7.41(d, J=8.2Hz, 2H, Ar-H), 5.31(s, 2H, CH 2 ). 13 C NMR (126MHz, DMSO) δ177.42(s), 166.49(s), 156.44(s), 143.32(s), 137.19(s), 134.13(s), 133.35(s), 128.96( s), 127.75(s), 125.59(s), 124.77(s), 121.40(s), 106.86(s), 105.51(s), 69.66(s), 20.71(s).MS(ESI) m/z 308.1[MH] - ; HRMS(ESI) calcd for C 18 H 14 NO 4 [MH] - 308.0928, found 308.0931.
实施例3Example 3
6-((2-氟苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7c)的合成Synthesis of 6-((2-fluorobenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7c)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为2-氟苄基溴，得到化合物7c，白色固体，产率88.6％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 2-fluorobenzyl bromide to obtain compound 7c as a white solid with a yield of 88.6%.
mp>250℃.IR(KBr):3439,2901,1694,1622,1492,1387cm -1. 1H NMR(500MHz,DMSO)δ15.50(s,1H,COOH),13.57(s,1H,NH),8.81(d,J＝6.3Hz,1H,CH),7.83(d,J＝9.1Hz,1H,Ar-H),7.78(d,J＝2.8Hz, 1H,Ar-H),7.63–7.55(m,2H,Ar-H),7.44(dd,J＝9.1,2.8Hz,1H,Ar-H),7.32–7.20(m,2H,Ar-H),5.30(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ177.98(s),167.01(s),161.93(s),159.97(s),156.78(s),143.96(s),134.86(s),131.17(dd,J＝21.6,6.1Hz),126.14(s),124.82–124.31(m),123.76(d,J＝14.6Hz),122.06(s),116.02(s),115.86(s),107.46(s),105.98(s),64.70(d).MS(ESI)m/z 336.0[M+Na] +；HRMS(ESI)calcd for C 17H 12FNO 4[M+Na] +336.0643,found 336.0641. mp>250℃.IR(KBr):3439,2901,1694,1622,1492,1387cm -1 . 1 H NMR(500MHz,DMSO)δ15.50(s,1H,COOH),13.57(s,1H,NH ),8.81(d,J＝6.3Hz,1H,CH),7.83(d,J＝9.1Hz,1H,Ar-H),7.78(d,J＝2.8Hz, 1H,Ar-H),7.63– 7.55(m,2H,Ar-H),7.44(dd,J=9.1,2.8Hz,1H,Ar-H),7.32–7.20(m,2H,Ar-H),5.30(s,2H,CH 2 ). 13 C NMR (126MHz, DMSO) δ177.98(s), 167.01(s), 161.93(s), 159.97(s), 156.78(s), 143.96(s), 134.86(s), 131.17(dd , J＝21.6, 6.1Hz), 126.14(s), 124.82–124.31(m), 123.76(d, J＝14.6Hz), 122.06(s), 116.02(s), 115.86(s), 107.46(s) ,105.98(s),64.70(d).MS(ESI)m/z 336.0[M+Na] + ; HRMS(ESI)calcd for C 17 H 12 FNO 4 [M+Na] + 336.0643,found 336.0641.
实施例4Example 4
6-(3,5-二(三氟甲基)苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7d)的合成Synthesis of 6-(3,5-bis(trifluoromethyl)benzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7d)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为3,5-双(三氟甲基)苄基溴，得到化合物7d，白色固体，产率90.6％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 3,5-bis(trifluoromethyl)benzyl bromide to obtain compound 7d, a white solid, with a yield of 90.6 %.
mp>250℃.IR(KBr):3405,1621,1585,1496,1368cm -1. 1H NMR(500MHz,DMSO)δ15.5(s,1H,COOH),13.82(s,1H,NH),8.78(d,J＝5.7Hz,1H,CH),8.24(s,2H,Ar-H),8.11(s,1H,Ar-H),7.88(d,J＝9.1Hz,1H,Ar-H),7.78(d,J＝2.1Hz,1H,Ar-H),7.67(dd,J＝9.1,2.1Hz,1H,Ar-H),5.47(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ177.44(s),166.46(s),156.00(s),143.37(s),140.09(s),134.50(s),130.23(s),128.30(s),125.59(s),124.75(s),124.33(s),122.37(m),121.87(m),106.96(s),105.52(s),68.15(s).MS(ESI)m/z 430.0[M-H] -；HRMS(ESI)calcd for C 19H 10F 6NO 4[M-H] -430.052,found 430.0523. mp>250℃.IR(KBr):3405,1621,1585,1496,1368cm -1 . 1 H NMR(500MHz,DMSO)δ15.5(s,1H,COOH),13.82(s,1H,NH), 8.78(d, J=5.7Hz, 1H, CH), 8.24(s, 2H, Ar-H), 8.11(s, 1H, Ar-H), 7.88(d, J=9.1Hz, 1H, Ar-H ), 7.78 (d, J=2.1Hz, 1H, Ar-H), 7.67 (dd, J=9.1, 2.1Hz, 1H, Ar-H), 5.47 (s, 2H, CH 2 ). 13 C NMR ( 126MHz, DMSO) δ177.44(s), 166.46(s), 156.00(s), 143.37(s), 140.09(s), 134.50(s), 130.23(s), 128.30(s), 125.59(s) ,124.75(s),124.33(s),122.37(m),121.87(m),106.96(s),105.52(s),68.15(s).MS(ESI) m/z 430.0[MH] - ; HRMS (ESI) calcd for C 19 H 10 F 6 NO 4 [MH] - 430.052, found 430.0523.
实施例5Example 5
6-((2-三氟甲基苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7e)的合成Synthesis of 6-((2-trifluoromethylbenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7e)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为2-(三氟甲基)苄基溴，得到化合物7e，白色固体，产率73.2％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 2-(trifluoromethyl)benzyl bromide to obtain compound 7e as a white solid with a yield of 73.2%.
mp>250℃.IR(KBr):3423,3079,1693,1621,1487,1455,1394cm -1. 1H NMR(500MHz,DMSO)δ15.45(s,1H,COOH),13.59(s,1H,NH),8.81(d,J＝6.1Hz,1H,CH),7.83(dd,J＝9.1Hz,2.8 1H,Ar-H),7.78(d,J＝2.8Hz,1H,Ar-H)7.77–7.71(m,2H,Ar-H),7.65–7.56(m,2H,Ar-H),5.40(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ177.42(s),166.46(s),156.09(s),143.51(s),134.44(s),134.26(s),132.84(s),130.52(s),128.92(s),126.23(s),125.61(s),125.36(s),124.65(s),123.18(s),121.65(s),106.96(s),105.41(s),66.79(s).MS(ESI)m/z 386.1[M+Na] +；HRMS(ESI)calcd for C 18H 12F 3NO 4[M+Na] +386.0611,found 386.0610. mp>250℃.IR(KBr):3423,3079,1693,1621,1487,1455,1394cm -1 . 1 H NMR(500MHz,DMSO)δ15.45(s,1H,COOH),13.59(s,1H ,NH),8.81(d,J=6.1Hz,1H,CH),7.83(dd,J=9.1Hz,2.8 1H,Ar-H),7.78(d,J=2.8Hz,1H,Ar-H) 7.77–7.71(m,2H,Ar-H),7.65–7.56(m,2H,Ar-H),5.40(s,2H,CH 2 ). 13 C NMR(126MHz,DMSO)δ177.42(s) ,166.46(s),156.09(s),143.51(s),134.44(s),134.26(s),132.84(s),130.52(s),128.92(s),126.23(s),125.61(s) ,125.36(s),124.65(s),123.18(s),121.65(s),106.96(s),105.41(s),66.79(s).MS(ESI) m/z 386.1[M+Na] + ; HRMS (ESI) calcd for C 18 H 12 F 3 NO 4 [M+Na] + 386.0611, found 386.0610.
实施例6Example 6
6-((4-氟苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7f)的合成Synthesis of 6-((4-fluorobenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7f)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为4-氟苄基溴，得到化合物7f，白色固体，产率76.5％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced with 4-fluorobenzyl bromide to obtain compound 7f as a white solid with a yield of 76.5%.
mp>250℃.IR(KBr):3439,2901,1694,1622,1488,1387cm -1. 1H NMR(500MHz,DMSO)δ15.39(s,1H,COOH),13.68(s,1H,NH),8.78(d,J＝5.4Hz,1H,CH),7.84(d,J＝9.1Hz,1H,Ar-H),7.73(d,J＝2.8Hz,1H,Ar-H),7.59(dd,J＝9.1,2.8Hz,1H,Ar-H),7.55(d,J＝8.4Hz,2H,Ar-H),7.24(d,J＝8.4Hz,2H,Ar-H),5.25(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ177.45(s),166.52(s),162.79(s),160.86(s),156.35(s),143.29(s),134.25(s),132.67(d),130.01(d),125.61(s),124.77(s),121.48(s),115.35(s),115.18(s),106.89(s),105.50(s),69.06(s).MS(ESI)m/z 336.1([M+Na] +)；HRMS(ESI)calcd for C 17H 12FNO 4[M+Na] +336.0643,found 336.0641. mp>250℃.IR(KBr):3439,2901,1694,1622,1488,1387cm -1 . 1 H NMR(500MHz,DMSO)δ15.39(s,1H,COOH),13.68(s,1H,NH ), 8.78(d, J=5.4Hz, 1H, CH), 7.84(d, J=9.1Hz, 1H, Ar-H), 7.73(d, J=2.8Hz, 1H, Ar-H), 7.59( dd,J=9.1,2.8Hz,1H,Ar-H),7.55(d,J=8.4Hz,2H,Ar-H),7.24(d,J=8.4Hz,2H,Ar-H),5.25( s, 2H, CH 2 ). 13 C NMR (126MHz, DMSO) δ177.45(s), 166.52(s), 162.79(s), 160.86(s), 156.35(s), 143.29(s), 134.25( s), 132.67(d), 130.01(d), 125.61(s), 124.77(s), 121.48(s), 115.35(s), 115.18(s), 106.89(s), 105.50(s), 69.06( s). MS(ESI) m/z 336.1 ([M+Na] + ); HRMS(ESI) calcd for C 17 H 12 FNO 4 [M+Na] + 336.0643, found 336.0641.
实施例7Example 7
6-((3-甲基苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7g)的合成Synthesis of 6-((3-methylbenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7g)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为3-甲基苄基溴，得到化合物7g，白色固体，产率80.4％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 3-methylbenzyl bromide to obtain compound 7g as a white solid with a yield of 80.4%.
mp>250℃.IR(KBr):3450,3066,2857,1687,1620,1488,1394cm -1. 1H NMR(500MHz,DMSO)δ15.50(s,1H,COOH),13.59(s,1H,NH),8.78(d,J＝5.1Hz,1H,CH),7.82(d,J＝9.1Hz,1H,Ar-H),7.72(d,J＝2.5Hz,1H,Ar-H),7.58(dd,J＝9.1,2.6Hz,1H,Ar-H),7.28–7.30(m,3H,Ar-H),7.15(d,J＝5.5Hz,1H,Ar-H),5.21(s,2H,CH 2),2.32(s,3H,CH 3). 13C NMR(126MHz,DMSO)δ177.42(s),166.50(s),156.46(s),143.27(s),137.60(s),136.30(s),134.16(s),128.56(s),128.32(s),128.22(s),125.59(s),124.78(s),124.72(s),121.42(s),106.86(s),105.44(s),69.79(s),20.93(s).MS(ESI)m/z 308.1[M-H] -；HRMS(ESI)calcd for C 18H 14NO 4[M-H] -308.0928,found 308.0927. mp>250℃.IR(KBr):3450,3066,2857,1687,1620,1488,1394cm -1 . 1 H NMR(500MHz,DMSO)δ15.50(s,1H,COOH),13.59(s,1H ,NH),8.78(d,J=5.1Hz,1H,CH),7.82(d,J=9.1Hz,1H,Ar-H),7.72(d,J=2.5Hz,1H,Ar-H), 7.58(dd, J=9.1, 2.6Hz, 1H, Ar-H), 7.28–7.30(m, 3H, Ar-H), 7.15(d, J=5.5Hz, 1H, Ar-H), 5.21(s , 2H, CH 2 ), 2.32(s, 3H, CH 3 ). 13 C NMR (126MHz, DMSO) δ177.42(s), 166.50(s), 156.46(s), 143.27(s), 137.60(s ),136.30(s),134.16(s),128.56(s),128.32(s),128.22(s),125.59(s),124.78(s),124.72(s),121.42(s),106.86(s ),105.44(s),69.79(s),20.93(s).MS(ESI)m/z 308.1[MH] - ; HRMS(ESI)calcd for C 18 H 14 NO 4 [MH] - 308.0928,found 308.0927 .
实施例8Example 8
6-((3-三氟甲基苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7h)的合成Synthesis of 6-((3-trifluoromethylbenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7h)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为3-(三氟甲基)苄基溴，得到化合物7h，白色固体，产率80％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 3-(trifluoromethyl)benzyl bromide to obtain compound 7h as a white solid with a yield of 80%.
mp>250℃.IR(KBr):3435,3069,2864,1695,1619,1489,1386cm -1. 1H NMR(500MHz,DMSO)δ15.44(s,1H,COOH),13.59(s,1H,NH),8.80(d,J＝6.0Hz,1H,CH),7.88(s,1H,Ar-H),7.84(d,J＝9.5Hz,1H,Ar-H),7.83(d,J＝6.9Hz,1H,Ar-H),7.76–7.67(m,3H,Ar-H),7.63(dd,J＝9.5,2.5Hz,1H,Ar-H),5.38(s,2H, CH 2). 13C NMR(126MHz,DMSO)δ177.44(s),166.46(s),156.20(s),143.42(s),137.98(s),134.34(s),131.68(s),129.56(s),124.75(s),125.60(s),124.65(s),124.62(s),124.08(s),124.05(s),121.52(s),106.93(s),105.52(s),68.87(s).MS(ESI)m/z 362.0[M-H] -；HRMS(ESI)calcd for C 18H 11F 3NO 4[M-H] -362.0646,found 362.0648. mp>250℃.IR(KBr):3435,3069,2864,1695,1619,1489,1386cm -1 . 1 H NMR(500MHz,DMSO)δ15.44(s,1H,COOH),13.59(s,1H ,NH),8.80(d,J=6.0Hz,1H,CH),7.88(s,1H,Ar-H),7.84(d,J=9.5Hz,1H,Ar-H),7.83(d,J =6.9Hz,1H,Ar-H),7.76–7.67(m,3H,Ar-H),7.63(dd,J=9.5,2.5Hz,1H,Ar-H),5.38(s,2H, CH2 ). 13 C NMR (126MHz, DMSO) δ177.44(s), 166.46(s), 156.20(s), 143.42(s), 137.98(s), 134.34(s), 131.68(s), 129.56(s ),124.75(s),125.60(s),124.65(s),124.62(s),124.08(s),124.05(s),121.52(s),106.93(s),105.52(s),68.87(s ).MS(ESI)m/z 362.0[MH] - ; HRMS(ESI)calcd for C 18 H 11 F 3 NO 4 [MH] - 362.0646,found 362.0648.
实施例9Example 9
6-((4-溴苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7i)的合成Synthesis of 6-((4-bromobenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7i)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为4-溴苄基溴，得到化合物7i，白色固体，产率81.1％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced with 4-bromobenzyl bromide to obtain compound 7i as a white solid with a yield of 81.1%.
mp>250℃.IR(KBr):3451,1623,1488,1391cm -1. 1H NMR(500MHz,DMSO)δ15.48(s,1H,COOH),13.58(s,1H,NH),8.80(s,1H,CH),7.83(d,J＝9.0Hz,1H,Ar-H),7.73(d,J＝2.8Hz,1H,Ar-H),7.68–7.54(m,3H,Ar-H),7.47(d,J＝8.4Hz,2H,Ar-H),5.26(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ177.43(s),166.48(s),156.24(s),143.42(s),135.92(s),134.27(s),131.37(s),129.78(s),125.60(s),124.76(s),121.50(s),121.05(s),106.91(s),105.60(s),68.94(s).MS(ESI)m/z 372.0[M-H] -；HRMS(ESI)calcd for C 17H 11BrNO 4[M-H] -371.9877,found 371.9879. mp>250℃.IR(KBr):3451,1623,1488,1391cm -1 . 1 H NMR(500MHz,DMSO)δ15.48(s,1H,COOH),13.58(s,1H,NH),8.80( s,1H,CH),7.83(d,J=9.0Hz,1H,Ar-H),7.73(d,J=2.8Hz,1H,Ar-H),7.68–7.54(m,3H,Ar-H ), 7.47(d, J=8.4Hz, 2H, Ar-H), 5.26(s, 2H, CH 2 ). 13 C NMR (126MHz, DMSO) δ177.43(s), 166.48(s), 156.24( s), 143.42(s), 135.92(s), 134.27(s), 131.37(s), 129.78(s), 125.60(s), 124.76(s), 121.50(s), 121.05(s), 106.91( s),105.60(s),68.94(s).MS(ESI)m/z 372.0[MH] - ; HRMS(ESI)calcd for C 17 H 11 BrNO 4 [MH] - 371.9877,found 371.9879.
实施例10Example 10
6-((3-硝基苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7j)的合成Synthesis of 6-((3-nitrobenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 7j)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为3-硝基苄基溴，得到化合物7j，棕色固体，产率87.4％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 3-nitrobenzyl bromide to obtain compound 7j as a brown solid with a yield of 87.4%.
mp>250℃.IR(KBr):3455,2928,1625,1528,1489,1389cm -1. 1H NMR(500MHz,DMSO)δ15.51(s,1H,COOH),13.44(s,1H,NH),8.83(s,1H,CH),8.37(s,1H,Ar-H),8.22(dd,J＝7.7,1.5Hz,1H,Ar-H),7.98(d,J＝7.7Hz,1H,Ar-H),7.82(d,J＝9.1Hz,1H,Ar-H),7.78(d,J＝2.9Hz,1H,Ar-H),7.66–7.81(m,1H,Ar-H),7.65(dd,J＝9.1,2.9Hz,1H,Ar-H),5.44(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ177.35(s),166.57(s),156.05(s),147.88(s),143.74(s),138.88(s),134.70(s),134.08(s),130.08(s),125.61(s),124.68(s),122.82(s),122.02(s),121.80(s),106.97(s),105.61(s),68.47(s).MS(ESI)m/z 339.0[M-H] -；HRMS(ESI)calcd for C 17H 11N 2O 6[M-H] -339.0623,found 339.0622. mp>250℃.IR(KBr):3455,2928,1625,1528,1489,1389cm -1 . 1 H NMR(500MHz,DMSO)δ15.51(s,1H,COOH),13.44(s,1H,NH ),8.83(s,1H,CH),8.37(s,1H,Ar-H),8.22(dd,J＝7.7,1.5Hz,1H,Ar-H),7.98(d,J＝7.7Hz,1H ,Ar-H),7.82(d,J=9.1Hz,1H,Ar-H),7.78(d,J=2.9Hz,1H,Ar-H),7.66–7.81(m,1H,Ar-H) ,7.65(dd,J＝9.1,2.9Hz,1H,Ar-H),5.44(s,2H,CH 2 ). 13 C NMR(126MHz,DMSO)δ177.35(s),166.57(s),156.05 (s), 147.88(s), 143.74(s), 138.88(s), 134.70(s), 134.08(s), 130.08(s), 125.61(s), 124.68(s), 122.82(s), 122.02 (s),121.80(s),106.97(s),105.61(s),68.47(s).MS(ESI)m/z 339.0[MH] - ; HRMS(ESI)calcd for C 17 H 11 N 2 O 6 [MH] - 339.0623, found 339.0622.
实施例11Example 11
6-((4-三氟甲基苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7k)的合成Synthesis of 6-((4-trifluoromethylbenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7k)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为4-三氟甲基苄基溴，得到化合物7k，白色固体，产率86.0％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced with 4-trifluoromethylbenzyl bromide to obtain compound 7k as a white solid with a yield of 86.0%.
mp>250℃.IR(KBr):3423,3069,2864,1695,1619,1489,1389cm -1. 1H NMR(500MHz,DMSO)δ15.47(s,1H,COOH),13.43(s,1H,NH),8.83(s,1H,CH),7.82(d,J＝9.2Hz,1H,Ar-H),7.79(d,J＝8.1Hz,2H,Ar-H),7.75(d,J＝1.8Hz,1H,Ar-H),7.73(d,J＝8.1Hz,2H,Ar-H),7.63(dd,J＝9.1,1.8Hz,1H,Ar-H),5.40(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ177.43(s),166.44(s),156.15(s),143.56(s),141.33(s),134.32(s),128.00(s),125.60(s),125.32(t),124.72(s),121.55(s),106.95(s),105.60(s),68.83(s).MS(ESI)m/z 362.1[M-H] -；HRMS(ESI)calcd for C 18H 12F 3NO 4[M-H] -362.0646,found 362.0647. mp>250℃.IR(KBr):3423,3069,2864,1695,1619,1489,1389cm -1 . 1 H NMR(500MHz,DMSO)δ15.47(s,1H,COOH),13.43(s,1H ,NH),8.83(s,1H,CH),7.82(d,J=9.2Hz,1H,Ar-H),7.79(d,J=8.1Hz,2H,Ar-H),7.75(d,J =1.8Hz,1H,Ar-H),7.73(d,J=8.1Hz,2H,Ar-H),7.63(dd,J=9.1,1.8Hz,1H,Ar-H),5.40(s,2H , CH 2 ). 13 C NMR (126MHz, DMSO) δ177.43(s), 166.44(s), 156.15(s), 143.56(s), 141.33(s), 134.32(s), 128.00(s), 125.60(s), 125.32(t), 124.72(s), 121.55(s), 106.95(s), 105.60(s), 68.83(s).MS(ESI) m/z 362.1[MH] - ; HRMS( ESI) calcd for C 18 H 12 F 3 NO 4 [MH] - 362.0646, found 362.0647.
实施例12Example 12
6-((3-氟苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7l)的合成Synthesis of 6-((3-fluorobenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 7l)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为3-氟苄基溴，得到化合物7l，白色固体，产率83.2％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 3-fluorobenzyl bromide to obtain compound 7l as a white solid with a yield of 83.2%.
mp>250℃.IR(KBr):3454,2903,1694,1623,1493,1389cm -1. 1H NMR(500MHz,DMSO)δ15.48(s,1H,COOH),13.43(s,1H,NH),8.82(d,J＝5.5Hz,1H,CH),7.81(d,J＝9.1Hz,1H,Ar-H),7.78(d,J＝2.9Hz,1H,Ar-H),7.63-7.55(m,2H,Ar-H),7.44(m,1H,Ar-H),7.31–7.20(m,2H,Ar-H),5.31(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ166.51(s),156.28(s),143.54(s),134.33(s),130.67(dd,J＝20.3,6.0Hz),124.72(s),124.55(d,J＝3.4Hz),121.54(s),115.51(s),105.50(s),64.18(s),40.02(s),39.85(s),39.60(d,J＝21.0Hz),39.35(s),39.19(s),39.02(s).MS(ESI)m/z 312.0[M-H] -；HRMS(ESI)calcd for C 17H 11FNO 4[M-H] -312.0678,found 312.0679. mp>250℃.IR(KBr):3454,2903,1694,1623,1493,1389cm -1 . 1 H NMR(500MHz,DMSO)δ15.48(s,1H,COOH),13.43(s,1H,NH ), 8.82(d, J=5.5Hz, 1H, CH), 7.81(d, J=9.1Hz, 1H, Ar-H), 7.78(d, J=2.9Hz, 1H, Ar-H), 7.63- 7.55(m,2H,Ar-H),7.44(m,1H,Ar-H),7.31–7.20(m,2H,Ar-H),5.31(s,2H,CH 2 ). 13 C NMR (126MHz , DMSO) δ166.51(s), 156.28(s), 143.54(s), 134.33(s), 130.67(dd, J=20.3, 6.0Hz), 124.72(s), 124.55(d, J=3.4Hz ), 121.54(s), 115.51(s), 105.50(s), 64.18(s), 40.02(s), 39.85(s), 39.60(d, J=21.0Hz), 39.35(s), 39.19(s ),39.02(s).MS(ESI)m/z 312.0[MH] - ; HRMS(ESI)calcd for C 17 H 11 FNO 4 [MH] - 312.0678,found 312.0679.
实施例13Example 13
6-((3-氨基苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7m)的合成Synthesis of 6-((3-aminobenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 7m)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为3-氨基苄基溴，得到化合物7m，棕色固体，产率59.2％。Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 3-aminobenzyl bromide to obtain compound 7m as a brown solid with a yield of 59.2%.
mp>250℃.IR(KBr):3441,1621,1488,1389cm-1.1H NMR(500MHz,DMSO)δ15.49(s,1H,COOH),13.95(s,1H,NH),8.91(s,2H,NH2),8.76(s,1H,CH),7.91(d,J＝9.1Hz,1H,Ar-H),7.75(d,J＝2.8Hz,1H,Ar-H),7.62(dd,J＝9.1,2.8Hz,1H,Ar-H),7.51–7.40(m,3H,Ar-H),7.29(d,J＝7.1Hz,1H,Ar-H),5.32(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.32(s),166.49(s),156.44(s),143.32(s),136.21(s),135.13(s),133.35(s),128.96(s),127.75(s),125.59(s),124.77(s),121.40(s),106.86(s),105.51(s),69.66(s).MS(ESI)m/z 311.1[M-H]-；HRMS(ESI)calcd for C 17H 15N 2O 4[M-H] -311.1026,found 311.1025. mp>250℃.IR(KBr):3441,1621,1488,1389cm-1.1H NMR(500MHz,DMSO)δ15.49(s,1H,COOH),13.95(s,1H,NH),8.91(s, 2H,NH2),8.76(s,1H,CH),7.91(d,J=9.1Hz,1H,Ar-H),7.75(d,J=2.8Hz,1H,Ar-H),7.62(dd, J=9.1,2.8Hz,1H,Ar-H),7.51–7.40(m,3H,Ar-H),7.29(d,J=7.1Hz,1H,Ar-H),5.32(s,2H,CH2 ).13C NMR (126MHz, DMSO) δ177.32(s), 166.49(s), 156.44(s), 143.32(s), 136.21(s), 135.13(s), 133.35(s), 128.96(s) ,127.75(s),125.59(s),124.77(s),121.40(s),106.86(s),105.51(s),69.66(s).MS(ESI) m/z 311.1[MH]-; HRMS (ESI) calcd for C 17 H 15 N 2 O 4 [MH] - 311.1026, found 311.1025.
实施例14Example 14
6-((2-甲基苄)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7n)的合成Synthesis of 6-((2-methylbenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 7n)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为2-甲基苄基溴，得到化合物7n，白色固体，产率为53.2％。mp 209-213℃.MS(ESI)m/z 310.1[M+H] +；HRMS(ESI)calcd for C 18H 15NNaO 4[M+Na] +332.0893,found332.0891. Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 2-methylbenzyl bromide to obtain compound 7n as a white solid with a yield of 53.2%. mp 209-213℃. MS(ESI) m/z 310.1[M+H] + ; HRMS(ESI) calcd for C 18 H 15 NNaO 4 [M+Na] + 332.0893, found 332.0891.
实施例15Example 15
6-((2-溴苄)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7o)的合成Synthesis of 6-((2-bromobenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7o)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为2-溴苄基溴，得到化合物7o，白色固体，产率为48.3％。mp 319-322℃.MS(ESI)m/z 395.9[M+Na] +；HRMS(ESI)calcd for C 17H 12BrNNaO 4[M+Na] +395.9842,found395.9842. Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced with 2-bromobenzyl bromide to obtain compound 7o as a white solid with a yield of 48.3%. mp 319-322℃. MS(ESI) m/z 395.9[M+Na] + ; HRMS(ESI) calcd for C 17 H 12 BrNNaO 4 [M+Na] + 395.9842, found 395.9842.
实施例16Example 16
6-((2-三氟甲氧基苄)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7p)的合成Synthesis of 6-((2-trifluoromethoxybenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Compound 7p)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为2-(三氟甲氧基)苄基溴，得到化合物7p，白色固体，产率为57.5％。mp 294-297℃.MS(ESI)m/z 380.1[M+H] +；HRMS(ESI)calcd for C 18H 12F 3NNaO 5[M+Na] +402.0560,found 402.0557. Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 2-(trifluoromethoxy)benzyl bromide to obtain compound 7p, a white solid, with a yield of 57.5% . mp 294-297℃. MS(ESI) m/z 380.1[M+H] + ; HRMS(ESI) calcd for C 18 H 12 F 3 NNaO 5 [M+Na] + 402.0560, found 402.0557.
实施例17Example 17
6-((2-甲氧基苄)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7q)的合成Synthesis of 6-((2-methoxybenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 7q)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为2-(甲氧基)苄基溴，得到化合物7q，白色固体，产率为50.12％。mp 296℃.MS(ESI)m/z 326.2[M+H] +；HRMS(ESI)calcd for C 18H 15NNaO 5[M+Na] +348.0842,found 348.0842. Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced by 2-(methoxy)benzyl bromide to obtain compound 7q as a white solid with a yield of 50.12%. mp 296°C. MS(ESI) m/z 326.2[M+H] + ; HRMS(ESI) calcd for C 18 H 15 NNaO 5 [M+Na] + 348.0842, found 348.0842.
实施例18Example 18
6-((4-甲氧基苄)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物7r)的合成Synthesis of 6-((4-methoxybenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 7r)
参照实施例1化合物7a的合成，仅将4-(三氟甲氧基)苄基溴替换为4-(甲氧基)苄基溴，得到化合物7r，白色固体，产率为52.4％。mp 282-284℃.MS(ESI)m/z 326.2[M+H] +；HRMS(ESI)calcd for C 18H 15NNaO 5[M+Na] +348.0842,found348.0838. Referring to the synthesis of compound 7a in Example 1, only 4-(trifluoromethoxy)benzyl bromide was replaced with 4-(methoxy)benzyl bromide to obtain compound 7r as a white solid with a yield of 52.4%. mp 282-284℃. MS(ESI) m/z 326.2[M+H] + ; HRMS(ESI) calcd for C 18 H 15 NNaO 5 [M+Na] + 348.0842, found 348.0838.
实施例19Example 19
Figure PCTCN2022088657-appb-000009
Figure PCTCN2022088657-appb-000009
6-(3,5(三氟苄基)氧基)-4-氧代-1,4-二氢喹啉(化合物7da)的合成Synthesis of 6-(3,5(trifluorobenzyl)oxy)-4-oxo-1,4-dihydroquinoline (compound 7da)
将化合物7d(0.5g，1.16mmol)加入二苯醚(4mL)中，220℃回流2小时，然后冷却至室温。加入石油醚，收集所得晶体并用大量石油醚洗涤，再采用丙酮加热至60℃，冷却至室温重结晶，过滤收集固体化合物7da(0.38g，产率为90％)，棕色固体。 Compound 7d (0.5 g, 1.16 mmol) was added into diphenyl ether (4 mL), refluxed at 220° C. for 2 hours, and then cooled to room temperature. Petroleum ether was added, the resulting crystals were collected and washed with a large amount of petroleum ether, heated to 60° C. with acetone, cooled to room temperature for recrystallization, and solid compound 7da (0.38 g, yield 90%) was collected by filtration as a brown solid.
mp>250℃.IR(KBr):3445,2867,1723,1483,1396cm -1. 1H NMR(500MHz,DMSO)δ12.23(s,1H,NH),8.22(s,2H,CH 2),8.09(s,1H,Ar-H),7.92(d,J＝7.2Hz,1H,Ar-H),7.69–7.58(m,2H,Ar-H),7.47(dd,J＝9.0,2.7Hz,1H,Ar-H),6.10(d,J＝7.2Hz,1H,Ar-H),5.41(s,2H,CH 2). 13C NMR(126MHz,DMSO)δ175.45(s),154.14(s),140.51(s),138.75(s),135.06(s),130.55(d,J＝32.9Hz),130.02(d,J＝32.9Hz),128.12(s),126.48(s),126.27(s),124.31(s),122.66(s),122.14(s),121.43(s),120.30(s),107.32(s),105.52(s),67.91(s).MS(ESI)m/z 388.1[M+H] +；HRMS(ESI)calcd for C 18H 11F 6NO 2[M+H] +388.0765,found 388.0767. mp>250℃.IR(KBr):3445,2867,1723,1483,1396cm -1 . 1 H NMR(500MHz,DMSO)δ12.23(s,1H,NH),8.22(s,2H,CH 2 ) ,8.09(s,1H,Ar-H),7.92(d,J=7.2Hz,1H,Ar-H),7.69–7.58(m,2H,Ar-H),7.47(dd,J=9.0,2.7 Hz,1H,Ar-H),6.10(d,J＝7.2Hz,1H,Ar-H),5.41(s,2H,CH 2 ). 13 C NMR(126MHz,DMSO)δ175.45(s), 154.14(s), 140.51(s), 138.75(s), 135.06(s), 130.55(d, J=32.9Hz), 130.02(d, J=32.9Hz), 128.12(s), 126.48(s), 126.27(s),124.31(s),122.66(s),122.14(s),121.43(s),120.30(s),107.32(s),105.52(s),67.91(s).MS(ESI)m /z 388.1[M+H] + ; HRMS(ESI) calcd for C 18 H 11 F 6 NO 2 [M+H] + 388.0765, found 388.0767.
化合物12a-12g的合成路线为：The synthetic route of compound 12a-12g is:
Figure PCTCN2022088657-appb-000010
Figure PCTCN2022088657-appb-000010
实施例20Example 20
2-(((4-氨基苯基)氨基)亚甲基)丙二酸二乙酯(化合物9)的合成Synthesis of Diethyl 2-(((4-aminophenyl)amino)methylene)malonate (Compound 9)
将对苯二胺(化合物8，3g,27.7mmol)加入到5mL乙醇中，再加入乙氧基亚甲基丙二酸二乙酯(化合物2，5.7mL,27.7mmol)，室温反应2小时，待反应完全，低压旋去乙醇，经硅胶柱层析(300-400目硅胶，洗脱剂为石油醚:乙酸乙酯＝4:1V/V)纯化得到2-(((4-氨基苯基)氨基)亚甲基)丙二酸二乙酯(化合物9)，暗黄色固体(5.4g，产率为70％)。Add p-phenylenediamine (compound 8, 3g, 27.7mmol) into 5mL of ethanol, then add ethoxymethylene malonate (compound 2, 5.7mL, 27.7mmol), and react at room temperature for 2 hours, After the reaction is complete, ethanol is removed under low pressure, and purified by silica gel column chromatography (300-400 mesh silica gel, eluent is petroleum ether:ethyl acetate=4:1V/V) to obtain 2-(((4-aminophenyl )amino)methylene)malonate (compound 9), dark yellow solid (5.4 g, yield 70%).
1H NMR(500MHz,CDCl 3)δ10.93(d,J＝13.6Hz,1H),8.40(d,J＝13.9Hz,1H),8.40(d,J＝13.9Hz,1H),6.95(d,J＝8.6Hz,2H),6.68(d,J＝8.6Hz,2H),4.29(q,J＝7.1Hz,2H),4.23(q,J＝7.1Hz,2H),3.72(s,2H),1.37(t,J＝7.1Hz,3H),1.31(t,J＝7.1Hz,3H). 1 H NMR (500MHz, CDCl 3 ) δ10.93(d, J=13.6Hz, 1H), 8.40(d, J=13.9Hz, 1H), 8.40(d, J=13.9Hz, 1H), 6.95(d ,J=8.6Hz,2H),6.68(d,J=8.6Hz,2H),4.29(q,J=7.1Hz,2H),4.23(q,J=7.1Hz,2H),3.72(s,2H ),1.37(t,J=7.1Hz,3H),1.31(t,J=7.1Hz,3H).
2-(((4-((3-(三氟甲基)苄基)氨基)苯基)氨基)亚甲基)丙二酸二乙酯(化合物10a)的合成Synthesis of Diethyl 2-(((4-((3-(trifluoromethyl)benzyl)amino)phenyl)amino)methylene)malonate (Compound 10a)
称取化合物9(400mg，1.44mmol)、醋酸(95mg，90.4μL，1.58mmol)，随后加入无水甲醇(8mL)溶解，加入3-三氟甲基苯甲醛(1.44mmol)，加热到45℃反应3小时，随后加入氰基硼氢化钠NaCNBH 3(135.5mg,2.16mmol)，45℃反应过夜。待反应结束，低压旋去甲醇，经硅胶柱层析(300-400目硅胶，洗脱剂为石油醚:乙酸乙酯＝4:1V/V)纯化得到化合物10a，暗黄色固体，产率为89％。 Weigh compound 9 (400mg, 1.44mmol), acetic acid (95mg, 90.4μL, 1.58mmol), then add anhydrous methanol (8mL) to dissolve, add 3-trifluoromethylbenzaldehyde (1.44mmol), heat to 45°C After reacting for 3 hours, sodium cyanoborohydride NaCNBH 3 (135.5 mg, 2.16 mmol) was added and reacted overnight at 45°C. After the reaction was completed, methanol was spun off under low pressure, and purified by silica gel column chromatography (300-400 mesh silica gel, eluent: petroleum ether: ethyl acetate = 4:1 V/V) to obtain compound 10a, a dark yellow solid, with a yield of 89%.
1H NMR(500MHz,DMSO)δ10.67(d,J＝14.2Hz,1H),8.24(d,J＝14.2Hz,1H),7.70(s,1H),7.66(d,J＝7.2Hz,1H),7.57(m,7.7Hz,2H),7.08(d,J＝8.8Hz,2H),6.61(d,J＝8.8Hz,2H),6.46(m,1H),4.38(d,J＝6.0Hz,2H),4.16(q,J＝7.0Hz,2H),4.08(q,J＝7.1Hz,2H),1.24(t,J＝6.3Hz,3H),1.21(t,J＝6.3Hz,3H). 1 H NMR (500MHz, DMSO) δ10.67(d, J=14.2Hz, 1H), 8.24(d, J=14.2Hz, 1H), 7.70(s, 1H), 7.66(d, J=7.2Hz, 1H), 7.57(m, 7.7Hz, 2H), 7.08(d, J=8.8Hz, 2H), 6.61(d, J=8.8Hz, 2H), 6.46(m, 1H), 4.38(d, J= 6.0Hz, 2H), 4.16(q, J=7.0Hz, 2H), 4.08(q, J=7.1Hz, 2H), 1.24(t, J=6.3Hz, 3H), 1.21(t, J=6.3Hz ,3H).
化合物11a的合成Synthesis of Compound 11a
称取化合物10a(450mg)，加入二苯醚(5mL)，在氩气保护下回流2h；冷却至室温后，加入15mL石油醚，沉淀析出，过滤沉淀固体，用乙酸乙酯加热至50℃溶解，再冷却至室温重结晶得到目标化合物11a，黄色固体(产率为42％)。Weigh compound 10a (450 mg), add diphenyl ether (5 mL), and reflux for 2 h under the protection of argon; after cooling to room temperature, add 15 mL of petroleum ether, precipitate out, filter the precipitated solid, and heat to 50 ° C with ethyl acetate to dissolve , and then cooled to room temperature for recrystallization to obtain the target compound 11a as a yellow solid (yield 42%).
1H NMR(500MHz,DMSO)δ12.08(d,J＝6.6Hz,1H),8.33(d,J＝6.6Hz,1H),7.72(s,1H),7.68(d,J＝7.1Hz,1H),7.62–7.53(m,2H),7.40(d,J＝9.3Hz,1H),7.17–7.09(m,2H),6.78(t,J＝6.0Hz,1H),4.45(d,J＝6.0Hz,2H),4.17(q,J＝7.1Hz,1H),1.25(t,J＝7.1Hz,3H). 1 H NMR (500MHz, DMSO) δ12.08(d, J=6.6Hz, 1H), 8.33(d, J=6.6Hz, 1H), 7.72(s, 1H), 7.68(d, J=7.1Hz, 1H),7.62–7.53(m,2H),7.40(d,J=9.3Hz,1H),7.17–7.09(m,2H),6.78(t,J=6.0Hz,1H),4.45(d,J =6.0Hz, 2H), 4.17(q, J=7.1Hz, 1H), 1.25(t, J=7.1Hz, 3H).
4-氧代-6-((3-(三氟甲基)苄基)氨基)1,4-二氢喹啉-3-羧酸(化合物12a)的合成Synthesis of 4-oxo-6-((3-(trifluoromethyl)benzyl)amino)1,4-dihydroquinoline-3-carboxylic acid (compound 12a)
化合物11a(120mg)在10％氢氧化钠溶液(10mL)中回流3h，在室温下冷却后，用盐酸调节pH到2，待析 出沉淀，过滤，收集固体，用水和石油醚依次洗涤，收集的固体用无水乙醇加热至50℃溶解，冷却至室温重结晶，过滤得到化合物12a，产率为92％，黄色固体。Compound 11a (120mg) was refluxed in 10% sodium hydroxide solution (10mL) for 3h. After cooling at room temperature, the pH was adjusted to 2 with hydrochloric acid. After precipitation, the solid was collected by filtration, washed with water and petroleum ether successively, and the collected The solid was dissolved by heating to 50°C with absolute ethanol, cooled to room temperature for recrystallization, and filtered to obtain compound 12a with a yield of 92% as a yellow solid.
mp>250℃.IR(KBr):3370,2069,1686,1621,1500cm -1.1H NMR(500MHz,DMSO)δ13.28(d,J＝5.4Hz,1H,NH),8.60(d,J＝6.7Hz,1H,CH),7.74(s,1H,NH),7.70(d,J＝7.2Hz,1H,Ar-H),7.55–7.65(m,3H,Ar-H),7.33(dd,J＝9.0,2.6Hz,1H,Ar-H),7.15(d,J＝2.4Hz,1H,Ar-H),4.50(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.06(s),166.88(s),146.87(s),141.07(s),140.90(s),131.26(d,J＝8.0Hz),129.36(s),125.96(s),123.53(d,J＝3.8Hz),122.39(s),120.56(s),106.23(s),101.46(s),45.80(s).HRMS(ESI)calcd for C 18H 14F 3N 2O 3[M+H] +363.0592,found 363.0591. mp>250℃.IR(KBr):3370,2069,1686,1621,1500cm -1 .1H NMR(500MHz,DMSO)δ13.28(d,J＝5.4Hz,1H,NH),8.60(d,J =6.7Hz,1H,CH),7.74(s,1H,NH),7.70(d,J=7.2Hz,1H,Ar-H),7.55–7.65(m,3H,Ar-H),7.33(dd ,J=9.0,2.6Hz,1H,Ar-H),7.15(d,J=2.4Hz,1H,Ar-H),4.50(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.06 (s), 166.88(s), 146.87(s), 141.07(s), 140.90(s), 131.26(d, J=8.0Hz), 129.36(s), 125.96(s), 123.53(d, J= 3.8Hz), 122.39(s), 120.56(s), 106.23(s), 101.46(s), 45.80(s).HRMS(ESI) calcd for C 18 H 14 F 3 N 2 O 3 [M+H] + 363.0592,found 363.0591.
实施例21Example 21
3-(((4-((3,5-双(三氟甲基)苄基)氨基)苯基)氨基)亚甲基)丙二酸二乙酯(化合物10b)的合成Synthesis of Diethyl 3-(((4-((3,5-bis(trifluoromethyl)benzyl)amino)phenyl)amino)methylene)malonate (Compound 10b)
参照实施例20化合物10a的合成，仅将3-三氟甲基苯甲醛替换为3,5-双三氟甲基苯甲醛，得到化合物10b，淡黄色固体，产率为87％。Referring to the synthesis of compound 10a in Example 20, only 3-trifluoromethylbenzaldehyde was replaced by 3,5-bistrifluoromethylbenzaldehyde to obtain compound 10b as a light yellow solid with a yield of 87%.
1H NMR(500MHz,DMSO)δ10.66(d,J＝14.2Hz,1H),8.25(d,J＝14.2Hz,1H),8.05(s,2H),7.94(s,1H),7.09(d,J＝8.8Hz,2H),6.63(d,J＝8.8Hz,2H),6.53(s,1H),4.48(d,J＝4.3Hz,2H),4.17(q,J＝7.1Hz,2H),4.08(q,J＝7.1Hz,2H),1.24(t,J＝6.6Hz,3H),1.21(t,J＝6.5Hz,3H). 1 H NMR (500MHz, DMSO) δ10.66(d, J=14.2Hz, 1H), 8.25(d, J=14.2Hz, 1H), 8.05(s, 2H), 7.94(s, 1H), 7.09( d,J=8.8Hz,2H),6.63(d,J=8.8Hz,2H),6.53(s,1H),4.48(d,J=4.3Hz,2H),4.17(q,J=7.1Hz, 2H), 4.08(q, J=7.1Hz, 2H), 1.24(t, J=6.6Hz, 3H), 1.21(t, J=6.5Hz, 3H).
6-((3,5-三氟甲基)氨基)-4-氧代-1,4-二氢喹啉-3-羧酸乙酯(化合物11b)的合成Synthesis of ethyl 6-((3,5-trifluoromethyl)amino)-4-oxo-1,4-dihydroquinoline-3-carboxylate (compound 11b)
参照实施例20化合物11a的制备方法，得到化合物11b，黄色固体，产率为42％。Referring to the preparation method of compound 11a in Example 20, compound 11b was obtained as a yellow solid with a yield of 42%.
1H NMR(500MHz,DMSO)δ12.10(s,1H),8.33(d,J＝5.8Hz,1H),8.07(s,2H),7.96(d,J＝11.3Hz,1H),7.42(d,J＝9.5Hz,1H),7.15(d,J＝2.5Hz,2H),6.83(q,J＝9.1Hz,2.5Hz,1H),4.56(d,J＝6.0Hz,2H),4.17(q,J＝7.1Hz,2H),1.25(t,J＝7.1Hz,3H). 1 H NMR (500MHz, DMSO) δ12.10(s, 1H), 8.33(d, J=5.8Hz, 1H), 8.07(s, 2H), 7.96(d, J=11.3Hz, 1H), 7.42( d,J=9.5Hz,1H),7.15(d,J=2.5Hz,2H),6.83(q,J=9.1Hz,2.5Hz,1H),4.56(d,J=6.0Hz,2H),4.17 (q,J=7.1Hz,2H),1.25(t,J=7.1Hz,3H).
6-((3,5-三氟甲基)氨基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物12b)的合成Synthesis of 6-((3,5-trifluoromethyl)amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 12b)
参照实施例20化合物12a的制备方法，得到化合物12b，黄色固体，产率为86％。Referring to the preparation method of compound 12a in Example 20, compound 12b was obtained as a yellow solid with a yield of 86%.
mp>250℃.IR(KBr):3383,3069,1689,1621,1503cm -1.1H NMR(500MHz,DMSO)δ13.52(s,1H,NH),8.58(d,J＝6.4Hz,1H,CH),8.1(s,2H,Ar-H),7.98(s,1H,Ar-H),7.67(d,J＝9.0Hz,1H,Ar-H),7.36(dd,J＝9.0,2.4Hz,1H,Ar-H),7.17(d,J＝2.4Hz,1H,Ar-H),4.50(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.06(s),166.88(s),146.58(s),143.31(s),141.11(s),131.50(s),130.32(s),130.06(s),127.94(s),125.93(s),124.39(s),122.30(d,J＝18.9Hz),120.68(s),106.26(s),101.65(s),45.39(s).HRMS(ESI)calcd for C 19H 13F 6N 2O 3[M+H] +431.0821,found 431.0825。 mp>250℃.IR(KBr):3383,3069,1689,1621,1503cm -1 .1H NMR(500MHz,DMSO)δ13.52(s,1H,NH),8.58(d,J＝6.4Hz,1H ,CH),8.1(s,2H,Ar-H),7.98(s,1H,Ar-H),7.67(d,J=9.0Hz,1H,Ar-H),7.36(dd,J=9.0, 2.4Hz, 1H, Ar-H), 7.17(d, J=2.4Hz, 1H, Ar-H), 4.50(s, 2H, CH2).13C NMR(126MHz, DMSO) δ177.06(s), 166.88 (s), 146.58(s), 143.31(s), 141.11(s), 131.50(s), 130.32(s), 130.06(s), 127.94(s), 125.93(s), 124.39(s), 122.30 (d, J=18.9Hz), 120.68(s), 106.26(s), 101.65(s), 45.39(s).HRMS(ESI) calcd for C 19 H 13 F 6 N 2 O 3 [M+H] + 431.0821,found 431.0825.
实施例22Example 22
2-(((4-((噻吩-2-基甲基)氨基)苯基)氨基)亚甲基)丙二酸二乙酯(化合物10c)的合成Synthesis of Diethyl 2-(((4-((thiophen-2-ylmethyl)amino)phenyl)amino)methylene)malonate (Compound 10c)
参照实施例20化合物10a的合成，仅3-三氟甲基苯甲醛替换为2-噻吩甲醛，得到化合物10c，淡黄色固体，产率为86％。Referring to the synthesis of compound 10a in Example 20, only 3-trifluoromethylbenzaldehyde was replaced by 2-thiophenecarbaldehyde to obtain compound 10c as a light yellow solid with a yield of 86%.
1H NMR(500MHz,DMSO)δ10.67(d,J＝14.2Hz,1H),8.25(d,J＝14.2Hz,1H),7.47(dd,J＝4.9,3.0Hz,1H),7.37–7.28(m,1H),7.08(dd,J＝4.8,3.6Hz,4H),6.64(d,J＝8.7Hz,2H),6.18(t,J＝5.9Hz,1H),4.24(d,J＝5.9Hz,2H),4.17(q,J＝7.1Hz,2H),4.09(q,J＝7.1Hz,2H),1.24(t,J＝6.1Hz,3H),1.22(t,J＝6.1Hz,3H). 1 H NMR (500MHz, DMSO) δ10.67 (d, J = 14.2Hz, 1H), 8.25 (d, J = 14.2Hz, 1H), 7.47 (dd, J = 4.9, 3.0Hz, 1H), 7.37– 7.28(m,1H),7.08(dd,J=4.8,3.6Hz,4H),6.64(d,J=8.7Hz,2H),6.18(t,J=5.9Hz,1H),4.24(d,J =5.9Hz, 2H), 4.17(q, J=7.1Hz, 2H), 4.09(q, J=7.1Hz, 2H), 1.24(t, J=6.1Hz, 3H), 1.22(t, J=6.1 Hz,3H).
4-氧代-6-((3-(三氟甲基)苄基)氨基)1,4-二氢喹啉-3-羧酸乙酯(化合物11c)的合成Synthesis of ethyl 4-oxo-6-((3-(trifluoromethyl)benzyl)amino)1,4-dihydroquinoline-3-carboxylate (compound 11c)
参照实施例20化合物11a的制备方法，得到化合物11c，黄色固体，产率为44％。Referring to the preparation method of compound 11a in Example 20, compound 11c was obtained as a yellow solid with a yield of 44%.
1H NMR(500MHz,DMSO)δ12.05(s,1H),8.32(s,1H),7.48(dd,J＝4.8,3.0Hz,1H),7.38(d,J＝8.8Hz,1H),7.35(s,1H),7.21(d,J＝2.4Hz,1H),7.12(dd,J＝8.5,3.2Hz,2H),6.52(m,1H),4.31(d,J＝5.8Hz,2H),4.18(q,J＝7.1Hz,2H),1.26(t,J＝7.1Hz,3H). 1 H NMR (500MHz,DMSO)δ12.05(s,1H),8.32(s,1H),7.48(dd,J=4.8,3.0Hz,1H),7.38(d,J=8.8Hz,1H), 7.35(s,1H),7.21(d,J=2.4Hz,1H),7.12(dd,J=8.5,3.2Hz,2H),6.52(m,1H),4.31(d,J=5.8Hz,2H ), 4.18(q, J=7.1Hz, 2H), 1.26(t, J=7.1Hz, 3H).
4-氧代-6-((3-(三氟甲基)苄基)氨基)1,4-二氢喹啉-3-羧酸(化合物12c)的合成Synthesis of 4-oxo-6-((3-(trifluoromethyl)benzyl)amino)1,4-dihydroquinoline-3-carboxylic acid (compound 12c)
参照实施例20化合物12a的制备方法，得到化合物12c，黄色固体，产率为90％。Referring to the preparation method of compound 12a in Example 20, compound 12c was obtained as a yellow solid with a yield of 90%.
mp>250℃. 1H NMR(500MHz,DMSO)δ15.88(s,1H,COOH),13.30(s,1H,NH),8.59(d,J＝5.3Hz,1H,CH),7.59(dd,J＝20.8,8.8Hz,1H,Ar-H),7.50(s,1H,Ar-H),7.35(d,J＝24.2Hz,2H,Ar-H),7.20(d,J＝12.6Hz,1H,Ar-H),7.13(s,1H,Ar-H),6.87(s,1H,NH),4.36(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.11(s),166.94(s),147.17(s),140.86(s),140.16(s),131.11(s),127.34(s),126.22(s),125.99(s),122.38(s),121.58(s),120.36(s),106.17(s),101.29(s),42.12(s)IR(KBr):3405,3070,1701,1619,1475cm-1.13C NMR(126MHz,DMSO)δ177.11(s),166.94(s),147.17(s),140.86(s),140.16(s),131.11(s),127.34(s),126.22(s),125.99(s),122.38(s),121.58(s),120.36(s),106.17(s),101.29(s),42.12(s).HRMS(ESI)calcd for C 19H 13F 6N 2O 3[M+H] +431.0821,found 431.0825. mp>250℃. 1 H NMR (500MHz, DMSO) δ15.88(s, 1H, COOH), 13.30(s, 1H, NH), 8.59(d, J＝5.3Hz, 1H, CH), 7.59(dd ,J=20.8,8.8Hz,1H,Ar-H),7.50(s,1H,Ar-H),7.35(d,J=24.2Hz,2H,Ar-H),7.20(d,J=12.6Hz ,1H,Ar-H),7.13(s,1H,Ar-H),6.87(s,1H,NH),4.36(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.11(s) ,166.94(s),147.17(s),140.86(s),140.16(s),131.11(s),127.34(s),126.22(s),125.99(s),122.38(s),121.58(s) ,120.36(s),106.17(s),101.29(s),42.12(s)IR(KBr):3405,3070,1701,1619,1475cm-1.13C NMR(126MHz,DMSO)δ177.11(s), 166.94(s), 147.17(s), 140.86(s), 140.16(s), 131.11(s), 127.34(s), 126.22(s), 125.99(s), 122.38(s), 121.58(s), 120.36(s), 106.17(s), 101.29(s), 42.12(s). HRMS(ESI) calcd for C 19 H 13 F 6 N 2 O 3 [M+H] + 431.0821, found 431.0825.
实施例23Example 23
3-(((4-((2-三氟甲基苄基)氨基)苯基)氨基)亚甲基)丙二酸二乙酯(化合物10d)的合成Synthesis of Diethyl 3-(((4-((2-trifluoromethylbenzyl)amino)phenyl)amino)methylene)malonate (Compound 10d)
参照实施例20化合物10a的合成，仅将3-三氟甲基苯甲醛替换为2-三氟甲基苯甲醛，得到化合物10d，淡黄色固体，产率为92％。Referring to the synthesis of compound 10a in Example 20, only 3-trifluoromethylbenzaldehyde was replaced by 2-trifluoromethylbenzaldehyde to obtain compound 10d as a pale yellow solid with a yield of 92%.
4-氧代-6-((2-(三氟甲基)苄基)氨基)1,4-二氢喹啉-3-羧酸乙酯(化合物11d)的合成Synthesis of ethyl 4-oxo-6-((2-(trifluoromethyl)benzyl)amino)1,4-dihydroquinoline-3-carboxylate (compound 11d)
参照实施例20化合物11a的制备方法，得到化合物11d，黄色固体，产率为52％。Referring to the preparation method of compound 11a in Example 20, compound 11d was obtained as a yellow solid with a yield of 52%.
1H NMR(500MHz,DMSO)δ12.12(s,1H),8.33(s,1H),7.76(d,J＝7.8Hz,1H),7.66–7.55(m,2H),7.50–7.40(m,2H),7.12(dd,J＝8.8,2.6Hz,1H),7.06(d,J＝2.5Hz,1H),6.82(s,1H),4.52(s,2H),4.17(q,t,J＝7.1Hz,2H),1.24(t,J＝7.1Hz,3H). 1 H NMR (500MHz, DMSO) δ12.12(s, 1H), 8.33(s, 1H), 7.76(d, J=7.8Hz, 1H), 7.66–7.55(m, 2H), 7.50–7.40(m ,2H),7.12(dd,J=8.8,2.6Hz,1H),7.06(d,J=2.5Hz,1H),6.82(s,1H),4.52(s,2H),4.17(q,t, J＝7.1Hz, 2H), 1.24(t, J＝7.1Hz, 3H).
4-氧代-6-((2-(三氟甲基)苄基)氨基)1,4-二氢喹啉-3-羧酸(化合物12d)的合成Synthesis of 4-oxo-6-((2-(trifluoromethyl)benzyl)amino)1,4-dihydroquinoline-3-carboxylic acid (compound 12d)
参照实施例20化合物12a的制备方法，得到化合物12d，黄色固体，产率为90％。Referring to the preparation method of compound 12a in Example 20, compound 12d was obtained as a yellow solid with a yield of 90%.
mp>250℃.IR(KBr):3369,1689,1621,1499cm -1. 1H NMR(500MHz,DMSO)δ13.48(s,1H,NH),8.58(d,J＝6.6Hz,1H,CH),7.77(d,J＝7.8Hz,1H,Ar-H),7.66(d,J＝9.0Hz,1H,Ar-H),7.64–7.57(m,2H,Ar-H),7.48(m,1H,Ar-H),7.33(dd,J＝9.0,2.6Hz,1H,Ar-H),7.05(d,J＝2.5Hz,1H,Ar-H),4.55(s,2H,CH2).13C NMR(126MHz,DMSO)δ176.98(s),166.88(s),146.72(s),140.98(s),137.49(s),132.63(s),131.39(s),128.27(s),127.39(s),126.67(s),125.98(s),122.35(s),120.65(s),106.24(s),100.97(s),42.98(s).HRMS(ESI)calcd for C18H14F3N2O3[M+H]+363.0950,found 363.0951. mp>250℃.IR(KBr):3369,1689,1621,1499cm -1 . 1 H NMR(500MHz,DMSO)δ13.48(s,1H,NH),8.58(d,J＝6.6Hz,1H, CH), 7.77(d, J=7.8Hz, 1H, Ar-H), 7.66(d, J=9.0Hz, 1H, Ar-H), 7.64–7.57(m, 2H, Ar-H), 7.48( m, 1H, Ar-H), 7.33(dd, J=9.0, 2.6Hz, 1H, Ar-H), 7.05(d, J=2.5Hz, 1H, Ar-H), 4.55(s, 2H, CH2 ).13C NMR (126MHz, DMSO) δ176.98(s), 166.88(s), 146.72(s), 140.98(s), 137.49(s), 132.63(s), 131.39(s), 128.27(s) ,127.39(s),126.67(s),125.98(s),122.35(s),120.65(s),106.24(s),100.97(s),42.98(s).HRMS(ESI)calcd for C18H14F3N2O3[M +H]+363.0950,found 363.0951.
实施例24Example 24
2-(((4-((3-(三氟甲氧基)苄基)氨基)苯基)氨基)亚甲基)丙二酸二乙酯(化合物10e)的合成Synthesis of Diethyl 2-(((4-((3-(trifluoromethoxy)benzyl)amino)phenyl)amino)methylene)malonate (Compound 10e)
参照实施例20化合物10a的合成，仅将3-三氟甲基苯甲醛替换为3-三氟甲氧基苯甲醛，得到化合物10e，淡黄色固体，产率为91％。Referring to the synthesis of compound 10a in Example 20, only 3-trifluoromethylbenzaldehyde was replaced by 3-trifluoromethoxybenzaldehyde to obtain compound 10e as a pale yellow solid with a yield of 91%.
1H NMR(500MHz,DMSO)δ10.66(d,J＝14.2Hz,1H),8.24(d,J＝14.2Hz,1H),7.45(t,J＝7.9Hz,1H),7.39(d,J＝7.7Hz,1H),7.31(s,1H),7.21(d,J＝8.0Hz,1H),7.08(d,J＝8.8Hz,2H),6.60(d,J＝8.8Hz,2H),6.38–6.49(m,1H),4.33(d,J＝6.1Hz,2H),4.17(q,J＝7.1Hz,2H),4.08(q,J＝7.1Hz,2H),1.24(t,J＝6.4Hz,3H),1.21(t,J＝7.0Hz,3H). 1 H NMR (500MHz, DMSO) δ10.66(d, J=14.2Hz, 1H), 8.24(d, J=14.2Hz, 1H), 7.45(t, J=7.9Hz, 1H), 7.39(d, J＝7.7Hz, 1H), 7.31(s, 1H), 7.21(d, J＝8.0Hz, 1H), 7.08(d, J＝8.8Hz, 2H), 6.60(d, J＝8.8Hz, 2H) ,6.38–6.49(m,1H),4.33(d,J=6.1Hz,2H),4.17(q,J=7.1Hz,2H),4.08(q,J=7.1Hz,2H),1.24(t, J＝6.4Hz, 3H), 1.21(t, J＝7.0Hz, 3H).
4-氧代-6-((3-(三氟甲氧基)苄基)氨基)1,4-二氢喹啉-3-羧酸乙酯(化合物11e)的合成Synthesis of ethyl 4-oxo-6-((3-(trifluoromethoxy)benzyl)amino)1,4-dihydroquinoline-3-carboxylate (compound 11e)
参照实施例20化合物11a的制备方法，得到化合物11e，黄色固体，产率为42％。Referring to the preparation method of compound 11a in Example 20, compound 11e was obtained as a yellow solid with a yield of 42%.
1H NMR(500MHz,DMSO)δ12.07(d,J＝6.8Hz,1H),8.32(d,J＝6.8Hz,1H),7.47(t,J＝7.9Hz,1H),7.45–7.36(m,3H),7.33(s,1H),7.21(d,J＝7.9Hz,1H),7.18–7.07(m,2H),7.00(d,J＝8.0Hz,1H),6.75(m,1H),4.41(d,J＝6.0Hz,2H),4.17(q,J＝7.1Hz,2H),1.25(t,J＝7.1Hz,3H). 1 H NMR (500MHz, DMSO) δ12.07(d, J=6.8Hz, 1H), 8.32(d, J=6.8Hz, 1H), 7.47(t, J=7.9Hz, 1H), 7.45–7.36( m,3H),7.33(s,1H),7.21(d,J=7.9Hz,1H),7.18–7.07(m,2H),7.00(d,J=8.0Hz,1H),6.75(m,1H ), 4.41(d, J=6.0Hz, 2H), 4.17(q, J=7.1Hz, 2H), 1.25(t, J=7.1Hz, 3H).
4-氧代-6-((3-(三氟甲氧基)苄基)氨基)1,4-二氢喹啉-3-羧酸(化合物12e)的合成Synthesis of 4-oxo-6-((3-(trifluoromethoxy)benzyl)amino)1,4-dihydroquinoline-3-carboxylic acid (compound 12e)
参照实施例20化合物12a的制备方法，得到化合物12e，黄色固体，产率为93％。Referring to the preparation method of compound 12a in Example 20, compound 12e was obtained as a yellow solid with a yield of 93%.
mp>250℃.IR(KBr):3384,1699,1619,1488cm -1. 1H NMR(500MHz,DMSO)δ13.47(s,1H,NH),8.57(d,J＝6.6Hz,1H,CH),7.64(d,J＝9.0Hz,1H,Ar-H),7.48(d,J＝7.8Hz,1H,Ar-H),7.43(d,J＝7.7Hz,1H,Ar-H),7.36(s,1H,Ar-H),7.33(dd,J＝9.1,2.6Hz,1H,Ar-H),7.23(d,J＝7.8Hz,1H,Ar-H),7.14(d,J＝2.5Hz,1H,Ar-H),4.46(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.03(s),166.89(s),148.55(s),146.84(s),142.38(s),140.91(s),131.29(s),130.24(s),126.01(d,J＝18.3Hz),122.38(s),120.51(s),119.29(s),119.09(s),106.19(s),101.48(s),45.71(s).HRMS(ESI)calcd for C 18H 13F 3N 2O 4[M+H] +379.0901,found 379.0900. mp>250℃.IR(KBr):3384,1699,1619,1488cm -1 . 1 H NMR(500MHz,DMSO)δ13.47(s,1H,NH),8.57(d,J＝6.6Hz,1H, CH),7.64(d,J=9.0Hz,1H,Ar-H),7.48(d,J=7.8Hz,1H,Ar-H),7.43(d,J=7.7Hz,1H,Ar-H) ,7.36(s,1H,Ar-H),7.33(dd,J=9.1,2.6Hz,1H,Ar-H),7.23(d,J=7.8Hz,1H,Ar-H),7.14(d, J＝2.5Hz, 1H, Ar-H), 4.46(s, 2H, CH2).13C NMR(126MHz, DMSO) δ177.03(s), 166.89(s), 148.55(s), 146.84(s), 142.38(s), 140.91(s), 131.29(s), 130.24(s), 126.01(d, J=18.3Hz), 122.38(s), 120.51(s), 119.29(s), 119.09(s), 106.19(s), 101.48(s), 45.71(s). HRMS(ESI) calcd for C 18 H 13 F 3 N 2 O 4 [M+H] + 379.0901, found 379.0900.
实施例25Example 25
2-(((4-((4-(三氟甲氧基)苄基)氨基)苯基)氨基)亚甲基)丙二酸二乙酯(化合物10f)的合成Synthesis of Diethyl 2-(((4-((4-(trifluoromethoxy)benzyl)amino)phenyl)amino)methylene)malonate (Compound 10f)
参照实施例20化合物10a的合成，仅将3-三氟甲基苯甲醛替换为4-三氟甲氧基苯甲醛，得到化合物10f，淡黄色固体，产率为93％。Referring to the synthesis of compound 10a in Example 20, only 3-trifluoromethylbenzaldehyde was replaced by 4-trifluoromethoxybenzaldehyde to obtain compound 10f as a pale yellow solid with a yield of 93%.
1H NMR(500MHz,DMSO)δ10.67(d,J＝14.2Hz,1H),8.24(d,J＝14.2Hz,1H),7.46(d,J＝8.4Hz,1H),7.35–7.25(m,3H),7.07(d,J＝8.7Hz,2H),6.59(d,J＝8.7Hz,2H),6.40(t,J＝6.2Hz,1H),4.30(d,J＝5.9Hz,2H),4.17(q,J＝7.1Hz,2H),4.08(q,J＝7.1Hz,2H),1.28–1.23(m,3H),1.21(d,J＝7.1Hz,3H). 1 H NMR (500MHz, DMSO) δ 10.67 (d, J = 14.2Hz, 1H), 8.24 (d, J = 14.2Hz, 1H), 7.46 (d, J = 8.4Hz, 1H), 7.35–7.25 ( m,3H),7.07(d,J=8.7Hz,2H),6.59(d,J=8.7Hz,2H),6.40(t,J=6.2Hz,1H),4.30(d,J=5.9Hz, 2H), 4.17(q, J=7.1Hz, 2H), 4.08(q, J=7.1Hz, 2H), 1.28–1.23(m, 3H), 1.21(d, J=7.1Hz, 3H).
4-氧代-6-((4-(三氟甲氧基)苄基)氨基)1,4-二氢喹啉-3-羧酸乙酯(化合物11f)的合成Synthesis of ethyl 4-oxo-6-((4-(trifluoromethoxy)benzyl)amino)1,4-dihydroquinoline-3-carboxylate (compound 11f)
参照实施例20化合物11a的制备方法，得到化合物11f，黄色固体，产率为41％。Referring to the preparation method of compound 11a in Example 20, compound 11f was obtained as a yellow solid with a yield of 41%.
1H NMR(500MHz,DMSO)δ12.05(d,J＝6.5Hz,1H),8.32(d,J＝6.8Hz,1H),7.49(d,J＝8.5Hz,2H),7.38(d,J＝8.8Hz,1H),7.31(d,J＝8.2Hz,2H),7.13(d,J＝2.4Hz,1H),7.10(dd,J＝8.8,2.5Hz,1H),6.72(t,J＝5.9Hz,1H),4.37(d,J＝5.9Hz,2H),4.17(d,J＝7.1Hz,2H),1.25(t,J＝7.1Hz,3H). 1 H NMR (500MHz, DMSO) δ12.05(d, J=6.5Hz, 1H), 8.32(d, J=6.8Hz, 1H), 7.49(d, J=8.5Hz, 2H), 7.38(d, J＝8.8Hz, 1H), 7.31(d, J＝8.2Hz, 2H), 7.13(d, J＝2.4Hz, 1H), 7.10(dd, J＝8.8, 2.5Hz, 1H), 6.72(t, J=5.9Hz, 1H), 4.37(d, J=5.9Hz, 2H), 4.17(d, J=7.1Hz, 2H), 1.25(t, J=7.1Hz, 3H).
4-氧代-6-((4-(三氟甲氧基)苄基)氨基)1,4-二氢喹啉-3-羧酸(化合物12f)的合成Synthesis of 4-oxo-6-((4-(trifluoromethoxy)benzyl)amino)1,4-dihydroquinoline-3-carboxylic acid (compound 12f)
参照实施例20化合物12a的制备方法，得到化合物12f，黄色固体，产率为85％。Referring to the preparation method of compound 12a in Example 20, compound 12f was obtained as a yellow solid with a yield of 85%.
mp>250℃.IR(KBr):3385,1702,1619,1485cm -1. 1H NMR(500MHz,DMSO)δ15.81(s,1H,COOH),13.25(d,J＝4.5Hz,1H,NH),8.60(d,J＝6.7Hz,1H Ar-H),7.61(d,J＝9.0Hz,1H Ar-H),7.50(d,J＝8.4Hz,2H Ar-H),7.32(m,3H,Ar-H),7.15(d,J＝2.4Hz,1H,Ar-H),7.05(s,1H,NH),4.42(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.07(s),166.89(s),147.18(s),146.98(s),141.00(s),138.74(s),131.20(s),128.87(s),125.99(s),122.35(s),120.88(s),120.50(s),106.21(s),101.31(s),45.56(s).HRMS(ESI)calcd for C 18H 13F 3N 2O 4[M+H] +379.0901,found 379.0900. mp>250℃.IR(KBr):3385,1702,1619,1485cm -1 . 1 H NMR(500MHz,DMSO)δ15.81(s,1H,COOH),13.25(d,J＝4.5Hz,1H, NH), 8.60(d, J=6.7Hz, 1H Ar-H), 7.61(d, J=9.0Hz, 1H Ar-H), 7.50(d, J=8.4Hz, 2H Ar-H), 7.32( m,3H,Ar-H),7.15(d,J=2.4Hz,1H,Ar-H),7.05(s,1H,NH),4.42(s,2H,CH2).13C NMR(126MHz,DMSO) δ177.07(s), 166.89(s), 147.18(s), 146.98(s), 141.00(s), 138.74(s), 131.20(s), 128.87(s), 125.99(s), 122.35(s) ),120.88(s),120.50(s),106.21(s),101.31(s),45.56(s).HRMS(ESI)calcd for C 18 H 13 F 3 N 2 O 4 [M+H] + 379.0901 , found 379.0900.
实施例26Example 26
2-(((4-((4-(三氟甲基)苄基)氨基)苯基)氨基)亚甲基)丙二酸二乙酯(化合物10g)的合成Synthesis of Diethyl 2-(((4-((4-(trifluoromethyl)benzyl)amino)phenyl)amino)methylene)malonate (Compound 10g)
参照实施例20化合物10a的合成，仅将3-三氟甲基苯甲醛替换为4-三氟甲基苯甲醛，得到化合物10g，淡黄色固体，产率为93％。Referring to the synthesis of compound 10a in Example 20, only 3-trifluoromethylbenzaldehyde was replaced by 4-trifluoromethylbenzaldehyde to obtain compound 10g as a pale yellow solid with a yield of 93%.
1H NMR(500MHz,DMSO)δ10.66(d,J＝14.2Hz,1H),8.24(d,J＝14.2Hz,1H),7.67(d,J＝8.1Hz,2H),7.56(d,J＝8.0Hz,2H),7.07(d,J＝8.7Hz,2H),6.58(d,J＝8.8Hz,2H),6.47(t,J＝6.1Hz,1H),4.16(q,J＝7.1Hz,2H),4.08(q,J＝7.1Hz,2H),1.24(t,J＝6.8Hz,1H),1.21(t,J＝6.7Hz,3H). 1 H NMR (500MHz, DMSO) δ10.66(d, J=14.2Hz, 1H), 8.24(d, J=14.2Hz, 1H), 7.67(d, J=8.1Hz, 2H), 7.56(d, J=8.0Hz, 2H), 7.07(d, J=8.7Hz, 2H), 6.58(d, J=8.8Hz, 2H), 6.47(t, J=6.1Hz, 1H), 4.16(q, J= 7.1Hz, 2H), 4.08(q, J=7.1Hz, 2H), 1.24(t, J=6.8Hz, 1H), 1.21(t, J=6.7Hz, 3H).
4-氧代-6-((4-(三氟甲基)苄基)氨基)1,4-二氢喹啉-3-羧酸乙酯(化合物11g)的合成Synthesis of ethyl 4-oxo-6-((4-(trifluoromethyl)benzyl)amino)1,4-dihydroquinoline-3-carboxylate (compound 11g)
参照实施例20化合物11a的制备方法，得到化合物11g，黄色固体，产率45.33％Referring to the preparation method of compound 11a in Example 20, compound 11g was obtained as a yellow solid with a yield of 45.33%
1H NMR(500MHz,DMSO)δ12.06(s,1H),8.32(s,1H),7.68(d,J＝8.1Hz,2H),7.58(d,J＝8.0Hz,2H),7.39(d,J＝9.5Hz,1H),7.19–7.03(m,2H),6.80(s,1H),4.45(d,J＝5.9Hz,2H),4.17(q,J＝7.1Hz,2H),1.25(t,J＝7.1Hz,3H). 1 H NMR (500MHz, DMSO) δ12.06(s, 1H), 8.32(s, 1H), 7.68(d, J=8.1Hz, 2H), 7.58(d, J=8.0Hz, 2H), 7.39( d,J=9.5Hz,1H),7.19–7.03(m,2H),6.80(s,1H),4.45(d,J=5.9Hz,2H),4.17(q,J=7.1Hz,2H), 1.25(t,J=7.1Hz,3H).
4-氧代-6-((4-(三氟甲基)苄基)氨基)1,4-二氢喹啉-3-羧酸(化合物12g)的合成Synthesis of 4-oxo-6-((4-(trifluoromethyl)benzyl)amino)1,4-dihydroquinoline-3-carboxylic acid (compound 12g)
参照实施例20化合物12a的制备方法，得到化合物12g，黄色固体，产率为90％。Referring to the preparation method of compound 12a in Example 20, compound 12g was obtained as a yellow solid with a yield of 90%.
mp>250℃.IR(KBr):3432,2916,1699,1619,1428cm -1. 1H NMR(500MHz,DMSO)δ13.44(s,1H,NH),8.60(d,J＝6.5Hz,1H,CH),7.72(d,J＝8.1Hz,2H,Ar-H),7.62–7.64(m,3H,Ar-H),7.35(dd,J＝9.0,2.5Hz,1H,Ar-H),7.14(d,J＝2.5Hz,1H,Ar-H),4.53(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.43(s),166.44(s),156.15(s),143.56(s),141.33(s),134.32(s),128.00(s),125.60(s),125.32(t),124.72(s),121.55(s),106.95(s),105.60(s),68.83(s).HRMS(ESI)calcd for C 18H 14F 3N 2O 3[M+H] +363.0948,found 363.0951. mp>250℃.IR(KBr):3432,2916,1699,1619,1428cm -1 . 1 H NMR(500MHz,DMSO)δ13.44(s,1H,NH),8.60(d,J＝6.5Hz, 1H, CH), 7.72 (d, J=8.1Hz, 2H, Ar-H), 7.62–7.64 (m, 3H, Ar-H), 7.35 (dd, J=9.0, 2.5Hz, 1H, Ar-H ),7.14(d,J=2.5Hz,1H,Ar-H),4.53(s,2H,CH2).13C NMR(126MHz,DMSO)δ177.43(s),166.44(s),156.15(s) ,143.56(s),141.33(s),134.32(s),128.00(s),125.60(s),125.32(t),124.72(s),121.55(s),106.95(s),105.60(s) ,68.83(s).HRMS(ESI)calcd for C 18 H 14 F 3 N 2 O 3 [M+H] + 363.0948,found 363.0951.
实施例27Example 27
2-(((4-(3,5-双(三氟甲基)苯甲酰氨基)苯基)氨基)亚甲基)丙二酸二乙酯(化合物13)的合成Synthesis of Diethyl 2-(((4-(3,5-bis(trifluoromethyl)benzamido)phenyl)amino)methylene)malonate (Compound 13)
Figure PCTCN2022088657-appb-000011
Figure PCTCN2022088657-appb-000011
将3,5-双(三氟甲基)苯甲酸(1.67g，1mmol)加入化合物9(1.52g,1.2mmol)的N,N-二甲基甲酰胺(10mL)溶液中，然后加入1-羟基苯并三唑(0.9g，5.8mmol)、1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐(1g，7.4mmol)，80℃搅拌4h，加水淬灭，过滤，得到化合物13，黄色固体，产率为64％。3,5-bis(trifluoromethyl)benzoic acid (1.67g, 1mmol) was added to a solution of compound 9 (1.52g, 1.2mmol) in N,N-dimethylformamide (10mL), and then 1- Hydroxybenzotriazole (0.9g, 5.8mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1g, 7.4mmol), stirred at 80°C for 4h, added water Quenching and filtration afforded compound 13 as a yellow solid in 64% yield.
1H NMR(300MHz,DMSO-d6)δ10.79(S,1H,NH)10.73(S,,1H,NH),8.61(d,J＝1.7Hz,2H,Ar-H),8.46–8.32(m,2H,Ar-H),7.86–7.73(m,2H,Ar-H),7.48-7.35(m,2H,Ar-H),4.18(dq,4H,2CH 2),1.26(td,6H,2CH 3). 1 H NMR (300MHz, DMSO-d6) δ10.79 (S, 1H, NH) 10.73 (S,, 1H, NH), 8.61 (d, J=1.7Hz, 2H, Ar-H), 8.46–8.32 ( m,2H,Ar-H),7.86–7.73(m,2H,Ar-H),7.48-7.35(m,2H,Ar-H),4.18(dq,4H,2CH 2 ),1.26(td,6H ,2CH 3 ).
6-(3,5-双(三氟甲基)苯甲酰氨基)-4-氧代-1,4-二氢喹啉-3-羧酸乙酯(化合物14)的合成Synthesis of ethyl 6-(3,5-bis(trifluoromethyl)benzamido)-4-oxo-1,4-dihydroquinoline-3-carboxylate (compound 14)
Figure PCTCN2022088657-appb-000012
Figure PCTCN2022088657-appb-000012
将化合物13(0.8g，1.69mmol)分批加入二苯醚(4mL)中，回流3小时，冷却至室温，加入石油醚，析出晶体，过滤，收集所得晶体，并用大量石油醚洗涤，采用丙酮加热至60℃溶解，再冷却至室温重结晶得到化合物14，白色固体，产率为64％。Add compound 13 (0.8g, 1.69mmol) to diphenyl ether (4mL) in batches, reflux for 3 hours, cool to room temperature, add petroleum ether, precipitate crystals, filter, collect the obtained crystals, wash with a large amount of petroleum ether, and use acetone Heat to 60°C to dissolve, then cool to room temperature and recrystallize to obtain compound 14 as a white solid with a yield of 64%.
1H NMR(300MHz,DMSO-d 6)δ12.42(s,1H,COOH),10.94(s,1H,NH),8.67(d,J＝1.7Hz,2H,Ar-H),8.54(d,J＝3.1Hz,2H,Ar-H),8.40(s,1H,Ar-H),8.23(dd,J＝8.9,2.5Hz,1H,Ar-H),7.68(d,J＝8.9Hz,1H,Ar-H),4.23(q,2H,CH 2),,1.29(t,3H,CH 3). 1 H NMR (300MHz, DMSO-d 6 )δ12.42(s, 1H, COOH), 10.94(s, 1H, NH), 8.67(d, J=1.7Hz, 2H, Ar-H), 8.54(d ,J=3.1Hz,2H,Ar-H),8.40(s,1H,Ar-H),8.23(dd,J=8.9,2.5Hz,1H,Ar-H),7.68(d,J=8.9Hz ,1H,Ar-H),4.23(q,2H,CH 2 ),,1.29(t,3H,CH 3 ).
6-((4-甲氧基苄基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物15a)的合成Synthesis of 6-((4-methoxybenzyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 15a)
Figure PCTCN2022088657-appb-000013
Figure PCTCN2022088657-appb-000013
将化合物14(0.2g，423umol)溶解在5mL等体积无水乙醇和10％氢氧化钾溶液的混合溶剂中，90℃加热3小时；冷却至室温，加入浓盐酸调节pH至2.0；过滤，沉淀用水洗涤并真空干燥，得到化合物15a，褐色固体，产率为84％。Dissolve compound 14 (0.2g, 423umol) in a mixed solvent of 5mL equal volume of absolute ethanol and 10% potassium hydroxide solution, heat at 90°C for 3 hours; cool to room temperature, add concentrated hydrochloric acid to adjust the pH to 2.0; filter and precipitate Washing with water and drying in vacuo afforded compound 15a as a tan solid in 84% yield.
mp 196-200℃.IR(KBr):3445,3274,3067,2967,1679,1373,1279cm -1. 1H NMR(400MHz,DMSO)δ15.42(s,1H,COOH),11.00(s,1H,NH),8.84(s,1H,C＝CH),8.71(d,J＝2.4Hz,1H,Ar-H),8.65(d,J＝1.7Hz,2H,Ar-H),8.39(s,1H,Ar-H),8.31(dd,J＝9.0,2.5Hz,1H,Ar-H),7.84(d,J＝9.0Hz,1H,Ar-H). 13C NMR(101MHz,DMSO)δ177.61(s),166.91(s),157.32(s),156.34(s),148.77(s),133.43(s),130.31(s),128.89(s),127.63(s),124.33(s),122.98(s),121.16(s),120.65(s),111.85(s),108.80((s),106.79(s),56.04(s),53.22δ178.43(s),166.96(s),163.16(s),144.88(s),137.16(s),137.06(s),136.55(s),131.13(s),130.79(s),129.10(s),127.40(s),125.84(s),125.28(s),124.93(s),122.22(s),120.89(s),115.22(s),107.66(s).MS(ESI)m/z 445.1[M+H] -；HRMS(ESI)calcd for C 19H 10F 6N 2NaO 4[M+Na] +467.0437,found467.0439. mp 196-200℃.IR(KBr):3445,3274,3067,2967,1679,1373,1279cm -1 . 1 H NMR(400MHz,DMSO)δ15.42(s,1H,COOH),11.00(s, 1H, NH), 8.84(s, 1H, C=CH), 8.71(d, J=2.4Hz, 1H, Ar-H), 8.65(d, J=1.7Hz, 2H, Ar-H), 8.39( s, 1H, Ar-H), 8.31 (dd, J＝9.0, 2.5Hz, 1H, Ar-H), 7.84 (d, J＝9.0Hz, 1H, Ar-H). 13 C NMR (101MHz, DMSO )δ177.61(s),166.91(s),157.32(s),156.34(s),148.77(s),133.43(s),130.31(s),128.89(s),127.63(s),124.33( s), 122.98(s), 121.16(s), 120.65(s), 111.85(s), 108.80((s), 106.79(s), 56.04(s), 53.22δ178.43(s), 166.96(s ),163.16(s),144.88(s),137.16(s),137.06(s),136.55(s),131.13(s),130.79(s),129.10(s),127.40(s),125.84(s ), 125.28(s), 124.93(s), 122.22(s), 120.89(s), 115.22(s), 107.66(s). MS(ESI) m/z 445.1[M+H] - ; HRMS(ESI )calcd for C 19 H 10 F 6 N 2 NaO 4 [M+Na] + 467.0437,found 467.0439.
实施例28Example 28
6-(((3,5-双(三氟甲基)苯基)磺酰基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物15b)的合成Synthesis of 6-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 15b)
Figure PCTCN2022088657-appb-000014
Figure PCTCN2022088657-appb-000014
化合物6(1mmol)、三甲胺(0.2mmol)和3,5-双(三氟甲基)苯磺酰氯(1.1mmol)在二氯甲烷(5ml)中的混合物在室温下保持4小时，减压蒸发，过滤，收集沉淀的固体，用水洗涤，用无水乙醇加热至60℃溶解，再冷却至室温重结晶，得到化合物15b，黄色固体，产率60.4％。A mixture of compound 6 (1 mmol), trimethylamine (0.2 mmol) and 3,5-bis(trifluoromethyl)benzenesulfonyl chloride (1.1 mmol) in dichloromethane (5 ml) was kept at room temperature for 4 hours and reduced pressure Evaporated, filtered, collected the precipitated solid, washed with water, heated to 60°C with absolute ethanol to dissolve, and then cooled to room temperature for recrystallization to obtain compound 15b as a yellow solid with a yield of 60.4%.
mp 260℃.IR(KBr):3446,3223,3094,1713,1619,1397,1280,1144cm -1. 1H NMR(400MHz,DMSO)δ14.93(s,1H,COOH),13.59(s,1H,NH),8.95(s,1H,C＝C-H),8.73(s,1H,Ar-H),8.54(d,J＝1.6Hz,2H,Ar-H),7.98(d,J＝2.7Hz,1H,Ar-H),7.89(d,J＝9.1Hz,1H,Ar-H),7.71(dd,J＝9.1,2.8Hz,1H,Ar-H). 13C NMR(101MHz,DMSO)δ177.97(s),166.43(s),146.61(s),146.13(s),139.11(s),136.96(s),132.95(s),132.61(s),132.26(s),128.88(s),126.79,125.81(s),124.07(s),123.06(s),121.35(s),118.52(s),108.21(s),[M+H] +；MS(ESI)m/z 482.0[M+H]+；HRMS(ESI)calcd for C 18H 10F 6NO 6S[M+H] +482.0129,found 482.0128. mp 260℃.IR(KBr):3446,3223,3094,1713,1619,1397,1280,1144cm -1 . 1 H NMR(400MHz,DMSO)δ14.93(s,1H,COOH),13.59(s, 1H, NH), 8.95(s, 1H, C=CH), 8.73(s, 1H, Ar-H), 8.54(d, J=1.6Hz, 2H, Ar-H), 7.98(d, J=2.7 Hz, 1H, Ar-H), 7.89(d, J=9.1Hz, 1H, Ar-H), 7.71(dd, J=9.1, 2.8Hz, 1H, Ar-H). 13 C NMR (101MHz, DMSO ( s), 126.79, 125.81(s), 124.07(s), 123.06(s), 121.35(s), 118.52(s), 108.21(s), [M+H] + ; MS(ESI) m/z 482.0 [M+H]+; HRMS(ESI) calcd for C 18 H 10 F 6 NO 6 S[M+H] + 482.0129, found 482.0128.
实施例29Example 29
6-(((3,5-双(三氟甲基)苯基)乙酰基)氧基)-4-氧代-1,4-二氢喹啉-3-羧酸(化合物15c)的合成Synthesis of 6-(((3,5-bis(trifluoromethyl)phenyl)acetyl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 15c)
Figure PCTCN2022088657-appb-000015
Figure PCTCN2022088657-appb-000015
参照实施例28化合物15b的制备方法，把3,5-双(三氟甲基)苯磺酰氯换成3,5-双(三氟甲基)苯甲酰氯，得到化合物15c，白色固体，产率为62.1％。Referring to the preparation method of compound 15b in Example 28, 3,5-bis(trifluoromethyl)benzenesulfonyl chloride was replaced with 3,5-bis(trifluoromethyl)benzoyl chloride to obtain compound 15c as a white solid, producing The rate is 62.1%.
mp 298-301℃.IR(KBr):3444,3228,3066,1752,1693,1397,1485,1290,1279cm -1. 1H NMR(400MHz,DMSO)δ15.39(s,1H,COOH),8.95(s,1H,C＝CH),8.70(s,2H,Ar-H),8.58(s,1H,Ar-H),8.27(d,J＝1.8Hz,1H,Ar-H),7.92(s,2H,Ar-H). 13C NMR(101MHz,DMSO)13C NMR(101MHz,DMSO-d6)δ177.95(s),167.04(s),162.84(s),148.21(s),146.51(s),139.08(s),132.31(s),131.64(s),131.30(s),130.81(s),128.68(s),127.91(s),125.75(s),124.69(s),122.73(s),121.98(s),117.56(s),107.92(s).MS(ESI)m/z 446.1[M+H] +；HRMS(ESI)calcd for C 19H 9F 6NO 5[M+H] +446.0457,found 446.0458. mp 298-301℃.IR(KBr):3444,3228,3066,1752,1693,1397,1485,1290,1279cm -1 . 1 H NMR(400MHz,DMSO)δ15.39(s,1H,COOH), 8.95(s,1H,C=CH),8.70(s,2H,Ar-H),8.58(s,1H,Ar-H),8.27(d,J=1.8Hz,1H,Ar-H),7.92 (s,2H,Ar-H) .13C NMR(101MHz,DMSO)13C NMR(101MHz,DMSO-d6)δ177.95(s),167.04(s),162.84(s),148.21(s),146.51 (s), 139.08(s), 132.31(s), 131.64(s), 131.30(s), 130.81(s), 128.68(s), 127.91(s), 125.75(s), 124.69(s), 122.73 (s), 121.98(s), 117.56(s), 107.92(s). MS(ESI) m/z 446.1[M+H] + ; HRMS(ESI) calcd for C 19 H 9 F 6 NO 5 [M +H] + 446.0457,found 446.0458.
实施例30Example 30
Figure PCTCN2022088657-appb-000016
Figure PCTCN2022088657-appb-000016
2-[(2-苄氧基-苯基氨基)-亚甲基]-丙二酸二乙酯(化合物4b)合成Synthesis of 2-[(2-benzyloxy-phenylamino)-methylene]-diethyl malonate (compound 4b)
将化合物3(1.52g，10mmol)溶于10mL乙腈中，加入碳酸钾(1.65g，12mmol)，室温搅拌1小时，加入3,5-双三氟甲基苄基溴(化合物4a，1.3mL，11mmol)，回流10小时。反应结束后，减压旋蒸除去乙腈，水洗，过滤，沉淀即为化合物4b，白色絮状物，产率为94％。Dissolve compound 3 (1.52g, 10mmol) in 10mL of acetonitrile, add potassium carbonate (1.65g, 12mmol), stir at room temperature for 1 hour, add 3,5-bistrifluoromethylbenzyl bromide (compound 4a, 1.3mL, 11mmol), refluxed for 10 hours. After the reaction was completed, the acetonitrile was removed by rotary evaporation under reduced pressure, washed with water, filtered, and compound 4b was precipitated as a white floc with a yield of 94%.
1H NMR(500MHz,DMSO)δ10.69(d,J＝13.2Hz,1H,NH),8.32(d,J＝13.4Hz,1H,CH),8.16(s,2H,Ar-H),8.08(s,1H,Ar-H),7.34(d,J＝9.0Hz,2H,Ar-H),7.09(d,J＝9.0Hz,2H,Ar-H),5.31(s,2H,CH 2),4.19(dd,J＝14.0,7.0Hz,2H,CH 2),4.11(q,J＝7.0Hz,2H,CH 2),1.25(t,J＝7.0Hz,3H,CH 3),1.23(t,J＝7.0Hz,3H,CH 3). 1 H NMR (500MHz, DMSO) δ10.69 (d, J = 13.2Hz, 1H, NH), 8.32 (d, J = 13.4Hz, 1H, CH), 8.16 (s, 2H, Ar-H), 8.08 (s,1H,Ar-H),7.34(d,J=9.0Hz,2H,Ar-H),7.09(d,J=9.0Hz,2H,Ar-H),5.31(s,2H,CH 2 ), 4.19 (dd, J=14.0, 7.0Hz, 2H, CH 2 ), 4.11 (q, J=7.0Hz, 2H, CH 2 ), 1.25 (t, J=7.0Hz, 3H, CH 3 ), 1.23 (t,J=7.0Hz,3H,CH 3 ).
6-(((3,5-双(三氟甲基)苯基))氧基)--4-氧代-1,4-二氢喹啉-3-羧酸乙酯(化合物5b)合成Synthesis of 6-(((3,5-bis(trifluoromethyl)phenyl))oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl ester (compound 5b)
将化合物4b(0.756g，3.50mmol)加入二苯醚(4mL)中，240℃回流1小时，然后冷却至室温，加入石油醚，待析出晶体，收集晶体并用大量石油醚洗涤；固体加入5mL丙酮加热至60℃溶解，随后冷却到室温，待析出沉淀，过滤，得到白色固体，即为化合物5b，产率为55％。Compound 4b (0.756g, 3.50mmol) was added to diphenyl ether (4mL), refluxed at 240°C for 1 hour, then cooled to room temperature, and petroleum ether was added to precipitate crystals, which were collected and washed with a large amount of petroleum ether; the solid was added to 5mL of acetone Heat to 60° C. to dissolve, then cool to room temperature, wait for precipitation and filter to obtain a white solid, which is compound 5b, with a yield of 55%.
mp 272℃.IR(KBr):3413,3157,3078,1817,1396,1277cm-1.1H NMR(400MHz,DMSO-d6)δ12.35(s,1H，NH),8.51(d,J＝4.4Hz,1H，C＝CH),8.24(s,2H,Ar-H),8.12(s,1H,Ar-H),7.70(d,J＝3.0Hz,1H,Ar-H),7.63(d,J＝9.0Hz,1H,Ar-H),7.49(dd,J＝9.1,2.9Hz,1H,Ar-H),5.42(s,2H,CH 2),4.22(q,J＝7.1Hz,2H,CH 2),1.28(t,J＝7.1Hz,3H,CH 3). 13C NMR(101MHz,DMSO-d6)δ173.29(s),155.67(s),144.30(s),140.87(s),134.23(s),130.98(s),130.66(s),128.89(s),123.26(s),121.20(s),109.26(s),107.23(s),68.43(s),59.98(s),14.81(s).MS(ESI)m/z 460.1[M+H] +；HRMS(ESI)calcd for C 21H 15F 6NNaO 4[M+H] +482.0797,found482.0799. mp 272℃.IR(KBr):3413,3157,3078,1817,1396,1277cm-1.1H NMR(400MHz,DMSO-d6)δ12.35(s,1H,NH),8.51(d,J＝4.4Hz ,1H,C=CH),8.24(s,2H,Ar-H),8.12(s,1H,Ar-H),7.70(d,J=3.0Hz,1H,Ar-H),7.63(d, J=9.0Hz, 1H, Ar-H), 7.49(dd, J=9.1, 2.9Hz, 1H, Ar-H), 5.42(s, 2H, CH 2 ), 4.22(q, J=7.1Hz, 2H , CH 2 ), 1.28(t, J=7.1Hz, 3H, CH 3 ). 13 C NMR (101MHz, DMSO-d6) δ173.29(s), 155.67(s), 144.30(s), 140.87(s ),134.23(s),130.98(s),130.66(s),128.89(s),123.26(s),121.20(s),109.26(s),107.23(s),68.43(s),59.98(s ), 14.81(s). MS(ESI) m/z 460.1[M+H] + ; HRMS(ESI) calcd for C 21 H 15 F 6 NNaO 4 [M+H] + 482.0797, found 482.0799.
6-(((3,5-双(三氟甲基)苯基))氧基)-1-甲基-4-氧代-1,4-二氢喹啉-3-羧酸乙酯(化合物16a)的合成6-(((3,5-bis(trifluoromethyl)phenyl))oxy)-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl ester ( Synthesis of compound 16a)
Figure PCTCN2022088657-appb-000017
Figure PCTCN2022088657-appb-000017
将化合物5b(1mmol)、碳酸铯(0.2mmol)和碘甲烷(1.1mmol)加入N,N-二甲基甲酰胺(5mL)中，60℃反应4小时，反应结束后，加入10mL水，乙酸乙酯萃取3次，收集有机层，减压蒸去乙酸乙酯，硅胶柱层析(300-400目硅胶，洗脱剂为二氯甲烷:甲醇＝15:1V/V)，纯化得到化合物16a，白色固体，产率为67.2％。Add compound 5b (1mmol), cesium carbonate (0.2mmol) and iodomethane (1.1mmol) into N,N-dimethylformamide (5mL), react at 60°C for 4 hours, after the reaction, add 10mL of water, acetic acid Ethyl extraction was performed 3 times, the organic layer was collected, ethyl acetate was evaporated under reduced pressure, silica gel column chromatography (300-400 mesh silica gel, eluent was dichloromethane:methanol=15:1V/V), purified to obtain compound 16a , white solid, yield 67.2%.
1H NMR(300MHz,DMSO-d6)δ8.64(s,1H,C＝CH),8.25(s,2H,Ar-H),8.12(s,1H,Ar-H),7.83–7.72(m,2H,Ar-H),7.58(dd,J＝9.2,3.0Hz,1H,Ar-H),5.45(s,2H,CH 2),4.24(t,J＝7.1Hz,2H,CH 2),3.94(s,3H,CH 3),1.29(t,J＝7.1Hz,3H,CH 3). 1 H NMR (300MHz, DMSO-d6) δ8.64(s,1H,C=CH),8.25(s,2H,Ar-H),8.12(s,1H,Ar-H),7.83–7.72(m ,2H,Ar-H),7.58(dd,J=9.2,3.0Hz,1H,Ar-H),5.45(s,2H,CH 2 ),4.24(t,J=7.1Hz,2H,CH 2 ) ,3.94(s,3H,CH 3 ),1.29(t,J=7.1Hz,3H,CH 3 ).
6-(((3,5-双(三氟甲基)苯基))氧基)-1-甲基-4-氧代-1,4-二氢喹啉-3-羧酸(化合物17a)的合成6-(((3,5-bis(trifluoromethyl)phenyl))oxy)-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 17a )Synthesis
Figure PCTCN2022088657-appb-000018
Figure PCTCN2022088657-appb-000018
参照实施例20化合物12a的制备方法，得到化合物17a，白色固体，产率为68.2％。Referring to the preparation method of compound 12a in Example 20, compound 17a was obtained as a white solid with a yield of 68.2%.
mp>350℃.IR(KBr):3435,3047,1726,1626,1518cm -1. 1H NMR(400MHz,DMSO-d6)δ15.39(s,1H，COOH),8.99(s,1H，C＝CH),8.27(s,2H，Ar-H),8.14(s,1H，Ar-H),7.99(d,J＝9.4Hz,1H，Ar-H),7.88(d,J＝3.0Hz,1H，Ar-H),7.77(dd,J＝9.3,3.0Hz,1H，Ar-H),5.51(s,2H，CH 2),4.12(s,3H,CH 3). 13C NMR(101MHz,DMSO-d6)δ177.43(s),166.74(s),156.58(s),149.09(s),140.54(s),135.78(s),131.02(s),128.83(s),126.97(s),125.13(s),124.93(s),120.89(s),107.28(s),106.85(s),68.62(s),46.14(s),42.32(s).MS(ESI) m/z 446.1[M+H] +；HRMS(ESI)calcd for C 20H 13F 6NNaO 4[M+Na] +468.0641,found 468.0638. mp>350℃.IR(KBr):3435,3047,1726,1626,1518cm -1 . 1 H NMR(400MHz,DMSO-d6)δ15.39(s,1H,COOH),8.99(s,1H,C =CH), 8.27(s, 2H, Ar-H), 8.14(s, 1H, Ar-H), 7.99(d, J=9.4Hz, 1H, Ar-H), 7.88(d, J=3.0Hz , 1H, Ar-H), 7.77 (dd, J=9.3, 3.0Hz, 1H, Ar-H), 5.51 (s, 2H, CH 2 ), 4.12 (s, 3H, CH 3 ). 13 C NMR ( 101MHz,DMSO-d6)δ177.43(s),166.74(s),156.58(s),149.09(s),140.54(s),135.78(s),131.02(s),128.83(s),126.97( s),125.13(s),124.93(s),120.89(s),107.28(s),106.85(s),68.62(s),46.14(s),42.32(s).MS(ESI) m/z 446.1[M+H] + ; HRMS(ESI) calcd for C 20 H 13 F 6 NNaO 4 [M+Na] + 468.0641, found 468.0638.
实施例31Example 31
6-(((3,5-双(三氟甲基)苯基))氧基)-1-乙基-4-氧代-1,4-二氢喹啉-3-羧酸乙酯(化合物16b)的合成6-(((3,5-bis(trifluoromethyl)phenyl))oxy)-1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl ester ( Synthesis of compound 16b)
Figure PCTCN2022088657-appb-000019
Figure PCTCN2022088657-appb-000019
参照实施例30化合物16a的制备方法，由碘乙烷替换碘甲烷，得到化合物16b，白色固体，产率68.7％。Referring to the preparation method of compound 16a in Example 30, methyl iodide was replaced by ethyl iodide to obtain compound 16b as a white solid with a yield of 68.7%.
1H NMR(300MHz,DMSO-d 6)δ8.65(s,1H,C＝CH),8.25(d,J＝1.7Hz,2H,Ar-H),8.12(s,1H,Ar-H),7.89–7.78(m,2H,Ar-H),7.56(dd,J＝9.2,3.1Hz,1H,Ar-H),5.45(s,2H,CH 2),4.42(q,J＝7.1Hz,2H,CH 2),4.23(q,J＝7.1Hz,2H,CH 2),1.37(t,J＝7.0Hz,3H,CH 3),1.29(t,J＝7.1Hz,3H,CH 3). 1 H NMR (300MHz, DMSO-d 6 )δ8.65(s, 1H, C=CH), 8.25(d, J=1.7Hz, 2H, Ar-H), 8.12(s, 1H, Ar-H) ,7.89–7.78(m,2H,Ar-H),7.56(dd,J＝9.2,3.1Hz,1H,Ar-H),5.45(s,2H,CH 2 ),4.42(q,J＝7.1Hz ,2H,CH 2 ), 4.23(q,J=7.1Hz,2H,CH 2 ),1.37(t,J=7.0Hz,3H,CH 3 ),1.29(t,J=7.1Hz,3H,CH 3 ).
6-(((3,5-双(三氟甲基)苯基))氧基)-1-乙基-4-氧代-1,4-二氢喹啉-3-羧酸(化合物17b)的合成6-(((3,5-bis(trifluoromethyl)phenyl))oxy)-1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 17b )Synthesis
Figure PCTCN2022088657-appb-000020
Figure PCTCN2022088657-appb-000020
参照实施例20化合物12a的制备方法，得到化合物17b，白色固体，产率为60.8％。Referring to the preparation method of compound 12a in Example 20, compound 17b was obtained as a white solid with a yield of 60.8%.
mp 265-268℃.IR(KBr):3430,3052,2978,1708,1616,1395cm -1. 1H NMR(400MHz,DMSO-d6)δ15.37(s,1H，COOH),9.01(s,1H，C＝CH),8.27(d,J＝1.7Hz,2H，Ar-H),8.13(s,1H，Ar-H),8.08(d,J＝9.4Hz,1H，Ar-H),7.90(d,J＝3.0Hz,1H，Ar-H),7.75(dd,J＝9.4,3.0Hz,1H，Ar-H),5.51(s,2H，CH 2),4.62(q,J＝7.1Hz,2H，CH 2),1.42(t,J＝7.1Hz,3H，CH 3). 13C NMR(101MHz,DMSO-d6)δ177.37(s),166.69(s),156.47(s),148.13(s),140.54(s),134.48(s),131.03(s),130.70(s),128.88(s),127.41(s),125.12(s),122.42(s),122.28(s),120.73(s),107.61(s),107.13(s),68.65(s),49.65(s),15.21(s).MS(ESI)m/z 460.1[M+H] +；HRMS(ESI)calcd for C 21H 15F 6NNaO 4[M+Na] +482.0797,found 482.0796. mp 265-268℃.IR(KBr):3430,3052,2978,1708,1616,1395cm -1 . 1 H NMR(400MHz,DMSO-d6)δ15.37(s,1H,COOH),9.01(s, 1H, C=CH), 8.27(d, J=1.7Hz, 2H, Ar-H), 8.13(s, 1H, Ar-H), 8.08(d, J=9.4Hz, 1H, Ar-H), 7.90(d, J=3.0Hz, 1H, Ar-H), 7.75(dd, J=9.4, 3.0Hz, 1H, Ar-H), 5.51(s, 2H, CH 2 ), 4.62(q, J= 7.1Hz, 2H, CH 2 ), 1.42(t, J=7.1Hz, 3H, CH 3 ). 13 C NMR (101MHz, DMSO-d6) δ177.37(s), 166.69(s), 156.47(s) ,148.13(s),140.54(s),134.48(s),131.03(s),130.70(s),128.88(s),127.41(s),125.12(s),122.42(s),122.28(s) ,120.73(s),107.61(s),107.13(s),68.65(s),49.65(s),15.21(s).MS(ESI) m/z 460.1[M+H] + ; HRMS(ESI) calcd for C 21 H 15 F 6 NNaO 4 [M+Na] + 482.0797, found 482.0796.
实施例32Example 32
6-(((3,5-双(三氟甲基)苯基))氧基)-1-异丙基-4-氧代-1,4-二氢喹啉-3-羧酸乙酯(化合物16c)的合成6-(((3,5-bis(trifluoromethyl)phenyl))oxy)-1-isopropyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl ester Synthesis of (Compound 16c)
Figure PCTCN2022088657-appb-000021
Figure PCTCN2022088657-appb-000021
参照实施例30化合物16a的制备方法，由2-碘代丙烷替换碘甲烷，得到化合物16c，白色固体，产率为60％。Referring to the preparation method of compound 16a in Example 30, 2-iodopropane was used to replace methyl iodide to obtain compound 16c as a white solid with a yield of 60%.
1H NMR(300MHz,DMSO-d 6)δ8.57(s,1H,C＝CH),8.25(s,2H,Ar-H),8.12(s,1H,Ar-H),7.99(d,J＝9.5Hz,1H,Ar-H),7.84(d,J＝3.1Hz,1H,Ar-H),7.57(dd,J＝9.3,3.1Hz,1H,Ar-H),5.45(s,2H,CH 2),5.09(p,J＝6.6Hz,1H,CH),4.24(q,J＝7.1Hz,2H,CH 2),1.51(d,J＝6.4Hz,6H,2CH 3),1.29(t,J＝7.1Hz,3H,CH 3). 1 H NMR (300MHz, DMSO-d 6 )δ8.57(s,1H,C=CH),8.25(s,2H,Ar-H),8.12(s,1H,Ar-H),7.99(d, J＝9.5Hz, 1H, Ar-H), 7.84(d, J＝3.1Hz, 1H, Ar-H), 7.57(dd, J＝9.3, 3.1Hz, 1H, Ar-H), 5.45(s, 2H, CH 2 ), 5.09 (p, J=6.6Hz, 1H, CH), 4.24 (q, J=7.1Hz, 2H, CH 2 ), 1.51 (d, J=6.4Hz, 6H, 2CH 3 ), 1.29(t,J=7.1Hz,3H,CH 3 ).
6-(((3,5-双(三氟甲基)苯基))氧基)-1-异丙基-4-氧代-1,4-二氢喹啉-3-羧酸(化合物17c)的合成6-(((3,5-bis(trifluoromethyl)phenyl))oxy)-1-isopropyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 17c) Synthesis
Figure PCTCN2022088657-appb-000022
Figure PCTCN2022088657-appb-000022
参照实施例20化合物12a的制备方法，得到化合物17c，白色固体，产率为63.4％。Referring to the preparation method of compound 12a in Example 20, compound 17c was obtained as a white solid with a yield of 63.4%.
mp 260-265℃.IR(KBr):3434,2975,2677,1710,1508cm -1.(300MHz,DMSO-d 6)δ15.37(s,1H,COOH),9.01(s,1H,C＝CH),8.27(s,2H,Ar-H),8.13(s,1H,Ar-H),8.08(d,J＝9.4Hz,1H,Ar-H),7.90(d,J＝3.0Hz,1H,Ar-H),7.75(dd,J＝9.4,3.0Hz,1H,Ar-H),5.51(s,2H,CH 2),4.62(q,J＝7.1Hz,2H,CH 2),1.42(t,J＝7.1Hz,3H,CH 3). 1H NMR(400MHz,DMSO-d6)δ9.15(s,1H,C＝CH),8.28(d,J＝1.8Hz,2H,Ar-H),8.22 (d,J＝9.3Hz,1H,Ar-H),8.13(s,1H,Ar-H),7.88(dd,J＝9.2,2.8Hz,1H,Ar-H),7.67(d,J＝2.8Hz,1H,Ar-H),5.59(s,2H,CH 2),,4.88(p,J＝6.1Hz,1H,CH),1.31(s,6H,2xCH 3). 13C NMR(101MHz,DMSO-d6)δ165.48(s),164.79(s),157.71(s),147.44(s),140.50(s),131.12(s),130.80(s),128.80(s),127.15(s),126.45(s),126.30(s),125.10(s),122.39(s),122.31(s),116.51(s),103.99(s),81.79(s),68.77(s),22.52(s).MS(ESI)m/z 474.1[M+H] +；HRMS(ESI)calcd for C 22H 18F 6NO 4[M+H] +474.1135,found 474.1135. mp 260-265℃.IR(KBr):3434,2975,2677,1710,1508cm -1 .(300MHz,DMSO-d 6 )δ15.37(s,1H,COOH),9.01(s,1H,C= CH),8.27(s,2H,Ar-H),8.13(s,1H,Ar-H),8.08(d,J=9.4Hz,1H,Ar-H),7.90(d,J=3.0Hz, 1H, Ar-H), 7.75(dd, J=9.4, 3.0Hz, 1H, Ar-H), 5.51(s, 2H, CH 2 ), 4.62(q, J=7.1Hz, 2H, CH 2 ), 1.42(t, J=7.1Hz, 3H, CH 3 ). 1 H NMR (400MHz, DMSO-d6) δ9.15(s, 1H, C=CH), 8.28(d, J=1.8Hz, 2H, Ar -H), 8.22 (d, J=9.3Hz, 1H, Ar-H), 8.13 (s, 1H, Ar-H), 7.88 (dd, J=9.2, 2.8Hz, 1H, Ar-H), 7.67 (d,J=2.8Hz,1H,Ar-H),5.59(s,2H,CH 2 ),,4.88(p,J=6.1Hz,1H,CH),1.31(s,6H,2xCH 3 ). 13 C NMR (101MHz, DMSO-d6) δ165.48(s), 164.79(s), 157.71(s), 147.44(s), 140.50(s), 131.12(s), 130.80(s), 128.80(s ),127.15(s),126.45(s),126.30(s),125.10(s),122.39(s),122.31(s),116.51(s),103.99(s),81.79(s),68.77(s ), 22.52(s). MS(ESI) m/z 474.1[M+H] + ; HRMS(ESI) calcd for C 22 H 18 F 6 NO 4 [M+H] + 474.1135, found 474.1135.
实施例33Example 33
6-(((3,5-双(三氟甲基)苯基))氧基)-1-羟基-4-氧代-1,4-二氢喹啉-3-羧酸乙酯(化合物16d)的合成6-(((3,5-bis(trifluoromethyl)phenyl))oxy)-1-hydroxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl ester (compound 16d) Synthesis
Figure PCTCN2022088657-appb-000023
Figure PCTCN2022088657-appb-000023
参照实施例30化合物16a的制备方法，由2-碘乙醇替换碘甲烷，得到化合物16d，白色固体，产率为62％。Referring to the preparation method of compound 16a in Example 30, 2-iodoethanol was substituted for methyl iodide to obtain compound 16d as a white solid with a yield of 62%.
1H NMR(400MHz,DMSO-d 6)δ8.54(s,1H，C＝CH),8.24(d,J＝1.7Hz,2H,Ar-H),8.12(s,1H,Ar-H),7.88–7.79(m,2H,Ar-H),7.53(dd,J＝9.3,3.1Hz,1H,Ar-H),5.44(s,2H,CH 2),5.03(t,J＝5.4Hz,1H,OH),4.43(t,J＝4.9Hz,2H,CH 2),4.23(q,J＝7.1Hz,2H,CH 2),3.73(q,J＝5.8,5.2Hz,2H,CH 2),1.31–1.27(t,3H,CH 3). 1 H NMR (400MHz, DMSO-d 6 ) δ8.54(s, 1H, C=CH), 8.24(d, J=1.7Hz, 2H, Ar-H), 8.12(s, 1H, Ar-H) ,7.88–7.79(m,2H,Ar-H),7.53(dd,J＝9.3,3.1Hz,1H,Ar-H),5.44(s,2H,CH 2 ),5.03(t,J＝5.4Hz ,1H,OH),4.43(t,J=4.9Hz,2H,CH 2 ),4.23(q,J=7.1Hz,2H,CH 2 ),3.73(q,J=5.8,5.2Hz,2H,CH 2 ),1.31–1.27(t,3H,CH 3 ).
6-(((3,5-双(三氟甲基)苯基))氧基)-1-羟基-4-氧代-1,4-二氢喹啉-3-羧酸(化合物17d)的合成6-(((3,5-bis(trifluoromethyl)phenyl))oxy)-1-hydroxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound 17d) Synthesis
Figure PCTCN2022088657-appb-000024
Figure PCTCN2022088657-appb-000024
参照实施例20化合物12a的制备方法，得到化合物17c，白色固体，产率为66.1％。Referring to the preparation method of compound 12a in Example 20, compound 17c was obtained as a white solid with a yield of 66.1%.
mp 258-263℃.IR(KBr):3463,3365,1715,1616cm -1. 1H NMR(400MHz,DMSO-d6)δ15.40(s,1H,COOH),8.85(s,1H,C＝CH),8.27(s,2H,Ar-H),8.14–8.06(m,2H,Ar-H),7.90(d,J＝3.0Hz,1H,Ar-H),7.75–7.69(m,1H,Ar-H),5.51(s,2H,CH 2),5.05(d,J＝5.5Hz,1H,OH),4.65(t,J＝4.9Hz,2H,CH 2),3.77(q,J＝5.1Hz,2H,CH 2). 13C NMR(101MHz,DMSO-d6)δ177.49(s),166.81,156.43(s),149.34(s),140.56(s),134.89(s),131.03(s),130.70(s),128.85(s),127.38(s),125.14(s),124.86(s),122.26(s),120.86(s),107.06(s),107.00(s),68.64(s),59.07(s),56.61(s).MS(ESI)m/z 476.1[M+H] +；HRMS(ESI)calcd for C 21H 15F 6NNaO 4[M+Na] +498.0747,found498.0749. mp 258-263℃.IR(KBr):3463,3365,1715,1616cm -1 . 1 H NMR(400MHz,DMSO-d6)δ15.40(s,1H,COOH),8.85(s,1H,C= CH),8.27(s,2H,Ar-H),8.14–8.06(m,2H,Ar-H),7.90(d,J＝3.0Hz,1H,Ar-H),7.75–7.69(m,1H ,Ar-H),5.51(s,2H,CH 2 ),5.05(d,J=5.5Hz,1H,OH),4.65(t,J=4.9Hz,2H,CH 2 ),3.77(q,J =5.1Hz, 2H, CH 2 ). 13 C NMR (101MHz, DMSO-d6) δ177.49(s), 166.81, 156.43(s), 149.34(s), 140.56(s), 134.89(s), 131.03 (s), 130.70(s), 128.85(s), 127.38(s), 125.14(s), 124.86(s), 122.26(s), 120.86(s), 107.06(s), 107.00(s), 68.64 (s), 59.07(s), 56.61(s). MS(ESI) m/z 476.1[M+H] + ; HRMS(ESI) calcd for C 21 H 15 F 6 NNaO 4 [M+Na] + 498.0747 , found 498.0749.
实施例34Example 34
化合物7a-7r，12a-12g，15a-c、17a-d、7da、5b以及阳性对照药BP-1-102的抗肿瘤细胞增殖活性评价Anti-tumor cell proliferation activity evaluation of compounds 7a-7r, 12a-12g, 15a-c, 17a-d, 7da, 5b and positive control drug BP-1-102
1.1细胞系培养和细胞活力测定1.1 Cell line culture and cell viability assay
所有细胞系均购自中国科学院上海生化与细胞生物学研究所细胞库(中国上海)。人结肠癌细胞(HT-29)在DMEM培养基中培养；非小细胞肺癌细胞(A549)、人骨肉瘤细胞(U2OS)、人乳腺癌细胞(MDA-MB-231和MDA-MB-468)四种细胞在RPMI-1640培养基中培养。所有细胞培养基都含有50μg/mL青霉素、50μg/mL链霉素、10％胎牛血清。细胞在组织培养瓶中于37℃、含5％CO 2的湿润环境中生长至80％汇合，然后用1×Trypsin-Versene进行胰蛋白酶消化并分裂。 All cell lines were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human colon cancer cells (HT-29) were cultured in DMEM medium; non-small cell lung cancer cells (A549), human osteosarcoma cells (U2OS), human breast cancer cells (MDA-MB-231 and MDA-MB-468) four The cells were cultured in RPMI-1640 medium. All cell culture media contained 50 μg/mL penicillin, 50 μg/mL streptomycin, 10% fetal bovine serum. Cells were grown to 80% confluency in tissue culture flasks at 37°C in a humidified atmosphere with 5% CO2 , then trypsinized with 1× Trypsin-Versene and split.
癌细胞(MDA-MB-231、MDA-MB-468、U2OS、A549、HT-29)以每孔4000-6000个细胞的密度接种在96孔板中，在37℃、加湿的5％CO 2培养箱中孵育过夜；去除培养基，采用生物级的99.9％DMSO配制不同浓度(5、10μM)的待测化合物，随后一式三份加入含有3％胎牛血清的200μL新鲜培养基中(HT-29细胞采用DMEM培养基，其他细胞系采用RPMI-1640培养基)，37℃孵育72小时。培养基中DMSO的百分比不超过0.1％。添加3-(4,5-二甲基噻唑基)-2,5-二苯基溴化四唑(MTT)评估细胞活力。通过ELISA读数器(SpectraMax Plus384，Molecular Devices，Sunnyvale，CA)在570nm的测试波长和630nm的参考波长下读取吸光度。通过以下公式计算细胞活力： Cancer cells (MDA-MB-231, MDA-MB-468, U2OS, A549, HT-29) were seeded in 96-well plates at a density of 4000-6000 cells per well at 37°C in a humidified 5% CO2 Incubate overnight in the incubator; remove the medium, use biological grade 99.9% DMSO to prepare different concentrations (5, 10 μM) of the compounds to be tested, and then add them in triplicate to 200 μL of fresh medium containing 3% fetal bovine serum (HT- 29 cells use DMEM medium, other cell lines use RPMI-1640 medium), and incubate at 37°C for 72 hours. The percentage of DMSO in the medium does not exceed 0.1%. Cell viability was assessed by addition of 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT). Absorbance was read by an ELISA reader (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA) at a test wavelength of 570 nm and a reference wavelength of 630 nm. Cell viability was calculated by the following formula:
细胞活力＝(At/As)×100％；其中，At和as分别表示待测化合物和溶剂对照的吸光度。Cell viability=(At/As)×100%; wherein, At and as represent the absorbance of the test compound and the solvent control, respectively.
结果见图1和图2。图1为所有合成化合物在(10μM)下抑制A549细胞增殖活性初筛，阳性对照 为BP-1-102，大多数化合物抑制活性优于BP-1-102。图2为初筛活性较优的合成化合物在(5μM)下抑制A549细胞增殖活性复筛。The results are shown in Figures 1 and 2. Figure 1 is a preliminary screening of all synthetic compounds inhibiting the proliferation of A549 cells at (10 μM). The positive control is BP-1-102, and most of the compounds have better inhibitory activity than BP-1-102. Figure 2 is a re-screening of the activity of synthetic compounds with better primary screening activity in inhibiting the proliferation of A549 cells at (5 μM).
1.2采用MTT法测试化合物7d对肿瘤细胞的抑制活性1.2 MTT method was used to test the inhibitory activity of compound 7d on tumor cells
MTT实验：细胞培养按照实施例34 1.1进行培养，将MDA-MB-231,MDA-MB-468,U2OS,A549,HT-29细胞传代后计数，按照6000个/孔的密度配制细胞悬浮液，混匀后，每孔吸取100μL细胞悬浮液接种到96孔板中，贴壁过夜，次日细胞给药，采用生物级别的99.9％DMSO配制10mMOL的7d化合物母液，用对应的培养基稀释化合物，终浓度分别为0、0.125、0.25、0.5、1、2、4、8、16、32μM共10个浓度梯度，吸出96孔板中的细胞培养液，按每孔100μL加入含不同浓度化合物7d的培养基到96孔板中，孵育24小时。然后每孔加入10μLMTT，避光孵育4小时。结束后取出96孔板，小心吸去上清后，每孔加入150μLDMSO复溶，荧光酶标仪测量每孔的吸光值，检测波长为570nm，参照波长为630nm。MTT experiment: Cell culture was carried out according to Example 34 1.1, MDA-MB-231, MDA-MB-468, U2OS, A549, HT-29 cells were counted after passage, and cell suspension was prepared according to the density of 6000 cells/well. After mixing, pipette 100 μL of cell suspension into a 96-well plate, adhere to the wall overnight, and administer the cells the next day. Prepare 10 mMOL of the 7d compound stock solution with biological grade 99.9% DMSO, and dilute the compound with the corresponding medium. The final concentrations were 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM, a total of 10 concentration gradients. The cell culture solution in the 96-well plate was aspirated, and 100 μL of compound 7d containing different concentrations were added to each well. Culture medium into 96-well plate and incubate for 24 hours. Then add 10 μL MTT to each well and incubate for 4 hours in the dark. After the end, take out the 96-well plate, carefully absorb the supernatant, add 150 μ LDMSO to each well to redissolve, measure the absorbance value of each well with a fluorescent microplate reader, the detection wavelength is 570nm, and the reference wavelength is 630nm.
肿瘤细胞的生长抑制率：增殖抑制率(％)＝(1﹣A 实验组/A 对照组)×100％。 Growth inhibition rate of tumor cells: proliferation inhibition rate (%)=(1—A experimental group /A control group )×100%.
表1为复筛后的化合物对A549细胞的抑制活性结果，其中化合物7a、7d、7h、12d、12f抑制活性显著，显著优于阳性药BP-1-102；而化合物7da显示去除喹诺酮母核的羧基后，活性显著下降。Table 1 shows the results of the inhibitory activity of the compounds after re-screening on A549 cells, among which compounds 7a, 7d, 7h, 12d, and 12f have significant inhibitory activity, which is significantly better than the positive drug BP-1-102; and compound 7da shows the removal of quinolone core After the carboxyl group, the activity decreased significantly.
采用HT-29细胞对化合物7a、7d、7h、12d、12f进行进一步活性筛选，见表2，化合物7d在候选化合物中活性最佳。 Compounds 7a, 7d, 7h, 12d, and 12f were further screened for activity using HT-29 cells, as shown in Table 2. Compound 7d had the best activity among the candidate compounds.
以阳性药BP-1-102作为对照，筛选化合物7d在不同细胞系中的抑制活性，结果见表3，显示化合物7d的活性显著优于阳性对照BP-1-102。The positive drug BP-1-102 was used as a control to screen the inhibitory activity of compound 7d in different cell lines. The results are shown in Table 3, which shows that the activity of compound 7d is significantly better than that of the positive control BP-1-102.
表1.化合物对A549细胞的抑制活性Table 1. Inhibitory activity of compounds on A549 cells
Figure PCTCN2022088657-appb-000025
Figure PCTCN2022088657-appb-000025
a数据是来自至少三个独立实验的平均值。 a Data are mean values from at least three independent experiments.
表2.化合物对HT-29细胞的抑制活性Table 2. Inhibitory activity of compounds on HT-29 cells
Figure PCTCN2022088657-appb-000026
Figure PCTCN2022088657-appb-000026
a数据是来自至少三个独立实验的平均值。 a Data are mean values from at least three independent experiments.
表3.化合物7d对不同的癌细胞的抑制活性Table 3. Inhibitory activity of compound 7d on different cancer cells
Figure PCTCN2022088657-appb-000027
Figure PCTCN2022088657-appb-000027
a数据是来自至少三个独立实验的平均值。 a Data are mean values from at least three independent experiments.
实施例35化合物7d抑制STAT3蛋白活Example 35 Compound 7d inhibits STAT3 protein activity
蛋白质印迹实验Western blot experiment
细胞培养见实施例34 1.1，采用99.9％生物级DMSO配制10mmol/L的7d化合物母液，选择RPMI-1640培养基稀释化合物7d至0、2.5、5、10、20μM浓度，将MDA-MB-231和A549细胞分别在含不同浓度的化合物7d(0、2.5、5、10、20μM)与0.1％DMSO的RPMI-1640培养基(培养基中添加10％(v/v)FBS、50 μg/mL青霉素和50μg/mL链霉素)中，在37℃、含有5％CO 2的湿润气氛中孵育24小时。胰蛋白酶消化后收获，细胞用1×RIPA裂解缓冲液(50mM Tris-HCl，pH 7.4，150mM NaCl，0.25％脱氧胆酸，1％NP-40，1mM EDTA和蛋白酶抑制剂配制而成)(Amresco，Solon，USA)裂解30分钟提取总蛋白质；离心后测试样品的蛋白质浓度。将等量的蛋白质与上样缓冲液混合并进行丙烯酰胺凝胶电泳(SDS-PAGE，BioRad Laboratories，Hercules，CA)，从总细胞裂解液(30至60μg/泳道)中分离出一份蛋白质，转移到NC膜(BioRad Laboratories，Hercules，CA)并特异于p-Src、Src、P-ERK、AKT、p-AKT(图3A、B)、Cleaved Caspase-3、Caspase-3、PARP、Cleaved PARP、Bcl-2、GAPDH(图4A、B)、STAT1、p-STAT1、STAT3、p-STAT3、STAT5、p-STAT5(图5A、B、C)。 See Example 34 1.1 for cell culture, use 99.9% biological grade DMSO to prepare 10mmol/L 7d compound mother solution, select RPMI-1640 medium to dilute compound 7d to 0, 2.5, 5, 10, 20μM concentration, MDA-MB-231 and A549 cells in RPMI-1640 medium containing different concentrations of compound 7d (0, 2.5, 5, 10, 20 μM) and 0.1% DMSO (medium supplemented with 10% (v/v) FBS, 50 μg/mL penicillin and 50 μg/mL streptomycin) for 24 hours at 37°C in a humidified atmosphere containing 5% CO 2 . After trypsinization, cells were harvested and cells were lysed with 1×RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA and protease inhibitors) (Amresco , Solon, USA) was lysed for 30 minutes to extract the total protein; the protein concentration of the sample was tested after centrifugation. Aliquots of protein were separated from total cell lysates (30 to 60 μg/lane) by mixing equal amounts of protein with loading buffer and performing acrylamide gel electrophoresis (SDS-PAGE, BioRad Laboratories, Hercules, CA), Transferred to NC membranes (BioRad Laboratories, Hercules, CA) and specific for p-Src, Src, P-ERK, AKT, p-AKT (Fig. 3A,B), Cleaved Caspase-3, Caspase-3, PARP, Cleaved PARP , Bcl-2, GAPDH (Fig. 4A, B), STAT1, p-STAT1, STAT3, p-STAT3, STAT5, p-STAT5 (Fig. 5A, B, C).
随后使用TBST中的5％牛血清白蛋白(BSA)封闭膜，然后将其与一抗在4℃下孵育过夜，然后与二抗在室温下孵育2小时。使用ChemiDOC TM XRS+系统(BioRad Laboratories,Hercules,CA)检测结合的免疫复合物。 Membranes were subsequently blocked with 5% bovine serum albumin (BSA) in TBST, and then incubated with primary antibodies overnight at 4°C, followed by secondary antibody incubation for 2 hours at room temperature. Bound immune complexes were detected using the ChemiDOC ™ XRS+ system (BioRad Laboratories, Hercules, CA).
图3A(MDA-MB-231细胞)、B(A549细胞)显示，使用GAPDH作对照，p-Src、p-AKT、p-ERK的相对表达水平不受化合物7d影响，说明化合物7d对STAT3相关激酶影响较小。Figure 3A (MDA-MB-231 cells), B (A549 cells) show that using GAPDH as a control, the relative expression levels of p-Src, p-AKT, and p-ERK are not affected by compound 7d, indicating that compound 7d is related to STAT3 Kinases are less affected.
图4A(MDA-MB-231细胞)、B(A549细胞)显示，使用GAPDH作为对照，在化合物7d作用下，细胞系Cleaved Caspase-3、Cleaved PARP、Bcl-2表达明显增加，说明化合物7d能够诱导细胞凋亡。Figure 4A (MDA-MB-231 cells), B (A549 cells) show that using GAPDH as a control, the expression of Cleaved Caspase-3, Cleaved PARP, and Bcl-2 in cell lines significantly increased under the action of compound 7d, indicating that compound 7d can Induces apoptosis.
图5A(MDA-MB-231细胞)、B(A549细胞)、C(MDA-MB-231细胞)显示，使用GAPDH作为对照，化合物7d选择性的抑制p-STAT3的形成，对其他同家族蛋白表达水平影响较小。Figure 5A (MDA-MB-231 cells), B (A549 cells), C (MDA-MB-231 cells) show that, using GAPDH as a control, compound 7d selectively inhibits the formation of p-STAT3, and has no effect on other proteins of the same family Expression levels are less affected.
流式细胞术检测细胞凋亡Detection of apoptosis by flow cytometry
将MDA-MB-231细胞接种在六孔板中(RPMI-1640培养基)，约40万细胞/孔，贴壁过夜。次日，分别加入化合物7d至终浓度为0、5、10μM，孵育24小时；收集细胞沉淀并以200μL buffer充分重悬后，Annexin V-FITC凋亡试剂盒(Bio-Vision)的说明书操作进行处理加入5μL Annexin V-FITC和10μL碘化丙啶，室温避光孵育15分钟；然后使用BD FACSCalibur流式细胞仪(Becton&Dickinson Company，Franklin Lakes，NJ)通过流式细胞术(488nm激发和600nm发射滤光片)分析细胞。化合物7d为5μM时，MDA-MB-231细胞凋亡比例约为37％；当化合物7d为10μM时，MDA-MB-231细胞凋亡比例约为42％。结果如图4C所示，结果表明，化合物7d可以诱导MDA-MB-23细胞的凋亡。MDA-MB-231 cells were seeded in six-well plates (RPMI-1640 medium), about 400,000 cells/well, and adhered overnight. On the next day, compound 7d was added to the final concentration of 0, 5, and 10 μM, and incubated for 24 hours; after the cell pellet was collected and fully resuspended in 200 μL buffer, the instructions of the Annexin V-FITC Apoptosis Kit (Bio-Vision) were followed. Add 5 μL Annexin V-FITC and 10 μL propidium iodide, and incubate at room temperature for 15 minutes in the dark; light sheet) to analyze the cells. When compound 7d was 5 μM, the apoptotic ratio of MDA-MB-231 cells was about 37%; when compound 7d was 10 μM, the apoptotic ratio of MDA-MB-231 cells was about 42%. The results are shown in Figure 4C, and the results indicated that compound 7d could induce the apoptosis of MDA-MB-23 cells.
将A549细胞接种在六孔板中，约40万细胞/孔，贴壁过夜。次日，分别加入化合物7d至终浓度为0、1、5、10μM，孵育24小时；收集细胞沉淀并以200μL buffer充分重悬后，Annexin V-FITC凋亡试剂盒(Bio-Vision)的说明书操作进行处理加入5μL Annexin V-FITC和10μL碘化丙啶，室温避光孵育15分钟；然后使用BD FACSCalibur流式细胞仪(Becton&Dickinson Company，Franklin Lakes，NJ)通过流式细胞术(488nm激发和600nm发射滤光片)分析细胞。结果如图4D所示，化合物7d为1μM时，A549细胞凋亡比例约为16％；当化合物7d为5μM时，A549细胞凋亡比例约为54％；当化合物7d为10μM时，A549细胞凋亡比例约为80％。结果表明，化合物7d可以诱导A549细胞的凋亡。A549 cells were seeded in a six-well plate, about 400,000 cells/well, and adhered overnight. On the next day, compound 7d was added to the final concentration of 0, 1, 5, and 10 μM, and incubated for 24 hours; after the cell pellet was collected and fully resuspended in 200 μL buffer, the instructions of the Annexin V-FITC apoptosis kit (Bio-Vision) Add 5 μL Annexin V-FITC and 10 μL propidium iodide, and incubate for 15 minutes at room temperature in the dark; emission filter) to analyze the cells. The results are shown in Figure 4D, when the compound 7d was 1 μM, the apoptosis ratio of A549 cells was about 16%; when the compound 7d was 5 μM, the apoptosis ratio of A549 cells was about 54%; The death rate is about 80%. The results showed that compound 7d could induce the apoptosis of A549 cells.
免疫荧光分析Immunofluorescence analysis
MDA-MB-231细胞培养如实施例34 1.1所述，随后接种到96孔板中，使得每孔的细胞数均匀一致，且在12000-15000之间。接种完毕后，将96孔板置于细胞培养孵箱中孵育过夜，使细胞充分贴壁。配制10μM化合物7d的培养基处理24h，随后细胞在4％PFA/PBS中固定15分钟，并储存在4℃的PBS中。MDA-MB-231细胞在PBS中用0.2％TritonX-100透化20分钟，用5％牛奶，2.5％BSA，10％血清，PBS中的0.2％Triton X-100封闭30分钟。与相应的一抗4C孵化过夜随后加入二抗，在封闭缓冲液中稀释并在黑暗中孵育1小时。使用DAPI(蓝色)对细胞核进行复染，滴加DAPI避光孵育5min，对标本进行染核，PBST 5minx4次洗去多余的DAPI；之后我们在ImageXpress高内涵成像分析系统下对细胞进行荧光成像。通过免疫荧光染色分析亚细胞定位和p-STAT1(图5D)、p-STAT3(图5E)和p-STAT5(图5F)的表达。结果如图5D、E、F所示，经过化合物7d处理后，磷酸化的STAT3蛋白显著减少，而磷酸化的STAT1、STAT5蛋白变化不明显，因此化合物7d选择性抑制细胞STAT3蛋白磷酸化。MDA-MB-231 cells were cultured as described in Example 34 1.1, and then seeded into 96-well plates so that the number of cells in each well was uniform and between 12,000-15,000. After inoculation, the 96-well plate was incubated overnight in a cell culture incubator to allow the cells to fully adhere to the wall. The culture medium prepared with 10 μM compound 7d was treated for 24 hours, and then the cells were fixed in 4% PFA/PBS for 15 minutes, and stored in PBS at 4°C. MDA-MB-231 cells were permeabilized with 0.2% TritonX-100 in PBS for 20 minutes and blocked with 5% milk, 2.5% BSA, 10% serum, 0.2% Triton X-100 in PBS for 30 minutes. Incubate overnight with corresponding primary antibody 4C followed by addition of secondary antibody, diluted in blocking buffer and incubated for 1 hr in the dark. Use DAPI (blue) to counterstain the nucleus, add DAPI dropwise and incubate in the dark for 5 minutes, then stain the nucleus of the specimen, wash off excess DAPI with PBST 5minx4 times; then we perform fluorescence imaging on the cells under the ImageXpress high-content imaging analysis system . Subcellular localization and expression of p-STAT1 (Fig. 5D), p-STAT3 (Fig. 5E) and p-STAT5 (Fig. 5F) were analyzed by immunofluorescence staining. The results are shown in Figure 5D, E, and F. After treatment with compound 7d, the phosphorylated STAT3 protein was significantly reduced, while the phosphorylated STAT1 and STAT5 proteins did not change significantly. Therefore, compound 7d selectively inhibited the phosphorylation of cellular STAT3 protein.
DNA结合活性的电泳迁移率变化分析(EMSA)实验Electrophoretic mobility shift assay (EMSA) assay of DNA binding activity
MDA-MB-231细胞在培养皿中培养过夜，然后在RPMI-1640培养基中用10μM的化合物7d处理0-24小时。如前所述，收获细胞并进行核提取物制备和EMSA分析。具体步骤见实施例34 1.1细胞系培养与提取，其他操作根据根据EMSA/Gel-Shift试剂盒(碧云天)的说明书完成。 32P标记的寡核苷酸hSIE探针用于结合STAT3，MGFe探针用于结合STAT1和STAT5。选择浓度8％的非变性聚丙烯酰胺凝胶并在0.5×TBE电泳缓冲液中进行目标蛋白的分离，电泳好的凝胶经干胶仪干燥后进行放射自显影。结果如图5G显示，0-24小时，STAT3二聚化蛋白显著减少，而同源的STAT1,STAT5二聚化蛋白影响甚微，说明化合物7d在10μM下可以选择性抑制STAT3二聚化的形成。 MDA-MB-231 cells were cultured overnight in culture dishes and then treated with 10 μM compound 7d in RPMI-1640 medium for 0–24 hours. Cells were harvested and subjected to nuclear extract preparation and EMSA analysis as previously described. For specific steps, see Example 34. 1.1 Cell line cultivation and extraction, and other operations were completed according to the instructions of the EMSA/Gel-Shift kit (Beiyuntian). 32 P-labeled oligonucleotide hSIE probes were used to bind STAT3, and MGFe probes were used to bind STAT1 and STAT5. A non-denaturing polyacrylamide gel with a concentration of 8% was selected and the target protein was separated in 0.5×TBE electrophoresis buffer, and the electrophoresed gel was dried by a dry gel apparatus for autoradiography. The results are shown in Figure 5G, 0-24 hours, STAT3 dimerization protein was significantly reduced, while the homologous STAT1, STAT5 dimerization protein had little effect, indicating that compound 7d can selectively inhibit the formation of STAT3 dimerization at 10 μM .
实施例36化合物7d靶向结合SH2结构域Example 36 Compound 7d Targeted Binding to SH2 Domain
表面等离子共振实验Surface Plasmon Resonance Experiment
使用BIAcore T200分子互作仪(GE Healthcare)在25℃下进行动力学亲和力测定。在预实验中，配制运行缓冲液(10mM HEPES、pH 7.4、150mM NaCl、3mM EDTA和0.005％Tween-20与5％DMSO)。运行缓冲液用于空白进样，并使用溶剂校正来校正样品中的参照错误。依次在pH 4.0、4.5、5.0和5.5的10mM醋酸盐缓冲液中进样，选择响应值与富集量最高的PH缓冲条件，合适的条件是pH 5.0，重组STAT3通过标准胺偶联程序在pH5.0下共价固定到CM5传感器芯片上，响应值大约为20000RU。随后将化合物7d以30μL/min的流速注射到STAT3固定的流动池中，采用99.9％生物级DMSO配制10mmol/L的化合物7d母液，在用运行缓冲液稀释7d母液，浓度为0.078、0.156、0.312、0.625、1.25、5、10μM分别进样，结合时间120s，并使其解离120s。使用BIAcore T200评估软件计算稳态K D值。结果如图6所示，K D值为460nM，表明化合物7d对STAT3蛋白有很强的亲和力。 Kinetic affinity measurements were performed at 25°C using a BIAcore T200 Molecular Interaction Instrument (GE Healthcare). In a preliminary experiment, a running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% Tween-20 with 5% DMSO) was prepared. Running buffer was used for blank injections, and solvent correction was used to correct for reference errors in the samples. Samples were injected in 10mM acetate buffer at pH 4.0, 4.5, 5.0 and 5.5 in sequence, and the pH buffer condition with the highest response value and enrichment was selected. The suitable condition was pH 5.0. Recombinant STAT3 was obtained by standard amine coupling procedures in Covalently immobilized on the CM5 sensor chip at pH5.0, the response value is about 20000RU. Subsequently, compound 7d was injected into the fixed flow cell of STAT3 at a flow rate of 30 μL/min, and 10 mmol/L compound 7d stock solution was prepared with 99.9% biological grade DMSO, and the 7d stock solution was diluted with running buffer at a concentration of 0.078, 0.156, and 0.312 , 0.625, 1.25, 5, and 10 μM were injected respectively, the binding time was 120 s, and the dissociation time was 120 s. Steady-state KD values were calculated using BIAcore T200 evaluation software. The results are shown in Figure 6, the K D value is 460nM, indicating that compound 7d has a strong affinity for STAT3 protein.
实施例37化合物7d体内肿瘤实验Example 37 Compound 7d tumor experiment in vivo
成年雌性裸鼠(4-6周龄)购自南京大学(中国南京)模型动物研究中心。根据标准，喂养小鼠标准饮食和水，并在给药前使其适应5天。在右侧腹区域向无胸腺balb/c裸鼠(15-18g)注射100μLPBS缓冲液悬浮的5×10 6人源肺癌A549细胞。接种肿瘤细胞三天后，肿瘤体积达到约70mm 3。将小鼠随机分为三组(10mg/kg化合物7d、20mg/kg化合物7d、生理盐水)，每组五只。将药物溶解在40％聚乙二醇和60％生理盐水的混合溶剂中，以进行体内给药。荷瘤小鼠接受一次10或20mg/kg的化合物7d或生理盐水(CT,对照)的腹腔注射。每周3次用卡尺测量肿瘤大小，记录肿瘤生长情况，按公式：长×宽×宽/2计算，测量并记录体重。治疗21天后，将所有小鼠杀死，分离肿瘤，称重并保存在-80℃中以备后用。对肿瘤组织细胞进行实施例34的细胞培养，使用蛋白质印迹法评估肿瘤切片中p-STAT1、p-STAT3、p-STAT5、Bcl-2和细胞周期蛋白D1蛋白的表达水平。 Adult female nude mice (4-6 weeks old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Mice were fed standard diet and water according to the standard and allowed to acclimatize for 5 days before dosing. Athymic balb/c nude mice (15-18 g) were injected with 5×10 6 human lung cancer A549 cells suspended in 100 μL PBS buffer in the right abdominal region. Three days after tumor cell inoculation, the tumor volume reached about 70 mm 3 . The mice were randomly divided into three groups (10 mg/ kg compound 7d, 20 mg/kg compound 7d, normal saline), five in each group. The drug was dissolved in a mixed solvent of 40% polyethylene glycol and 60% physiological saline for in vivo administration. Tumor-bearing mice received an intraperitoneal injection of 10 or 20 mg/kg compound 7d or normal saline (CT, control). The size of the tumor was measured with a caliper three times a week, the growth of the tumor was recorded, calculated according to the formula: length×width×width/2, and the body weight was measured and recorded. After 21 days of treatment, all mice were sacrificed, tumors were isolated, weighed and stored at -80°C for future use. The tumor tissue cells were cultured according to Example 34, and the expression levels of p-STAT1, p-STAT3, p-STAT5, Bcl-2 and cyclin D1 proteins in the tumor slices were evaluated by Western blotting.
实验结果如图7所示。结果显示当化合物7d给药量为10mg/kg，20mg/kg时，小鼠的体重与对照组相比没有明显变化，说明化合物7d的毒性较小(图7B)。此外，在给药量为20mg/kg时，与对照组相比肿瘤几乎消失，说明7d体内抑制肿瘤作用明显(图7A、C)；磷酸化的STAT3蛋白含量明显降低，而磷酸化的STAT1、STAT5蛋白含量没有明显变化，说明化合物7d可以选择性抑制STAT3蛋白的磷酸化，进一步的Bcl-2、Cyclin D1的表达水平显著降低(图7D)。因此，化合物7d具有显著的体内抗肿瘤活性，且毒性较低，选择性抑制JAK-STAT3细胞通路。The experimental results are shown in Figure 7. The results showed that when compound 7d was administered at 10 mg/kg and 20 mg/kg, the body weight of the mice had no significant change compared with the control group, indicating that compound 7d was less toxic (Fig. 7B). In addition, when the dose was 20 mg/kg, compared with the control group, the tumor almost disappeared, indicating that the tumor inhibition effect was obvious in vivo at 7 days (Figure 7A, C); the phosphorylated STAT3 protein content was significantly reduced, while the phosphorylated STAT1, The content of STAT5 protein did not change significantly, indicating that compound 7d could selectively inhibit the phosphorylation of STAT3 protein, and further the expression levels of Bcl-2 and Cyclin D1 were significantly reduced (Figure 7D). Therefore, compound 7d has significant in vivo antitumor activity with low toxicity and selectively inhibits the JAK-STAT3 cellular pathway.
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also possible. It should be regarded as the protection scope of the present invention.

Claims (8)

  1. 式I所示的喹诺酮衍生物或其药学上可接受的盐或酯：The quinolone derivative shown in formula I or its pharmaceutically acceptable salt or ester:
    Figure PCTCN2022088657-appb-100001
    Figure PCTCN2022088657-appb-100001
    其中，A环选自苯基、含1～2个杂原子的五、六元饱和或不饱和杂环，杂原子选自N、O、S；Among them, ring A is selected from phenyl, five- and six-membered saturated or unsaturated heterocycles containing 1 to 2 heteroatoms, and the heteroatoms are selected from N, O, and S;
    R 1每次出现独立的选自氢、C1～C3烷基、C1～C3烷氧基、卤素取代的C1～C3烷基、卤素取代的C1～C3烷氧基、硝基、氨基、卤素；m为1～5的整数； Each occurrence of R1 is independently selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl, halogen-substituted C1-C3 alkoxy, nitro, amino, halogen; m is an integer from 1 to 5;
    Y选自NH、O，X选自亚甲基、羰基、
    Figure PCTCN2022088657-appb-100002
    n＝1～3的整数；     Y is selected from NH, O, X is selected from methylene, carbonyl,
    Figure PCTCN2022088657-appb-100002
    n=1～3 integer;
    R 2选自氢、甲基、乙基、异丙基、
    Figure PCTCN2022088657-appb-100003
     R is selected from hydrogen, methyl, ethyl, isopropyl,
    Figure PCTCN2022088657-appb-100003
  2. 根据权利要求1所述的喹诺酮衍生物，其特征在于：A环选自苯基、吡啶、嘧啶、噻吩、吡咯、呋喃；quinolone derivative according to claim 1, is characterized in that: ring A is selected from phenyl, pyridine, pyrimidine, thiophene, pyrrole, furan;
    R 1每次出现独立的选自氢、甲基、硝基、氨基、三氟甲基、三氟甲氧基、F、Br；m为1～2的整数； Each occurrence of R1 is independently selected from hydrogen, methyl, nitro, amino, trifluoromethyl, trifluoromethoxy, F, Br; m is an integer of 1 to 2;
    Y选自NH、O，X选自亚甲基，n＝1；Y is selected from NH, O, X is selected from methylene, n=1;
    R 2选自氢。 R2 is selected from hydrogen.
  3. 喹诺酮衍生物或其药学上可接受的盐或酯，其特征在于：喹诺酮衍生物选自以下化合物：A quinolone derivative or a pharmaceutically acceptable salt or ester thereof, characterized in that the quinolone derivative is selected from the following compounds:
    Figure PCTCN2022088657-appb-100004
    Figure PCTCN2022088657-appb-100004
    Figure PCTCN2022088657-appb-100005
    Figure PCTCN2022088657-appb-100005
  4. 如下式所示的喹诺酮衍生物或其药学上可接受的盐或酯：A quinolone derivative or a pharmaceutically acceptable salt or ester thereof shown in the following formula:
    Figure PCTCN2022088657-appb-100006
    Figure PCTCN2022088657-appb-100006
  5. 权利要求1-4任一项所述的喹诺酮衍生物或其药学上可接受的盐或酯在制备STAT3抑制剂中的应用。Use of the quinolone derivatives or pharmaceutically acceptable salts or esters of any one of claims 1-4 in the preparation of STAT3 inhibitors.
  6. 权利要求1-4任一项所述的喹诺酮衍生物或其药学上可接受的盐或酯在制备预防和/或治疗与肿瘤有关疾病的药物中的应用。Use of the quinolone derivatives or pharmaceutically acceptable salts or esters thereof according to any one of claims 1-4 in the preparation of medicaments for preventing and/or treating tumor-related diseases.
  7. 根据权利要求6所述的应用，其特征在于：所述的与肿瘤有关疾病为结肠癌、骨肉瘤、肺癌、乳腺癌。The application according to claim 6, characterized in that: said tumor-related diseases are colon cancer, osteosarcoma, lung cancer, and breast cancer.
  8. 一种药物组合物，其特征在于：含有治疗有效量的权利要求1-4任一项所述的喹诺酮 衍生物或其药学上可接受的盐或酯。A pharmaceutical composition, characterized in that: containing the quinolone derivative or pharmaceutically acceptable salt or ester thereof described in any one of claims 1-4 in a therapeutically effective dose.
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