WO2024011663A1 - 着丝粒蛋白CENP-M小分子抑制剂cenpemlin及其制备方法与应用 - Google Patents

着丝粒蛋白CENP-M小分子抑制剂cenpemlin及其制备方法与应用 Download PDF

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WO2024011663A1
WO2024011663A1 PCT/CN2022/108196 CN2022108196W WO2024011663A1 WO 2024011663 A1 WO2024011663 A1 WO 2024011663A1 CN 2022108196 W CN2022108196 W CN 2022108196W WO 2024011663 A1 WO2024011663 A1 WO 2024011663A1
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cenpemlin
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姚雪彪
刘行
杨丰瑞
马明明
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University of Science and Technology of China USTC
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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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
    • C07D251/40—Nitrogen atoms
    • C07D251/54—Three nitrogen atoms
    • C07D251/70—Other substituted melamines

Definitions

  • the invention belongs to the field of medicine, and specifically relates to cenpemlin, a small molecule inhibitor of the key mitotic centromere protein CENP-M and its preparation method and application.
  • Kinetochores are an essential element of this molecular machinery and can be viewed as lamellar structures with inner and outer layers in direct contact with centromeric chromatin and spindle microtubules, respectively.
  • the inner kinetochore is composed of at least 16 centromeric proteins, which are assembled into multi-component complexes and directly interact with centromeric chromatin.
  • complexes include: CENP-H, I, K, M complex, CENP -O, P, Q, U, R complexes, CENP-T, W, S, X complexes, CENP-L, N complexes and CENP-C proteins, which further recruit other kinetochore proteins after assembly of these complexes .
  • interactions between centromere proteins have been extensively studied, the specific biological functions of some of these proteins remain to be further explored.
  • the CCAN complex appears in the shape of a "herringbone", with CENP-L/N dimers forming an arch, and DNA passing through the bottom of the arch.
  • the CENP-M protein interacts with three other centromere proteins, CENP-H, I, and K, to form a complex that becomes part of the inner layer of the kinetochore.
  • CENP-M binds near the CENP-L/N dimer at the top of the vault, and has a spatial interaction interface with both CENP-L and CENP-N.
  • CENP-M has dynamic positioning throughout the cell cycle, and its function may be constitutive, but the specific molecular mechanism has not been fully elucidated.
  • CENP-M was originally named PANE1 (proliferation associated nuclear element 1) and is only conserved in metazoans. Subsequent studies demonstrated that CENP-M is closely related to CENP-A, CENP-L, CENP-N and CENP-T. Loss of CENP-M leads to mislocalization of other CCAN proteins, suggesting that CENP-M is critical for the assembly and stabilization of inner kinetochores. However, although CENP-M is structurally and evolutionarily related to GTP hydrolases, it is not a true enzyme.
  • the object of the present invention is to provide a compound, its isomer and its pharmaceutically acceptable salt, as well as its preparation method and application.
  • the compound represented by the above formula I specifically targets the key centromere protein CENP-M in mitosis, and as a CENP-M inhibitor, it is named cenpemlin.
  • CENP-M is an important member of the inner kinetochore CCAN complex and was named PANE1 because it was identified in rapidly proliferating cells. Subsequent studies found that CENP-M is a pseudo-GTP hydrolase. In recent years, studies on the inner kinetochore CCAN complex have emerged one after another, but its fine structure is still not fully resolved. We use cryo-EM-based structural biology methods to answer scientific questions. The CCAN complex protein was expressed and purified in an insect system, and then recombined in vitro to obtain a complete CCAN complex. Three-dimensional images of the complex were collected by cryo-electron microscopy, followed by three-dimensional reconstruction. Finally, we obtained the structural information of the CCAN complex containing CENP-M protein.
  • CENP-M Based on the fine structure of the protein interaction interface between CENP-M and CENP-L, we identified specific CENP-M targeting small molecules through a combination of molecular docking analysis, virtual screening, cell phenotype analysis and protein interaction experiments.
  • the compound represented by formula I is prepared according to the synthesis route shown in Figure 1 by a method including the following steps:
  • the molar ratio of cyanuric chloride to morpholine can be 1:2-2.5, specifically 1:2;
  • reaction time can be 1-1.5h, specifically 1h;
  • the alkaline condition is provided by sodium bicarbonate; the molar ratio of sodium bicarbonate to morpholine can be 1:1.0-1.2, specifically 1:1;
  • step 2) of the above method the molar ratio of the cyanogen chloride intermediate product 1 containing bis 1,4-oxaza heterocycle and hydrazine hydrate is 1:1-2;
  • the temperature of the reaction is 40°C-60°C, and the time is 3-4h, specifically 3h;
  • step 3 of the above method the ratio of 1,3(1,4-oxazine heterocycle)-5-hydrazine polycyanide and six-carbon monosaccharide can be 10mmol:1.8-2.0g, specifically 10mmol:1.8g ;
  • Six-carbon monosaccharides that can be used as raw materials include but are not limited to D-glucose, D-galactose, and D-mannose;
  • the reaction temperature is 40°C-60°C, and the reaction time is 4-5h, specifically 4h.
  • inhibiting cell mitosis is achieved by interfering with CENP-M-mediated kinetochore assembly, specifically inhibiting the interaction between CENP-M and CENP-L;
  • the tumor cells can be epithelial cancer cells; specifically, they can be cervical cancer cells, liver cancer cells, breast cancer cells, etc.;
  • the cancer may specifically be epithelial cancer, specifically cervical cancer, liver cancer, breast cancer, etc.
  • the present invention also provides a reagent for inhibiting cell mitosis, which contains a compound represented by formula I, an isomer thereof or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a product for inhibiting tumor cell proliferation.
  • the product for inhibiting tumor cell proliferation contains a compound represented by Formula I, an isomer thereof or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a product for preventing and/or treating cancer, which contains the compound represented by formula I, its isomer or a pharmaceutically acceptable salt thereof.
  • the invention also provides a method for inhibiting the interaction between CENP-M and CENP-L, which is: adding the compound represented by formula I, its isomer or its pharmaceutically acceptable compound to a system containing CENP-M and CENP-L. of salt, culture, and that's it.
  • the CENP-M inhibitory organic small molecule compound provided by the present invention after being added to the culture medium and combined with the CENP-M protein, causes the chromosomes to be unable to be arranged correctly and further causes the cells to experience delayed mitosis and lagging chromosome phenotypes.
  • Cenpemlin can inhibit the interaction between CENP-M and CENP-L at a concentration of 1 ⁇ M.
  • cenpemlin can inhibit the interaction between CENP-M and CENP-L at a concentration of 1 ⁇ M.
  • CENP-M small molecule inhibitor cenpemlin of the present invention will play an important role in cell biology research, and its effect in regulating tumor cell proliferation can lay the foundation for the development of new chemotherapy drugs.
  • the present invention analyzes the protein plane information of the fine structure of CENP-M and CENP-L and targets the small molecule compound cenpemlin.
  • Biochemical and cytological experimental results show that cenpemlin inhibits the proliferation of cancer cells by interfering with the interaction between CENP-M and CENP-L, providing a new compound for analyzing and interfering with the rapid proliferation of cancer cells.
  • Figure 1 is a synthesis route diagram of the CENP-M inhibitor represented by Formula 1 of the present invention.
  • Figure 2 shows the NMR identification results of the small molecule compound cenpemlin.
  • Figure 3 shows the phenotypic analysis of cell mitosis by the small molecule compound cenpemlin.
  • Figure 4 is a real-time imaging analysis of the small molecule compound cenpemlin blocking cell mitosis.
  • Figure 5 shows the elution effect analysis of the small molecule compound cenpemlin.
  • Figure 6 shows that the small molecule compound cenpemlin inhibits the interaction between CENP-M and CENP-L.
  • Figure 7 shows the statistical analysis of the small molecule compound cenpemlin inhibiting the proliferation of cancer cells.
  • Figure 8 shows the experiment of the small molecule compound cenpemlin inhibiting the proliferation of liver cancer cells in mice.
  • accession number of CENP-M used in the following examples in the NCBI database is Accession: NP_076958.1 (Update: 27-JUN-2022)
  • accession number of CENP-L used in the following examples in the NCBI database is Accession: NP_001164653.1 (update: 30-JAN-2022)
  • the Bruker AV-500 nuclear magnetic resonance spectrometer was used to identify the structural formula of the small molecule. According to the chemical shift of the hydrogen spectrum and the integrated area of the peak, it was found that the structural formula of the small molecule conformed to the formula I structure.
  • Figure 2 shows the NMR identification results of the small molecule compound cenpemlin.
  • transfect plasmid DNA GFP-tubulin and mCherry-H2B (transfection plasmid cell density is 70%-80%);
  • transfect plasmid DNA GFP-tubulin and mCherry-H2B (transfection plasmid cell density is 70%-80%);
  • the cells treated with cenpemlin exited mitosis after 45 minutes, but the cells showed a multicellular phenotype, that is, the genome was unequally distributed. The reason was that the cells treated with cenpemlin showed a multipolar spindle phenomenon.
  • Example 5 The small molecule compound cenpemlin affects the interaction between CENP-M and CENP-L
  • transfect plasmid DNA GFP-CENP-M and FLAG-CENP-L (transfection plasmid cell density is 70%-80%);
  • HeLa cells cervical cancer cells
  • MDA-MB-231 cells triple-negative breast cancer cells
  • HepG2 cells liver cancer cells
  • Example 8 The small molecule compound cenpemlin inhibits the proliferation of liver cancer cells.
  • liver cancer cells MHCC97-H cells (5x10 6 in 0.1mL normal saline) stably expressing luciferase for 6 weeks. in the liver of aged female NOD/SCID mice.
  • the experiment was divided into three groups, namely DMSO control group, paclitaxel group and cenpemlin group (2mg/kg), with 10 mice in each group.
  • A is the fluorescein imaging picture on the 11th day after administration.
  • B is the statistical analysis chart. The results showed that the number of cancer cells in the liver of mice in the paclitaxel and small molecule compound cenpemlin groups was significantly reduced. The above results show that the small molecule compound cenpemlin can effectively inhibit the proliferation of liver cancer cells in mice, providing new vision and targets for the screening and development of anti-cancer drugs.
  • the present invention analyzes the protein plane information of the fine structure of CENP-M and CENP-L and targets the small molecule compound cenpemlin. Biochemical and cytological experimental results have shown that cenpemlin inhibits the proliferation of cancer cells by interfering with the interaction between CENP-M and CENP-L, providing a new compound for analyzing and interfering with the rapid proliferation of cancer cells.

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Abstract

公开了着丝粒蛋白CENP-M小分子抑制剂cenpemlin及其制备方法与应用。通过解析CENP-M与CENP-L精细结构的蛋白位面信息，靶向筛选到小分子化合物cenpemlin。生物化学与细胞学实验结果均表明，cenpemlin通过干预CENP-M与CENP-L的相互作用，抑制了癌细胞的增殖，为解析和干预癌细胞的快速增殖提供了新型化合物。

Description

着丝粒蛋白CENP-M小分子抑制剂cenpemlin及其制备方法与应用 技术领域
本发明属于医药领域，具体涉及有丝分裂关键着丝粒蛋白CENP-M小分子抑制剂cenpemlin及其制备方法与应用。
背景技术
细胞精准的有丝分裂保证了基因组的稳定及细胞稳态的维持。有丝分裂通过姐妹染色单体的平等分离产生两个基因相同的子细胞。驱动染色体分离的分子机制在真核生物进化过程中相对保守。动粒是此分子机制的基本要素，可视为层状结构，其内层和外层分别直接接触着丝粒染色质和纺锤体微管。动粒内层由至少16个着丝粒蛋白质组成，这些蛋白质组装成多元复合物并直接与着丝粒染色质相互作用，这些复合物包括：CENP-H、I、K、M复合物、CENP-O、P、Q、U、R复合物，CENP-T、W、S、X复合物、CENP-L、N复合物和CENP-C蛋白，这些复合物组装完成后进一步招募其余动粒蛋白。虽然着丝粒蛋白之间的相互作用研究广泛，但其中一些蛋白的具体生物学功能仍待进一步探索。
近来，我们研究组利用冷冻电镜解析了CCAN复合物的结构，CCAN复合物呈现为“人”字形，CENP-L/N二聚体构成了拱门，DNA从拱底处穿过。CENP-M蛋白与其他三种着丝粒蛋白CENP-H、I、K相互作用形成复合物，成为动粒内层的一部分。CENP-M在拱顶顶端的CENP-L/N二聚体附近结合，与CENP-L和CENP-N在空间上都存在相互作用界面。同时，CENP-M在整个细胞周期中都存在动点定位，其功能可能是组成性的，但具体的分子机制仍未完全解析。
CENP-M最初被命名为PANE1(proliferation associated nuclear element 1)，仅在后生动物中保守。随后的研究证明，CENP-M与CENP-A、CENP-L、CENP-N和CENP-T密切相关。CENP-M的缺失导致其他CCAN蛋白的错误定位，从而表明CENP-M对于内层动粒的组装和稳定至关重要。然而，尽管CENP-M在结构和进化上与GTP水解酶相关，但它不是一种真正意义上的酶。通过与进化保守的CENP-H、I、K蛋白形成四元复合物，在招募染色质重塑FACT蛋白复合物、促进CENP-A核小体重构和招募纺锤体检验点相关蛋白等一系列重要生物学进程中发挥至关重要的作用。
目前没有有关CENP-M抑制剂的相关报道。
发明公开
本发明的目的是提供一种化合物、其异构体及其药学上可接受的盐及其制备方法与应用。
本发明所提供的化合物，其结构式如式I所示：
Figure PCTCN2022108196-appb-000001
上述式I所示化合物特异性地靶向有丝分裂关键着丝粒蛋白CENP-M，作为CENP-M抑制剂，名为cenpemlin。
CENP-M是内层动粒CCAN复合物中的重要一员，因在快速增殖的细胞中被鉴定而命名为PANE1，随后的研究发现CENP-M是一种假GTP水解酶。近年来，关于内层动粒CCAN复合物的研究层出不穷，但是其精细结构仍然没有完全解析。我们采用基于冷冻电镜的结构生物学方法来回答科学问题。利用昆虫体系表达纯化CCAN复合物蛋白，随后在体外重组，进而得到了完整的CCAN复合物。通过冷冻电镜收集复合物的三维图像，随后进行三维重构，最终，我们得到了包含CENP-M蛋白的CCAN复合物的结构信息。基于CENP-M和CENP-L蛋白相互作用界面的精细结构，我们通过分子对接分析、虚拟筛选、细胞表型分析及蛋白质相互作用实验相结合，鉴定出具有特异性的CENP-M靶向小分子化合物，式I所示化合物，cenpemlin。
式I所示化合物根据图1所示合成路线图通过包括如下步骤的方法制备得到：
1)使得三聚氯氰和吗啉在碱性条件下反应，生成含双1,4-氧氮杂环的氯氰中间产物1；
Figure PCTCN2022108196-appb-000002
2)将含双1,4-氧氮杂环的氯氰中间产物1与水合肼发生取代反应，生成1,3(1,4-氧氮杂环)-5-肼聚氰(化合物2)；
Figure PCTCN2022108196-appb-000003
3)在冰醋酸中使得1,3(1,4-氧氮杂环)-5-肼聚氰与六碳单糖发生缩合反应，得到式I所示化合物。
上述方法步骤1)中，三聚氯氰与吗啉的摩尔比可为1:2-2.5，具体可为1:2；
所述反应在冰水浴作用下进行，所述反应的时间可为1-1.5h，具体可为1h；
所述碱性条件由碳酸氢钠提供；碳酸氢钠与吗啉的摩尔比可为1:1.0-1.2，具体可为1:1；
上述方法步骤2)中，含双1,4-氧氮杂环的氯氰中间产物1与水合肼的摩尔比为1:1-2；
所述反应的温度为40℃-60℃，时间为3-4h，具体可为3h；
上述方法步骤3)中，1,3(1,4-氧氮杂环)-5-肼聚氰与六碳单糖的配比可为10mmol:1.8-2.0g，具体可为10mmol:1.8g；
可作为原料的六碳单糖包括但不限于D-葡萄糖，D-半乳糖，D-甘露糖；
所述反应的温度为40℃-60℃，时间为4-5h，具体可为4h。
式I所示化合物、其异构体及其药学上可接受的盐在制备具有如下功能的产品中的应用也属于本发明的保护范围：
1)着丝粒蛋白CENP-M抑制剂；
2)抑制细胞有丝分裂的试剂；
3)抑制肿瘤细胞增殖的产品；
4)预防和/或治疗癌症的产品；
所述2)中，抑制细胞有丝分裂是通过干扰CENP-M介导的动粒组装实现的，具体为抑制CENP-M与CENP-L相互作用；
3)中，所述肿瘤细胞可为上皮癌细胞；具体可为宫颈癌细胞、肝癌细胞、乳腺癌细胞等；
4)所述癌症具体可为上皮癌，具体可为宫颈癌、肝癌、乳腺癌等。
本发明还提供一种抑制细胞有丝分裂的试剂，所述试剂含有式I所示化合物、其异构体或其药学上可接受的盐。
本发明还提供一种抑制肿瘤细胞增殖的产品，所述抑制肿瘤细胞增殖的产品含有式I所示化合物、其异构体或其药学上可接受的盐。
本发明还提供一种预防和/或治疗癌症的产品，所述预防和/或治疗癌症的产品含有式I所示化合物、其异构体或其药学上可接受的盐。
本发明还提供一种抑制CENP-M与CENP-L相互作用的方法，为：向含有CENP-M和CENP-L的体系中加入式I所示化合物、其异构体或其药学上可接受的盐，培养，即可。
本发明提供的CENP-M抑制型的有机小分子化合物，在加入培养基中与CENP-M蛋白结合后，使得染色体不能正确排列并进一步导致细胞出现有丝分裂的延迟及滞后染色体的表型。
实时活细胞拍摄发现cenpemlin处理的细胞，会产生染色体错误排列和纺锤体不稳定，还会导致有丝分裂延迟的表型。这些诱导细胞有丝分裂异常的表型与CENP-M敲低的表型是一致的。
Cenpemlin在1μM浓度的处理下可以抑制CENP-M与CENP-L的相互作用。利用cenpemlin对CENP-M/CENP-L的有效抑制作用，可以进一步研究CENP-M在有丝分裂动粒组装及染色体排列中的功能。
本发明的CENP-M小分子抑制剂cenpemlin将在细胞生物学研究中发挥重要作用，同时其调控肿瘤细胞增殖的功效可为新型化疗药物的研制奠定基础。
本发明通过解析CENP-M与CENP-L精细结构的蛋白位面信息，靶向筛选到小分子化合物cenpemlin。生物化学与细胞学实验结果均表明，cenpemlin通过干预CENP-M与CENP-L的相互作用，抑制了癌细胞的增殖，为解析和干预癌细胞的快速增殖提供了新型化合物。
附图说明
图1为本发明式1所示CENP-M抑制剂的合成路线图。
图2为小分子化合物cenpemlin的核磁鉴定结果。
图3为小分子化合物cenpemlin对细胞有丝分裂的表型分析。
图4为小分子化合物cenpemlin阻滞细胞有丝分裂的实时成像分析。
图5为小分子化合物cenpemlin的洗脱效果分析。
图6表明小分子化合物cenpemlin抑制CENP-M与CENP-L相互作用。
图7为小分子化合物cenpemlin抑制癌细胞增殖的统计分析。
图8为小分子化合物cenpemlin抑制小鼠体内肝癌细胞增殖的实验。
实施发明的最佳方式
下述实施例中的实验方法，如无特别说明，均为常规方法
下面结合实施例对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所做的任何变更或改进，均属于本发明的保护范围。
下述实施例中采用的CENP-M在NCBI数据库中的登录号Accession:NP_076958.1(Update：27-JUN-2022)
下述实施例中采用的CENP-L在NCBI数据库中的登录号Accession:NP_001164653.1(update：30-JAN-2022)
实施例1、小分子化合物cenpemlin的合成
将三聚氯氰(10.0g,0.054mol)溶于150mL二氯甲烷，将溶液在冰水浴中冷却到低于5℃，将吗啉(9.50mL,0.108mol)和碳酸氢钠(9.1g,0.108mol)溶于100mL水，将溶液在冰水浴中冷却到低于5℃。冰水浴中，在快速搅拌下，将水溶液滴加到二氯甲烷溶液中，并继续搅拌反应1h。将两相分开，用50mL水洗涤有机相，洗涤两次。通过旋蒸浓缩有机相至20mL，向有机相中加入50mL石油醚，将产生的白色沉淀过滤并用冷水和冷石油醚洗涤，干燥后得14.7g白色固体，即中间产物1，产率95％。核磁氢谱：1H NMR(400MHz,CDCl3)δ3.80-3.68(m,16H)。
将中间产物1(5.7g,20mmol)加入25mL乙醇，加入5mL水合肼，将溶液加热至40℃-60℃回流3小时，然后冷却到室温，白色固体逐渐析出。过滤，并用20mL乙醇洗涤，洗涤两次，干燥后得5.2g白色固体产物，即化合物2，产率93％。核磁氢谱：1H NMR(400MHz,CDCl3)δ3.80-3.68(m,16H)，4.77(d,2H),9.39(t,1H).
将化合物2(2.8g,10mmol)加入25mL乙醇，加入10mmol六碳糖(1.8g)，并加入0.2mL冰醋酸，将溶液加热至40℃-60℃回流4小时，然后冷却到室温，白色固体逐渐析出。过滤，并用20mL乙醇洗涤，洗涤两次，得白色固体产物，用乙醇/乙酸乙酯重结晶可纯化产物。根据所用六碳糖的种类不同，所得产物的产率在70-85％间。可作为原料的六碳单糖包括但不限于D-葡萄糖，D-半乳糖，D-甘露糖。
利用Bruker AV-500核磁共振波谱仪进行小分子结构式的鉴定，根据氢谱的化学 位移和峰的积分面积得知该小分子的结构式符合式Ⅰ结构。
图2示小分子化合物cenpemlin的核磁鉴定结果。
化合物的结构式和其600MHz 1HNMR谱图的归属情况如下：
Figure PCTCN2022108196-appb-000004
其600MHz 1H谱图的归属情况如下：
1H NMR(600MHz,DMSO-d6)δ3.01(26,m,1CH),δ3.59(3,5,19,23,m,4CH2),δ3.67(3,5,19,23,m,4CH2),δ4.34(30,t,1CH2),δ4.43(28,m,1CH),δ4.62(26,m,1CH),δ4.87(16,d,1CH),δ6.25(15,d,1CH),δ8.05(13,s,1NH)
实施例2、小分子化合物cenpemlin造成染色体排列错误
1、实验步骤
1)将稳定表达GFP-CENP-N的HeLa细胞接种到直径为12mm的圆盖玻片上；
2)24h后加入终浓度为2mM的Thymidine处理细胞14-16h；
3)用37℃预热的PBS清洗细胞3次将Thymidine洗脱，换上新鲜培养基继续培养8h；
4)实验组加入终浓度为1μM的cenpemlin，对照组加入同等体积的DMSO，处理1h；
5)用含3.7％甲醛的PBS缓冲液固定细胞10min；
6)经过0.2％TritonX-100打孔和1％BSA封闭后，室温孵育ACA抗体1h；
7)DAPI染色3min后封片；
8)将玻片置于DV显微镜载物台上，在60×，NA＝1.42镜头下拍摄。
2、结果如图3所示。
cenpemlin处理HeLa细胞后可见大量未排列到赤道板的染色体，以GFP-CENP-N作为CENP-L/N复合物的表征，可见未排列至赤道板区域的动粒，即表明，cenpemlin处理细胞将导致细胞有丝分裂染色体队列异常。
实施例3、小分子化合物cenpemlin造成有丝分裂阻滞
1、实验步骤
1)将HeLa细胞接种到35mm活细胞培养皿中；
2)24h后转染质粒DNA：GFP-tubulin和mCherry-H2B(转染质粒细胞密度为70％-80％)；
3)转染6h后换上新鲜培养基并加入2mM的Thymidine处理细胞14-16h；
4)用37℃预热的PBS清洗细胞3次将细胞释放，随后换上新鲜培养基继续培养；
5)打开Applied Precision Personal DV显微镜及恒温装置使温度稳定在37℃；
6)释放后8-9h将细胞培养基换成CO 2-independent Medium，加入终浓度为1μM的cenpemlin或相同体积的DMSO；
7)将细胞置于DV显微镜37℃恒温载物台上，在60×，NA＝1.42镜头下实时拍摄，每隔3min拍摄一帧。
2、结果如图4所示。
从染色体运动的情况来看，cenpemlin处理后会致使细胞的部分染色体始终无法正确队列，还造成了明显的有丝分裂延迟，细胞在核膜破裂120min后仍未进入后期。该结论同样证实了之前一些研究所认为的CENP-M对纺锤体组装检验点的具有一定功能。
实施例4、小分子化合物cenpemlin洗脱效果检测
1、实验步骤
1)将HeLa细胞接种到35mm活细胞培养皿中；
2)24h后转染质粒DNA：GFP-tubulin和mCherry-H2B(转染质粒细胞密度为 70％-80％)；
3)转染6h后换上新鲜培养基并加入终浓度为20μM的MG132和cenpemlin或DMSO处理细胞1h；
4)打开Applied Precision Personal DV显微镜及恒温装置使温度稳定在37℃；
5)用37℃预热的PBS清洗细胞3次将细胞释放，随后换上新鲜培养基继续培养；
6)释放后立即将细胞培养基换成CO 2-independent Medium；
7)将细胞置于DV显微镜37℃恒温载物台上，在60×，NA＝1.42镜头下实时拍摄，每隔3min拍摄一帧。
2、结果如图5所示。
Cenpemlin处理后的细胞在45min后退出有丝分裂，但是细胞表现为多细胞表型，即基因组出现不均等分配，原因是cenpemlin处理后的细胞出现多极纺锤体现象。
实施例5、小分子化合物cenpemlin影响CENP-M与CENP-L的相互作用
1、实验步骤
1)将HEK293T细胞接种到6cm培养皿中；
2)24h后转染质粒DNA：GFP-CENP-M和FLAG-CENP-L(转染质粒细胞密度为70％-80％)；
3)转染4h后换上新鲜培养基并继续培养24h；
4)加入终浓度为1μM的cenpemlin或相同体积的DMSO处理细胞1h；
5)用细胞刮将细胞刮下，收集在离心管中，1,000rpm离心去除培养基；
6)加入细胞裂解buffer进行超声破碎，12,000rpm离心10min；
7)取上清与FLAG beads孵育4h；
8)细胞裂解buffer清洗beads 3次，sample buffer煮样，随后进行电泳和免疫印迹分析。
2、结果如图6所示。
Cenpemlin处理后(第三泳道)CENP-M和CENP-L的相互作用相对于对照组(第二泳道)减弱，表明1μM的cenpemlin足以破坏CENP-M与CENP-L的相互作用，这一结果呼应了前文中的细胞表型实验，说明cenpemlin抑制肿瘤细胞增殖是通过干扰CENP-M与CENP-L的相互作用而实现的。
实施例6、小分子化合物cenpemlin的广谱抑癌作用
1、实验步骤
1)将HeLa细胞(宫颈癌细胞)、MDA-MB-231细胞(三阴性乳腺癌细胞)和HepG2细胞(肝癌细胞)接种到6cm培养皿中；
2)分别加入终浓度为1μM的小分子化合物cenpemlin或DMSO处理细胞24h；
3)分别用胰酶消化细胞，取细胞悬液进行台盼蓝染色；
4)在普通光学显微镜下观察蓝色着色的细胞并进行计数。
2、结果如图7所示。
Cenpemlin处理后HeLa细胞、MDA-MB-231细胞和HepG2细胞都出现台盼蓝着色不同比例的升高，这一结果呼应了前文中的细胞表型实验，说明cenpemlin具有广谱的抑制癌细胞增殖的作用。
实施例8、小分子化合物cenpemlin抑制肝癌细胞增殖。
1、实验步骤
1)根据已发表的实验方案(Chen et al.,2011.Cancer Res)，我们把稳定表达荧光素酶(luciferase)的肝癌细胞MHCC97-H cells(5x10 6in 0.1mL生理盐水)种植在6周龄的雌性NOD/SCID小鼠肝内。实验共分三组，即DMSO对照组、紫杉醇组与cenpemlin组(2mg/kg)，每组10只小鼠。
2)造模14天后(荧光素测试造模成功)，DMSO对照组、紫杉醇组及cenpemlin实验组(2mg/kg)分别进行腹腔注射(每只0.2mL；两侧分别注射0.1mL)，每天一次共三天。
3)每隔一天注射，四次后(给药第11天)，进行荧光素测试。
2、结果如图8所示。
A为给药后第11天荧光素成像图。B为统计分析图。结果表明，紫杉醇和小分子化合物cenpemlin组的小鼠，肝内的癌细胞数显著降低。以上结果表明，小分子化合物cenpemlin可以有效地抑制小鼠体内肝癌细胞的增殖，为抗癌药物的筛选和研发提供了新的视野和靶标。
工业应用
本发明通过解析CENP-M与CENP-L精细结构的蛋白位面信息，靶向筛选到小分 子化合物cenpemlin。生物化学与细胞学实验结果均表明，cenpemlin通过干预CENP-M与CENP-L的相互作用，抑制癌细胞的增殖，为解析和干预癌细胞的快速增殖提供了新型化合物。

Claims (13)

  1. 式I所示化合物、其异构体及其药学上可接受的盐：
    Figure PCTCN2022108196-appb-100001
  2. 制备权利要求1中式I所示化合物的方法，包括如下步骤：
    1)使得三聚氯氰和吗啉在碱性条件下反应，生成含双1,4-氧氮杂环的氯氰中间产物1；
    Figure PCTCN2022108196-appb-100002
    2)将含双1,4-氧氮杂环的氯氰中间产物1与水合肼发生取代反应，生成化合物2；
    Figure PCTCN2022108196-appb-100003
    3)在冰醋酸中使得1,3(1,4-氧氮杂环)-5-肼聚氰与六碳单糖发生缩合反应，得到式I所示化合物。
  3. 根据权利要求2所述的方法，其特征在于：步骤1)中，三聚氯氰与吗啉的摩尔比为1:2-2.5；
    所述反应在冰水浴作用下进行，所述反应的时间为1-1.5h；
    所述碱性条件由碳酸氢钠提供。
  4. 根据权利要求2或3所述的方法，其特征在于：
    步骤2)中，含双1,4-氧氮杂环的氯氰中间产物1与水合肼的摩尔比为1:1-2；
    所述反应的温度为40℃-60℃，时间为3-4h。
  5. 根据权利要求2或3所述的方法，其特征在于：步骤3)中，1,3(1,4-氧氮杂环)-5-肼聚氰与六碳单糖的的配比为10mmol:1.8-2.0g；
    所述反应的温度为40℃-60℃，时间为4-5h。
  6. 权利要求1中式I所示化合物、其异构体及其药学上可接受的盐在制备具有如下功能的产品中的应用：
    1)着丝粒蛋白CENP-M抑制剂；
    2)抑制细胞有丝分裂的试剂；
    3)抑制肿瘤细胞增殖的产品；
    4)预防和/或治疗癌症的产品。
  7. 根据权利要求6所述的应用，其特征在于：所述2)中，抑制细胞有丝分裂是通过干扰CENP-M介导的动粒组装实现的。
  8. 根据权利要求6所述的应用，其特征在于：所述3)中，所述肿瘤细胞为上皮癌细胞；具体可为宫颈癌细胞、肝癌细胞、乳腺癌细胞。
  9. 根据权利要求9所述的应用，其特征在于：所述4)中，所述癌症为上皮癌，具体为宫颈癌、肝癌、乳腺癌。
  10. 一种抑制细胞有丝分裂的试剂，所述试剂含有权利要求1中式I所示化合物、其异构体及其药学上可接受的盐。
  11. 一种抑制肿瘤细胞增殖的产品，含有权利要求1中式I所示化合物、其异构体及其药学上可接受的盐。
  12. 一种预防和/或治疗癌症的产品，含有权利要求1中式I所示化合物、其异构体及其药学上可接受的盐。
  13. 一种抑制CENP-M与CENP-L相互作用的方法，为：向含有CENP-M和CENP-L的体系中加入权利要求1中式I所示化合物、其异构体及其药学上可接受的盐，培养，即可。
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