WO2016127681A1 - 4，4'-联吡啶桥联四核铂配合物在制备抗端粒酶阴性肿瘤药物中的应用 - Google Patents

4，4'-联吡啶桥联四核铂配合物在制备抗端粒酶阴性肿瘤药物中的应用 Download PDF

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WO2016127681A1
WO2016127681A1 PCT/CN2015/094908 CN2015094908W WO2016127681A1 WO 2016127681 A1 WO2016127681 A1 WO 2016127681A1 CN 2015094908 W CN2015094908 W CN 2015094908W WO 2016127681 A1 WO2016127681 A1 WO 2016127681A1
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cells
telomerase
platinum complex
bipyridine
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赵勇
郑小辉
毛宗万
夏立新
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中山大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • C—CHEMISTRY; METALLURGY
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    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table

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  • the invention belongs to the field of medicine, and particularly relates to the application of 4,4'-bipyridine bridged tetranuclear platinum complex in preparing anti-telomerase negative tumor drugs.
  • Telomere is a special structure at the end of a chromosome in a eukaryotic organism. It consists of repeating units of DNA and proteins bound to it, which maintains the structural and functional integrity of the chromosome. With each division of the cell, the telomeres are shortened a bit. When the telomeres of normal human cells are shortened to a certain extent, the cells will apoptosis and lose the ability to divide and proliferate.
  • telomere length stability Approximately 80% of cancer cells maintain telomere length stability by expressing telomerase, gaining the ability to divide indefinitely; in addition, approximately 20% of cancer cells do not express telomerase (telomerase-negative tumor cells), they pass The alternative pathway mechanism known as ALT (Alternative Lengthening of Telomeres) extends telomeres to maintain telomere length. Since telomerase is not present in normal human tissue cells, it is a specific anti-cancer target.
  • telomerase-negative tumors include: about 20% of human tumor cells; the tumor cells lack (or fail to detect) telomerase activity; the tumor cells extend telomeres using an ALT (Alternative Lengthening of Telomeres) mechanism; Tumor cells are rich in circular telomeric DNA (C-Circle DNA or T-Circle DNA); in most of the tumor cells, there is an observable APB (Associated Promyelocytic leukaemia Body).
  • ALT Alterative Lengthening of Telomeres
  • telomerase-targeting drugs allow tumor cells to activate ALT mechanisms. Therefore, inhibition of ALT is also an intrinsic requirement and ultimate solution for telomerase-targeted cancer therapy.
  • the object of the present invention is to provide a route capable of effectively inhibiting proliferation of telomerase-negative tumor cells in view of the above technical problems.
  • the present invention provides the use of a 4,4'-bipyridine bridged tetranuclear platinum complex of the following formula (I) for the preparation of a medicament for anti-telomerase-negative tumors:
  • the compound of the above formula (I) is known, and the anion is a nitrate (NO 3 - ) ion, which has extremely high water solubility and good thermal stability.
  • the telomerase-negative tumor is an osteosarcoma.
  • the medicament contains a pharmaceutically acceptable carrier or excipient.
  • Figure 1 is a cell proliferation curve.
  • Figure 2 shows the results of ⁇ -galactosidase staining experiments.
  • Figure 3 shows the results of flow cytometry for detecting apoptosis.
  • human osteosarcoma is taken as an example to illustrate the utility of the compound of formula I (4,4'-bipyridine bridged tetranuclear platinum complex) in inhibiting proliferation of telomerase-negative tumor cells, but it should be understood that although The use of human osteosarcoma as an example to illustrate its use for the preparation of a drug against telomerase-negative tumors, but the term "telomerase-negative tumor” cells as used herein refers to the lack (or inability to detect) telomerase activity, Tumor cells expressing telomerase include, but are not limited to, human osteosarcoma, glioma, and the like.
  • the compound of formula I (4,4'-bipyridyl bridged tetranuclear platinum complex) has a significant inhibitory effect on tumor cell proliferation for a variety of telomerase-negative tumor cells, including but not limited to, for example, human osteosarcoma. .
  • Pulmonary fibroblast (MRC-5, normal cell) cell line, human cervical cancer (HeLa, telomerase positive) cell line, human osteosarcoma (U2OS and SAOS2, telomerase negative) cell line were purchased from China National Species Collection ( CTCC).
  • each cell is counted by a cell counter for each passage, and then some cells are re-inoculated, and the seeding density is still 1 ⁇ 10 6 /dish until the total number of cells in the culture dish is ⁇ 1 ⁇ 10 6 can not be inoculated.
  • ⁇ -galactosidase staining experiment a) After the cell culture is terminated, the medium is directly drained, and then washed 3 times with 1 ⁇ PBS; b) a fixing solution is added to the cells, and fixed at room temperature for 15 minutes; c) After discarding the fixative, rinse it again with 1 ⁇ PBS for 5 min each time; d) the specific staining procedure was carried out according to the method provided on the Biyuntian X-gal staining kit; e) adding the staining solution, Incubate overnight at 37 ° C with CO 2 isolation; f) After the end of staining, continue to rinse 3 times with 1 ⁇ PBS, and observe the cells under a 10 ⁇ microscope and photograph.
  • Flow cytometry experiment a) Collect all cells (including floating cells), adherent cells were collected by 0.25% trypsin digestion, centrifuged, and washed twice with 1 ⁇ PBS, discarded. The supernatant, retaining the pellet (cell); b) collecting the cells Fluor 488annexin V and PI for double dyeing, the specific dyeing process according to Alexa The method provided on the 488annexin V/Dead Cell Apoptosis Kit is operated; c) the stained cell suspension is directly tested by flow cytometry, and the experimental data is fitted by FlowJo software.
  • telomerase-negative cells U2OS and SAOS 2.
  • telomerase-negative human osteosarcoma SAOS2 cells showed significant aging after treatment with the compound (Compound 4 of formula I, 4'-bipyridyl bridged tetranuclear platinum complex).
  • phosphatidylserine In normal cells, phosphatidylserine (PS) is only distributed in the inner side of the lipid bilayer of the cell membrane, but in the early stage of apoptosis The phosphatidylserine (PS) on the cell membrane is turned from the inside to the outside of the lipid membrane, so it is possible to determine whether the cell is in the early stage of apoptosis by detecting the presence of PS on the extracellular surface.
  • Annexin V has a high affinity for PS and can be used to determine the number of apoptotic cells.
  • Propidium Iodide is a nucleic acid dye that does not pass through the intact cell membrane of normal cells or early apoptotic cells, but for advanced cells in apoptosis, PI can pass through the cell membrane and stain the nucleus. .
  • Annexin V By matching Annexin V to PI, cells at different stages of apoptosis can be identified.
  • the lower left quadrant shows live cells
  • the lower right quadrant is the early apoptotic cell
  • the upper left quadrant is the metaphase apoptotic cell
  • the upper right quadrant is the late apoptotic cell.
  • the compound 4,4'-bipyridine bridged tetranuclear platinum complex of the formula I can effectively inhibit the proliferation of telomerase-negative tumor cells and inhibit tumor growth, and can be used for preparing an effective anti-telomerase-negative tumor. drug.

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Abstract

本发明公开了4,4'-联吡啶桥联四核铂配合物在制备抗端粒酶阴性肿瘤药物中的应用，能够有效抑制端粒酶阴性肿瘤细胞的增殖。

Description

4，4’-联吡啶桥联四核铂配合物在制备抗端粒酶阴性肿瘤药物中的应用 技术领域
本发明属于医药领域，具体涉及4,4’-联吡啶桥联四核铂配合物在制备抗端粒酶阴性肿瘤药物中的应用。
背景技术
端粒(telomere)是真核生物染色体末端的一种特殊结构，由重复单位的DNA及结合其上的蛋白质组成，其作用是保持染色体结构和功能的完整性。伴随着细胞每一次分裂，端粒都会缩短一点。正常人类细胞的端粒缩短到一定程度后，细胞就会凋亡，从而失去分裂增殖能力。大约80％的癌细胞通过表达端粒酶(telomerase)维持端粒长度的稳定，获得无限分裂的能力；另外大约20％的癌细胞不表达端粒酶(端粒酶阴性肿瘤细胞)，它们通过被称为ALT(Alternative Lengthening of Telomeres)的旁路途径机制延伸端粒，从而维持端粒长度。由于端粒酶在人类的正常组织细胞中不存在，它是特异的抗癌靶标。端粒酶阴性肿瘤的特征包括：占人体肿瘤细胞的20％左右；该肿瘤细胞缺乏(或检查不到)端粒酶活性；该肿瘤细胞采用ALT(Alternative Lengthening of Telomeres)机制延伸端粒；该肿瘤细胞中富含环状的端粒DNA(C-Circle DNA或T-Circle DNA)；在绝大部分的该肿瘤细胞中有可观察到的APB(Associated Promyelocytic leukaemia Body)。
越来越多的研究表明，采用ALT机制的癌细胞具有更强的侵润、转移能力，目前对该类癌症的治疗成功率极低，急需开发针对ALT肿瘤的治疗药物及方法。另外，最新的研究表明，靶向端粒酶的药物可以让肿瘤细胞激活ALT机制，因此，抑制ALT机制也是以端粒酶为靶标的癌症治疗的内在需求和终极方案。
发明内容
本发明的目的是针对以上要解决的技术问题，提供一种能够有效抑制端粒酶阴性肿瘤细胞增殖的途径。
为此，本发明提供了以下式(I)的4,4’-联吡啶桥联四核铂配合物在制备抗端粒酶阴性肿瘤药物中的应用：
Figure PCTCN2015094908-appb-000001
以上式(I)化合物为已知的，阴离子为硝酸根(NO3 -)离子，具有极高的水溶性，热稳定性良好。
根据本发明所述的应用，所述端粒酶阴性肿瘤为骨肉瘤。
根据本发明所述的应用，所述药物含有药学上可接受的载体或赋形剂。
实验证明，式I化合物4,4’-联吡啶桥联四核铂配合物对于多种端粒酶阴性肿瘤细胞，包括但不限于例如人骨肉瘤等，均有显著抑制肿瘤细胞增殖的效用。
附图说明
图1为细胞增殖曲线。
图2为β-半乳糖苷酶染色实验结果。
图3为流式细胞法检测细胞凋亡的结果。
具体实施方式
下面结合具体实施例进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的应用范围。
本实施例中，以人骨肉瘤为例说明式I化合物(4,4’-联吡啶桥联四核铂配合物)在抑制端粒酶阴性肿瘤细胞增殖方面的效用，但应理解的是，虽然仅以人骨肉瘤为例说明其用于制备抗端粒酶阴性肿瘤的药物的用途，但本发明所称的“端粒酶阴性肿瘤”细胞指缺乏(或检查不到)端粒酶活性、不表达端粒酶的肿瘤细胞，包括但不限于例如人骨肉瘤、胶质瘤等。实验证明，式I化合物(4,4’-联吡啶桥联四核铂配合物)对于多种端粒酶阴性肿瘤细胞，包括但不限于例如人骨肉瘤等，均有显著抑制肿瘤细胞增殖的效用。
1材料与方法
1.1实验材料
肺纤维母(MRC-5，正常细胞)细胞株、人宫颈癌(HeLa、端粒酶阳性)细胞株、人骨肉瘤(U2OS和SAOS2、端粒酶阴性)细胞株购自中国典型物种保藏中心(CTCC)。
1.2实验方法
1)将MRC-5、HeLa、U2OS和SAOS2细胞分别接种于10cm2的细胞培养皿中，接种密度为1×106/皿，等细胞6小时贴壁后，小心换掉培养基，加入10mL含有相应浓度式I化合物(4,4’-联吡啶桥联四核铂配合物)的培养基或者等体积的含0.1％DMSO的培养基。该操作每隔2天重复一次。
2)增殖过程中，每次传代时均需使用细胞计数器对每皿细胞进行计数，然后再从中取部分细胞重新接种，接种密度仍为1×106/皿，直到培养皿中细胞总数<1×106个无法接种为止。
3)取增殖结束的细胞分别进行β-半乳糖苷酶染色实验以及流式细胞凋亡实验。
4)β-半乳糖苷酶染色实验：a)细胞培养终止后，直接倒掉培养基，然后用1×PBS润洗3遍；b)往细胞中加入固定液，常温下固定15min；c)弃去固定液后，再用1×PBS润洗3遍，每次5min；d)具体的染色操作过程按照碧云天的X-gal染色试剂盒上提供的方法进行操作；e)加入染色液，37℃并隔离CO2孵育过夜；f)染色结束后，继续用1×PBS润洗3遍，将细胞置于10×显微镜下观察、照相。
5)流式细胞凋亡实验：a)收集好所有的细胞(包括浮起来的细胞)，贴壁的细胞用0.25％胰蛋白酶消化法收集，离心，并用1×PBS润洗2遍，弃去上清液，保留沉淀(细胞)；b)收集好的细胞用
Figure PCTCN2015094908-appb-000002
Fluor 488annexin V and PI进行双染，具体的染色操作过程按照Alexa 
Figure PCTCN2015094908-appb-000003
488annexin V/Dead Cell Apoptosis Kit试剂盒上提供的方法进行操作；c)染色好后的细胞悬浮液直接用流式细胞仪进行测试，实验数据由FlowJo软件进行处理拟合。
2结果
2.1细胞增殖曲线
细胞培养第32天时，4个细胞的增殖曲线如图1所示。实验结果表明，多个浓度的配合物(式I化合物4,4’-联吡啶桥联四核铂配合物)并不会对正常细胞(MRC-5)及端粒酶阳性细胞(HeLa)的增殖造成影响，但是却可以显著的降低端粒酶阴性细胞(U2OS和SAOS2)的增殖。
2.2β-半乳糖苷酶染色实验
细胞衰老时β-半乳糖苷酶SA-β-Gal活性水平上升，通过检测细胞内β-半乳糖苷酶的水平(Senescenceβ-Galactosidase Staining)能迅速确定衰老细胞的数量。如图2所示，端粒酶阴性的人骨肉瘤(SAOS2)细胞在经过化合物(式I化合物4,4’-联吡啶桥联四核铂配合物)处理后出现了明显的衰老现象。
2.3流式细胞法检测细胞凋亡
在正常细胞中，磷脂酰丝氨酸(PS)只分布在细胞膜脂质双层的内侧，而在细胞凋亡早 期，细胞膜上的磷脂酰丝氨酸(PS)由脂膜内侧翻向外侧，因此可以通过检测细胞外表面PS的存在确定细胞是否处于细胞凋亡早期。Annexin V与PS有高度亲和力，可用于确定凋亡细胞的数量。碘化丙啶(Propidium Iodide,PI)是一种核酸染料，它不能透过正常细胞或早期凋亡细胞的完整的细胞膜，但对凋亡中晚期的细胞，PI能够穿过细胞膜并使细胞核染色。将Annexin V与PI匹配使用，就可鉴定出处于不同凋亡时期的细胞。在双变量流式细胞仪的散点图上，左下象限显示活细胞，右下象限为早期凋亡细胞，左上象限是中期凋亡细胞，右上象限是凋亡晚期细胞。
由以上实验结果可见，式I化合物4,4’-联吡啶桥联四核铂配合物能够有效抑制端粒酶阴性肿瘤细胞的增殖，抑制肿瘤生长，可用于制备有效抗端粒酶阴性肿瘤的药物。

Claims (3)

  1. 式(I)的4,4’-联吡啶桥联四核铂配合物在制备抗端粒酶阴性肿瘤药物中的应用：
    Figure PCTCN2015094908-appb-100001
  2. 根据权利要求1所述的应用，其特征在于，所述端粒酶阴性肿瘤为骨肉瘤。
  3. 根据权利要求1所述的应用，其特征在于，所述药物含有药学上可接受的载体或赋形剂。
PCT/CN2015/094908 2015-02-09 2015-11-18 4，4'-联吡啶桥联四核铂配合物在制备抗端粒酶阴性肿瘤药物中的应用 WO2016127681A1 (zh)

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