CN106420741A - Novel application of compound 4-iodo-6-phenylpyrimidine - Google Patents
Novel application of compound 4-iodo-6-phenylpyrimidine Download PDFInfo
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- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
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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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
- A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
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Abstract
本发明涉及化合物4‑吲哚‑6‑苯基哌啶4‑Iodo‑6‑phenylpyrimidine的新用途。试验表明，该化合物4‑Iodo‑6‑phenylpyrimidine具有明显的保护神经细胞，促进神经细胞在各种神经系统疾病损害情况下的存活作用。因此，可以用于制备治疗神经系统疾病的药物。所制备的药物可以是以该化合物为活性成分，且含有药学上可以接受的载体的组合物，活性成分的重量含量为0.1‑99.95％。药物剂型可以是片剂、粉剂、粒剂和注射剂等。
The present invention relates to the novel use of compound 4-indole-6-phenylpiperidine 4-Iodo-6-phenylpyrimidine. Tests have shown that the compound 4-Iodo-6-phenylpyrimidine has obvious protective effects on nerve cells and promotes the survival of nerve cells under damage from various nervous system diseases. Therefore, it can be used to prepare medicines for treating nervous system diseases. The prepared medicine can be a composition with the compound as an active ingredient and a pharmaceutically acceptable carrier, and the weight content of the active ingredient is 0.1-99.95%. The pharmaceutical dosage form can be tablet, powder, granule and injection etc.
Description
技术领域technical field
本发明涉及一种化合物4-吲哚-6-苯基哌啶4-Iodo-6-phenylpyrimidine的新用途，特别是在制备治疗神经系统疾病药物中的应用。The present invention relates to a new application of compound 4-indole-6-phenylpiperidine 4-Iodo-6-phenylpyrimidine, especially the application in the preparation of medicines for treating nervous system diseases.
背景技术Background technique
神经系统的结构复杂性及神经元死亡后的不可替代性决定了在所有神经系统疾病治疗的终极目标都是尽可能的挽救神经元的存活，其直接关系到临床疗效和病人长期生存的质量。目前，缺血性中风，神经退行性病的发病率很高，常会严重剥夺患者的劳动和生活自理能力，已成为困扰国民生活和国家医疗财政的重大健康问题。在这些神经系统疾病的发生和进展中神经元死亡无疑是重要的病理改变。因此目前对于神经元死亡的分子机制的研究是国内外研究的一个热点， 同时研究如何阻止神经元的死亡也是治疗上述疾病的重要手段。缺血性中风和神经退行性疾病是两大类主要致死和致残性神经系统疾病，前者治疗的关键是挽救缺血-再灌注损伤区域的神经元，后者亦是阻止新病灶的发展并以挽救神经元为最终目的。目前发现脑缺血再灌注损伤,谷氨酸兴奋性毒性及各种炎性反应导致的神经死亡的主要原因之一是由聚腺苷酸二磷酸核糖转移酶-1（PARP-1）过度激活介导的区别于凋亡和坏死的细胞死亡，又称Parthanatos（PARP-1依赖性细胞死亡）。发生在神经细胞的parthanatos，可以用DNA 烷化剂甲基硝基亚硝基胍（N-Methyl-N′-nitro-N-nitrosoguanidine，MNNG) 处理肿瘤细胞Hela 细胞得到相同的结果，所有的细胞都通过PARP-1 的过度激活而造成parthanatos 死亡。我们利用这个细胞模型筛选化合物库发现化合物4-Iodo-6-phenylpyrimidine可以阻止MNNG处理的Hela的死亡, 化合物4-Iodo-6-phenylpyrimidine的分子式为C10H7IN2，英文缩写为4-IPP。进而我们发现其同样能阻止神经细胞的死亡，因为NMDA（N-methyl-D-aspartic acid，N-甲基-D-天冬氨酸）处理神经细胞可以在培养细胞水平上模拟缺血性中风及神经退行性病神经细胞死亡的过程，而500uM的NMDA处理不能杀死加了化合物4-IPP的神经细胞。因此化合物4-IPP可以通过阻止缺血性中风、神经退行性疾病等疾病中的parthanatos 细胞死亡来治疗这些神经系统疾病。The structural complexity of the nervous system and the irreplaceability of neurons after death determine that the ultimate goal of the treatment of all neurological diseases is to save the survival of neurons as much as possible, which is directly related to the clinical efficacy and the quality of long-term survival of patients. At present, ischemic stroke and neurodegenerative diseases have a high incidence rate, often severely depriving patients of their ability to work and take care of themselves, and have become a major health problem that plagues national life and national medical finances. Neuronal death is undoubtedly an important pathological change in the occurrence and progression of these neurological diseases. Therefore, the current research on the molecular mechanism of neuron death is a hot topic at home and abroad, and how to prevent neuron death is also an important means of treating the above diseases. Ischemic stroke and neurodegenerative diseases are two main types of fatal and disabling neurological diseases. The key to the treatment of the former is to save neurons in the area of ischemia-reperfusion injury, while the latter is to prevent the development of new lesions and The ultimate goal is to save neurons. It has been found that one of the main causes of neuronal death caused by cerebral ischemia-reperfusion injury, glutamate excitotoxicity and various inflammatory reactions is the excessive activation of polyadenylate diphosphate ribosyltransferase-1 (PARP-1). Cell death mediated by differentiation from apoptosis and necrosis, also known as Parthanatos (PARP-1-dependent cell death). The parthanatos that occur in nerve cells can be treated with DNA alkylating agent methyl nitrosonitrosoguanidine (N-Methyl-N′-nitro-N-nitrosoguanidine, MNNG) to treat tumor cells Hela cells to obtain the same results, all cells Both cause the death of parthanatos through the overactivation of PARP-1. We used this cell model to screen the compound library and found that the compound 4-Iodo-6-phenylpyrimidine can prevent the death of Hela treated with MNNG. The molecular formula of the compound 4-Iodo-6-phenylpyrimidine is C 10 H 7 IN 2 , and the English abbreviation is 4-IPP . Furthermore, we found that it can also prevent the death of nerve cells, because NMDA (N-methyl-D-aspartic acid, N-methyl-D-aspartic acid) treatment of nerve cells can simulate ischemic stroke at the level of cultured cells And the process of nerve cell death in neurodegenerative diseases, while 500uM NMDA treatment could not kill the nerve cells added with compound 4-IPP. Therefore compound 4-IPP can treat neurological diseases such as ischemic stroke and neurodegenerative diseases by preventing parthanatos cell death in these diseases.
发明内容Contents of the invention
本发明的目的之一在于提供化合物4-Iodo-6-phenylpyrimidine在制备治疗神经系统疾病药物中的应用。One of the objects of the present invention is to provide the application of the compound 4-Iodo-6-phenylpyrimidine in the preparation of drugs for treating nervous system diseases.
本发明的目的之二在于提供化合物4-Iodo-6-phenylpyrimidine在制备神经退行性疾病药物中的应用。The second object of the present invention is to provide the application of the compound 4-Iodo-6-phenylpyrimidine in the preparation of drugs for neurodegenerative diseases.
本发明的目的之三在于提供化合物4-Iodo-6-phenylpyrimidine在制备阻止神经细胞的parthanatos死亡药物中的应用。The third object of the present invention is to provide the application of the compound 4-Iodo-6-phenylpyrimidine in the preparation of the drug for preventing the death of parthanatos of nerve cells.
本发明的目的之四在于提供化合物4-Iodo-6-phenylpyrimidine作为神经细胞保护剂在制备治疗缺血性中风药物中的应用。The fourth object of the present invention is to provide the application of the compound 4-Iodo-6-phenylpyrimidine as a nerve cell protective agent in the preparation of a medicament for treating ischemic stroke.
试验表明，该化合物4-Iodo-6-phenylpyrimidine 具有明显的保护神经细胞，促进神经细胞在各种神经系统疾病损害情况下的存活作用。因此，可以用于制备治疗神经系统疾病的药物。所制备的药物可以是以该化合物为活性成分，且含有药学上可以接受的载体的组合物，活性成分的重量含量为0.1-99.95％。药物剂型可以是片剂、粉剂、粒剂和注射剂等。Tests have shown that the compound 4-Iodo-6-phenylpyrimidine has the obvious effect of protecting nerve cells and promoting the survival of nerve cells under the damage of various nervous system diseases. Therefore, it can be used to prepare medicines for treating nervous system diseases. The prepared medicine can be a composition with the compound as an active ingredient and a pharmaceutically acceptable carrier, and the weight content of the active ingredient is 0.1-99.95%. The pharmaceutical dosage form can be tablet, powder, granule and injection etc.
附图说明Description of drawings
图1，MNNG处理不能杀死加了化合物4-IPP的Hela细胞。(A) 20倍相差显微镜下典型图片显示加了50uM的化合物4-IPP的Hela细胞经50uM MNNG处理15分钟再培养24小时后细胞基本不死（右上图），对照组的Hela细胞经同样条件处理后细胞完全死亡（右下图）。（B）柱状统计图说明经4次重复实验后统计经50uM MNNG处理15分钟再培养24小时后，加了50uM的化合物4-IPP的Hela细胞与对照组的存活率，差异极其显著，Mean±SEM, P<0.001。Figure 1, MNNG treatment cannot kill Hela cells added with compound 4-IPP. (A) A typical picture under a 20-fold phase contrast microscope shows that Hela cells added with 50uM compound 4-IPP were treated with 50uM MNNG for 15 minutes and then cultured for 24 hours, and the cells were basically immortal (upper right picture). Hela cells in the control group were treated under the same conditions Afterwards the cells die completely (lower right panel). (B) Histogram shows that after 4 repeated experiments, the statistics show that after 50uM MNNG treatment for 15 minutes and then cultured for 24 hours, the survival rate of Hela cells added with 50uM compound 4-IPP and the control group is extremely significant, Mean± SEM, P<0.001.
图2，500uM的NMDA处理不能杀死加了化合物4-IPP的神经细胞。Figure 2, 500uM NMDA treatment can not kill the nerve cells added with compound 4-IPP.
(A) PI/Hochest活细胞染色典型图片显示加了50uM的化合物4-IPP的神经细胞经500uM NMDA处理15分钟再培养24小时后细胞死亡率相对于照组大大减少，对照组（加DMSO组）的神经细胞经同样条件处理后细胞大部分完全死亡，显示为PI染色细胞增多。（B）柱状统计图说明经4次重复实验后统计经500uM NMDA处理15分钟再培养24小时后，加了50uM的化合物4-IPP的神经细胞与对照组的存活率，差异极其显著，Mean±SEM, P<0.001。(A) A typical picture of PI/Hochest live cell staining shows that the neurons added with 50uM compound 4-IPP were treated with 500uM NMDA for 15 minutes and then cultured for 24 hours. Compared with the control group, the cell death rate was greatly reduced. ) nerve cells treated under the same conditions, most of the cells died completely, showing increased PI staining cells. (B) Histogram shows that after 4 repeated experiments, after 4 repeated experiments, the survival rate of nerve cells added with 50uM compound 4-IPP and the control group after being treated with 500uM NMDA for 15 minutes and then cultured for 24 hours, the difference is extremely significant, Mean± SEM, P<0.001.
具体实施方式detailed description
下面的实施例可以使本专业技术人员更全面地理解本发明，但不以任何方式限制本发明。The following examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.
实施例1：Hela细胞的培养及MNNG的处理Embodiment 1: the processing of the culture of Hela cell and MNNG
Hela细胞用含10%胎牛血清的DMEM正常传代培养，待其长到90%融合度时，用50uM MNNG37度细胞培养箱中处理15分钟（对照组加等体积二甲基亚砜DMSO，筛选组或处理组加溶于DMSO的化合物），换回正常培养基再培养24小时后，对照组100%的细胞都已经死亡并悬浮在培养基中。Hela cells were normally subcultured with DMEM containing 10% fetal bovine serum. When they grew to 90% confluence, they were treated with 50uM MNNG in a 37-degree cell culture incubator for 15 minutes (add an equal volume of dimethyl sulfoxide DMSO to the control group, and select group or treatment group plus compound dissolved in DMSO), and after changing back to normal culture medium for another 24 hours, 100% of the cells in the control group had died and were suspended in the culture medium.
实施例2：神经细胞的培养Example 2: Culture of nerve cells
C57BL6小鼠，胎鼠（E12-14）取出大脑皮层组织，在解剖液中先剪碎，用0.125%胰蛋白酶在37℃孵育30min，移入接种液（MEM含1%谷氨酰胺，另加入10%马血清），停止消化，并洗去胰蛋白酶液，用细口吸管吹打细胞悬液，使其充分分散，如此多次，待沉淀后吸出上层细胞悬液，计数，预置细胞密度，移入接种液接种于预先涂有多聚赖氨酸的康宁12孔培养板，隔夜换维持培养液（MEM中含5%马血清，1%谷氨酰胺）每周换液两次，每次换1/2。培养3-5天后，用阿糖胞苷，或5-FU抑制神经胶质细胞的生长。C57BL6 mice, fetal mice (E12-14) took out the cerebral cortex tissue, cut it into pieces in the dissection solution, incubated with 0.125% trypsin at 37°C for 30min, and transferred it into the inoculation solution (MEM containing 1% glutamine, and added 10 %horse serum), stop digestion, and wash away the trypsin solution, pipette the cell suspension with a narrow mouth pipette to make it fully dispersed, so many times, after precipitation, suck out the upper layer of cell suspension, count, preset cell density, transfer into The inoculum was inoculated on Corning 12-well culture plates pre-coated with polylysine, and the maintenance medium (5% horse serum and 1% glutamine in MEM) was changed overnight, and the medium was changed twice a week, 1 /2. After 3-5 days of culture, the growth of glial cells was inhibited with cytarabine or 5-FU.
实施例3： 神经细胞存活实验Example 3: Nerve cell survival experiment
具体实验过程为：取实施例2中的小鼠神经细胞，培养两周后，用500uM NMDA 37度细胞培养箱中处理15分钟（对照组加等体积二甲基亚砜DMSO，处理组加溶于DMSO的化合物），换回正常培养基再培养24-48小时。The specific experimental process is as follows: take the mouse nerve cells in Example 2, and after culturing them for two weeks, treat them with 500uM NMDA in a 37-degree cell incubator for 15 minutes (the control group adds an equal volume of dimethyl sulfoxide DMSO, and the treatment group adds dissolved Compounds in DMSO), replaced with normal medium and cultured for another 24-48 hours.
实施例4：Hoechst/PI双染检测神经细胞坏死Example 4: Hoechst/PI double staining detection of nerve cell necrosis
因为坏死细胞细胞膜的完整性被破坏，细胞膜的通透性增强，因此进入坏死细胞中的Hoechst 33342与PI染料比正常细胞的多。而PI等染料是不能进入细胞膜完整的活细胞中，即正常细胞在不经固定的情况下对这些染料是拒染，坏死细胞由于膜完整性在早期即已破损，可被这些染料染色。根据这些特性，用Hoechst 33342结合PI等染料对坏死细胞进行双染色，就可在荧光显微镜上将正常细胞和坏死细胞区别开来，并可以标定细胞总数目与坏死细胞数目。正常细胞为高蓝色/低红色（Hoechst 33342+/PI-），坏死细胞为高蓝色/高红色（Hoechst 33342+/PI++）。通过这种方法对坏死神经细胞进行检测，并统计存活率。Because the integrity of the necrotic cell membrane was destroyed, the permeability of the cell membrane was enhanced, so more Hoechst 33342 and PI dyes entered the necrotic cells than normal cells. However, dyes such as PI cannot enter living cells with intact cell membranes, that is, normal cells are resistant to these dyes without fixation, and necrotic cells can be stained by these dyes because their membrane integrity has been damaged in the early stage. According to these characteristics, double staining of necrotic cells with Hoechst 33342 combined with dyes such as PI can distinguish normal cells from necrotic cells on a fluorescence microscope, and can calibrate the total number of cells and the number of necrotic cells. Normal cells are high blue/low red (Hoechst 33342+/PI-), and necrotic cells are high blue/high red (Hoechst 33342+/PI++). By this method, the necrotic nerve cells were detected, and the survival rate was counted.
具体实验过程为：取实施例3中的小鼠神经细胞，使用凯基生物的“KGA-212荧光Hoechst33342/PI双染检测试剂盒。终止培养后吸除培养液，用冷Buffer A洗涤细胞二次，滴加150 μlhoechst33342工作液和75ulPI工作液(原液稀释10倍)，室温避光染色10 min，在相应波长激发荧光倒置显微镜下观察细胞核形态。The specific experimental process is as follows: take the mouse nerve cells in Example 3, and use KGA-212 Fluorescent Hoechst33342/PI Double Staining Detection Kit of KGI Biotech. After the culture is terminated, the culture medium is aspirated, and the cells are washed with cold Buffer A for 2 Once, 150 μl of hoechst33342 working solution and 75ul of PI working solution were added dropwise (the original solution was diluted 10 times), stained at room temperature in the dark for 10 min, and the morphology of cell nuclei was observed under an inverted microscope with fluorescence excited at the corresponding wavelength.
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