WO2021190142A1 - 一类抗肿瘤化合物及其制备与用途 - Google Patents

一类抗肿瘤化合物及其制备与用途 Download PDF

Info

Publication number
WO2021190142A1
WO2021190142A1 PCT/CN2021/074623 CN2021074623W WO2021190142A1 WO 2021190142 A1 WO2021190142 A1 WO 2021190142A1 CN 2021074623 W CN2021074623 W CN 2021074623W WO 2021190142 A1 WO2021190142 A1 WO 2021190142A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
amino
naphthyridine
chlorophenyl
benzo
Prior art date
Application number
PCT/CN2021/074623
Other languages
English (en)
French (fr)
Inventor
苟少华
王园江
Original Assignee
东南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 东南大学 filed Critical 东南大学
Publication of WO2021190142A1 publication Critical patent/WO2021190142A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the technical field of medicine, and relates to a new type of compound with both CK2 protein kinase and cancer cell stemness inhibitory ability; also relates to the preparation method of this type of compound and their application in the preparation of anti-tumor drugs.
  • CK2 Casein Kinase II
  • CK2 ⁇ /CK2 ⁇ ' two catalytic subtypes
  • ⁇ regulatory subtypes
  • CK2 has different key roles in different stages of the cell cycle.
  • the addition of CK2 inhibitors can inhibit cell cycle progression, indicating that the cycle activities of animal cells require the participation and regulation of CK2.
  • over-expression of CK2 can inhibit programmed death caused by anti-cancer drugs.
  • CK2 is overexpressed in a variety of drug-resistant cancer cells, inhibiting CK2 can not only promote the apoptosis of cancer cells, but also increase the sensitivity of cancer cells to anticancer drugs.
  • cancers such as head and neck cancer, breast cancer, colon cancer, kidney cancer, lung cancer, leukemia and prostate cancer
  • CK2 is overexpressed in cancer cells.
  • CK2 is directly related to the poor prognosis of cancer and the deterioration of the disease. Therefore, CK2 has become a very promising cancer treatment target.
  • the homology of the entire human protein kinase family is relatively high, especially the core part of its catalytic domain.
  • Most small molecule protein kinase inhibitors achieve their inhibitory effect mainly by interacting with the conservative ATP binding site of the kinase target. Due to the large amino acid residues, the ATP binding site of CK2 is relatively small compared to the binding sites of other kinases. This makes the design and synthesis of ATP competitive inhibitors of CK2 must meet two requirements, namely, high specificity and Smaller molecular weight.
  • CK2 protein was first publicly reported in the literature in 1954, elucidating the important role of CK2 protein in cell growth regulation (J. Biol. Chem. 1954, 211, 969-980). On this basis, it was not until 1986 that small molecule inhibitors targeting the protein, such as benzimidazole derivatives (J. Biol. Chem. 1986, 261, 3414-3419; Bioorg. Med. Chem. 2003, 11, 3997-4002), flavonoid derivatives (Curr. Top. Med. Chem. 2011, 11, 1340-1351), coumarin derivatives (Curr. Pharmaceut. Des.
  • small molecule inhibitors targeting the protein such as benzimidazole derivatives (J. Biol. Chem. 1986, 261, 3414-3419; Bioorg. Med. Chem. 2003, 11, 3997-4002), flavonoid derivatives (Curr. Top. Med. Chem. 2011, 11, 1340-1351), coumarin derivatives (Curr. Pharmaceut. Des.
  • CX-4945 (formula I) developed by Cylene Pharmaceuticals based on benzonaphthyridine is currently in phase I/II clinical studies.
  • CX-4945 exhibits broad-spectrum anti-tumor activity and superior CK2 kinase inhibitory activity, it still has some obvious shortcomings.
  • CX-4945 has an inhibitory rate (IC 50 ) of 1 nM for CK2, but it has resistance to other 12 kinases. It also has a nanomolar level (IC 50 ) inhibitory effect.
  • IC 50 inhibitory rate
  • its inhibitory effect on CLK2 is even stronger than that on CK2, which can easily lead to severe off-target toxicity. Therefore, it is of great significance to develop new CK2 inhibitors with excellent activity and high selectivity on the basis of CX-4945.
  • Cancer stem cells also known as cancer stem cells or cancer stem cells, refer to cells in tumors that have the ability to self-renew and produce heterogeneous tumor cells. They are also considered to be the "power" in tumor tissues that really drives the occurrence and development of tumors. It is also the root cause of tumor recurrence and metastasis.
  • cancer stem cell populations Due to the presence of cancer stem cell populations in tumor tissues, they have strong self-renewal and differentiation capabilities, and can maintain the malignant proliferation and invasion of tumors by regulating multiple signal pathways such as Wnt/ ⁇ -catenin, Notch, Hedgehog, BMI-1, etc. , Drug resistance, metastasis and recurrence, etc.
  • multiple signal pathways such as Wnt/ ⁇ -catenin, Notch, Hedgehog, BMI-1, etc.
  • Drug resistance, metastasis and recurrence etc.
  • cancer stem cells have been successfully isolated from hematomas and various solid tumors. More and more evidence shows that conventional chemotherapy drugs, including targeted drugs, can effectively kill differentiated cancer cells at effective doses, but are ineffective against cancer stem cells, and some even promote the growth of cancer stem cells.
  • the purpose of the present invention is to provide a class of anti-tumor compounds and their preparation and use, that is, a class of compounds that can inhibit both CK2 protein kinase and cancer cell stemness and their preparation and use.
  • the present invention introduces some aliphatic chain or heterocyclic structure fragments containing amino, hydroxyl, sulfhydryl or halogen atoms into the parent structure of known CK2 inhibitor (CX-4945) via amide bond, ester bond, thioester bond, etc., to provide A new class of compounds.
  • This type of compound has obvious anti-cancer activity, and the in vitro anti-cancer activity and enzyme activity of some compounds are better than the parent compound, and it has been found to effectively inhibit the stemness of cancer cells.
  • the present invention provides compounds of the structures shown in Formula II-A, Formula II-B, Formula II-C, Formula II-D and II-E:
  • R 1 is -NH 2 , -OH, -SO 2 CH 3 , -CH 2 CHF 2 , -CH 2 C ⁇ CH, -(CH 2 ) 2 OH, -(CH 2 ) 2 NH 2 , -(CH 2 ) 2 Br, -(CH 2 ) 2 CO 2 CH 3 , -(CH 2 ) 2 CO 2 H, -(CH 2 ) 2 N(CH 3 ) 2 , -(CH 2 ) 3 OH , -(CH 2 ) 3 NH 2 , -(CH 2 ) 3 CO 2 CH 3 ,
  • R 2 is -CH 2 CHF 2 , -(CH 2 ) 2 OH, -(CH 2 ) 2 F, -(CH 2 ) 2 I, -(CH 2 ) 2 CHCl 2 , -(CH 2 O) 2 (CH 2 ) 2 OH,
  • R 3 is -(CH 2 ) 2 SH
  • R 4 is -C 2 H 5 , -(CH 2 ) 2 SCH 3 , -OCH 2 CH(CH 3 ) 2 ,
  • any one or more of the following compounds 1 to 31 is preferred:
  • the synthesis method of the above compound is mainly synthesized by using CX-4945 or its precursor as a raw material.
  • CX-4945 was synthesized by the reference method. Firstly, 3-bromoisonicotinic acid was used as the starting material. After esterification, the Suzuki coupling reaction with 2-amino-4-carbomethoxyphenyl borate was carried out to obtain the intermediate Form 5,6-dihydrobenzo[c][2,6]naphthyridine-8-methyl carboxylate, then undergoes chlorination reaction with phosphorus oxychloride, and then undergoes substitution reaction with m-chloroaniline, and finally obtains by hydrolysis product.
  • the specific synthesis route is as follows:
  • the compound of formula II-A can be obtained by exchange reaction or condensation reaction.
  • the specific synthesis route is as follows:
  • the compound of formula II-B can be obtained by esterification reaction, and the specific synthesis route is as follows:
  • the compound of formula II-C can be obtained by the esterification reaction of thiol, and the specific synthesis route is as follows:
  • the compound of formula II-D can be obtained by the amide condensation reaction of hydrazide and carboxylic acid.
  • the specific synthesis route is as follows:
  • the compound of formula II-E can be obtained by the amide condensation reaction of hydrazide and sulfonic acid.
  • the specific synthesis route is as follows:
  • HATU represents the condensation agent 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyl Urea hexafluorophosphate
  • TBTU stands for condensing agent O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate
  • DIPEA stands for N,N-diisopropylethylamine
  • DMF represents the solvent N,N-dimethylformamide
  • Reflux represents reflux
  • rt represents room temperature.
  • Compound 1 obtained by introducing hydrazine group into CX-4945 parent skeleton through amide bond has higher cytotoxic activity on cancer cells, inhibition of CK2 protein kinase and selectivity to CK2 protein than CX-4945;
  • the obtained compound 2 has higher cytotoxic activity, but the enzyme activity is poor;
  • the methanesulfonamide group is introduced, the cytotoxic activity and enzyme activity of the obtained compound 3 are lower than that of CX-4945. Therefore, introducing a hydrogen bond donor of a smaller group through an amide bond can increase the cytotoxic activity, enzyme activity and selectivity to CK2 protein of the parent compound.
  • the cytotoxic activity of compounds with C2 aliphatic chain introduced through amide bonds in the CX-4945 matrix is generally higher than that of compounds with C3 aliphatic chain (such as compounds 11, 12, 13, 14, 15), and the end group of the C2 aliphatic chain is the hydroxyl group, which has the best activity.
  • compound 4 cytotoxic activity, enzyme activity and selectivity to CK2 protein are all improved compared with CX-4945, and it also reduces the toxicity to normal liver cell LO2; when it is amino, although it increases the resistance of related compounds CK2 is active, but its cytotoxic activity and selectivity to CK2 protein are poor.
  • compounds obtained by introducing N-containing heterocycles through C2 aliphatic chains, such as morpholine ring, pyrrole ring and piperidine ring, can all improve the cytotoxic activity of the compound.
  • the compound has high cytotoxic activity, enzyme activity and selectivity for CK2 protein. Therefore, when the CX-4945 carboxyl terminal is introduced into the C2 fatty chain through an amide bond, and the fatty chain terminal contains a hydrogen bond acceptor group, the resulting compound has higher cytotoxic activity, enzymatic activity and selectivity to CK2 protein.
  • C2 aliphatic chain end is hydroxyl, while retaining the cytotoxic activity of the original parent compound, it also increases the activity and selectivity to CK2 protein.
  • the introduction of the pyridine ring through the C2 aliphatic chain can also increase the inhibitory activity and selectivity of the compound on the CK2 protein.
  • the atomic radius of sulfur is larger than that of oxygen, and the thioester bond is less stable than the oxygen ester bond. Therefore, the compound obtained by introducing thiol through the thioester bond (compound 26) only retains the cytotoxic activity of the original parent compound, but The enzyme activity is poor.
  • Compound 20 is also better than CX-4945 in inhibiting Wnt signaling pathway and Hedgehog signaling pathway.
  • Compound 4 inhibited stem cell-related genes OCT4, SOX2 and Nanog higher than CX-4945 and BBI608, while compound 20 inhibited stem cell genes as much as BBI608, but better than CX-4945.
  • the compounds of the present invention have obvious anti-cancer activity. Some compounds have better cytotoxic activity on cancer cells, inhibition of CK2 protein kinase and selectivity to CK2 protein than parent compounds, and can effectively inhibit the stemness of cancer cells. For the preparation of anti-tumor drugs.
  • Figure 1 The inhibitory activity of some compounds on CK2 and CLK2 protein kinases.
  • Figure 2 The inhibition of representative compounds on the stem cell marker CD44/CD133 in HCT-116 primary cancer cells, (a) flow cytometry of the sample, (b) inhibition rate of the sample.
  • FIG. 1 The effects of representative compounds on Wnt and Hedgehog signaling pathways and stem cell-related gene expression.
  • the anti-tumor compound of the present invention introduces aliphatic chains containing amino, hydroxyl, sulfhydryl or halogen atoms into the parent structure of a known CK2 inhibitor (CX-4945) via an amide bond, an ester bond, or a thioester bond, or a hetero Ring structure fragments provide a new type of compound as shown in formula II;
  • R 1 is -NH 2 , -OH, -SO 2 CH 3 , -CH 2 CHF 2 , -CH 2 C ⁇ CH, -(CH 2 ) 2 OH, -(CH 2 ) 2 NH 2 , -(CH 2 ) 2 Br, -(CH 2 ) 2 CO 2 CH 3 , -(CH 2 ) 2 CO 2 H, -(CH 2 ) 2 N(CH 3 ) 2 , -(CH 2 ) 3 OH , -(CH 2 ) 3 NH 2 , -(CH 2 ) 3 CO 2 CH 3 ,
  • R 2 is -CH 2 CHF 2 , -(CH 2 ) 2 OH, -(CH 2 ) 2 F, -(CH 2 ) 2 I, -(CH 2 ) 2 CHCl 2 , -(CH 2 O) 2 (CH 2 ) 2 OH,
  • R 3 is -(CH 2 ) 2 SH
  • R 4 is -C 2 H 5 , -(CH 2 ) 2 SCH 3 , -OCH 2 CH(CH 3 ) 2 ,
  • the known compound CX-4945 and its precursor compound are prepared by the method reported in the reference (J. Med. Chem. 2011, 54, 635-654), and verified by the hydrogen nuclear magnetic spectrum.
  • the MTT method was used to test the cytotoxic activity of the compounds of the present invention. Count the cells in the logarithmic growth phase and inoculate them in a 96-well culture plate with about 8000-10000 cells per well. After the cells were cultured overnight, they were administered after the cells adhered to the wall. The administration group and the control group were set up respectively.
  • the compound to be tested is prepared as a stock solution with DMSO solution, and diluted to a series of concentrations with cell culture medium before use, wherein the final concentration of DMSO does not exceed 4 ⁇ (the following experiment is similar). Set 3 multiple holes for each concentration.
  • the compounds of the present invention have been tested for their cytotoxic activity against human prostate cancer cell PC-3, colon cancer cell HCT-116, breast cancer cell MCF-7, colon cancer cell HT-29, bladder cancer cell T24 and normal liver cell LO2, CX -4945 was used as a positive control. Observe the compound's inhibition of tumor cell growth at different concentrations, calculate the inhibition rate and its IC 50 value to evaluate the compound's cytotoxic activity, and the results are shown in Table 1.
  • CK2 kit Commercialized human casein kinase 2 (CK2) and human cell cycle-like kinase 2 (CLK2) kits were used to test the inhibitory activity of the compounds of the present invention and CX-4945 on CK2 and CLK2 proteins.
  • CK2 kit, CLK2 kit, CK2 protein and CLK2 protein were purchased from Shanghai Fusheng Industrial Co., Ltd. According to the experimental methods provided in the instructions of the CK2 and CLK2 kits, the compounds were tested for CK2 and CLK2 enzyme activity, and the results are shown in Table 1 and Figure 1.
  • a .nd means not detected
  • SF represents the selection factor, IC 50 (CLK2)/IC 50 (CK2).
  • acetaldehyde dehydrogenase 1 (ALDH1) kit and CD44 and CD133 antibodies
  • flow cytometry was used to detect the inhibitory activity of some compounds of the present invention on the stemness of HCT-116 primary cancer cells.
  • ALDH1 kit, CD44 and CD133 antibodies were purchased from Shanghai Biyuntian Biotechnology Co., Ltd., and HCT-116 primary tumor was provided by the local hospital.
  • the primer sequence is designed using Primer5, ⁇ -actin is used as an internal reference, and the primer information is shown in Table 3:
  • RT-PCR was used to detect the expression of related genes in the Wnt/ ⁇ -catenin and Hedgehog signaling pathways and stem cell-related genes OCT4, SOX2 and Nanog after HCT-116 primary cancer cells were incubated with the sample for 24 hours.
  • the catalog number is K3102
  • the total RNA in the cell is extracted, and the RNA purity and concentration are determined by UV spectrophotometer: take 1 ⁇ L RNA solution, dilute 100 times, and UV spectrophotometer Measure the light absorption values at 260nm and 280nm to calculate the OD260/280 ratio and solution concentration.
  • the ratio is required to be between 1.8-2.0, and the RNA concentration is greater than 500 ⁇ g/mL (reagents, utensils, and consumables are treated without RNase during operation).
  • OligdT as the downstream primer to perform conventional reverse transcription reaction; perform reverse transcription to synthesize the first strand of cDNA according to the following reaction system:
  • the Wnt and Hedgehog signaling pathways and stem cell-related gene expression in HCT-116 primary cancer cells were quantitatively analyzed according to the following reaction system, with ⁇ -actin as the internal reference gene, and ddH 2 O as a template to set up a negative control (taking ⁇ -catenin as an example):
  • Real-time fluorescence quantitative analysis uses the 2- ⁇ CT method. Four replicate experiments are carried out for each sample and each gene. The selection of experimental data is to discard values with large errors, and take the average of the remaining values as the final experiment. Keep the data, the experimental data is shown in Figure 3.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)

Abstract

本发明涉及一类抗肿瘤化合物及其制备与用途，是一类兼具抑制CK2蛋白激酶和癌细胞干性的化合物，通过在已知CK2抑制剂(CX-4945)母体结构中经酰胺键、酯键、硫酯键的方式引入含有氨基、羟基、巯基或卤素原子的脂肪链或者杂环结构片段，提供一类如式II所示的新型的化合物；该类化合物具有明显的抗癌活性，部分化合物对癌细胞的细胞毒活性、对CK2蛋白激酶的抑制能力和对CK2蛋白的选择性优于其母体化合物；除此之外，一些化合物对癌细胞干性表现出强的抑制能力，有利于克服药物耐药；式II-A、式II-B、式II-C、式II-D和II-E所示结构的化合物：本发明的化合物对癌细胞的增殖具有良好抑制作用，还能够抑制癌细胞干性，可用于制备抗肿瘤药物。

Description

一类抗肿瘤化合物及其制备与用途 技术领域
本发明属于医药技术领域，涉及一类新型的兼具CK2蛋白激酶与癌细胞干性抑制能力的化合物；还涉及该类化合物的制备方法和它们在制备抗肿瘤药物方面的应用。
背景技术
蛋白激酶2又被称为酪蛋白激酶2(Casein kinase II，CK2)，是一种多效且高度保守的第二信使非依赖性丝氨酸/苏氨酸蛋白激酶，广泛存在于真核细胞的细胞质及细胞核中。CK2由两个催化亚型(CK2α/CK2α’)和两个调节亚型(β)组成，形成三种四聚体(ααββ、αα’ββ、α’α’ββ)。有大量证据表明，CK2在多种细胞活动中具有非常重要的作用，其磷酸化底物超过300种，在细胞生长、增殖、凋亡和癌变的过程中扮演着重要的角色，它调节多条抗凋亡通路及前导信号级联，包括PI3K/AKT信号通路、Wnt信号级联、NF-κB转录和DNA损伤应答等。
CK2在细胞周期的不同阶段具有不同的关键性作用。在体外动物细胞周期实验中，加入CK2抑制剂能够抑制细胞周期进程，表明动物细胞的周期活动需要CK2的参与和调节。同时实验证明，过表达的CK2可抑制由抗癌药物引起的程序性死亡。由于多种耐药癌细胞均存在CK2过表达的现象，因此抑制CK2不仅能够促使癌细胞凋亡，而且能够增加癌细胞对抗癌药物的敏感度。在多种癌症如头颈癌、乳腺癌、结肠癌、肾癌、肺癌、白血病和前列腺癌中，癌细胞内CK2是过表达的。此外，CK2与癌症的不良预后和病情恶化有直接的联系。因此，CK2已经成为一个非常有前景的癌症治疗靶点。
人类整个蛋白激酶家族的同源性相对较高，尤其是其催化域的核心部分。大部分小分子蛋白激酶抑制剂主要是通过与激酶靶点的保守ATP结合位点发生相互作用达到抑制效果。由于存在较大的氨基酸残基，CK2的ATP结合位点相对于其它激酶的结合位点来说较小，这使得设计合成CK2的ATP竞争性抑制剂必须满足两个要求，即高特异性和较小分子量。
1954年文献首次公开报道了CK2蛋白，阐明了CK2蛋白对细胞生长调控的重要作用(J.Biol.Chem.1954，211，969-980)。在此基础上，直到1986年后才相继出现靶向该蛋白的小分子抑制剂，如苯并咪唑类衍生物(J.Biol.Chem.1986，261，3414-3419；Bioorg.Med.Chem.2003，11，3997-4002)、类黄酮类衍生物(Curr.Top.Med.Chem.2011，11，1340-1351)、香豆素类衍生物(Curr.Pharmaceut.Des.2004，10，3797-3811)、蒽醌类衍生物(Eur.J.Med.Chem.2010，45，292-297)、吡唑啉三嗪类衍生物(Bioorg.Med.Chem.Lett.2007，17，4191-4195)、羧酸类衍生物(ChemBioChem2007，8，129-139)、非ATP竞争型CK2抑制剂(J.Med.Chem.2019，62，1817-1836)和第二代CK2α抑制剂CAM4066(Chem.Sci.2018，9，3041-3049)等。不过迄今为止，仅有Cylene制药公司开发的以苯并萘啶为母体的CK2小分子抑制剂CX-4945(式I)目前正处于I/Ⅱ期临床研究。虽然CX-4945表现出广谱的抗肿瘤活性和优越的CK2激酶抑制活性，但仍存在一些明显的缺陷，如CX-4945对CK2的抑制率(IC 50)为1nM，但对其它12种激酶也具有纳摩尔级(IC 50)的抑制作用，此外它对CLK2的抑制作用甚至强于对CK2的抑制作用，易导致严重的脱靶毒性。因此，在CX-4945基础上开发新的具有优良活性和高选择性的CK2抑制剂是很有意义的。
Figure PCTCN2021074623-appb-000001
近年来，随着肿瘤生物学的发展，肿瘤的临床治疗取得了显著进步，但是绝大多数的恶性肿瘤仍然无法根除，其中转移和复发是当前肿瘤治疗的最主要挑战。癌症干细胞(CSC)又称为癌干细胞或肿瘤干细胞，是指肿瘤中具有自我更新能力并能产生异质性肿瘤细胞的细胞，也被认为是肿瘤组织内真正驱动肿瘤发生和发展的“动力”，同时也是肿瘤复发和转移的根源。由于肿瘤组织中存在癌症干细胞群体，它们具有很强的自我更新和分化能力，可通过调节多条信号通路如Wnt/β-catenin、Notch、Hedgehog、BMI-1等来维持肿瘤的恶性增殖、侵袭、耐药、转移和复发等。近来肿瘤干细胞学说受到越来越多的关注，并且已在血液瘤和多种实体瘤中成功分离出癌症干细胞。越来越多的证据表明，常规的化疗药物包括靶向药物，在有效剂量下虽然都能够有效的杀死分化的癌细胞，但对癌症干细胞无效，有的甚至会促进癌症干细胞的生长，而这极少量的癌症干细胞又能够导致药物耐药和肿瘤的复发与转移，降低了许多抗癌药物的疗效。因此，研发一类能够同时抑制正常癌细胞和癌症干细胞的药物是十分有益的。
发明内容
技术问题：本发明的目的是提供一类抗肿瘤化合物及其制备与用途，即是一类可兼具抑制CK2蛋白激酶和癌细胞干性的化合物及其制备与用途。
本发明通过在已知CK2抑制剂(CX-4945)母体结构中经酰胺键、酯键、硫酯键等方式引入一些含有氨基、羟基、巯基或卤素原子的脂肪链或者杂环结构片段，提供一类新型的化合物。该类化合物具有明显的抗癌活性，部分化合物的体外抗癌活性和酶活性均优于母体化合物，且被发现可以有效抑制癌细胞干性。
技术方案：本发明提供了式II-A、式II-B、式II-C、式II-D和II-E所示结构的化合物：
Figure PCTCN2021074623-appb-000002
其中：
式II-A中R 1为-NH 2、-OH、-SO 2CH 3、-CH 2CHF 2、-CH 2C≡CH、-(CH 2) 2OH、-(CH 2) 2NH 2、-(CH 2) 2Br、-(CH 2) 2CO 2CH 3、-(CH 2) 2CO 2H、-(CH 2) 2N(CH 3) 2、-(CH 2) 3OH、-(CH 2) 3NH 2、-(CH 2) 3CO 2CH 3、
Figure PCTCN2021074623-appb-000003
式II-B中R 2为-CH 2CHF 2、-(CH 2) 2OH、-(CH 2) 2F、-(CH 2) 2I、-(CH 2) 2CHCl 2、 -(CH 2O) 2(CH 2) 2OH、
Figure PCTCN2021074623-appb-000004
式II-C中R 3为-(CH 2) 2SH；
式II-D中R 4为-C 2H 5、-(CH 2) 2SCH 3、-OCH 2CH(CH 3) 2、
Figure PCTCN2021074623-appb-000005
式II-E中R 5为
Figure PCTCN2021074623-appb-000006
本发明所制备的化合物中优选下列化合物1～31中的任意一种或多种：
化合物1：5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰肼
Figure PCTCN2021074623-appb-000007
化合物2：N-羟基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000008
化学物3：N-甲磺酰基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000009
化合物4：N-(2-羟乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000010
化合物5：N-(2-氨乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000011
化合物6：N-(2-溴乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000012
化合物7：3-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-酰氨基)丙酸甲酯
Figure PCTCN2021074623-appb-000013
化合物8：3-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-酰氨基)丙酸
Figure PCTCN2021074623-appb-000014
化合物9：N-(2-(二甲氨基)乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000015
化合物10：N-(2,2-二氟乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000016
化合物11：N-(2-丙基-1-炔)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000017
化合物12：N-(3-羟丙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000018
化合物13：N-(3-氨丙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000019
化合物14：4-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-酰胺基)丁酸甲酯
Figure PCTCN2021074623-appb-000020
化合物15：4-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-酰胺基)丁酸
Figure PCTCN2021074623-appb-000021
化合物16：N-(2-吗啉乙烷基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000022
化合物17：N-(2-(1-吡咯烷基)乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000023
化合物18：N-(1-哌啶基)乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
Figure PCTCN2021074623-appb-000024
化合物19：2-羟乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
Figure PCTCN2021074623-appb-000025
化合物20：2-氟乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
Figure PCTCN2021074623-appb-000026
化合物21：2-碘乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
Figure PCTCN2021074623-appb-000027
化合物22：2,2-二氟乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
Figure PCTCN2021074623-appb-000028
化合物23：3,3-二氯丙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
Figure PCTCN2021074623-appb-000029
化合物24：2-(2-(2-羟乙氧基)乙氧基)乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
Figure PCTCN2021074623-appb-000030
化合物25：2-(2-哌啶基)乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
Figure PCTCN2021074623-appb-000031
化合物26：S-(2-巯基乙醇基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸硫酯
Figure PCTCN2021074623-appb-000032
化合物与27：N’-丙酰基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰肼
Figure PCTCN2021074623-appb-000033
化合物28：N’-(3-(甲硫基)氯丙酰基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰肼
Figure PCTCN2021074623-appb-000034
化合物29：异丁基2-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰基)联氨-1-羧酸酯
Figure PCTCN2021074623-appb-000035
化合物30：N'-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰基)-3-吗啉丙烷-1-磺酰肼
Figure PCTCN2021074623-appb-000036
化合物31：N'-(5-(3-1,2-二硫戊环基)戊醇基)-5-((3-氯苯基)氨基)苯并[2,6]萘啶-8-碳酰肼
Figure PCTCN2021074623-appb-000037
上述化合物的合成方法主要是以CX-4945或其前体为原料合成得到。
CX-4945参考文献方法合成，首先以3-溴异烟酸为起始原料，经酯化反应后与2-氨基-4-甲酯基苯基硼酸盐酸盐发生Suzuki偶联反应得到中间体5,6-二氢苯并[c][2,6]萘啶-8-羧酸甲酯，然后与三氯氧磷发生氯代反应，再与间氯苯胺发生取代反应，最后水解得到产物。具体合成路线如下：
Figure PCTCN2021074623-appb-000038
a.EtOH,H 2SO 4,reflux,24～48h；b.Cs 2CO 3,Pd(dppf)Cl 2,N 2,100℃,30～48h；
c.POCl 3,110℃,4h；d.NMP,110℃,8h；e.1.5N NaOH,70℃。
式II-A化合物可通过交换反应或缩合反应得到，具体合成路线如下：
Figure PCTCN2021074623-appb-000039
Figure PCTCN2021074623-appb-000040
式II-B化合物可通过酯化反应得到，具体合成路线如下：
Figure PCTCN2021074623-appb-000041
式II-C化合物可通过硫代醇的酯化反应得到，具体合成路线如下：
Figure PCTCN2021074623-appb-000042
式II-D化合物可通过酰肼与羧酸的酰胺缩合反应得到，具体合成路线如下：
Figure PCTCN2021074623-appb-000043
式II-E化合物可通过酰肼与磺酸的酰胺缩合反应得到，具体合成路线如下：
Figure PCTCN2021074623-appb-000044
其中R 1、R 2、R 3、R 4、R 5如前所述，HATU代表缩合剂2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐，TBTU代表缩合剂O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸，DIPEA代表N，N-二异丙基乙胺，DMF代表溶剂N,N-二甲基甲酰胺，Reflux代表回流，r.t.代表室温。
有益效果：
(一)、化合物的体外抗肿瘤活性和酶活性
测定了本发明化合物针对多种癌细胞及正常细胞的细胞毒活性，并检测了它们对CK2和CLK2蛋白的抑制活性。由表1和图1可知：
(1)在CX-4945母体骨架中通过酰胺键引入肼基得到的化合物1对癌细胞的细胞毒活性、对CK2蛋白激酶的抑制能力和对CK2蛋白的选择性较CX-4945均有提高；当引入羟胺基时，得到的化合物2虽然有较高的细胞毒活性，但酶活性较差；当引入甲磺酰胺基团，得到的化合物3的细胞毒活性和酶活性较CX-4945降低。因此，通过酰胺键引入较小基团的氢键供体可增加母体化合物的细胞毒活性、酶活性和对CK2蛋白的选择性。
(2)在CX-4945母体骨架中通过酰胺键引入C2脂肪链的化合物(如化合物4、5、7、9)的细胞毒活性普遍高于引入C3脂肪链的化合物(如化合物11、12、13、14、15)，且C2脂肪链末端基团为羟基的活性最好。如化合物4，其细胞毒活性、酶活性和对CK2蛋白的选择性较CX-4945均有所提高，同时还降低了对正常肝细胞LO2的毒性；当为氨基时，虽然提高了相关化合物对CK2的活性，但是其细胞毒活性和对CK2蛋白的选择性较差。此外，通过C2脂肪链引入含N杂环所得的化合物(化合物16、17、18)，如吗啉环、吡咯环和哌啶环，均可提高化合物的细胞毒活性，其中引入吗啉环所得的化合物有较高的细胞毒活性、酶活性和对CK2蛋白的选择性。因此，在CX-4945羧基末端通过酰胺键引入C2脂肪链，且脂肪链末端含有可提供氢键受体基团时，所得化合物的细胞毒活性、酶活性和对CK2蛋白的选择性较高。
(3)在CX-4945羧酸末端通过酯键引入C2脂肪链的化合物，可普遍提高母体化合物的细胞毒活性。当引入的C2脂肪链末端基团越小且可作为氢键受体时，所得化合物的细胞毒活性、酶活性和对CK2蛋白的选择性较好。如化合物20，C2脂肪链末端为原子半径小、电负性强、可作为氢键受体的氟原子，其活性明显优于母体化合物和其它化合物。化合物19，C2脂肪链末端为羟基，在保留原有母体化合物细胞毒活性的同时，增加了对CK2蛋白的活性和选择性。同样，通过C2脂肪链引入吡啶环，也可增加化合物对CK2蛋白的抑制活性和选择性。硫的原子半径要大于氧原子，硫酯键也较氧酯键不太稳定，因此通过硫酯键引入硫醇得到的化合物(化合物26)，仅保留了原有母体化合物的细胞毒活性，但酶活性较差。
(4)在化合物1的基础上，对活性基团酰肼基进行结构修饰得到的化合物(化合物26、27、28、29、30、31)的细胞毒性和酶活性一般。
(二)、化合物抑制癌细胞干性的能力
(1)采用流式细胞仪检测了本发明部分化合物对HCT-116原代癌细胞干性的抑制能力，表2数据表明，所测化合物能够有效抑制HCT-116原代癌细胞内的ALDH1活性，且明显优于CX-4945，并且化合物4和20对ALDH1的抑制率高于癌细胞干性抑制剂BBI 608。
(2)测定了化合物4和20对HCT-116原代癌细胞内干细胞标志物CD44和CD133的表达情况。由图2流式分析结果可知，HCT-116原代癌细胞与样品孵育24h后，化合物4和20对干细胞标志物CD44/CD133的抑制率分别达到为86.97％和76.05％，明显高于化合物CX-4945的56.70％，尤其是化合物4的抑制率还高于癌细胞干性抑制剂BBI608(82.26％)，表现出很强的抑制癌细胞干性的活性。
(3)通过qRT-PCR分析了化合物4和20对干细胞抑制通路Wnt/β-catenin和Hedgehog中的相关基因。由图3可知，化合物4在HCT-116原代癌细胞内可以较好地 促进Wnt信号通路中DKK1基因的表达，同时抑制下游的β-catenin基因，进而抑制Wnt信号通路，并且抑制效果明显优于化合物20，远优于CX-4945。在Hedgehog信号通路中，化合物4可以同时抑制上游的PTCH1基因和下游Gli1基因，进而抑制Hedgehog信号通路，并且抑制效果要优于化合物20和CX-4945。化合物20对Wnt信号通路和Hedgehog信号通路的抑制也优于CX-4945。化合物4对干细胞相关基因OCT4、SOX2和Nanog的抑制高于CX-4945和BBI608，而化合物20对干性基因的抑制与BBI 608相当，但优于CX-4945。这些结果说明化合物4和20可通过Wnt信号通路和Hedgehog信号通路抑制癌细胞干性，两者的抑制能力明显优于其母体化合物CX-4945，其中化合物4对癌细胞干性的抑制甚至优于癌细胞干性抑制剂BBI608。
综上，通过在CX-4945母体结构末端引入不同活性基团，得到了一系列对CK2抑制活性高于CX-4945的化合物，如化合物1、4、5、7、12、16、19、20、21、25。其中，化合物1、4、16、19、20、21和25对CLK2的抑制活性低于CX-4945，它们具有较好的选择性，尤其是化合物20，选择因子高达5.28。大部分受试化合物对5种癌细胞的增殖都具有良好的抑制作用，优于CX-4945或与之相当，而对正常肝细胞LO2的毒性大多都低于母体化合物。通过对CK2选择性高于CLK2的7个化合物对癌细胞干性抑制的测定，发现化合物4和20可有效抑制原代癌细胞干性，其活性甚至高于已知的癌细胞干性抑制剂BBI 608。
研究表明，本发明化合物具有明显的抗癌活性，部分化合物对癌细胞的细胞毒活性、对CK2蛋白激酶的抑制和对CK2蛋白的选择性优于母体化合物，且可有效抑制癌细胞干性，用于制备抗肿瘤药物。
附图说明
图1.部分化合物对CK2和CLK2蛋白激酶的抑制活性。
图2.代表性化合物对HCT-116原代癌细胞内干细胞标志物CD44/CD133的抑制情况，(a)样品的流式细胞图，(b)样品的抑制率。
图3.代表性化合物对Wnt和Hedgehog信号通路以及干细胞相关基因表达量的影响。
具体实施方式
下面结合实施例对本发明做进一步详细说明，本发明的保护范围包括但不仅限于这些实施例。
本发明的一类抗肿瘤化合物通过在已知CK2抑制剂(CX-4945)母体结构中经酰胺键、酯键、硫酯键的方式引入含有氨基、羟基、巯基或卤素原子的脂肪链或者杂环结构片段，提供一类如式II所示的新型的化合物；
式II-A、式II-B、式II-C、式II-D和II-E所示结构的化合物：
Figure PCTCN2021074623-appb-000045
其中：
式II-A中R 1为-NH 2、-OH、-SO 2CH 3、-CH 2CHF 2、-CH 2C≡CH、-(CH 2) 2OH、-(CH 2) 2NH 2、-(CH 2) 2Br、-(CH 2) 2CO 2CH 3、-(CH 2) 2CO 2H、-(CH 2) 2N(CH 3) 2、-(CH 2) 3OH、-(CH 2) 3NH 2、-(CH 2) 3CO 2CH 3、
Figure PCTCN2021074623-appb-000046
式II-B中R 2为-CH 2CHF 2、-(CH 2) 2OH、-(CH 2) 2F、-(CH 2) 2I、-(CH 2) 2CHCl 2、-(CH 2O) 2(CH 2) 2OH、
Figure PCTCN2021074623-appb-000047
式II-C中R 3为-(CH 2) 2SH；
式II-D中R 4为-C 2H 5、-(CH 2) 2SCH 3、-OCH 2CH(CH 3) 2、
Figure PCTCN2021074623-appb-000048
式II-E中R 5为
Figure PCTCN2021074623-appb-000049
下面实施例中所有试剂均为分析纯。化合物结构表征所用的核磁数据由Bruker ARX-600核磁共振仪测定，内标为TMS；高分辨质谱采用Agilent6224TOF LC/MS仪测定。
本发明中，已知化合物CX-4945及其前体化合物参考文献(J.Med.Chem.2011，54，635-654)所报道的方法制备，经核磁氢谱验证。
CX-4945： 1H NMR(600MHz,DMSO-d 6)δ12.04(s,1H),10.30(s,1H),10.16(s,1H),9.08(d,J＝5.8Hz,1H),9.00-8.97(m,1H),8.94(d,J＝8.5Hz,1H),8.31-8.28(m,2H),8.12(d,J＝8.2Hz,1H),8.03(dd,J＝8.4,1.5Hz,1H),7.45(t,J＝8.1Hz,1H),7.17(d,J＝7.9Hz,1H)ppm.
CX-4945前体： 1H NMR(600MHz,DMSO-d 6)δ10.09(s,1H),9.91(s,1H),8.94(d,J＝5.6Hz,1H),8.79(d,J＝8.5Hz,1H),8.77(d,J＝5.6Hz,1H),8.34(t,J＝1.9Hz,1H),8.17(dd,J＝6.3,1.4Hz,1H),7.90(dd,J＝8.4,1.7Hz,1H),7.42(t,J＝8.1Hz,1H),7.12(dd,J＝7.9,1.4Hz,1H),3.92(s,1H)ppm.
(一)、化合物的制备
实施例1：化合物1的制备
于50mL单口瓶中加入CX-4945前体1.09g(3mmol)和20mL甲醇，室温下缓慢滴加水合肼0.15g(3mmol)，加热回流72h至完全反应。冷却，析出大量固体，抽滤，滤饼用3×10mL冰甲醇冲洗，得亮黄色固体产物1.05g，收率97.1％。 1H NMR(600MHz,DMSO-d 6)δ10.14(s,1H),10.08(s,1H),9.63(s,1H),8.95(d,J＝6.0Hz,1H),8.80(d,J＝12.0Hz,1H),8.55(d,J＝6.0Hz,1H),8.33(t,J＝1.8Hz,1H),8.21(d,J＝1.6Hz,1H),8.07(d,J＝6.0Hz,1H),7.91(dd,J＝12.0,1.6Hz,1H),7.42(t,J＝6.0Hz,1H),7.12(dd,J＝8.0,1.4Hz,1H),4.63(s,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ165.77,150.47,148.13,147.69,143.76,142.40,134.73,133.35,130.52,127.52,126.26,124.17,123.00,122.83,122.60,121.47,120.57,119.56,116.79ppm.HR-MS(m/z)(ESI):calcd for C 19H 14ClN 5O[M+H] +:364.0965；found:364.0967.
实施例2：化合物2的制备
于200mL单口瓶中加入NH 2OH·HCl 29.17g(420mmol)和150mL甲醇。将KOH35.2g(627mmol)溶于75mL甲醇，在0℃下逐滴加入上述溶液，保持在0℃下搅拌30min，抽滤，除去不溶固体，得含有羟胺的甲醇溶液。于30mL单口瓶中加入CX-4945前体0.36g(1mmol)和10mL上述制备的含有羟胺的甲醇溶液，室温下搅拌1h，TLC检测至完全反应，旋干溶剂，加10mL水溶解固体，用2M HCl调节溶液pH＝7析出黄色固体。抽滤，滤饼用3×10mL去离子水洗三遍，得粗产物，重结晶，得黄色固体产物0.332g，收率91.2％。 1H NMR(600MHz,DMSO-d 6)δ11.52(s,1H),10.16(s,1H),9.65(s,1H),9.19(s,1H),8.97(d,J＝5.4Hz,1H),8.83(d,J＝8.4Hz,1H),8.57(d,J＝5.2Hz,1H),8.33(s,1H),8.15(d,J＝1.2Hz,1H),8.07(d,J＝8.0Hz,1H),7.85(d,J＝1.2Hz,1H),7.43(t,J＝8.0Hz,1H),7.13(dd,J＝7.8,1.2Hz,1H)ppm. 13C NMR(150MHz,DMSO-d 6)δ164.19,150.52,148.13,147.73,143.74,142.35,134.16,133.27,130.53,127.49,126.08,124.19,122.95,122.90,122.64,121.54,120.60,119.60,116.81ppm.HR-MS(m/z)(ESI):calcd for C 19H 13ClN 4O 2[M+H] +:365.0805；found:365.0800.
实施例3：化合物3的制备
于10mL单口烧瓶中分别加入CX-4945 0.174g(0.5mmol)、甲磺酰胺0.048g(0.5mmol)、HATU 0.228g(0.6mmol)、DIPEA 0.129g(1mmol)和无水DMF 5mL，45℃下搅拌24h，TLC监测至反应完全，真空旋干溶剂，硅胶柱层析纯化，洗脱剂为二氯甲烷和甲醇(100:1-50:1)，得黄色固体产物0.093g，收率43.5％。 1H NMR(600MHz,DMSO-d 6)δ13.26(s,1H),10.19(s,1H),9.69(s,1H),9.01(d,J＝5.5Hz,1H),8.87(d,J＝8.4Hz,1H),8.60(d,J＝5.6Hz,1H),8.33(s,1H),8.28(s,1H),8.10(d,J＝8.2Hz,1H),7.99(d,J＝8.1Hz,1H),7.45(t,J＝8.1Hz,1H),7.16-7.13(d,J＝7.8Hz,1H),2.51(s,3H)ppm. 13C NMR(150MHz,DMSO-d 6)δ150.71,150.60,148.30,148.09,143.76,142.33,133.24,130.62,128.72,127.42,124.70,124.47,123.13,123.08,122.80,122.67,120.57,119.61,116.86,63.26ppm.HR-MS(m/z)(ESI):calcd for C 20H 15ClN 4O 3S[M+H] +:427.0631；found:427.0643.
实施例4：化合物4的制备
采用CX-4945和2-氨基乙醇参考实施例3所述方法制备，得黄色固体产物，收率72.1％。 1H NMR(600MHz,DMSO-d 6)δ10.18(s,1H),9.89(s,1H),8.97(d,J＝5.4Hz,1H),8.84(t,J＝6.0Hz,2H),8.77(d,J＝5.4Hz,1H),8.35(s,1H),8.27(s,1H),8.21(d,J＝8.2Hz,1H),8.00(d,J＝8.2Hz,1H),7.45(t,J＝8.0Hz,1H),7.14(d,J＝7.6Hz,1H),4.91(t,J＝5.6Hz,1H),3.61(q,J＝5.8Hz,2H),3.43(dd,J＝11.2,5.4Hz,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.40,150.61,148.05,147.64,143.77,142.51,135.92,133.19,130.46,127.52,126.40,124.25,123.38,122.71,122.56,121.46,120.70,119.73,117.16,60.20,42.88ppm.HR-MS(m/z)(ESI):calcd for C 21H 17ClN 4O 2[M+H] +:393.1118；found:393.1120.
实施例5：化合物5的制备
采用CX-4945和乙二胺参照实施例3所述方法制备，得黄色固体产物，收率81.6％。 1H NMR(600MHz,DMSO-d 6)δ10.12(s,1H),9.60(s,1H),8.95(d,J＝5.4Hz,1H),8.81(dd,J＝11.2,7.0Hz,2H),8.53(d,J＝5.6Hz,1H),8.24(t,J＝1.8Hz,1H),8.20(d,J＝1.4Hz,1H),8.09(d,J＝8.2Hz,1H),7.93(dd,J＝8.2,1.4Hz,1H),7.42(t,J＝8.2Hz,1H),7.11(d,J＝7.8Hz,1H),3.45(dd,J＝12.6,6.4Hz,2H),3.35(m,2H),1.94-1.88(m,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.29,150.46,148.07,147.66,143.75,142.37,135.96,133.26,130.52,127.48,126.25,124.13,123.30,122.74,122.62,121.45,120.65,119.65,116.78,37.98,29.60ppm.HR-MS(m/z)(ESI):calcd for C 21H 18ClN 5O[M+H] +:393.1278；found:393.1280.
实施例6：化合物6的制备
于10mL单口烧瓶中加入CX-4945 0.174g(0.5mmol)、2-溴乙胺氢溴酸盐0.102g(0.5mmol)、HATU 0.228g(0.6mmol)、DIPEA 0.194g(1.5mmol)和无水DMF 5mL，50℃下搅拌24h，TLC监测至反应完全，浓缩溶剂，硅胶柱层析纯化，洗脱剂为二氯甲烷和甲醇(100:1-50:1)，得黄色固体产物0.139g，收率61.4％。 1H NMR(600MHz,DMSO-d 6)δ10.20(s,1H),9.67(s,1H),9.12(t,J＝5.0Hz,1H),9.00(d,J＝5.4Hz,1H),8.82(d,J＝3.8Hz,1H),8.59(d,J＝5.4Hz,1H),8.29(s,1H),8.28(s,1H),8.13(d,J＝8.0Hz,1H),7.97(d,J＝8.2Hz,1H),7.45(t,J＝8.0Hz,1H),7.15(d,J＝7.6Hz,1H),4.85(t,J＝5.2Hz,2H),3.88-3.84(m,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.69,152.29,150.61,148.16,147.77,143.79,142.37,135.05,133.28,130.55,127.50,126.41,124.25,123.36,122.87,122.71,120.76,119.76,116.84,79.74,38.61ppm.HR-MS(m/z)(ESI):calcd for C 21H 16BrClN 4O[M+H] +:455.0274；found:455.0272.
实施例7：化合物7的制备
采用CX-4945和β-丙氨酸甲酯盐酸盐参照实施例6所述方法制备，得淡黄色固体产物，收率65.4％。 1H NMR(600MHz,DMSO-d 6)δ10.18(s,1H),9.66(s,1H),8.99(d,J＝5.6Hz,1H),8.88(t,J＝5.4Hz,1H),8.85(d,J＝8.4Hz,1H),8.58(d,J＝5.6Hz,1H),8.28(t,J＝1.8Hz,1H),8.22(d,J＝1.4Hz,1H),8.10(dd,J＝8.2,1.2Hz,1H),7.93(dd,J＝8.4,1.6Hz,1H),7.45(t,J＝8.2Hz,1H),7.15(dd,J＝7.8,1.2Hz,1H),3.65(s,3H),3.58(dd,J＝12.6,6.8Hz,2H),2.68(t,J＝7.0Hz,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ172.28,166.39,150.55,148.15,147.73,143.76,142.37,135.72,133.27,130.55,127.51,126.31,124.21,123.33,122.81,122.68,121.58,120.71,119.71,116.82,51.89,36.13,34.02ppm.HR-MS(m/z)(ESI):calcd for C 19H 14ClN 5O[M+H] +:435.1224；found:435.1220.
实施例8：化合物8的制备
于30mL单口烧瓶中加入化合物7 0.434g(1mmol)、LiOH■H 2O 0.105g(2.5mmol)、甲醇10mL和纯水5mL，60℃搅拌过夜。旋干溶剂得到黄色固体，加入30mL水溶解固体，用1M HCl调节溶液pH到5，析出黄色固体，过滤、水洗(3×10mL)、烘干得黄色固体产物0.39g，收率91.0％。 1H NMR(600MHz,DMSO-d 6)δ12.37(s,1H),10.15(s,1H),9.63(s,1H),8.96(s,1H),8.84(s,1H),8.81(d,J＝7.4Hz,1H),8.55(s,1H),8.24(t,J＝1.8Hz,1H),8.21(d,J＝1.2Hz,1H),8.09(d,J＝6.8Hz,1H),7.92(dd,J＝7.2,1.8Hz,1H),7.42(d,J＝6.8Hz,1H),7.13(d,J＝6.4Hz,1H),3.54(dd,J＝10.6,5.8Hz,2H),2.58(t,J＝6.4Hz,2H)ppm. 13C NMR(150MHz,DMSO)δ173.63,166.29,150.52,148.12,147.69,143.75,142.37,135.77,133.27,130.54,127.50,126.30,124.18,123.32,122.76,122.65,121.53,120.68,119.68,116.80,36.31,34.40ppm.HR-MS(m/z)(ESI):calcd for C 22H 17ClN 4O 3[M+H] +:421.1067；found:421.1060.
实施例9：化合物9的制备
采用CX-4945和N,N-二甲基乙二胺参照实施例3所述方法制备，得黄色固体产物，收率69.8％。 1H NMR(600MHz,DMSO-d 6)δ10.18(s,1H),9.67(s,1H),8.98(d,J＝5.6Hz,1H),8.84(d,J＝8.4Hz,1H),8.72(t,J＝5.4Hz,1H),8.59(d,J＝5.6Hz,1H),8.28(t,J＝1.8Hz,1H),8.23(d,J＝1.4Hz,1H),8.11(dd,J＝8.4,1.0Hz,1H),7.94(dd,J＝8.4,1.6Hz,1H),7.45(t,J＝8.2Hz,1H),7.15(dd,J＝7.8,1.4Hz,1H),3.45(dd,J＝12.8,6.6Hz,2H),2.53-2.51(m,2H),2.25(s,6H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.19,150.54,148.13,147.70,143.77,142.39,135.94,133.27,130.54,127.53,126.29,124.20,123.35,122.76,122.66,121.50,120.71,119.70,116.83,58.59,45.64,37.92ppm.HR-MS(m/z)(ESI):calcd for C 23H 22ClN 5O[M+H] +:420.1591；found:420.1595.
实施例10：化合物10的制备
采用CX-4945和2,2-二氟乙胺参照实施例3所述方法制备，得黄色固体产物，收率61.8％。 1H NMR(600MHz,DMSO-d 6)δ10.18(s,1H),9.99(s,1H),9.28(s,1H),8.96(d,J＝4.6Hz,1H),8.86(d,J＝8.2Hz,1H),8.83(d,J＝4.6Hz,1H),8.37(s,1H),8.29(s,1H),8.22(d,J＝7.4Hz,1H),8.01(d,J＝7.8Hz,1H),7.44(t,J＝7.8Hz,1H),7.14(d,J＝7.2Hz,1H),6.23(t,J＝56.2Hz,1H),3.77(t,J＝14.8Hz,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.98,150.76,148.05,147.77,143.80,142.50,134.89,133.17,130.42,127.44,126.57,124.37,123.31,122.90,122.59,121.87,120.80,119.82,117.29,115.09(t,J＝240.2Hz),42.25(t,J＝26.6Hz)ppm.HR-MS(m/z)(ESI):calcd for C 21H 15ClF 2N 4O[M+H] +:413.0980；found:413.0967.
实施例11：化合物11的制备
采用CX-4945和炔丙胺参照实施例3所述方法制备，得黄色固体产物，收率76.4％。 1H NMR(600MHz,DMSO-d 6)δ10.17(s,1H),9.96(s,1H),9.36(t,J＝5.2Hz,1H),8.96(d,J＝5.4Hz,1H),8.85(d,J＝8.4Hz,1H),8.82(d,J＝5.5Hz,1H),8.40(s,1H),8.28(s,1H),8.23(d,J＝8.2Hz,1H),8.00(d,J＝8.3Hz,1H),7.45(t,J＝8.1Hz,1H),7.15(d,J＝7.8Hz,1H),4.18(d,J＝3.2Hz,2H),3.21(s,1H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.06,150.67,148.01,147.69,143.78,142.50,135.11,133.17,130.40,128.23,127.42,126.52,124.31,123.26,122.83,122.54,121.73,120.71,119.74,117.23,81.83,73.37,29.12ppm.HR-MS(m/z)(ESI):calcd for C 22H 15ClN 4O[M+H] +:387.1012；found:387.1011.
实施例12：化合物12的制备
采用CX-4945和3-氨基-1-丙醇参照实施例3所述方法制备，得黄色固体产物，收率79.8％。 1H NMR(600MHz,DMSO-d 6)δ10.19(s,1H),9.67(s,1H),8.99(d,J＝5.6Hz,1H),8.85(d,J＝8.4Hz,1H),8.77(t,J＝5.6Hz,1H),8.59(d,J＝5.6Hz,1H),8.28(s,1H),8.23(s,1H),8.12(dd,J＝8.2,1.0Hz,1H),7.95(dd,J＝8.4,1.6Hz,1H),7.45(t,J＝8.2Hz,1H),7.15(dd,J＝7.8,1.2Hz,1H),4.54(t,J＝5.2Hz,1H),3.52(q,J＝6.2Hz,2H),3.40(dd,J＝12.8,7.0Hz,2H),1.75(p,J＝6.6Hz,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.27,150.53,148.15,147.70,143.78,142.39,136.06,133.27,130.56,127.54,126.27,124.19,123.35,122.77,122.66,121.46,120.68,119.69,116.84,59.13,37.22,32.92ppm.HR-MS(m/z)(ESI):calcd for C 22H 19ClN 4O 2[M+H] +:407.1269；found:407.1264.
实施例13：化合物13的制备
采用CX-4945和1,3-二氨基丙烷参照实施例3所述方法制备，得黄色固体产物，收率57.3％。 1H NMR(600MHz,DMSO-d 6)δ10.19(s,1H),10.01(s,1H),8.96(d,J＝5.6Hz,1H),8.91(t,J＝5.4Hz,1H),8.87(d,J＝3.8Hz,1H),8.38(s,1H),8.25-8.21(m,2H),8.04(s,1H),7.99(d,J＝8.4Hz,1H),7.44(t,J＝8.2Hz,1H),7.14(d,J＝7.8Hz,1H),3.36(dd,J＝12.6,6.4Hz,2H),3.18(dd,J＝12.8,6.2Hz,2H),1.76-1.70(m,2H),1.22(s,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.25,161.58,150.71,148.07,147.68,143.82,142.56,135.91,133.16,130.41,127.55,126.34,124.30,123.21,122.79,122.56,120.77,119.82,117.38,37.53,35.51,29.61ppm.HR-MS(m/z)(ESI):calcd for C 22H 20ClN 5O[M+H] +:406.1434；found:406.1431.
实施例14：化合物14的制备
采用CX-4945与4-氨基丁酸甲酯盐酸参照实施例3所述方法制备，得橘黄色固体产物0.147g，收率65.8％。 1H NMR(600MHz,DMSO-d 6)δ10.17(s,1H),9.94(s,1H),8.95(d,J＝6.0Hz,1H),8.87-8.83(m,2H),8.81(d,J＝5.0Hz,1H),8.34(s,1H),8.22(s,1H),8.19(d,J＝8.0Hz,1H),7.95(d,J＝7.8Hz,1H),7.43(t,J＝7.8Hz,1H),7.13(d,J＝7.4Hz,1H),3.59(s,3H),3.35(dd,J＝12.6,6.8Hz,2H),2.41(t,J＝6.8Hz,2H),1.87-1.81(m,2H) ppm. 13C NMR(150MHz,DMSO-d 6)δ173.68,166.36,150.69,148.03,147.66,143.79,142.52,135.91,133.17,130.46,127.54,126.30,124.29,123.35,122.76,122.59,121.49,120.78,119.81,117.30,51.75,39.15,31.36,24.94ppm.HR-MS(m/z)(ESI):calcd for C 24H 21ClN 4O 3[M+H] +:449.1380；found:449.1386.
实施例15：化合物15的制备
参照实施例8所述方法制备，得黄色固体产物，收率89.6％。 1H NMR(600MHz,DMSO-d 6)δ12.43(s,1H),10.18(s,1H),9.66(d,J＝5.3Hz,1H),8.99(d,J＝5.4Hz,1H),8.84(d,J＝8.4Hz,1H),8.81(t,J＝6.4Hz,1H),8.58(d,J＝5.4Hz,1H),8.27(s,1H),8.24(s,1H),8.13(d,J＝8.5Hz,1H),7.96(d,J＝8.3Hz,1H),7.46(t,J＝8.1Hz,1H),7.15(d,J＝7.5Hz,1H),3.38(dd,J＝12.6,6.5Hz,2H),2.35(t,J＝7.3Hz,2H),1.87-1.81(m,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.06,150.67,148.01,143.78,142.50,135.11,133.17,130.40,128.23,127.42,126.52,124.31,123.26,122.83,122.54,121.73,120.71,119.74,117.23,81.83,73.37,29.12ppm.HR-MS(m/z)(ESI):calcd for C 23H 19ClN 4O 3[M+H] +:435.1224；found:435.1221.
实施例16：化合物16的制备
采用CX-4945和4-(2-氨基乙基)吗啉参照实施例3所述方法制备，得黄色固体产物，收率68.7％。 1H NMR(600MHz,DMSO-d 6)δ10.16(s,1H),9.98(s,1H),8.94(d,J＝5.6Hz,1H),8.84(d,J＝5.2Hz,2H),8.82(s,1H),8.38(s,1H),8.22(s,1H),8.19(d,J＝8.3Hz,1H),7.96(d,J＝8.2Hz,1H),7.42(t,J＝8.1Hz,1H),7.12(dd,J＝7.8,0.9Hz,1H),3.62(m,4H),3.49(m,2H),3.38(m,4H),2.61(m,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.30,150.71,148.04,147.67,143.80,142.56,135.89,133.16,130.42,127.53,126.36,124.31,123.32,122.79,122.55,121.53,120.79,119.81,117.37,66.36,63.29,57.60,53.54ppm.HR-MS(m/z)(ESI):calcd for C 25H 24ClN 5O 2[M+H] +:462.1697；found:462.1695.
实施例17：化合物17的制备
采用CX-4945和N-(2-氨基乙基)吡咯烷参照实施例3所述方法制备，得黄色固体产物，收率72.8％。 1H NMR(600MHz,DMSO-d 6)δ10.19(s,1H),9.69(s,1H),9.00(d,J＝5.6Hz,1H),8.97(d,J＝5.2Hz,1H),8.88(d,J＝8.4Hz,1H),8.58(d,J＝5.6Hz,1H),8.26(d,J＝4.2Hz,2H),8.05(d,J＝8.0Hz,1H),7.96(d,J＝8.2Hz,1H),7.44(t,J＝8.2Hz,1H),7.16(d,J＝7.8Hz,1H),3.66(dd,J＝10.8,4.8Hz,2H),3.37(m,6H),1.96(m,4H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.99,150.73,148.20,147.90,143.79,142.32,135.29,133.29,130.59,127.49,126.43,124.32,123.43,122.92,122.83,121.85,120.90,119.89,116.88,54.06,53.91,36.50,23.00ppm.HR-MS(m/z)(ESI):calcd for C 25H 24ClN 5O[M+H] +:446.1747；found:446.1750.
实施例18：化合物18的制备
采用CX-4945和N-(2-氨基乙基)哌啶参照实施例3所述方法制备，得黄色固体产物，收率77.1％。 1H NMR(600MHz,DMSO-d 6)δ10.19(s,1H),9.69(s,1H),9.00(d,J＝5.4Hz,2H),8.88(d,J＝8.4Hz,1H),8.59(d,J＝5.6Hz,1H),8.27(s,1H),8.25(d,J＝0.8Hz,1H),8.05(d,J＝8.2Hz,1H),7.96(dd,J＝8.4,1.0Hz,1H),7.45(t,J＝8.2Hz,1H),7.16(dd,J＝7.8,1.2Hz,1H),3.70(dd,J＝11.8,5.8Hz,2H),3.59(t,J＝5.8Hz,2H),3.30(t,J＝6.2Hz,2H),2.97(t,J＝6.4Hz,2H),1.91-1.61(m,6H)ppm. 13C NMR(150MHz,DMSO-d 6)δ167.00,151.59,148.20,147.90,143.79,142.31,135.24,133.28,130.59,129.32,126.43,124.32,123.39,122.96,122.83,121.19,120.89,119.88,116.88,55.62,52.81,34.77,22.99,21.68ppm.HR-MS(m/z)(ESI):calcd for C 26H 26ClN 5O[M+H] +:460.1904；found:460.1900.
实施例19：化合物19的制备
于10mL单口烧瓶中分别加入CX-4945 0.174g(0.5mmol)、TBTU 0.193g(0.6mmol)和无水DMF 5mL，50℃下搅拌15min，加入TEA 0.101g(1.0mmol)后继续搅拌15min，再加入乙二醇0.064g(1mmol)反应24h，TLC监测至反应完全，浓缩溶剂，硅胶柱层析纯化，洗脱剂为二氯甲烷和甲醇(100:1-50:1)，得黄色固体产物0.170g，收率86.5％。 1H NMR(600MHz,DMSO-d 6)δ10.17(s,1H),9.95(s,1H),8.99(d,J＝5.6Hz,1H),8.88(d,J＝8.5Hz,1H),8.79(d,J＝5.6Hz,1H),8.32(t,J＝2.0Hz,1H),8.27(d,J＝1.6Hz,1H),8.18(dd,J＝8.2,1.3Hz,1H),8.01(dd,J＝8.4,1.7Hz,1H),7.45(t,J＝8.1Hz,1H),7.15(dd,J＝7.9,1.4Hz,1H),5.12(t,J＝5.4Hz,1H),4.39-4.35(m,2H),3.78(dd,J＝9.4,4.8Hz,2H)ppm. 13C NMR(150MHz,DMSO-d 6) 13C NMR(151MHz,DMSO)δ166.18,150.85,148.21,148.16,143.76,142.34,133.15,131.11,130.52,128.59,127.27,124.62,124.41,123.23,123.15,122.71,120.79,119.85,117.26,67.36,59.52ppm.HR-MS(m/z)(ESI):calcd for C 21H 16ClN 3O 3[M+H] +:394.0958；found:394.0961.
实施例20：化合物20的制备
采用CX-4945和2-氟乙醇参照实施例19所述方法制备，得黄色固体产物，收率82.8％。 1H NMR(600MHz,DMSO-d 6)δ10.11(s,1H),9.64(s,1H),8.97(d,J＝5.4Hz,1H),8.82(d,J＝8.2Hz,1H),8.54(d,J＝5.2Hz,1H),8.22(s,1H),8.20(s,1H),8.07(d,J＝8.0Hz,1H),7.94(d,J＝8.2Hz,1H),7.44(t,J＝8.0Hz,1H),7.14(dd,J＝7.8,1.2Hz,1H),4.88-4.86(m,1H),4.80-4.77(m,1H),4.65-4.62(m,1H),4.59-4.57(m,1H)ppm. 13C NMR(150MHz,DMSO-d 6)δ165.79,150.59,148.15,148.11,143.62,142.15,133.19,130.51,130.46,128.53,127.08,124.44,124.18,123.20,123.18,122.70,120.64,119.64,116.72,82.22(d,J＝166.2Hz),64.72(d,J＝18.2Hz)ppm.HR-MS(m/z)(ESI):calcd for C 21H 15ClFN 3O 2[M+H] +:396.0915；found:396.0911.
实施例21：化合物21的制备
采用CX-4945和2-碘乙醇参照实施例19所述方法制备，得黄色固体产物，收率77.3％。 1H NMR(600MHz,DMSO-d 6)δ10.16(s,1H),10.04(s,1H),8.98(d,J＝5.6Hz,1H),8.82(d,J＝8.5Hz,1H),8.52(dd,J＝8.3,1.0Hz,1H),8.34(s,1H),8.26(d,J＝8.2Hz,1H),8.02(d,J＝1.4Hz,1H),7.73(dd,J＝8.4,1.5Hz,1H),7.48(t,J＝8.1Hz,1H),7.16(dd,J＝7.9,1.2Hz,1H),5.11-5.08(t,J＝8.2Hz,2H),4.78(t,J＝7.8Hz,2H). 13C NMR(150MHz,DMSO-d 6)δ165.62,152.34,150.89,148.19,143.68,142.39,133.15,130.50,130.26,128.49,127.20,124.66,124.04,123.26,123.17,122.71,120.89,119.95,117.39,79.06,63.61.HR-MS(m/z)(ESI):calcd for C 21H 15ClF 2N 4O[M+H] +:503.9975；found:503.9980.
实施例22：化合物22的制备
采用CX-4945和2,2-二氟乙醇参照实施例19所述方法制备，得土黄色固体产物，收率65.7％。 1H NMR(600MHz,DMSO-d 6)δ10.10(s,1H),9.90(s,1H),8.96(d,J＝5.6Hz,1H),8.83(d,J＝8.5Hz,1H),8.76(d,J＝5.4Hz,1H),8.31(d,J＝1.7Hz,1H),8.19(d,J＝1.7Hz,1H),8.16-8.14(m,1H),7.93(dd,J＝8.4,1.8Hz,1H),7.44(t,J＝8.2Hz,1H),7.14(dd,J＝7.8,1.2Hz,1H),6.51(tt,J＝54.2,3.2Hz,1H),4.68(td,J＝15.2,3.2Hz,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ165.21,150.86,148.21,148.12,143.70,142.25,133.12,130.41,129.78,128.69,127.07,124.63,124.17,123.51,123.37,122.69,120.82,119.82,117.19,114.12(t,J＝238.9Hz),63.04(t,J＝26.4Hz).HR-MS(m/z)(ESI):calcd for C 21H 14ClF 2N 3O 2[M+H] +:414.0821；found:414.0820.
实施例23：化合物23的制备
采用CX-4945和3,3-二氯丙醇参照实施例19所述方法制备，得黄色固体产物，收率61.5％。 1H NMR(600MHz,DMSO)δ10.14(s,1H),10.02(s,1H),8.97(d,J＝5.6Hz,1H),8.87(d,J＝3.6Hz,1H),8.86(s,1H),8.39(t,J＝1.8Hz,1H),8.24(d,J＝1.4Hz,1H),8.17(dd,J＝8.2,1.2Hz,1H),7.98(dd,J＝8.4,1.6Hz,1H),7.43(t,J＝8.2Hz,1H),7.14(dd,J＝7.8,1.2Hz,1H),4.84-4.80(m,1H),4.70(dd,J＝11.8,4.6Hz,1H),4.65(dd,J＝11.8,6.2Hz,1H),4.14(t,J＝5.2Hz,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ165.47,150.91,148.21,148.17,143.75,142.34,133.12,130.40,130.23,128.72,127.17,124.67,124.27,123.44,123.36,122.67,120.86,119.87,117.38,65.77,58.78,46.48ppm.HR-MS(m/z)(ESI):calcd for C 22H 16Cl 3N 3O 2[M+H] +:460.0386；found:460.0380.
实施例24：化合物24的制备
采用CX-4945和三缩乙二醇参照实施例19所述方法制备，得黄色固体产物，收率72.6％。 1H-NMR(600MHz,DMSO-d 6)δ10.12(s,1H),9.63(s,1H),8.98(d,J＝6.0Hz,1H),8.82(d,J＝6.0Hz,1H),8.54(d,J＝6.0Hz,1H),8.27(t,J＝2.0Hz,1H),8.20(d,J＝1.6Hz,1H),8.07(dd,J＝8.2,1.4Hz,1H),7.94(dd,J＝8.4,1.8Hz,1H),7.45(t,J＝8.1Hz,1H),7.15(dd,J＝6.0,1.4Hz,1H),4.57(t,J＝6.0Hz,1H),4.47(t,J＝6.0Hz,2H),3.83(t,J＝6.0Hz,2H),3.66(dd,J＝5.8,3.8Hz,2H),3.58(dd,J＝5.8,3.8Hz,2H),3.48(dd,J＝10.4,4.8Hz,2H),3.44(dd,J＝7.8,3.0Hz,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ165.97,150.65,148.22,148.16,143.67,142.19,133.22,130.88,130.54,128.53,127.19,124.49,124.25,123.21,123.15,122.73,120.67,119.67,116.78,72.86,70.41,70.20,68.84,64.79,60.69ppm.HR-MS(m/z)(ESI):calcd for C 25H 24ClN 3O 5[M+H] +:482.1482；found:482.1483.
实施例25：化合物25的制备
采用CX-4945和2-(2-羟基乙基)吡啶参照实施例19所述方法制备，得黄色固体产物，收率77.2％。 1H NMR(600MHz,DMSO-d 6)δ10.15(s,1H),10.02(s,1H),8.97(s,1H),8.87(d,J＝7.0Hz,1H),8.83(s,1H),8.61(s,1H),8.36(s,1H),8.27(s,1H),8.18(d,J＝6.1Hz,1H),8.00(d,J＝6.7Hz,1H),7.88(s,1H),7.57(d,J＝5.3Hz,1H),7.43(s,1H),7.39(s,1H),7.13(d,J＝5.9Hz,1H),5.50(s,2H),3.40(s,1H),3.18(s,1H)ppm. 13C NMR(150MHz,DMSO-d 6)δ165.78,156.03,150.96,149.74,148.25,143.82,142.36,137.58,133.11,130.64,130.47,128.69,127.23,124.71,124.32,123.63,123.44,122.68,122.50,122.10,120.83,119.88,118.27,117.41,67.41,49.03ppm.HR-MS(m/z)(ESI):calcd for C 26H 19ClN 4O 2[M+H] +:455.1275；found:455.1269.
实施例26：化合物26的制备
采用CX-4945和1,2-乙二硫醇参照实施例19所述方法制备，得黄色固体产物，收率42.7％。 1H NMR(600MHz,DMSO-d 6)δ10.15(s,1H),9.94(s,1H),8.93(d,J＝5.6Hz,1H),8.81(s,1H),8.80(d,J＝3.2Hz,1H),8.38(s,1H),8.19(dd,J＝8.2,1.2Hz,1H),7.70(d,J＝2.8Hz,1H),7.48(dd,J＝8.2,1.2Hz,1H),7.40(t,J＝8.1Hz,1H),7.11(dd,J＝7.8,0.8Hz,1H),3.16(d,J＝3.2Hz,1H),3.05(t,J＝5.2Hz,2H),2.99-2.96(m,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ170.11,150.64,147.87,147.45,143.71,142.50,138.26,133.13,130.43,127.57,125.65,124.10,123.16,122.88,122.48,120.60,120.01,119.66,117.24,49.03,35.23ppm.HR-MS(m/z)(ESI):calcd for C 21H 16ClN 3OS 2[M+H] +:426.0501；found:426.0507.
实施例27：化合物27的制备
于10mL单口烧瓶中分别加入化合物1 0.182g(0.5mmol)、丙酸0.037g(0.5mmol)、HATU 0.228g(0.6mmol)、DIPEA 0.129g(1.0mmol)和无水DMF 5mL，50℃下搅拌24h-48h，TLC监测至反应完全，浓缩溶剂，硅胶柱层析纯化，洗脱剂为二氯甲烷和甲醇 (80:1-30:1)，得黄色固体产物0.088g，收率43.1％。 1H NMR(600MHz,DMSO-d 6)δ10.58(s,1H),10.20(s,1H),10.03(s,2H),8.98(d,J＝5.4Hz,1H),8.89(t,J＝8.1Hz,2H),8.42(s,1H),8.29(s,1H),8.22(d,J＝8.0Hz,1H),7.96(d,J＝8.0Hz,1H),7.44(t,J＝8.0Hz,1H),7.14(d,J＝7.6Hz,1H),2.24(dd,J＝15.0,7.4Hz,2H),1.09(t,J＝7.5Hz,3H)ppm. 13C NMR(150MHz,DMSO-d 6)δ172.85,165.60,150.80,148.13,143.78,142.52,133.93,133.18,130.43,127.47,126.71,124.44,123.30,123.04,122.60,121.99,120.75,119.79,117.39,27.04,10.20ppm.HR-MS(m/z)(ESI):calcd for C 22H 18ClN 5O 2[M+H] +:420.1227；found:420.1219.
实施例28：化合物28的制备
采用化合物1和3-甲基硫代丙酸参照实施例27所述方法制备，得黄色固体产物，收率46.3％。 1H NMR(600MHz,DMSO-d 6)δ10.67(s,1H),10.18(s,2H),10.03(s,1H),8.96(d,J＝5.5Hz,1H),8.87(dd,J＝6.9,4.2Hz,2H),8.41(s,1H),8.28(d,J＝1.5Hz,1H),8.21(d,J＝7.3Hz,1H),7.95(dd,J＝8.4,1.5Hz,1H),7.42(t,J＝8.1Hz,1H),7.12(dd,J＝7.9,1.3Hz,1H),2.73(t,J＝7.3Hz,2H),2.55(t,J＝7.3Hz,2H),2.10(s,3H)ppm. 13C NMR(150MHz,DMSO-d 6)δ170.49,165.51,150.79,148.10,147.85,143.76,142.52,133.80,133.16,130.41,127.45,126.76,124.43,123.31,123.02,122.58,122.01,120.74,119.78,117.41,34.06,29.36,15.19ppm.HR-MS(m/z)(ESI):calcd for C 23H 20ClN 5O 2S[M+H] +:466.1104；found:466.1109.
实施例29：化合物29的制备
采用化合物1和氯甲酸异丁酯参照实施例27所述方法制备，得黄色固体产物，收率57.8％。 1H NMR(600MHz,DMSO-d 6)δ10.62(s,1H),10.20(s,1H),10.02(s,1H),9.33(s,1H),8.98(d,J＝4.5Hz,1H),8.90(d,J＝8.3Hz,1H),8.87(s,1H),8.43(s,1H),8.28(s,1H),8.21(d,J＝6.3Hz,1H),7.96(d,J＝6.7Hz,1H),7.44(t,J＝7.4Hz,1H),7.14(d,J＝7.2Hz,1H),3.86(d,J＝4.2Hz,2H),1.22(m,1H),0.94(d,J＝4.3Hz,4H),0.84(s,2H)ppm. 13C NMR(150MHz,DMSO-d 6)δ166.22,157.06,150.81,148.13,147.89,143.78,142.49,133.78,133.18,130.43,127.45,126.68,124.44,123.23,123.10,122.61,122.06,120.76,119.79,117.36,70.91,28.14,19.32ppm.HR-MS(m/z)(ESI):calcd for C 24H 22ClN 5O 3[M+H] +:464.1489；found:464.1476.
实施例30：化合物30的制备
采用化合物1和3-(N-吗啉)丙磺酸参照实施例27所述方法制备，得黄色固体产物，收率35.1％。 1H NMR(600MHz,DMSO-d 6)δ10.06(s,1H),9.94(s,1H),8.89(d,J＝5.5Hz,1H),8.78(s,1H),8.77(d,J＝2.0Hz,1H),8.30(s,1H),8.19(d,J＝8.1Hz,1H),7.98(d,J＝1.3Hz,1H),7.83(dd,J＝8.4,1.3Hz,1H),7.41(t,J＝8.1Hz,1H),7.11(dd,J＝7.8,1.2Hz,1H),3.39(m,6H),3.09(m,6H),1.23(t,J＝6.3Hz,2H). 13C NMR(150MHz,DMSO-d 6)δ165.09,158.34,150.87,147.79,147.52,144.16,142.41,133.11,130.42,127.41,125.42,124.09,123.71,123.38,122.58,120.93,120.80,119.82,117.29,49.03,46.39,38.17,29.48,19.02ppm.HR-MS(m/z)(ESI):calcd for C 26H 27ClN 6O 4S[M+H] +:555.1581；found:555.1584.
实施例31：化合物31的制备
采用化合物1和硫辛酸参照实施例27所述方法制备，得黄色固体产物，收率43.7％。 1H NMR(600MHz,DMSO-d 6)δ10.55(s,1H),10.20(s,1H),10.07(s,2H),8.97(d,J＝5.6Hz,1H),8.92(d,J＝5.7Hz,1H),8.89(d,J＝8.6Hz,1H),8.43(t,J＝1.9Hz,1H),8.29(d,J＝1.6Hz,1H),8.24(dd,J＝8.2,1.2Hz,1H),7.96(dd,J＝8.4,1.6Hz,1H),7.44(t,J＝8.1Hz,1H),7.14(dd,J＝7.9,1.4Hz,1H),3.65(ddd,J＝12.3,8.3,6.2Hz,1H),3.24-3.19(m, 1H),3.18-3.12(m,2H),2.45(td,J＝12.5,6.3Hz,1H),2.25(t,J＝7.2Hz,2H),1.91(dq,J＝13.5,6.8Hz,1H),1.77-1.70(m,1H),1.63-1.58(m,2H),1.49-1.42(m,2H)ppm.HR-MS(m/z)(ESI):calcd for C 27H 26ClN 5O 2S 2[M+H] +:552.1294；found:552.1300.
(二)、化合物的细胞毒活性测试
采用MTT方法对本发明的化合物进行了细胞毒活性测试。取对数生长期的细胞计数，接种于96孔培养板内，每孔约8000-10000个细胞。培养过夜，待细胞贴壁后进行给药，分别设给药组和对照组。待测的化合物用DMSO溶液配制成贮液，临用前用细胞培养基稀释成一系列浓度，其中DMSO的终浓度不超过4‰(下面实验类同)。每个浓度设3个复孔。加药后培养72h，加20μL浓度为5mg/mL的MTT，37℃孵育4h，去上清，加入150μL的DMSO溶解。用酶标仪在490波长下测定每孔的OD值，并计算抑制率，做浓度-抑制率曲线计算IC 50值。
测试了本发明化合物对人前列腺癌细胞PC-3、结肠癌细胞HCT-116、乳腺癌细胞MCF-7、结肠癌细胞HT-29、膀胱癌细胞T24和正常肝细胞LO2的细胞毒活性，CX-4945作为阳性对照。观察化合物在不同浓度下对肿瘤细胞生长的抑制情况，计算抑制率及其IC 50值来评价化合物的细胞毒活性，结果见表1。
(三)、化合物的体外酶活性测试
采用商业化的人酪蛋白激酶2(CK2)和人细胞分裂周期样激酶2(CLK2)试剂盒检测了本发明化合物和CX-4945对CK2和CLK2蛋白的抑制活性。CK2试剂盒、CLK2试剂盒、CK2蛋白和CLK2蛋白均购置于上海抚生实业有限公司。按照CK2和CLK2试剂盒说明书提供的实验方法，测试了化合物对CK2和CLK2酶活性，结果见表1与图1。
表1.化合物的细胞毒活性和酶活性
Figure PCTCN2021074623-appb-000050
Figure PCTCN2021074623-appb-000051
a.nd表示未检测；
b.SF表示选择因子，IC 50(CLK2)/IC 50(CK2)。
(四)、化合物抑制癌细胞干性的测试
采用商业化的乙醛脱氢酶1(ALDH1)试剂盒和CD44、CD133抗体，通过流式细胞仪检测了本发明部分化合物对HCT-116原代癌细胞干性抑制的活性。ALDH1试剂盒、CD44、CD133抗体购置于上海碧云天生物技术有限公司，HCT-116原代肿瘤由当地医院提供。
1.ALDH1酶活性检测
根据ALDH1检测试剂盒提供的实验方法，检测化合物1、4、16、19、20、21和25对ALDH1的抑制活性，同时选用CX-4945和已知的癌细胞干性抑制剂BBI 608作为阳性对照，结果见表2。
表2.部分化合物对HCT-116原代癌细胞干性的抑制
Figure PCTCN2021074623-appb-000052
2.干细胞标志物测定
取对数生长期的细胞计数，接种于6孔培养板内，培养至细胞贴壁后加样，空白组作为对照。24h后离心收集细胞，每个离心管中加入300μL PBS，1μL CD44-FITC抗体和1μL CD133-PE抗体,避光孵育30min，转移至2mL离心管中，用流式细胞仪测定。通过流式细胞仪测定了化合物4和20分别与HCT-116原代癌细胞共同孵育24h后细胞内干细胞标志物CD44和CD133的表达情况，选用CX-4945和BBI 608作为阳性对照，
3.实时荧光定量分析
通过qRT-PCR分析了化合物4和20对干细胞抑制通路Wnt/β-catenin和Hedgehog中的相关基因，以及对干细胞相关基因表达的影响，同时选用CX-4945和BBI 608作为阳性对照。
实验方法：
(1)引物序列应用Primer5设计，β-actin作为内参，引物信息如下表3：
表3.引物序列
Figure PCTCN2021074623-appb-000053
(2)采用RT-PCR检测HCT-116原代癌细胞与样品共同孵育24h后，细胞中Wnt/β-catenin和Hedgehog信号通路中的相关基因以及干细胞相关基因OCT4、SOX2和Nanog的表达情况。
实验方法：
(i)HCT-116原代癌细胞内总RNA抽提
根据Karrol RNA Reagent总RNA提取试剂目录号为K3102说明书上提供的实验方法提取细胞内的总RNA，并通过紫外分光光度计测定RNA纯度及浓度：取1μL RNA溶液，稀释100倍，紫外分光光度仪测量其260nm和280nm下的光吸收值，以此计算OD260/280比值以及溶液浓度。要求比值在1.8-2.0之间，RNA浓度大于500μg/mL(操作中试剂、器皿、耗材均经无RNA酶的处理)。
(ii)反转录合成cDNA
以OligdT为下游引物，进行常规逆转录反应；按照如下反应体系进行逆转录合成cDNA第一条链:
Figure PCTCN2021074623-appb-000054
采用无RNA酶去离子水定容至20μL；以上体系42℃反应1h，然后95℃灭活AMV5min,产物置于冰上进行后续试验。
(iii)实时荧光定量PCR(qRT-PCR)
以反转录合成的cDNA产物为模板，按如下反应体系对HCT-116原代癌细胞内的Wnt和Hedgehog信号通路以及干细胞相关基因表达量进行定量分析，以β-actin作为内参基因，并以ddH 2O为模板设阴性对照(以β-catenin为例)：
Figure PCTCN2021074623-appb-000055
以上体系在PCR仪上分别按照如下程序反应：β-catenin以及β-actin，94℃预变性5min，进入三步循环(94℃-40s、52℃-40s、72℃-40s共30cycles)，最后72℃延伸10min。
实时荧光定量分析是利用2 -△△CT方法，每种样本每个基因进行四个复孔平行实验，实验数据的选取，是舍去误差较大的数值，取剩余数值的平均值作为最终实验保留数据，实验数据见图3。

Claims (5)

  1. 一类抗肿瘤化合物，其特征在于通过在已知CK2抑制剂(CX-4945)母体结构中经酰胺键、酯键、硫酯键的方式引入含有氨基、羟基、巯基或卤素原子的脂肪链或者杂环结构片段，提供一类如式II所示的新型的化合物；
    式II-A、式II-B、式II-C、式II-D和II-E所示结构的化合物：
    Figure PCTCN2021074623-appb-100001
    其中：
    式II-A中R 1为-NH 2、-OH、-SO 2CH 3、-CH 2CHF 2、-CH 2C≡CH、-(CH 2) 2OH、-(CH 2) 2NH 2、-(CH 2) 2Br、-(CH 2) 2CO 2CH 3、-(CH 2) 2CO 2H、-(CH 2) 2N(CH 3) 2、-(CH 2) 3OH、-(CH 2) 3NH 2、-(CH 2) 3CO 2CH 3、
    Figure PCTCN2021074623-appb-100002
    式II-B中R 2为-CH 2CHF 2、-(CH 2) 2OH、-(CH 2) 2F、-(CH 2) 2I、-(CH 2) 2CHCl 2、-(CH 2O) 2(CH 2) 2OH、
    Figure PCTCN2021074623-appb-100003
    式II-C中R 3为-(CH 2) 2SH；
    式II-D中R 4为-C 2H 5、-(CH 2) 2SCH 3、-OCH 2CH(CH 3) 2、
    Figure PCTCN2021074623-appb-100004
    式II-E中R 5为
    Figure PCTCN2021074623-appb-100005
  2. 根据权利要求1所述的一类抗肿瘤化合物，其特征在于，所述的化合物优选下列化合物1～31中的任意一种或多种：
    化合物1：5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰肼
    Figure PCTCN2021074623-appb-100006
    化合物2：N-羟基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100007
    化合物3：N-甲磺酰基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100008
    化合物4：N-(2-羟乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100009
    化合物5：N-(2-氨乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100010
    化合物6：N-(2-溴乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100011
    化合物7：3-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-酰氨基)丙酸甲酯
    Figure PCTCN2021074623-appb-100012
    化合物8：3-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-酰氨基)丙酸
    Figure PCTCN2021074623-appb-100013
    化合物9：N-(2-(二甲氨基)乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100014
    化合物10：N-(2,2-二氟乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100015
    化合物11：N-(2-丙基-1-炔)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100016
    化合物12：N-(3-羟丙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100017
    化合物13：N-(3-氨丙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100018
    化合物14：4-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-酰胺基)丁酸甲酯
    Figure PCTCN2021074623-appb-100019
    化合物15：4-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-酰胺基)丁酸
    Figure PCTCN2021074623-appb-100020
    化合物16：N-(2-吗啉乙烷基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100021
    化合物17：N-(2-(1-吡咯烷基)乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100022
    化合物18：N-(1-哌啶基)乙基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-甲酰胺
    Figure PCTCN2021074623-appb-100023
    化合物19：2-羟乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
    Figure PCTCN2021074623-appb-100024
    化合物20：2-氟乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
    Figure PCTCN2021074623-appb-100025
    化合物21：2-碘乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
    Figure PCTCN2021074623-appb-100026
    化合物22：2,2-二氟乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
    Figure PCTCN2021074623-appb-100027
    化合物23：3,3-二氯丙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
    Figure PCTCN2021074623-appb-100028
    化合物24：2-(2-(2-羟乙氧基)乙氧基)乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
    Figure PCTCN2021074623-appb-100029
    化合物25：2-(2-哌啶基)乙基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸酯
    Figure PCTCN2021074623-appb-100030
    化合物26：S-(2-巯基乙醇基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-羧酸硫酯
    Figure PCTCN2021074623-appb-100031
    化合物与27：N’-丙酰基-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰肼
    Figure PCTCN2021074623-appb-100032
    化合物28：N’-(3-(甲硫基)氯丙酰基)-5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰肼
    Figure PCTCN2021074623-appb-100033
    化合物29：异丁基2-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰基)联氨-1-羧酸酯
    Figure PCTCN2021074623-appb-100034
    化合物30：N'-(5-((3-氯苯基)氨基)苯并[c][2,6]萘啶-8-碳酰基)-3-吗啉丙烷-1-磺酰肼
    Figure PCTCN2021074623-appb-100035
    化合物31：N'-(5-(3-1,2-二硫戊环基)戊醇基)-5-((3-氯苯基)氨基)苯并[2,6]萘啶-8-碳酰肼
    Figure PCTCN2021074623-appb-100036
  3. 一种如权利要求1或2所述的一类抗肿瘤化合物的制备方法，其特征在于，上述化合物的合成主要是以CX-4945或其前体为原料合成得到，
    Figure PCTCN2021074623-appb-100037
    CX-4945的合成是以3-溴异烟酸为起始原料，经酯化反应后与2-氨基-4-甲酯基苯基硼酸盐酸盐发生Suzuki偶联反应得到中间体5,6-二氢苯并[c][2,6]萘啶-8-羧酸甲酯，然后与三氯氧磷发生氯代反应，再与间氯苯胺发生取代反应，最后水解得到产物。具体合成路线如下：
    Figure PCTCN2021074623-appb-100038
    a.EtOH,H 2SO 4,reflux,24～48h；b.Cs 2CO 3,Pd(dppf)Cl 2,N 2,100℃,30～48h；
    c.POCl 3,110℃,4h；d.NMP,110℃,8h；e.1.5N NaOH,70℃。
  4. 如权利要求1所述的一类抗肿瘤化合物的制备方法，其特征在于，(i)式II-A化合物可通过交换反应或缩合反应得到，具体合成路线如下：
    Figure PCTCN2021074623-appb-100039
    (ii)式II-B化合物可通过酯化反应得到，具体合成路线如下：
    Figure PCTCN2021074623-appb-100040
    (iii)式II-C化合物可通过硫代醇的酯化反应得到，具体合成路线如下：
    Figure PCTCN2021074623-appb-100041
    (iv)式II-D化合物可通过酰肼与羧酸的酰胺缩合反应得到，具体合成路线如下：
    Figure PCTCN2021074623-appb-100042
    (v)式II-E化合物可通过酰肼与磺酸的酰胺缩合反应得到，具体合成路线如下：
    Figure PCTCN2021074623-appb-100043
    其中R 1、R 2、R 3、R 4、R 5如前所述，HATU代表缩合剂2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐，TBTU代表缩合剂O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸，DIPEA代表N，N-二异丙基乙胺，DMF代表溶剂N,N-二甲基甲酰胺，Reflux代表回流，r.t.代表室温。
  5. 一种如权利要求1所述的一类抗肿瘤化合物的用途，其特征在于，所述的化合物对癌细胞的增殖、对CK2蛋白激酶的抑制和对CK2蛋白的选择性具有良好的作用，同时还能够抑制癌细胞干性，用于制备抗肿瘤药物。
PCT/CN2021/074623 2020-03-26 2021-02-01 一类抗肿瘤化合物及其制备与用途 WO2021190142A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010224405.9A CN113444084A (zh) 2020-03-26 2020-03-26 一类抗肿瘤化合物及其制备与用途
CN202010224405.9 2020-03-26

Publications (1)

Publication Number Publication Date
WO2021190142A1 true WO2021190142A1 (zh) 2021-09-30

Family

ID=77807174

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/074623 WO2021190142A1 (zh) 2020-03-26 2021-02-01 一类抗肿瘤化合物及其制备与用途

Country Status (2)

Country Link
CN (1) CN113444084A (zh)
WO (1) WO2021190142A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114436925A (zh) * 2022-01-14 2022-05-06 河北大学 间二苯酚醚类化合物及其制备方法与应用
CN115124511A (zh) * 2022-07-28 2022-09-30 中国人民解放军北部战区总医院 他克林衍生物及其制备方法与作为cdk2/9抑制剂的应用
CN115160310A (zh) * 2022-05-10 2022-10-11 中国人民解放军北部战区总医院 作为CDK2/Topo I抑制剂的白叶藤碱衍生物及其制备方法和抗肿瘤的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101528704A (zh) * 2006-09-01 2009-09-09 赛林药物股份有限公司 丝氨酸-苏氨酸蛋白激酶和parp调节剂
CN102036561A (zh) * 2008-02-29 2011-04-27 赛林药物股份有限公司 蛋白激酶调节剂
WO2012054493A1 (en) * 2010-10-22 2012-04-26 Cylene Pharmaceuticals, Inc. Deuterated serine-threonine protein kinase modulators
CN102481289A (zh) * 2009-04-17 2012-05-30 赛林药物股份有限公司 治疗与蛋白激酶ck2活性有关的疾病的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101528704A (zh) * 2006-09-01 2009-09-09 赛林药物股份有限公司 丝氨酸-苏氨酸蛋白激酶和parp调节剂
CN102036561A (zh) * 2008-02-29 2011-04-27 赛林药物股份有限公司 蛋白激酶调节剂
CN102481289A (zh) * 2009-04-17 2012-05-30 赛林药物股份有限公司 治疗与蛋白激酶ck2活性有关的疾病的方法
WO2012054493A1 (en) * 2010-10-22 2012-04-26 Cylene Pharmaceuticals, Inc. Deuterated serine-threonine protein kinase modulators

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN HONG, FEIHONG CHEN, NANNAN LIU, XINYI WANG, SHAOHUA GOU: "Chemically induced degradation of CK2 by proteolysis targeting chimeras based on a ubiquitin–proteasome pathway", BIOORGANIC CHEMISTRY, vol. 81, 12 September 2018 (2018-09-12), pages 536 - 544, XP055853274, DOI: 10.1016/j.bioorg.2018.09.005 *
FABRICE PIERRE; ERIC STEFAN; ANNE-SOPHIE NDELLEC; MARIE-CLAIRE CHEVREL; COLLIN F. REGAN; ADAM SIDDIQUI-JAIN; DIWATA MACALINO; NICO: "7-(4-1,2,4-Triazol-3-yl)benzo[][2,6]naphthyridines: A novel class of Pim kinase inhibitors with potent cell antiproliferative activity", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 21, no. 22, 15 September 2011 (2011-09-15), AMSTERDAM, NL, pages 6687 - 6692, XP028320719, ISSN: 0960-894X, DOI: 10.1016/j.bmcl.2011.09.059 *
PIERRE F, CHUA P C, O'BRIEN S E, SIDDIQUI-JAIN A, BOURBON P, HADDACH M, MICHAUX J, NAGASAWA J, SCHWAEBE M K, STEFAN E, VIALETTES A: "Discovery and SAR of 5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic Acid (CX-4945), the First Clinical Stage Inhibitor of Protein Kinase CK2 for the treatment of Cancer", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 54, no. 2, 27 January 2011 (2011-01-27), US, pages 635 - 654, XP002715731, ISSN: 0022-2623, DOI: 10.1021/jm101251q *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114436925A (zh) * 2022-01-14 2022-05-06 河北大学 间二苯酚醚类化合物及其制备方法与应用
CN114436925B (zh) * 2022-01-14 2023-08-15 河北大学 间二苯酚醚类化合物及其制备方法与应用
CN115160310A (zh) * 2022-05-10 2022-10-11 中国人民解放军北部战区总医院 作为CDK2/Topo I抑制剂的白叶藤碱衍生物及其制备方法和抗肿瘤的应用
CN115160310B (zh) * 2022-05-10 2023-06-23 中国人民解放军北部战区总医院 作为CDK2/Topo I抑制剂的白叶藤碱衍生物及其制备方法和抗肿瘤的应用
CN115124511A (zh) * 2022-07-28 2022-09-30 中国人民解放军北部战区总医院 他克林衍生物及其制备方法与作为cdk2/9抑制剂的应用
CN115124511B (zh) * 2022-07-28 2023-09-26 中国人民解放军北部战区总医院 他克林衍生物及其制备方法与作为cdk2/9抑制剂的应用

Also Published As

Publication number Publication date
CN113444084A (zh) 2021-09-28

Similar Documents

Publication Publication Date Title
WO2021190142A1 (zh) 一类抗肿瘤化合物及其制备与用途
ES2871949T3 (es) N-[4-fluoro-5-[[(2S,4S)-2-metil-4-[(5-metil-1,2,4-oxadiazol-3-il)metoxi]-1-piperidilmetil]tiazol-2-il]acetamida como inhibidor de OGA
BR112015000615B1 (pt) Purinas macrocíclicas para o tratamento de infecções virais e composição farmacêutica que as compreende
IL233984A (en) The history of tetrahydro-quinazolinone, their preparation and their pharmaceutical preparations
EP2152693A1 (en) 2-morpholin-4-yl-pyrimidines as pi3k inhibitors
US20060264439A1 (en) Inhibitors of polo-like kinase-1
US9233995B2 (en) Quinazoline derivatives and quinazoline complex protein kinase inhibitor for inhibiting multiplication of tumor cells and preparation method thereof
CN115353508B (zh) 5-吡啶-1h-吲唑类化合物、药物组合物和应用
WO2021023272A1 (zh) 一种atr抑制剂的晶型及其应用
OuYang et al. Design, synthesis, antiproliferative activity and docking studies of quinazoline derivatives bearing oxazole or imidazole as potential EGFR inhibitors
CN109280032B (zh) 一种哒嗪酮母核结构的组蛋白去乙酰化酶抑制剂及其制备方法和用途
WO2023041059A1 (zh) 八氢吡嗪并二氮杂萘啶二酮化合物及其晶型
CN115286583B (zh) 一种含二苯基氨基嘧啶类化合物、制备及其作为HDACs酶抑制剂的应用
WO2019185033A1 (zh) 用作fgfr不可逆抑制剂的酰胺基吡唑类化合物
WO2020077944A1 (zh) 嘌呤系列衍生物及其制备方法和用途
CA3125505C (en) Fluorine-containing substituted benzothiophene compound, and pharmaceutical composition and application thereof
CN111247143B (zh) 可用作蛋白激酶抑制剂的吡啶并喹唑啉衍生物
WO2021078141A1 (zh) 新型嘌呤衍生物及其中间体与制备抗癌症药物的应用
CN115340502B (zh) Bcl-xl抑制剂及其制备方法和用途
CN115340499B (zh) Bcl-xl抑制剂及其用途
WO2023131305A1 (zh) Prmt5抑制剂和抗癌治疗剂的组合
CN110526907B (zh) 苯并噁嗪酮类衍生物及其应用
CN110950868B (zh) 吡唑并嘧啶化合物及制备方法与制备抗癌症药物的应用
WO2023125947A1 (zh) 四氢异喹啉类化合物的可药用盐、晶型及其用途
CN110294745B (zh) 五元杂环类衍生物及其制备方法和其在医药上的用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21775308

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21775308

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 21775308

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC, EPO FORM 1205A DATED 13.07.2023