WO2022166994A1 - 一种萘基脲类化合物、其制备方法及应用 - Google Patents

一种萘基脲类化合物、其制备方法及应用 Download PDF

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WO2022166994A1
WO2022166994A1 PCT/CN2022/077027 CN2022077027W WO2022166994A1 WO 2022166994 A1 WO2022166994 A1 WO 2022166994A1 CN 2022077027 W CN2022077027 W CN 2022077027W WO 2022166994 A1 WO2022166994 A1 WO 2022166994A1
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nmr
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徐学军
杨玉坡
杨争艳
徐红运
段超群
张尊
张韶华
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河南省锐达医药科技有限公司
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Priority to US18/313,365 priority Critical patent/US20230271928A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/155—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with 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
    • C07D213/72—Nitrogen atoms
    • C07D213/75—Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/14—Nitrogen atoms not forming part of a nitro radical

Definitions

  • the invention belongs to the field of tumor targeted therapy, and particularly relates to a naphthylurea compound, a preparation method and application thereof.
  • the cell cycle consists of a series of relatively independent phases, including four phases G1, S, G2 and M that proceed in sequence.
  • the cell cycle process is tightly organized, and the initiation of each phase must depend on the completion of the previous phase.
  • This orderly process is precisely regulated by cycle checkpoints, which can temporarily arrest or delay the cell cycle in the event of damage from various stresses such as oxygen free radicals, UV radiation, chemicals and heavy metals, allowing cells to win Opportunity to repair damage.
  • the treatment options for many tumors include radiotherapy and some chemotherapeutic drugs to kill tumor cells by destroying the genetic structure, enhancing the instability of the genome, and triggering cell death.
  • these damages can also lead to cell cycle arrest, repairing the damage and triggering tumor resistance to therapy.
  • mutations in genes or proteins frequently occur in malignant tumors the absence of cell cycle checkpoints is common.
  • Tumor cells lacking the G1/S checkpoint mainly rely on the G2/M checkpoint to repair damage. Therefore, selectively inhibiting the expression of cycle checkpoints can enhance the sensitivity of tumors to injury, and has become an important tumor suppressor strategy.
  • the present invention aims to reveal the anti-tumor effect of a new class of naphthyl urea compounds and derivatives thereof, as well as their potential targets and tumor-inhibiting mechanisms.
  • the object of the present invention is to provide a naphthyl urea compound, its preparation method and application.
  • a naphthylurea compound the structural formula is shown in general formula I:
  • R is selected from hydrogen, C1-C5 straight-chain alkyl, C1-C5 straight-chain alkyl substituted by halogen at the end, 5-8 membered cycloalkyl,
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 are each independently selected from H, F, Cl, Br, -CN, -CH 3 , -CF 3 , -OCH 3 , -OCF 3 , R 5 is also phenyl, M is H or -CH 3 ,
  • n represents the number of CH 2 substituents, n is 1, 2, 3, 4...10;
  • A is Wherein p represents the number of CH 2 substituents, p is 1, 2, 3;
  • X is O or S.
  • naphthyl urea compound is specifically the compound of following structure:
  • naphthylurea compounds are combined with acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, Biologically acceptable salts of at least one of tartaric acid, ascorbic acid, boric acid, lactic acid, and ethylenediaminetetraacetic acid.
  • the preparation method of above-mentioned naphthylamine compound comprises the following steps:
  • the The preparation process is as follows:
  • step (1) The molar ratio with NaH is 1:1.2:2; in step (2) The molar ratio with iron powder is 1:5, and the volume ratio of ethanol and saturated aqueous ammonium chloride solution is 1:1;
  • step (3) The molar ratio of R-based isocyanate or R-based isothiocyanate and N,N-diisopropylethylamine is 1:1.2:2.0.
  • step (a) The molar ratio of triphenylphosphine and diisopropyl azodicarboxylate is 1:1.2:1.2:1.2;
  • step (b) The molar ratio of tetrahydroaluminum lithium is 1:1.
  • the antitumor drugs refer to drugs for the treatment of liver cancer, breast cancer, lung cancer and leukemia.
  • Another object of the present invention is to provide a class of small molecule compounds with antitumor activity.
  • the tumor may specifically be a tumor with high CyclinB1 expression or vigorous proliferation, including but not limited to liver cancer, breast cancer, lung cancer, tyrosine kinase inhibitor (TKI)-resistant lung cancer, colon cancer, and leukemia.
  • TKI tyrosine kinase inhibitor
  • the present invention has synthesized a class of naphthyl urea compounds ID1120B-1 and its derivatives ID1214B-1, IY1214A-1 and IY1214B-2 and the like with a new structure.
  • the proliferation inhibitory effect of these compounds on various cancer cells was detected by MTT method; the effects of the compounds on the cell cycle and apoptosis of tumor cells were detected by flow cytometry.
  • the present invention provides a novel naphthylurea compound and its derivatives in the use and potential molecular mechanism of tumor therapy.
  • Figure 1 and Figure 2 are the MTT detection of ID1120B-1 and its derivatives ID1120B-P, ID1214B-1, IY1214A-1 and IY1214B-2 on the proliferation inhibition of liver cancer cells, breast cancer cells, non-small cell lung cancer cells and leukemia cells , the experimental results were characterized by IC50 ( ⁇ M) value, Sorafinib, WP1066 and Gefitinib were used as positive control drugs;
  • Figure 3 and Figure 4 are flow cytometry to detect the effect of compound ID1120B-1 and its derivatives ID1214B-1, IY1214A-1 and IY1214B-2 on the cell cycle of hepatoma cells HepG2;
  • Fig. 5 is the statistical analysis to the result of Fig. 3, Fig. 4;
  • Figure 6 is a flow cytometry detection of the effects of compounds IY1214A-1 and IY1214B-2 on the apoptosis of hepatoma cells HepG2;
  • Figure 7 is the effect of IY1214B-2 on the mRNA levels of cell cycle and autophagy-related molecules detected by Q-PCR.
  • various raw materials used in the reaction can be prepared by those skilled in the art according to the existing knowledge, or can be prepared by methods known in the literature, or can be purchased through commercial of.
  • the intermediates, raw materials, reagents, reaction conditions, etc. used in the above reaction scheme can all be appropriately changed according to the existing knowledge of those skilled in the art.
  • the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature; more specifically, the room temperature refers to 20-30°C;
  • the organic solvent is usually The drying method is dry, and the solvent is evaporated under reduced pressure using a rotary evaporator, and the bath temperature is not higher than 50 ° C; the developing solvent and the eluent are both in volume ratio;
  • the reaction process is tracked by thin layer chromatography (TLC);
  • TLC thin layer chromatography
  • the final product has satisfactory proton nuclear magnetic resonance (1H-NMR).
  • Compound ID1120B-1 is named
  • the solid was dissolved in ethyl acetate, adjusted to pH 1 with 1N aqueous hydrochloric acid, extracted three times with ethyl acetate, the aqueous phase was adjusted to pH 8 with solid sodium bicarbonate, and then acetic acid Ethyl ester was extracted three times, and the organic phase was dried and spin-dried to obtain 1.5 g of white solid methyl 4-(2-(pipidin-1-yl)ethoxy)benzoate (2) with a yield of 86.7%.
  • reaction solution was cooled to 0°C, 1 mL of NaOH (15wt%) aqueous solution and 1 mL of water were added in sequence; celite was filtered, and the filtrate was spin-dried to obtain 680 mg of (4-(2-(pipidin-1-yl)ethoxy)phenyl)methanol(3 ), white solid, yield 88.7%.
  • Step 5 Compound (5)( 200mg, 0.53mmol, 1.0eq), benzyl isocyanate (84.9mg, 0.64mmol, 1.2eq) and DIEA (137mg, 1.06mmol, 2.0eq) were dissolved in 25ml of 1,2-dichloroethane and reacted at 85°C 12 hours.
  • Example 2 For the synthesis methods of other compounds, refer to Example 1, except that the structure of compound 1 can be changed in step 1 or the corresponding isocyanate can be replaced in step 5.
  • compound ID1120B-P is 1-benzyl-3-(4-((4-(2-(pipidin-1-yl)ethoxy)benzyl)oxy)naphthalen-1-yl)ureaphosphate
  • ID1120B-1 (2100 mg, 0.20 mmol, 1.0 eq) was dissolved in 10 ml of DMSO, 85% aqueous phosphoric acid solution (45 mg, 0.40 mmol, 2.0 eq) was added, and the reaction was carried out at 50° C. for 2 hours. After monitoring the reaction by TLC, pour it into 50 ml of water, extract twice with dichloromethane:methanol 10:1, combine the organic phases, dry with anhydrous sodium sulfate and spin dry to obtain 115 mg
  • Example 2 Inhibitory effect of the above compound and phosphate ID1120B-P on the proliferation of liver cancer cells, breast cancer, lung cancer, gefitinib or afatinib-resistant lung cancer and leukemia cells
  • HepG2 HepG2, SMMC-7721, HuH-7, MCF-7, MDA-MB-231, MDA-MB-468, PC9, PC9-AR, PC9-GR, Jurkat and Molt-13 cells in logarithmic growth phase were collected, respectively, Count, adjust the concentration of cell suspension to 5 ⁇ 10 4 cells/mL, add to 96-well cell culture plate, and the volume of each well is 100ul.
  • ID1120B-1 and its derivatives ID1214B-1, IY1214A-1 and IY1214B-2 have good proliferation inhibition effects on liver cancer cells, breast cancer, lung cancer and leukemia cells, especially in the inhibition of tumor growth of liver cancer cells With stronger activity, we focused on further studies on the antitumor effects of these four compounds.
  • HepG2 cells in logarithmic growth phase were taken, digested and centrifuged to make single cell suspension. After counting, cells were plated into a 12-well plate, 2 ⁇ 10 5 cells per well, and 3 wells were plated for parallel control. After 16 h of plating, cells were individually treated with graded concentrations of compounds. After 48 hours, the cells were digested with trypsin, resuspended and counted, and the cell concentration was adjusted to 5 ⁇ 10 5 cells.
  • Figures 3 and 4 are the results of analyzing the effects of ID1120B-1 and its derivatives ID1214B-1, IY1214A-1 and IY1214B-2 on the cycle distribution of HepG2 hepatoma cells using ModFit software.
  • Figure 5 is a further quantitative analysis of the results of Figures 3 and 4 by Graphpad prism 6.0.
  • the results of Figure 3, Figure 4 and Figure 5 show that, compared with the solvent control group (DMSO), compound ID1120B-1 and its derivatives ID1214B-1, IY1214A-1 and IY1214B-2 can induce hepatoma cells in a dose-dependent manner.
  • the ratio of G2/M phase increased significantly, and the ratio of G1/S phase decreased accordingly.
  • the G2 phase ratio of ID1120B-1-induced hepatoma cells increased from 8.72% to 15.9%; ID1214B-1 induced G2 phase from 10.6% to 17.54%; IY1214A-1 induced G2 phase from 13.35% to 34.54%; IY1214B-2 induced The G2 period was extended from 9.6% to 21.71%.
  • HepG2 cells in logarithmic growth phase were taken, digested and centrifuged to make single cell suspension. After counting, cells were plated into a 12-well plate, 2 ⁇ 10 5 cells per well, and 3 wells were plated for parallel control. After 16 h of plating, cells were treated with graded concentrations of compounds for 48 h, respectively. The cells were digested with EDTA-free trypsin, resuspended and counted, and the cell concentration was adjusted to 1 ⁇ 10 6 cells.
  • Follow-up operations were performed according to the instructions of the Annexin V FITC-PI apoptosis detection kit (product of Beijing Soleibao Company).
  • Figure 6 is a flow cytometry detection of the effects of IY1214A-1 and IY1214B-2 on the apoptosis of HepG2 hepatoma cells.
  • the results showed that both IY1214A-1 and IY1214B-2 could induce increased apoptosis in a dose-dependent manner compared with the control group.
  • the apoptotic rates of cells were 57.7% and 63%, respectively, which were more than 2 times higher than that in the control wells; Compared with the treated wells, the apoptosis rate was about 3.3 times higher.
  • Liver cancer HepG2 cells were seeded in 6-well plates, 1 ⁇ 10 6 cells per well. Compound IY1214B-2 (concentrations of 0 and 10 ⁇ M) was added for 24 h.
  • Total cell RNA was extracted according to the TRIzol one-step method, and the RNA concentration and purity were determined. Using total RNA as a template, cDNA was synthesized according to the instructions of Promega's reverse transcription kit. Semi-quantitative RT-PCR and real-time quantitative RT-PCR were used to detect CCNB1, CDK1 and SQSTM, with ACTB as the internal reference. The primers used are shown in Table 1.
  • the CT value of ⁇ -actin was used as the initial value for data analysis.
  • Figure 7 is the effect of IY1214B-2 on the mRNA levels of cell cycle and autophagy-related molecules detected by Q-PCR.
  • the results showed that after IY1214B-2 was treated with 0 and 10 ⁇ M for 24 h, compared with the expression level of the gene for the memory protein ⁇ -actin (ATCB), the G2 phase regulators of the cell cycle, Cyclin B1 (gene name: CCNB1) and CDC2 (gene name) : CDK1) mRNA level was down-regulated by about 20-25%, and the expression of autophagy-related marker P62 (gene name: SQSTM) was up-regulated by about 2.5 times.
  • ATCB memory protein ⁇ -actin
  • IY1214B-2 can induce G2/M phase arrest by down-regulating the expression of Cyclin B1 and CDC2 from the mRNA level or promote autophagy and inhibit the growth of tumor cells by up-regulating the expression of P62 from the mRNA level.
  • the compounds of the present invention can be applied to cancer therapeutic drugs related to abnormal cell proliferation. Salts or mixed with pharmaceutical carriers to prepare antitumor drugs.

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Abstract

本发明提供了一种萘基脲类化合物、其制备方法及应用，其含有的具有生物活性萘脲母核基团进一步化学修饰产生诸多生物活性更高化合物，拓展了此类化合物在生物医药上的广泛应用以及药物制剂开发前景。该类化合物在低剂量(亚微摩尔)即可显著抑制肝癌、乳腺癌、肺癌、耐药的肺癌和白血病等细胞的增殖，诱导细胞周期G2/M期阻滞，并促进细胞凋亡，表明该类化合物具有开发为抗肿瘤药物的前景。

Description

一种萘基脲类化合物、其制备方法及应用 技术领域
本发明属于肿瘤靶向治疗领域，具体涉及一种萘基脲类化合物、其制备方法及应用。
背景技术
根据2020年国际癌症研究机构发布的报告，全球癌症负担持续增长，2020年有18094716例新发和9894402例死亡癌症患者。其中乳腺癌患者的新增发病率最高，肺癌导致的死亡病例最多，此外，结直肠癌、胃癌和肝癌的发病和死亡率也很高。传统的化疗药物紫杉醇、顺铂和阿霉素等，在早期可以有效抑制肿瘤生长，但是治疗一段时间后往往会发生耐药，失去治疗效果。而曲妥珠单抗、吉非替尼、索拉替尼等靶向抗癌药物的治疗反应率和疗效均有待提高。因此迫切需要开发新的抗癌药物。
细胞周期由一系列相对独立的阶段构成，包括依次进行的G1,S,G2和M四期。细胞周期过程组织严密，每一期的开始都必须依赖前一期的完成。这一有序的进程受到周期检测点的精确调控，后者能使细胞周期在多种应激如氧自由基、紫外线辐射、化学药物和重金属等损伤的情况下暂时停滞或延迟，为细胞赢得修复损伤的机会。细胞周期过程中有两个重要的检测点，即G1/S期和G2/M期检测点，它们负责调控DNA复制，蛋白质的合成和细胞分裂等重大事件，对于维持基因组结构和功能的完整性至关重要。
许多肿瘤的治疗方案包括放疗和一些化疗药物通过破坏基因结构，增强基因组的不稳定性，引发细胞死亡而达到杀伤肿瘤细胞的目的。然而，这些损伤也会导致细胞周期阻滞，修复损伤，而引发肿瘤对治疗的抵抗。由于基因或蛋白的突变在恶性肿瘤中经常出现，细胞周期检测点的缺失具有一定的普遍性。缺乏G1/S期检测点的肿瘤细胞主要依靠G2/M期检测点来修复损伤的。因此，选择性的抑制周期检测点的表达，能增强肿瘤对损伤的敏感性，已成为一种重要的抑瘤策略。
为了抑制恶性肿瘤细胞的增殖和发展，我们近期合成了一类具有全新结构式的萘基脲类化合物。通过一些生物学技术分析，发现该类化合物能够显著抑制肝癌、乳腺癌、肺癌和白血病细胞株的细胞增殖，诱导细胞发生G2/M期阻滞和细胞凋亡。因此，对此类化合物进一步开发，将在肿瘤治疗的应用方面具有重要的意义。
本发明旨在揭示一类新型萘基脲类化合物及其衍生物的抗肿瘤作用及其潜在作用靶点和抑瘤机制。
发明内容
本发明的目的是提供一种萘基脲类化合物、其制备方法及应用。
一种萘基脲类化合物，结构式如通式I所示：
Figure PCTCN2022077027-appb-000001
其中，R选自氢、C1～C5直链烷基、末端被卤素取代的C1～C5直链烷基、5～8元环烷基、
Figure PCTCN2022077027-appb-000002
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9各自独立地选自H、F、Cl、Br、-CN、-CH 3、-CF 3、-OCH 3、-OCF 3，R 5还为苯基，M为H或-CH 3，
n代表CH 2取代基的个数，n为1、2、3、4...10；
A为
Figure PCTCN2022077027-appb-000003
其中p代表CH 2取代基的个数，p为1、2、3；
X为O或S。
上述萘基脲类化合物，具体为如下结构的化合物：
Figure PCTCN2022077027-appb-000004
Figure PCTCN2022077027-appb-000005
上述萘基脲类化合物与乙酸、二氢叶酸、苯甲酸、柠檬酸、山梨酸、丙酸、草酸、富马酸、马来酸、盐酸、苹果酸、磷酸、亚硫酸、硫酸、香草酸、酒石酸、抗坏血酸、硼酸、乳酸和乙二胺四乙酸中的至少一种形成的生物学可接受的盐。
上述萘胺类化合物的制备方法，包括以下步骤：
(1)将
Figure PCTCN2022077027-appb-000006
溶于四氢呋喃中，-5℃～5℃分批加入NaH，再加入
Figure PCTCN2022077027-appb-000007
室温搅拌反应至完全，后处理得到
Figure PCTCN2022077027-appb-000008
(2)将
Figure PCTCN2022077027-appb-000009
溶于乙醇和饱和氯化铵水溶液的混合溶液中，40～50℃下加入铁粉，50～60℃搅拌反应至完全，经后处理得到
Figure PCTCN2022077027-appb-000010
(3)将化合物
Figure PCTCN2022077027-appb-000011
R基异氰酸酯或R基异硫氰酸酯和N,N-二异丙基乙胺溶于1,2-二氯乙烷中，80～90℃搅拌反应至完全，柱层析得到
Figure PCTCN2022077027-appb-000012
优选地，所述
Figure PCTCN2022077027-appb-000013
的制备过程如下：
(a)将
Figure PCTCN2022077027-appb-000014
和三苯基膦溶于四氢呋喃中，-5℃～5℃保护气氛下加入 偶氮二甲酸二异丙酯，室温搅拌反应至完全，经后处理得到
Figure PCTCN2022077027-appb-000015
(b)将化合物
Figure PCTCN2022077027-appb-000016
溶于四氢呋喃中，-5℃～5℃分批加入四氢铝锂，室温搅拌至反应完全，经后处理得到
Figure PCTCN2022077027-appb-000017
优选地，所述步骤(1)中
Figure PCTCN2022077027-appb-000018
与NaH的摩尔比为1:1.2:2；步骤(2)中
Figure PCTCN2022077027-appb-000019
与铁粉的摩尔比为1:5，乙醇和饱和氯化铵水溶液的体积比为1:1；
步骤(3)中，
Figure PCTCN2022077027-appb-000020
R基异氰酸酯或R基异硫氰酸酯和N,N-二异丙基乙胺的摩尔比为1:1.2:2.0。
优选地，步骤(a)中，
Figure PCTCN2022077027-appb-000021
三苯基膦、偶氮二甲酸二异丙酯的摩尔比为1:1.2:1.2:1.2；
步骤(b)中，
Figure PCTCN2022077027-appb-000022
和四氢铝锂的摩尔比为1:1。
上述的萘基脲类化合物及其生物学可接受的盐在制备抗肿瘤药物中的用途。
优选地，所述的抗肿瘤药物是指治疗肝癌、乳腺癌、肺癌和白血病的药物。
本发明的另一目的是提供一类具有抗肿瘤活性的小分子化合物。
所述肿瘤具体可为CyclinB1高表达或增殖旺盛的肿瘤，包括但不限于肝癌、乳腺癌、肺癌、酪氨酸激酶抑制剂(TKI)耐药的肺癌、结肠癌和白血病等。
具体的说，本发明合成了一类具有全新结构的萘基脲类化合物ID1120B-1及其衍生物ID1214B-1，IY1214A-1和IY1214B-2等。通过MTT法检测此类化合物对多种癌细胞的增殖抑制作用；通过流式细胞术检测化合物对肿瘤细胞的细胞周期和凋亡的影响。
结果表明，本发明的化合物ID1120B-1，ID1214B-1，IY1214A-1和IY1214B-2等，可以有效抑制肝癌、乳腺癌、肺癌和白血病细胞的增殖，诱导细胞G2/M期阻滞和细胞凋亡。
总之，本发明提供了一种新的萘基脲类化合物以及它的衍生物在肿瘤治疗上的用途和潜在分子机制。
附图说明
图1、图2是MTT检测ID1120B-1及其衍生物ID1120B-P、ID1214B-1，IY1214A-1和IY1214B-2对肝癌细胞、乳腺癌细胞、非小细胞肺癌细胞和白血病细胞的增殖抑制作用，实验结果以IC50(μM)值进行表征，Sorafinib，WP1066和Gefitinib作为阳性对照药物；
图3、图4是通过流式细胞术检测化合物ID1120B-1及其衍生物ID1214B-1，IY1214A-1和IY1214B-2对肝癌细胞HepG2的细胞周期的影响；
图5是对图3、图4结果的统计分析；
图6是通过流式细胞术检测化合物IY1214A-1和IY1214B-2对肝癌细胞HepG2的细胞凋亡的影响；
图7是通过Q-PCR检测IY1214B-2对细胞周期和自噬相关分子的mRNA水平的影响。
具体实施方式
为了使本发明的技术目的、技术方案和有益效果更加清楚，下面结合附图和具体实施例对本发明的技术方案作出进一步的说明。
在本发明合成式I化合物的方法中，反应所用的各种原材料是本领域技术人员根据已有知识可以制备得到的，或者是可以通过文献公知的方法制得的，或者是可以通过商业购得的。以上反 应方案中所用的中间体、原材料、试剂、反应条件等均可以根据本领域技术人员已有知识做适当改变的。
在本发明中，除非另外说明，其中：(i)温度以摄氏度(℃)表示，操作在室温环境下进行；更具体地，所述室温是指20-30℃；(ii)有机溶剂用常用干燥方法干燥，溶剂的蒸发使用旋转蒸发仪减压蒸发，浴温不高于50℃；展开剂和洗脱剂均为体积比；(iii)反应过程用薄层色谱(TLC)跟踪；(iv)终产物具有满意的质子核磁共振(1H-NMR)。
实施例1:化合物的合成
Figure PCTCN2022077027-appb-000023
ID1120B-1：
Figure PCTCN2022077027-appb-000024
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000025
X＝O；
ID1120C-1：
Figure PCTCN2022077027-appb-000026
R 1＝H，R 2＝Cl，n＝2，
Figure PCTCN2022077027-appb-000027
X＝O
ID1120D-1：
Figure PCTCN2022077027-appb-000028
R 1＝CN，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000029
X＝O；
IY210119B-1：
Figure PCTCN2022077027-appb-000030
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000031
X＝O；
IY210115B-1：
Figure PCTCN2022077027-appb-000032
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000033
X＝O；
IY210118B-1：
Figure PCTCN2022077027-appb-000034
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000035
X＝O；
IY210113D-1：
Figure PCTCN2022077027-appb-000036
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000037
X＝O；
IY1210B-1：
Figure PCTCN2022077027-appb-000038
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000039
X＝O；
ID210106D-1：
Figure PCTCN2022077027-appb-000040
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000041
X＝O；
ID210118D-1：
Figure PCTCN2022077027-appb-000042
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000043
X＝O；
ID210113C-1：
Figure PCTCN2022077027-appb-000044
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000045
X＝O；
IY210113C-1：
Figure PCTCN2022077027-appb-000046
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000047
X＝O；
ID210118C-1：
Figure PCTCN2022077027-appb-000048
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000049
X＝O；
ID210115B-1：
Figure PCTCN2022077027-appb-000050
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000051
X＝O；
ID210114B-1：
Figure PCTCN2022077027-appb-000052
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000053
X＝O；
ID1210B-1：
Figure PCTCN2022077027-appb-000054
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000055
X＝O；
IY1207A-1：
Figure PCTCN2022077027-appb-000056
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000057
X＝S；
IY1223B-1：
Figure PCTCN2022077027-appb-000058
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000059
X＝O；
IY1214A-1：
Figure PCTCN2022077027-appb-000060
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000061
X＝O；
ID1214B-1：
Figure PCTCN2022077027-appb-000062
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000063
X＝O；
IY1225B-1：
Figure PCTCN2022077027-appb-000064
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000065
X＝O；
IY1210A-1：
Figure PCTCN2022077027-appb-000066
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000067
X＝O；
IY1226B-1：
Figure PCTCN2022077027-appb-000068
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000069
X＝O；
IY1229C-1：
Figure PCTCN2022077027-appb-000070
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000071
X＝O；
ID1229C-1：
Figure PCTCN2022077027-appb-000072
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000073
X＝O；
ID1229D-1：
Figure PCTCN2022077027-appb-000074
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000075
X＝O；
ID1224D-1：
Figure PCTCN2022077027-appb-000076
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000077
X＝O；
ID1231B-1：
Figure PCTCN2022077027-appb-000078
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000079
X＝O；
IY1214B-2：
Figure PCTCN2022077027-appb-000080
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000081
X＝O；
ID1224C-1：
Figure PCTCN2022077027-appb-000082
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000083
X＝O；
IY1229D-1：
Figure PCTCN2022077027-appb-000084
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000085
X＝O；
IY210103B-1：
Figure PCTCN2022077027-appb-000086
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000087
X＝O；
IY210105B-1：
Figure PCTCN2022077027-appb-000088
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000089
X＝O；
IY210105C-1：
Figure PCTCN2022077027-appb-000090
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000091
X＝O；
ID210105C-1：
Figure PCTCN2022077027-appb-000092
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000093
X＝O；
IY210105D-1：
Figure PCTCN2022077027-appb-000094
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000095
X＝O；
IY210105A-1：
Figure PCTCN2022077027-appb-000096
R 1＝H，R 2＝Br，n＝2，
Figure PCTCN2022077027-appb-000097
X＝O；
IY210106D-1：
Figure PCTCN2022077027-appb-000098
R 1＝H，R 2＝F，n＝2，
Figure PCTCN2022077027-appb-000099
X＝O；
ID210110C-1：
Figure PCTCN2022077027-appb-000100
R 1＝H，R 2＝Cl，n＝2，
Figure PCTCN2022077027-appb-000101
X＝O；
IY210110D-1：
Figure PCTCN2022077027-appb-000102
R 1＝H，R 2＝OMe，n＝2，
Figure PCTCN2022077027-appb-000103
X＝O；
ID1207B-1：
Figure PCTCN2022077027-appb-000104
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000105
X＝O；
ID1217B-1：
Figure PCTCN2022077027-appb-000106
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000107
X＝O；
ID1223A-1：R＝H，R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000108
X＝O；
ID1215B-1：
Figure PCTCN2022077027-appb-000109
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000110
X＝O；
ID1215C-1：
Figure PCTCN2022077027-appb-000111
R 1＝Cl，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000112
X＝O；
IY1215C-1：
Figure PCTCN2022077027-appb-000113
R 1＝F，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000114
X＝O；
ID1215A-1：
Figure PCTCN2022077027-appb-000115
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000116
X＝O；
IY1215D-1：
Figure PCTCN2022077027-appb-000117
R 1＝CN，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000118
X＝O；
IY210122C-1：
Figure PCTCN2022077027-appb-000119
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000120
X＝O；
ID210119B-1：
Figure PCTCN2022077027-appb-000121
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000122
X＝O；
IY210128B-1：
Figure PCTCN2022077027-appb-000123
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000124
X＝O；
ID210127B-1：
Figure PCTCN2022077027-appb-000125
R 1＝H，R 2＝H，n＝2，
Figure PCTCN2022077027-appb-000126
X＝O；
具体合成方法，以化合物ID1120B-1和ID1120B-P为例，结构式分别如下：
Figure PCTCN2022077027-appb-000127
化合物ID1120B-1的名称为
1-benzyl-3-(4-((4-(2-(piperidin-1-yl)ethoxy)benzyl)oxy)naphthalen-1-yl)urea，
其合成路线如下：
Figure PCTCN2022077027-appb-000128
步骤1.methyl4-(2-(piperidin-1-yl)ethoxy)benzoate(2)
将4-羟基苯甲酸甲酯(1.0g,6.57mmol,1.0eq)，N-羟乙基哌啶(1.02g,7.89mmol,1.2eq)和三苯基膦(2.07g,7.89mmol,1.2eq)溶解到30mL无水四氢呋喃中，降温到0℃，氮气保护下慢慢滴加偶氮二甲酸二异丙酯(1.59g,7.89mmol,1.2eq),然后室温反应16小时。TLC监测反应完毕后，减压浓缩除去四氢呋喃，固体用乙酸乙酯溶解，用1N的盐酸水溶液调节pH到1，乙酸乙酯萃取三次，水相用碳酸氢钠固体调节pH到8，再用乙酸乙酯萃取三次，有机相干燥旋干得到1.5g白色固体methyl 4-(2-(piperidin-1-yl)ethoxy)benzoate(2)，收率86.7％。
1H NMR(CDCl 3,300MHz)δ:8.0(d,J＝9.0Hz,2H),6.93(d,J＝9.0Hz,2H),4.17(t,J＝6.0Hz,2H),3.90(s,3H),2.82(t,J＝6.0Hz,2H),2.58-2.55(m,4H),1.66-1.61(m,4H),1.50(t,J＝3.0Hz,2H)
步骤2.(4-(2-(piperidin-1-yl)ethoxy)phenyl)methanol(3)
将化合物(2)(1.00g,3.80mmol,1.0eq)溶于40mL无水四氢呋喃，冷却到0℃，分批加入四氢铝锂(144mg,3.80mmol,1.0eq)，自然升温到室温反应0.5小时。TLC监测显示原料反应完毕，并有新点产生。将反应液冷却到0℃，依次加入1mLNaOH(15wt％)水溶液，1mL水；硅藻土过滤，滤液旋干得到680mg(4-(2-(piperidin-1-yl)ethoxy)phenyl)methanol(3)，白色固体，收率88.7％。
1H NMR(CDCl 3,300MHz)δ:7.30(d,J＝6.0Hz,2H),6.92(d,J＝6.0Hz,2H),4.64(s,2H),4.17(t,J＝6.0Hz,2H),2.98(t,J＝6.0Hz,2H),2.74(m,4H),1.89-1.86(m,6H)
步骤3. 1-(2-(4-(((4-nitronaphthalen-1-yl)oxy)methyl)phenoxy)ethyl)piperidine(4)
将化合物(3)(1.03g,4.39mmol,1.2eq)溶解于30mL无水四氢呋喃中，冷却到0℃，分批加入NaH(293mg,7.32mmol,2eq)，半小时后加入1-氟-4-硝基萘(700mg,3.66mmol,1.0eq)，继续室温反应12小时。TLC监测反应完毕后，倒入100mL饱和氯化铵水溶液中，用乙酸乙酯萃取3次(100mL*3)，将有机相合并，用无水硫酸钠干燥，旋干过柱(二氯甲烷：甲醇＝60:1～20:1)得到710mg 1-(2-(4-(((4-nitronaphthalen-1-yl)oxy)methyl)phenoxy)ethyl)piperidine(4),黄色固体，收率47.6％。
1H NMR(CDCl 3,400MHz)δ:8.81(d,J＝8.0Hz,2H),8.45-8.41(m,2H),7.77-7.75(m,1H),7.63-7.60(m,1H),7.47(d,J＝8.0Hz,2H),7.0(d,J＝8.0Hz,2H),6.93-6.90(m,1H),5.32(s,2H),1.89-1.86(m,6H),4.37(t,J＝6.0Hz,2H),3.55-3.30(m,4H),2.97(t,J＝6.0Hz,2H),1.79-1.67(m,4H),1.65(m,4H),1.39-1.20(m,2H).
步骤4. 4-((4-(2-(piperidin-1-yl)ethoxy)benzyl)oxy)naphthalen-1-amine(5)
将化合物(4)(700mg,1.72mmol,1.0eq)溶解于25mL乙醇和25mL饱和氯化铵水溶液中，升温至45℃，分批缓慢加入铁粉(480mg,8.61mmol,5.0eq)，升温至55℃反应2小时。TLC监测反应完毕后，硅藻土过滤，滤液用乙酸乙酯萃取3次(100mL*3)，将有机相合并，用无水硫酸钠干燥，旋干过柱(二氯甲烷：甲醇＝60:1～20:1)得到350mg4-((4-(2-(piperidin-1-yl)ethoxy)benzyl)oxy)naphthalen-1-amine(5),紫红色油状物，收率54％。
1H NMR(CDCl 3,300MHz)8.20(d,J＝9.0Hz,1H),8.13(d,J＝9.0Hz,2H),7.63-7.52(m,2H),7.34-7.21(m,3H),6.92(d,J＝9.0Hz,1H),6.82(d,J＝9.0Hz,1H),4.50(s,2H),4.37(t,J＝6.0Hz,2H),3.55-3.30(m,4H),2.97(t,J＝6.0Hz,2H),1.79-1.67(m,4H),1.65(m,4H),1.39-1.20(m,2H).
步骤5. 1-benzyl-3-(4-((4-(2-(piperidin-1-yl)ethoxy)benzyl)oxy)naphthalen-1-yl)urea(ID1120B-1)将化合物(5)(200mg,0.53mmol,1.0eq)，苄基异氰酸酯(84.9mg,0.64mmol,1.2eq)和DIEA(137mg,1.06mmol,2.0eq)溶于25毫升1,2-二氯乙烷中，85℃反应12小时。TLC监测反应完毕后，直接旋干过柱(二氯甲烷：甲醇＝50:1～15:1)得到210mg1-benzyl-3-(4-((4-(2-(piperidin-1-yl)ethoxy)benzyl)oxy)naphthalen-1-yl)urea(ID1120B-1),棕色固体，收率77.8％。
1H NMR(DMSO-d6,300MHz)δ:8.32(s,1H),8.19(d,J＝8.0Hz,1H),8.01(d,J＝8.0Hz,2H),7.68(d,J＝8.0Hz,2H),7.58-7.26(m,8H),7.05-6.98(m,3H),6.82(m,1H),5.20(s,2H),4.34(d,J＝4.0Hz,2H),4.11(m,2H),2.52(m,2H),1.53(m,4H),1.40(m,2H),1.39-1.20(m,2H).
其它化合物的合成方法参照实施例1，区别在于，在步骤1改变化合物1的结构或在步骤5 中换成相应的异氰酸酯即可。
化合物ID1120B-P的名称为1-benzyl-3-(4-((4-(2-(piperidin-1-yl)ethoxy)benzyl)oxy)naphthalen-1-yl)ureaphosphate，
其合成路线如下：
Figure PCTCN2022077027-appb-000129
将ID1120B-1(2100mg,0.20mmol,1.0eq)溶于10毫升DMSO，加入85％磷酸水溶液(45mg,0.40mmol,2.0eq)，50℃反应2小时。TLC监测反应完毕后，倒入50毫升水中，用二氯甲烷：甲醇10:1萃取两次，有机相合并，用无水硫酸钠干燥旋干得到115毫克
1-benzyl-3-(4-((4-(2-(piperidin-1-yl)ethoxy)benzyl)oxy)naphthalen-1-yl)ureaphosphate
(ID1120B-P)，棕色固体，收率90％。
其他化合物的核磁如下：
ID1120C-1  1H NMR(DMSO-d6,300MHz)δ:8.35(s,1H),8.18(d,J＝8.0Hz,1H),8.02(d,J＝8.0Hz,2H),7.69(d,J＝8.0Hz,2H),7.58-7.26(m,7H),7.05-6.98(m,3H),6.82(m,1H),5.20(s,2H),4.34(d,J＝4.0Hz,2H),4.11(m,2H),2.52(m,2H),1.53(m,4H),1.40(m,2H),1.39-1.20(m,2H).
ID1120D-1  1H NMR(DMSO-d6,300MHz)δ:8.33(s,1H),8.20(d,J＝8.0Hz,1H),8.03(d,J＝8.0Hz,2H),7.71(d,J＝8.0Hz,2H),7.58-7.26(m,7H),7.05-6.98(m,3H),6.82(m,1H),5.20(s,2H),4.34(d,J＝4.0Hz,2H),4.11(m,2H),2.52(m,2H),1.53(m,4H),1.40(m,2H),1.39-1.20(m,2H).
IY210119B-1  1H NMR(DMSO-d6,300MHz)δ:8.55(s,1H),8.19(d,J＝6.0Hz,1H),8.09(d,J＝6.0Hz,1H),7.67(d,J＝6.0Hz,1H),7.57-7.36(m,6H),7.20-7.12(m,2H),7.06-7.02(m,6H),5.23(s,2H),4.42-4.40(m,2H),4.32(d,J＝3.0Hz,2H),3.62-3.60(m,4H),3.13-3.11(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
IY210115B-1  1H NMR(DMSO-d6,300MHz)δ:8.59(s,1H),8.19(d,J＝6.0Hz,1H),8.08(d,J＝6.0Hz,1H),7.63(d,J＝6.0Hz,1H),7.52-7.50(m,4H),7.17-7.03(m,7H),5.23(s,2H),4.36-4.34(m,4H),3.62-3.60(m,4H),3.13-3.11(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
IY210118B-1  1H NMR(DMSO-d6,300MHz)δ:8.65(s,1H),8.21(d,J＝6.0Hz,1H),8.12(d,J＝ 6.0Hz,1H),7.70(d,J＝6.0Hz,1H),7.58-7.34(m,6H),7.20-7.12(m,2H),7.06-7.02(m,6H),5.23(s,2H),4.42-4.40(m,2H),4.32(d,J＝3.0Hz,2H),3.62-3.60(m,4H),3.13-3.11(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
IY210113D-1  1H NMR(DMSO-d6,300MHz)δ:8.57(s,1H),8.19(d,J＝6.0Hz,1H),8.08(d,J＝6.0Hz,1H),7.71(d,J＝6.0Hz,1H),7.56-7.51(m,6H),7.17(m,1H),7.04-7.02(m,3H),5.22(s,2H),4.43-4.41(m,4H),3.62-3.60(m,4H),3.13-3.11(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
IY1210B-1  1H NMR(DMSO-d6,300MHz)δ:8.37(s,1H),8.18(d,J＝6.0Hz,1H),8.05(d,J＝6.0Hz,2H),7.70(d,J＝6.0Hz,1H),7.57-7.25(m,10H),7.06-7.00(m,4H),5.19(s,2H),4.88-4.84(m,1H),4.25(t,J＝3.0Hz,2H),2.30(t,J＝3.0Hz,2H),1.64-1.51(m,4H),1.40-1.30(m,5H).
ID210106D-1  1H NMR(DMSO-d6,300MHz)δ:8.47(s,1H),8.19(d,J＝6.0Hz,1H),8.04(d,J＝6.0Hz,1H),7.64(d,J＝6.0Hz,1H),7.58-7.51(m,4H),7.38-7.29(m,4H),7.06-7.03(m,4H),5.26(s,2H),4.39-4.33(m,4H),3.49-3.43(m,4H),3.01-2.99(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID210118D-1  1H NMR(DMSO-d6,300MHz)δ:8.62(s,1H),8.19(d,J＝6.0Hz,1H),8.11(d,J＝6.0Hz,1H),7.68(d,J＝6.0Hz,1H),7.56-7.24(m,9H),7.05-7.02(m,3H),5.22(s,2H),4.42-4.40(m,2H),4.34(d,J＝3.0Hz,2H),3.62-3.60(m,4H),3.13-3.11(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID210113C-1  1H NMR(DMSO-d6,300MHz)δ:8.41(s,1H),8.19(d,J＝6.0Hz,1H),8.04(d,J＝6.0Hz,1H),7.68(d,J＝6.0Hz,1H),7.58-7.49(m,4H),7.27-7.25(m,2H),7.06-7.02(m,3H),6.93-6.90(m,3H),5.23(s,2H),4.41(t,J＝3.0Hz,2H),4.26(d,J＝6.0Hz,2H),3.74(s,3H),3.52-3.49(m,4H),3.13-3.11(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
IY210103C-1  1H NMR(DMSO-d6,300MHz)δ:8.23-8.21(m,2H),8.08(d,J＝6.0Hz,1H),7.74(d,J＝6.0Hz,1H),7.64-7.44(m,5H),7.40-7.37(m,8H),7.23-7.21(m,1H),7.08-7.04(m,3H),5.25(s,2H),4.38(m,2H),3.49-3.43(m,4H),3.13-3.01(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID210118C-1  1H NMR(DMSO-d6,300MHz)δ:8.23(m,2H),8.09(d,J＝6.0Hz,1H),7.70(d,J＝6.0Hz,1H),7.54-7.47(m,5H),7.42-7.39(m,7H),7.23-7.21(m,1H),7.10-7.06(m,3H),5.25(s,2H),4.38(m,2H),3.49-3.43(m,4H),3.13-3.01(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID210115B-1  1H NMR(DMSO-d6,300MHz)δ:8.62(s,1H),8.19(d,J＝6.0Hz,1H),8.09(d,J＝ 6.0Hz,1H),7.65(d,J＝6.0Hz,1H),7.56-7.35(m,8H),7.05-7.02(m,3H),5.22(s,2H),4.39-4.30(m,4H),3.62-3.60(m,4H),3.13-3.11(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID210114B-1  1H NMR(DMSO-d6,300MHz)δ:8.68(s,1H),8.19(d,J＝6.0Hz,1H),8.08(d,J＝6.0Hz,1H),7.81-7.50(m,8H),7.30(brs,1H),7.05-7.02(m,3H),5.22(s,2H),4.40-4.38(m,4H),3.62-3.60(m,4H),3.13-3.11(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID1210B-1  1H NMR(DMSO-d6,300MHz)δ:8.34(s,1H),8.18(d,J＝6.0Hz,1H),8.00(d,J＝6.0Hz,2H),7.50(d,J＝6.0Hz,1H),7.33-7.26(m,8H),7.04-7.01(m,3H),6.47(t,J＝6.0Hz,1H),5.22(s,2H),4.34(m,2H),3.58(t,J＝3.0Hz,2H),2.30(t,J＝3.0Hz,2H),1.53-1.51(m,4H),1.40-1.39(m,2H).
IY1207A-1  1H NMR(CDCl3,300MHz)δ:8.26(d,J＝6.0Hz,1H),7.66(d,J＝6.0Hz,1H),8.19(d,J＝6.0Hz,1H),7.46(s,1H),7.34-7.07(m,10H),6.87(d,J＝6.0Hz,2H),6.77(d,J＝6.0Hz,1H),5.83(brs,1H),5.09(s,2H),4.75(d,J＝3.0Hz,2H),4.13(t,J＝3.0Hz,2H),2.81(t,J＝3.0Hz,2H),2.56(m,4H),1.61(t,J＝6.0Hz,4H),1.40-1.39(m,2H).
IY1223B-1  1H NMR(DMSO-d6,300MHz)δ:8.56(s,1H),8.22(d,J＝6.0Hz,1H),8.07(d,J＝6.0Hz,2H),7.71(d,J＝6.0Hz,1H),7.61-7.48(m,4H),7.29(t,J＝6.0Hz,2H),7.09-6.96(m,3H),5.25(s,2H),4.41(m,2H),3.50-3.45(m,2H),3.02(m,2H),1.68-1.27(m,6H).
IY1214A-1  1H NMR(DMSO-d6,300MHz)δ:8.69(s,1H),8.26(d,J＝6.0Hz,1H),8.19(d,J＝6.0Hz,2H),7.76(d,J＝6.0Hz,1H),7.59-7.56(m,4H),7.45(d,J＝6.0Hz,2H),7.13-7.09(m,3H),6.92(d,J＝6.0Hz,2H),5.30(s,2H),4.45(m,2H),3.55-3.54(m,4H),3.17(t,J＝3.0Hz,2H),1.84-1.80(m,4H),1.32-1.28(m,2H).
ID1214B-1  1H NMR(DMSO-d6,300MHz)δ:9.75(brs,1H),8.85(brs,1H),8.22(d,J＝6.0Hz,1H),8.11(d,J＝6.0Hz,2H),8.10(s,1H),7.69-7.56(m,5H),7.38-7.35(m,1H),7.07(t,J＝3.0Hz,3H),5.26(s,2H),4.38(m,2H),3.61-3.50(m,4H),3.00(t,J＝3.0Hz,2H),1.80-1.71(m,4H),1.29-1.26(m,2H).
IY1225B-1  1H NMR(DMSO-d6,300MHz)δ:8.72(s,1H),8.20(d,J＝6.0Hz,1H),8.10(d,J＝6.0Hz,2H),7.71(d,J＝6.0Hz,1H),7.60-7.53(m,4H),7.31-7.29(m,1H),7.08(t,J＝6.0Hz,3H),5.25(s,2H),4.40(m,2H),3.81(s,3H),3.51-3.45(m,4H),3.02(m,2H),1.80-1.72(m,4H),1.39-1.27(m,2H).
IY1210A-1  1H NMR(DMSO-d6,300MHz)δ:9.98(brs,1H),8.95(m,1H),8.23-8.06(m,2H),7.70-7.49(m,4H),7.09-6.99(m,2H),7.85-6.59(m,1H),5.20(s,2H),4.33(d,J＝3.0Hz,2H),4.11(t,J＝3.0Hz,2H),2.30(t,J＝3.0Hz,2H),1.53-1.51(m,4H),1.40-1.39(m,2H).
IY1226B-1  1H NMR(DMSO-d6,300MHz)δ:8.80(s,1H),8.22(d,J＝6.0Hz,1H),8.13(d,J＝6.0Hz,2H),7.70(d,J＝6.0Hz,1H),7.61-7.47(m,4H),7.30-7.28(m,1H),7.08-7.01(m,4H),5.26(s,2H),4.39(m,2H),3.82(s,3H),3.50-3.45(m,4H),3.02(m,2H),1.81-1.72(m,4H),1.40-1.27(m,2H).
IY1229C-1  1H NMR(DMSO-d6,300MHz)δ:8.22-8.17(m,2H),7.74-7.71(m,2H),7.69-7.44(m,5H),7.08-7.04(m,4H),5.25(s,2H),4.41(m,2H),3.48-3.39(m,4H),3.14-3.12(m,2H),1.79-1.71(m,4H),1.27-1.23(m,2H).
ID1229C-1  1H NMR(DMSO-d6,300MHz)δ:8.21(d,J＝6.0Hz,1H),8.13(d,J＝6.0Hz,2H),7.93(s,1H),7.68(d,J＝6.0Hz,1H),7.69-7.43(m,5H),7.08-7.04(m,3H),5.25(s,2H),4.39(m,2H),3.48-3.39(m,4H),3.14-3.12(m,2H),1.79-1.71(m,4H),1.27-1.23(m,2H).
ID1229D-1  1H NMR(DMSO-d6,300MHz)δ:8.21(d,J＝6.0Hz,1H),8.07(s,1H),7.53(d,J＝6.0Hz,2H),7.48-7.47(m,6H),7.09-7.04(m,3H),5.26(s,2H),4.42(m,2H),3.61-3.59(m,4H),3.40(s,3H),3.12-3.11(m,2H),1.79-1.60(m,6H).
ID1224D-1  1H NMR(DMSO-d6,300MHz)δ:8.74(s,1H),8.22(d,J＝6.0Hz,1H),8.11(d,J＝6.0Hz,2H),7.70(d,J＝6.0Hz,1H),7.61-7.52(m,4H),7.30-7.28(m,1H),7.07(t,J＝6.0Hz,3H),5.26(s,2H),4.39(m,2H),3.82(s,3H),3.50-3.45(m,4H),3.02(m,2H),1.81-1.72(m,4H),1.40-1.27(m,2H).
ID1231B-1  1H NMR(DMSO-d6,300MHz)δ:8.80(s,1H),8.21(d,J＝6.0Hz,1H),8.10(d,J＝6.0Hz,2H),7.39(d,J＝6.0Hz,1H),7.51-7.48(m,4H),7.32-7.29(m,1H),7.07(t,J＝6.0Hz,3H),5.22(s,2H),4.40(m,2H),3.81(s,3H),3.51-3.46(m,4H),3.01(m,2H),1.81-1.72(m,4H),1.40-1.27(m,2H).
IY1214B-2  1H NMR(DMSO-d6,300MHz)δ:8.75(s,1H),8.20(d,J＝6.0Hz,1H),8.15(d,J＝6.0Hz,2H),7.41(d,J＝6.0Hz,1H),7.35-7.30(m,5H),7.30-7.28(m,1H),7.02(t,J＝6.0Hz,3H),5.23(s,2H),4.38(m,2H),3.80(s,3H),3.51-3.46(m,4H),3.01(m,2H),1.81-1.72(m,4H),1.40-1.27(m,2H).
ID1224C-1  1H NMR(DMSO-d6,300MHz)δ:8.59(s,1H),8.21(d,J＝6.0Hz,1H),8.11(d,J＝6.0Hz,2H),7.71(d,J＝6.0Hz,1H),7.60-7.50(m,4H),7.26-7.25(m,1H),7.07-7.02(m,3H),6.91-6.88(m,2H),5.24(s,2H),4.30(m,2H),3.74(s,3H),3.71(s,3H),3.60(m,2H),3.14(m,2H),1.68-1.18(m,6H).
IY1229D-1  1H NMR(DMSO-d6,300MHz)δ:8.50(s,1H),8.19(d,J＝6.0Hz,1H),8.05(d,J＝6.0Hz,1H),7.51(d,J＝6.0Hz,1H),7.40-7.37(m,8H),7.04-7.00(m,4H),5.23(s,2H),4.39(m, 2H),3.48-3.39(m,4H),3.14-3.12(m,2H),1.79-1.71(m,4H),1.27-1.23(m,2H).
IY210103B-1  1H NMR(DMSO-d6,300MHz)δ:8.84(brs,1H),8.21(d,J＝6.0Hz,1H),8.13(d,J＝6.0Hz,1H),7.84(d,J＝6.0Hz,1H),7.69-7.51(m,5H),7.34-7.33(m,2H),7.08-7.04(m,3H),5.25(s,2H),4.39(m,2H),3.48-3.43(m,4H),3.01-2.99(m,2H),1.79-1.72(m,4H),1.27-1.23(m,2H).
IY210105B-1  1H NMR(DMSO-d6,300MHz)δ:8.70(brs,1H),8.21(d,J＝6.0Hz,1H),8.12(d,J＝6.0Hz,1H),7.70(d,J＝6.0Hz,1H),7.54-7.52(m,5H),7.13-6.94(m,5H),5.26(s,2H),4.39(m,2H),3.81(s,3H),3.49-3.43(m,4H),3.01-2.99(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
IY210105C-1  1H NMR(DMSO-d6,300MHz)δ:8.83(s,1H),8.22(d,J＝6.0Hz,1H),8.10(d,J＝6.0Hz,1H),7.69(d,J＝6.0Hz,1H),7.60-7.55(m,11H),5.25(s,2H),4.40(t,J＝6.0Hz,2H),3.82(s,3H),3.49-3.43(m,4H),3.01-2.99(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID210105C-1  1H NMR(DMSO-d6,300MHz)δ:8.80(s,1H),8.21(d,J＝6.0Hz,1H),8.11(d,J＝6.0Hz,1H),7.70(d,J＝6.0Hz,1H),7.74-7.05(m,11H),5.26(s,2H),4.40(t,J＝6.0Hz,2H),3.49-3.43(m,4H),3.01-2.99(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
IY210105D-1  1H NMR(DMSO-d6,300MHz)δ:8.78(brs,1H),8.25(d,J＝6.0Hz,1H),8.17(d,J＝6.0Hz,1H),7.75(d,J＝6.0Hz,1H),7.50-7.49(m,5H),7.10-6.96(m,5H),5.25(s,2H),4.41(m,2H),3.40-3.38(m,4H),3.01-2.99(m,2H),1.81-1.72(m,4H),1.27-1.23(m,2H).
IY210105A-1  1H NMR(DMSO-d6,300MHz)δ:8.76(brs,1H),8.23(d,J＝6.0Hz,1H),8.16(d,J＝6.0Hz,1H),7.73(d,J＝6.0Hz,1H),7.51-7.49(m,4H),7.10-6.92(m,5H),5.23(s,2H),4.37(m,2H),3.45-3.43(m,4H),3.01-2.99(m,2H),1.84-1.72(m,4H),1.27-1.23(m,2H).
IY210106D-1  1H NMR(DMSO-d6,300MHz)δ:8.62(brs,1H),8.19(d,J＝6.0Hz,1H),8.11(d,J＝6.0Hz,1H),7.69(d,J＝6.0Hz,1H),7.52-7.49(m,4H),7.11-6.94(m,5H),5.22(s,2H),4.39(m,2H),3.49-3.43(m,4H),3.01-2.99(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID210110C-1  1H NMR(DMSO-d6,300MHz)δ:8.60(s,1H),8.18(d,J＝6.0Hz,1H),8.13(d,J＝6.0Hz,1H),7.71(d,J＝6.0Hz,1H),7.55-7.51(m,4H),7.11-6.94(m,5H),5.22(s,2H),4.39(m,2H),3.81(s,3H),3.49-3.43(m,4H),3.01-2.99(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
IY210110D-1  1H NMR(DMSO-d6,300MHz)δ:8.58(s,1H),8.16(d,J＝6.0Hz,1H),8.11(d,J＝6.0Hz,1H),7.69(d,J＝6.0Hz,1H),7.55-7.51(m,4H),7.11-6.94(m,5H),5.22(s,2H),4.39(m,2H),3.81(s,3H),3.49-3.43(m,4H),3.01-2.99(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID1207B-1  1H NMR(DMSO-d6,300MHz)δ:8.18(d,J＝6.0Hz,1H),8.10(s,1H),8.01(d,J＝6.0Hz,2H),7.71(d,J＝6.0Hz,1H),7.59-7.46(m,4H),7.03-6.99(m,3H),6.42(d,J＝3.0Hz,1H), 5.19(s,2H),4.00(m,2H),3.99-3.95(m,1H),2.33-2.30(m,2H),1.87-1.84(m,2H),1.68-1.40(m,8H),1.27-1.24(m,2H).
ID1217B-1  1H NMR(DMSO-d6,300MHz)δ:8.20(d,J＝6.0Hz,1H),8.11(s,1H),8.05(d,J＝6.0Hz,2H),7.70(d,J＝6.0Hz,1H),7.56-7.48(m,4H),7.05-6.98(m,3H),6.48(d,J＝3.0Hz,1H),5.20(s,2H),4.00(m,2H),3.99-3.97(m,1H),2.34-2.32(m,2H),1.85-1.84(m,2H),1.68-1.40(m,10H),1.27-1.24(m,2H).
ID1223A-1  1H NMR(DMSO-d6,300MHz)δ:8.28(s,1H),8.18(d,J＝6.0Hz,1H),8.00(d,J＝6.0Hz,2H),7.64(d,J＝6.0Hz,2H),7.45-7.47(m,4H),7.03-6.98(m,3H),5.93(s,2H),5.19(s,2H),4.11(t,J＝3.0Hz,2H),2.74(t,J＝3.0Hz,2H),1.53-1.51(m,4H),1.41-1.39(m,2H).
ID1215B-1  1H NMR(DMSO-d6,300MHz)δ:8.20(s,1H),8.18(d,J＝6.0Hz,1H),8.00(d,J＝6.0Hz,2H),7.66(d,J＝6.0Hz,1H),7.48-7.46(m,3H),7.03-6.99(m,3H),6.38(t,J＝3.0Hz,1H),5.20(s,2H),4.12(m,2H),2.70-2.68(m,4H),1.54-1.27(m,8H).
ID1215C-1  1H NMR(DMSO-d6,300MHz)δ:8.22(s,1H),8.18(d,J＝6.0Hz,1H),8.01(d,J＝6.0Hz,1H),7.69(d,J＝6.0Hz,1H),7.51-7.48(m,3H),7.03-7.01(m,3H),6.53-6.51(m,1H),5.21(s,2H),4.42-4.40(m,2H),3.86-3.83(m,2H),3.70-3.68(m,2H),3.41-3.38(m,4H),1.85-1.24(m,13H).
IY1215C-1  1H NMR(DMSO-d6,300MHz)δ:8.19(d,J＝6.0Hz,1H),8.13(s,1H),8.00(d,J＝6.0Hz,2H),7.72(d,J＝6.0Hz,1H),7.56-7.44(m,3H),7.01-6.99(m,3H),6.40(d,J＝3.0Hz,1H),5.19(s,2H),4.01(m,2H),4.00-3.95(m,1H),2.30-2.28(m,2H),1.86-1.84(m,2H),1.67-1.40(m,10H),1.27-1.24(m,2H).
ID1215A-1  1H NMR(DMSO-d6,300MHz)δ:8.18(s,1H),8.10(d,J＝6.0Hz,1H),8.02(d,J＝6.0Hz,1H),7.70(d,J＝6.0Hz,1H),7.50-7.48(m,3H),7.02-7.01(m,3H),6.53-6.51(m,1H),5.20(s,2H),4.43-4.40(m,2H),3.85-3.83(m,2H),3.69-3.68(m,2H),3.41-3.38(m,4H),1.85-1.24(m,13H).
IY1215D-1  1H NMR(DMSO-d6,300MHz)δ:8.24(s,1H),8.19(d,J＝6.0Hz,1H),8.03(d,J＝6.0Hz,1H),7.68(d,J＝6.0Hz,1H),7.50-7.48(m,3H),7.04-7.01(m,3H),6.52-6.50(m,1H),5.20(s,2H),4.41-4.39(m,2H),3.85-3.83(m,2H),3.72-3.68(m,2H),3.40-3.37(m,4H),1.84-1.24(m,13H).
IY210122C-1  1H NMR(DMSO-d6,300MHz)δ:8.56(brs,1H),8.18(d,J＝6.0Hz,1H),8.11(d,J＝6.0Hz,1H),7.69(d,J＝6.0Hz,1H),7.52-7.50(m,5H),7.14-7.06(m,5H),5.26(s,2H),4.40(t,J＝6.0Hz,2H),3.49-3.43(m,4H),3.01-2.99(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID210119B-1  1H NMR(DMSO-d6,300MHz)δ:8.75(brs,1H),8.21(d,J＝6.0Hz,1H),8.14(d,J＝6.0Hz,1H),7.70(d,J＝6.0Hz,1H),7.54-7.51(m,5H),7.15-7.05(m,5H),5.26(s,2H),4.40(t,J＝6.0Hz,2H),3.49-3.43(m,4H),3.01-2.99(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
ID210106C-1  1H NMR(DMSO-d6,300MHz)δ:8.51(s,1H),8.18(d,J＝6.0Hz,1H),8.06(d,J＝6.0Hz,1H),7.66(d,J＝6.0Hz,1H),7.57-7.34(m,7H),7.07-7.02(m,3H),5.22(s,2H),4.39-4.31(m,4H),3.62-3.60(m,4H),3.14-3.11(m,2H),1.80-1.72(m,4H),1.27-1.23(m,2H).
IY210128B-1  1H NMR(DMSO-d6,300MHz)δ:8.75(s,1H),8.22(d,J＝6.0Hz,1H),8.14(d,J＝6.0Hz,1H),7.74(d,J＝6.0Hz,1H),7.65-7.32(m,10H),7.09-7.05(m,3H),5.26(s,2H),4.39(m,2H),3.49-3.45(m,4H),3.02-3.00(m,2H),1.80-1.71(m,4H),1.29-1.24(m,2H).
ID210127B-1  1H NMR(DMSO-d6,300MHz)δ:8.25(s,1H),8.17(d,J＝6.0Hz,1H),8.02(d,J＝6.0Hz,1H),7.70(d,J＝6.0Hz,1H),7.50-7.48(m,4H),7.04-7.01(m,3H),6.53-6.51(m,1H),5.20(s,2H),4.41-4.40(m,2H),3.84-3.83(m,2H),3.71-3.68(m,2H),3.41-3.38(m,4H),1.84-1.24(m,13H).
实施例2、将上述化合物及磷酸盐ID1120B-P对肝癌细胞、乳腺癌、肺癌、吉非替尼或阿法替尼耐药的肺癌和白血病细胞的增殖抑制作用
分别收集对数生长期的HepG2，SMMC-7721，HuH-7，MCF-7，MDA-MB-231，MDA-MB-468，PC9，PC9-AR，PC9-GR，Jurkat和Molt-13细胞，计数，调整细胞悬液浓度为5×10 4个/mL，加入96孔细胞培养板，每孔体积100ul。以DMSO为溶剂对照，Sorafinib、WP1066(中文名称：(2E)-3-(6-溴-2-吡啶基)-2-氰基-N-[(1S)-1-苯基乙基]-2-丙烯酰胺,CAS:857064-38-1，结构为
Figure PCTCN2022077027-appb-000130
)或Gefitinib为阳性对照，将上述化合物及磷酸盐ID1120B-P用DMSO稀释后加入培养孔，使体系中化合物的终浓度分别为0.1、0.3、1、3、10、30、100和300(μmol/L)。继续培养48h后，每孔加入MTT溶剂(5mg/ml)10μL，37℃孵育4h，吸弃培养上清，每孔加入150μl DMSO，摇床震荡脱色10min，酶标仪读值，测定在吸收波长为490nm下的OD值，记录结果，以化合物的剂量为横坐标，吸光值为纵坐标绘制细胞生长曲线。肿瘤细胞的半数抑制率(IC50值)的统计结果如下表及图1和图2所示：
Figure PCTCN2022077027-appb-000131
Figure PCTCN2022077027-appb-000132
Figure PCTCN2022077027-appb-000133
Figure PCTCN2022077027-appb-000134
表中显示：ID1120B-1及其衍生物ID1214B-1，IY1214A-1和IY1214B-2等对肝癌细胞、乳腺癌、肺癌和白血病细胞均有良好的增殖抑制作用，特别是在肝癌细胞的抑瘤活性更强，我们重点对这四种化合物的抗肿瘤效应进行了进一步研究。
实施例2、ID1120B-1及其衍生物ID1214B-1，IY1214A-1和IY1214B-2诱导肝癌细胞的G2/M周期阻滞。
取对数生长期的HepG2细胞，消化后离心并将细胞制成单细胞悬液。计数后将细胞铺入1个12孔板，每孔2×10 5个细胞，铺3个孔做平行对照。铺板16h后，用梯度浓度的化合物分别处理细胞。48h后，胰酶消化细胞，重悬之后计数，将细胞浓度调整为5×10 5个。消化完成后离心弃上清，再用PBS洗细胞两遍(2000rpm，离心5min)，之后弃尽上清，每管加入980μl的70％冷乙醇和20μl的5％BSA(添加少量BSA可以减少操作过程中的细胞损失)在4℃条件下固定过夜。弃固定液，用PBS洗3遍以去除残余的固定液(1000rpm，离心3min)。细胞洗涤完成后，按DNA含量检测试剂盒(北京索莱宝公司产品)的说明书的要求进行后续操作。每个样品分别用100μl Rnase A于37度孵育30min，然后每个样 品中加入500μl已经配制好的PI(碘化丙啶)工作液，室温避光孵育30min。最后，通过流式细胞仪测定细胞周期。采用ModFit软件对实验结果进行分析，通过Graphpadprism 6.0进一步分析得到两种细胞各自的细胞周期比例。
图3、图4是用ModFit软件分析ID1120B-1及其衍生物ID1214B-1，IY1214A-1和IY1214B-2对HepG2肝癌细胞的周期分布的影响的结果。图5是通过Graphpad prism 6.0对图3、图4结果的进一步定量分析。图3、图4和图5结果表明，与溶剂对照组(DMSO)相比，化合物ID1120B-1及其衍生物ID1214B-1，IY1214A-1和IY1214B-2均可以剂量依赖的方式诱导肝癌细胞的G2/M期比率显著增加，G1/S期的比率相应减少。ID1120B-1诱导肝癌细胞的G2期比率由8.72％延长至15.9％；ID1214B-1诱导G2期由10.6％延长至17.54％；IY1214A-1诱导G2期由13.35％延长至34.54％；IY1214B-2诱导G2期由9.6％延长至21.71％。
实施例3、IY1214A-1和IY1214B-2诱导肝癌细胞凋亡
取对数生长期的HepG2细胞，消化后离心并将细胞制成单细胞悬液。计数后将细胞铺入1个12孔板，每孔2×10 5个细胞，铺3个孔做平行对照。铺板16h后，用梯度浓度的化合物分别处理细胞48h。用不含EDTA的胰酶消化细胞，重悬之后计数，将细胞浓度调整为1×10 6个。用Annexin V FITC-PI细胞凋亡检测试剂盒(北京索莱宝公司产品)的说明书的要求进行用后续操作。具体为：1×PBS洗细胞2遍(6000rpm，离心0.5min)，用1×Binding buffer洗细胞1遍(6000rpm，离心0.5min)之后弃尽上清，以300μl的1×Binding buffer重悬细胞，每管加入5μlAnnexinV-FITC，避光孵育10min。随后，每管加入5μl的PI，避光孵育5min。避光上机检测。
图6是流式细胞术检测IY1214A-1和IY1214B-2对HepG2肝癌细胞凋亡的影响。结果显示，与对照组相比，IY1214A-1和IY1214B-2均可以剂量依赖的方式诱导细胞凋亡增加。IY1214A-14μM和8μM处理48h后，细胞的凋亡率分别为57.7％和63％，均较对照孔升高2倍以上；IY1214B-28μM处理48h后，细胞的凋亡率为47.1％，与未处理孔比较，凋亡率约升高3.3倍。
实施例4、IY1214B-2影响细胞周期调控分子和自噬相关基因的表达
将肝癌HepG2细胞接种于6孔板，每孔1×10 6个细胞。加化合物IY1214B-2(浓度为0和10μM)处理24h。按照TRIzol一步法提取细胞总RNA，测定RNA浓度和纯度。以总RNA为模板，按Promega公司反转录试剂盒说明书合成cDNA。半定量RT-PCR和实时定量RT-PCR扩增检测CCNB1、CDKl和SQSTM，以ACTB为内参照。所用引物见表1。
表1:
Figure PCTCN2022077027-appb-000135
实时定量RT-PCR
反应体系:
Figure PCTCN2022077027-appb-000136
每组样品设3个复孔。
反应条件：
95℃预变性5min。
Figure PCTCN2022077027-appb-000137
扩增40个循环，以β-actin的CT值作为初始值进行数据分析。
图7是通过Q-PCR检测IY1214B-2对细胞周期和自噬相关分子的mRNA水平的影响。结果表明，IY1214B-2以0和10μM处理24h后，与内存蛋白β-actin的基因(ATCB)的表达水平相比，细胞周期G2期调控分子Cyclin B1(基因名：CCNB1)和CDC2(基因名：CDK1)的mRNA水平约下调20-25％，细胞自噬相关标志物P62(基因名：SQSTM)的表达上调约2.5倍。表明IY1214B-2可以通过从mRNA水平下调Cyclin B1和CDC2的表达，诱导G2/M期阻滞或通过从mRNA水平上调P62的表达，促进自噬，抑制肿瘤细胞生长。
综上结果表明，ID1120B-1及其衍生物ID1214B-1，IY1214A-1和IY1214B-2等化合物可 以显著抑制肝癌细胞、乳腺癌、肺癌、吉非替尼或阿法替尼耐药的肺癌和白血病细胞的增殖，诱导肝癌HepG2细胞发生G2/M周期阻滞和细胞凋亡，显示了良好的抗癌作用。
按照药物开发的一般途径(先进行常规的抗肿瘤体外筛选，然后进行针对性的研究)，本发明的化合物可以应用到与细胞增殖异常相关的癌症治疗药物中，可通过与人体可接受的成盐或与药用载体混合制备抗肿瘤药物。
最后所应说明的是：上述实施例仅用于说明而非限制本发明的技术方案，任何对本发明进行的等同替换及不脱离本发明精神和范围的修改或局部替换，其均应涵盖在本发明权利要求保护的范围之内。

Claims (9)

  1. 一种萘基脲类化合物，其特征在于，结构式如通式I所示：
    Figure PCTCN2022077027-appb-100001
    其中，R选自氢、C1～C5直链烷基、末端被卤素取代的C1～C5直链烷基、5～8元环烷基、
    Figure PCTCN2022077027-appb-100002
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9各自独立地选自H、F、Cl、Br、-CN、-CH 3、-CF 3、-OCH 3、-OCF 3，R 5还为苯基，M为H或-CH 3，
    m代表CH 2取代基的个数，m为0、1；
    n代表CH 2取代基的个数，n为1、2、3、4；
    A为
    Figure PCTCN2022077027-appb-100003
    其中p代表CH 2取代基的个数，p为2；
    X为O或S。
  2. 根据权利要求1所述的萘基脲类化合物，其特征在于，具体为如下结构的化合物：
    Figure PCTCN2022077027-appb-100004
    Figure PCTCN2022077027-appb-100005
    Figure PCTCN2022077027-appb-100006
  3. 权利要求1或2所述的萘基脲类化合物与乙酸、二氢叶酸、苯甲酸、柠檬酸、山梨酸、丙酸、草酸、富马酸、马来酸、盐酸、苹果酸、磷酸、亚硫酸、硫酸、香草酸、酒石酸、抗坏血酸、硼酸、乳酸和乙二胺四乙酸中的至少一种形成的生物学可接受的盐。
  4. 权利要求1或2所述的萘基脲类化合物的制备方法，其特征在于，包括以下步骤：
    (1)将
    Figure PCTCN2022077027-appb-100007
    溶于四氢呋喃中，-5℃～5℃分批加入NaH，再加入
    Figure PCTCN2022077027-appb-100008
    室温搅拌反应至完全，后处理得到
    Figure PCTCN2022077027-appb-100009
    (2)将
    Figure PCTCN2022077027-appb-100010
    溶于乙醇和饱和氯化铵水溶液的混合溶液中，40～50℃下加入铁粉，50～60℃搅拌反应至完全，经后处理得到
    Figure PCTCN2022077027-appb-100011
    (3)将化合物
    Figure PCTCN2022077027-appb-100012
    R基异氰酸酯或R基异硫氰酸酯和N,N-二异丙基乙胺溶于1,2-二氯乙烷中，80～90℃搅拌反应至完全，柱层析得到
    Figure PCTCN2022077027-appb-100013
  5. 根据权利要求4所述的萘基脲类化合物的制备方法，其特征在于，所述
    Figure PCTCN2022077027-appb-100014
    的制备过程如下：
    (a)将
    Figure PCTCN2022077027-appb-100015
    和三苯基膦溶于四氢呋喃中，-5℃～5℃保护气氛下加入偶氮二甲酸二异丙酯，室温搅拌反应至完全，经后处理得到
    Figure PCTCN2022077027-appb-100016
    (b)将化合物
    Figure PCTCN2022077027-appb-100017
    溶于四氢呋喃中，-5℃～5℃分批加入四氢铝锂，室温搅拌至反应完全，经后处理得到
    Figure PCTCN2022077027-appb-100018
  6. 根据权利要求4所述的萘基脲类化合物的制备方法，其特征在于，所述步骤(1)中
    Figure PCTCN2022077027-appb-100019
    与NaH的摩尔比为1:1.2:2；
    步骤(2)中
    Figure PCTCN2022077027-appb-100020
    与铁粉的摩尔比为1:5，乙醇和饱和氯化铵水溶液的体积比为1:1；
    步骤(3)中，
    Figure PCTCN2022077027-appb-100021
    R基异氰酸酯或R基异硫氰酸酯和N,N-二异丙基乙胺的摩尔比为1:1.2:2.0。
  7. 根据权利要求5所述的萘基脲类化合物的制备方法，其特征在于，步骤(a)中，
    Figure PCTCN2022077027-appb-100022
    三苯基膦、偶氮二甲酸二异丙酯的摩尔比为1:1.2:1.2:1.2；步骤(b)中，
    Figure PCTCN2022077027-appb-100023
    和四氢铝锂的摩尔比为1:1。
  8. 权利要求1至3任一项所述的萘基脲类化合物及其生物学可接受的盐在制备抗肿瘤药物中 的用途。
  9. 根据要求8所述的用途，其特征在于：所述的抗肿瘤药物是指治疗肝癌、乳腺癌、肺癌和白血病的药物。
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