【作者】 王东；

【导师】 孙平华；

【作者基本信息】 暨南大学 ， 药物化学， 2022， 硕士

【摘要】 炎症是身体的一种保护性反应,但持续的炎症反应会损害组织,甚至导致功能丧失、肿瘤和死亡。遗憾的是,目前临床所用的抗炎药普遍具有选择性差以及临床上应用副作用明显,限制了传统抗炎药的临床应用,需要开发新的抗炎药。藁本内酯（ligustilide）是从传统伞形科中药当归中分离获得的具有较强抗炎止痛的中药提取物,但由于藁本内酯化学性质不稳定,其C-3位连接的丁烯基易被环境中的光、热以及酶等氧化或降解,成药性较差。本研究中通过对藁本内酯结构进行设计与合成,得到结构稳定的衍生物,对所得产物的抗炎活性进行评价,为后续的构效关系以及抗炎机理研究提供了科学基础。衍生物合成:本论文首先以苯酞为原料,通过亲核取代,Birch还原反应以及消除反应三步得到藁本内酯,进一步胺和藁本内酯发生亲核取代反应合成了系列一衍生物。根据活性结果筛选出系列一活性最优化合物4j,对化合物4j结构中苯环结构进行修饰得到系列二,进一步探讨该系列衍生物中苯环上连接不同基团以及基团在苯环的不同位置对抗炎活性的影响。一共得到36个藁本内酯衍生物,所有目标化合物结构都经过了¹H NMR、¹³C NMR和HRMS验证。体外抗炎活性评价:首先通过Griess试剂法对所有藁本内酯衍生物抗炎作用（LPS诱导RAW264.7细胞生成NO的抑制作用）进行初步活性筛选,根据实验结果得出:1.系列一藁本内酯衍生物N上连接的基团为六元环时可以提高部分化合物抗炎活性,尝试延长N原子与苯环之间的碳链时发现,碳链个数为一个C时活性最优,得到系列一中活性较优的化合物4j;2.系列二藁本内酯衍生物中苯环上连接强给电子基团时活性明显减弱,但当苯环上连接强吸电子基团三氟甲基（-CF₃）得到活性表现最优的化合物5f,其抑制NO的IC₅₀为1.61±0.11μM优于两种阳性对照药吲哚美辛IC₅₀（2.81±0.06μM）和地塞米松IC₅₀（2.22±0.02μM）。本论文以NO抑制率最优的化合物5f作为候选化合物,检测其对炎症因子IL-1β、TNF-α和IL-6的影响,化合物5f抑制IL-1β、TNF-α和IL-6的IC₅₀分别为18.22±0.12 n M、6.2±0.02μM和2.61±0.31Μm均高于阳性药地塞米松(IC₅₀分别为566.09±0.32 n M、65.34±0.17μM和15.03±0.36μM)。最后接着通过Western Blot等方法对该类化合物的抗炎机理进行研究,化合物5f抑制NLRP3、COX-2以及i NOS蛋白的表达;抑制NF-κB p65、IκB以及JNK-MAPK的磷酸化。另外通过MTT法检测了所有化合物对RAW264.7细胞、非洲绿猴肾细胞（Vero细胞）以及人正常肝细胞（LO2细胞）的细胞增殖活性的影响,藁本内酯衍生物在有效浓度范围内均未呈现细胞毒性。结论:本论文通过藁本内酯的内酯环结构进行改造得到系列藁本内酯衍生物。对该系列藁本内酯衍生物进行体外抗炎活性实验,筛选出了活性最优的化合物5f。通过进一步总结藁本内酯衍生物构效关系发现,藁本内酯的内酯环改为内酰胺环以及C-3位的丁烯基被加成后,化学稳定性显著提升,且内酰胺环N原子连接苄基时活性最好。苄基上苯环连接基团在对位时活性优于其他位置。炎症机制研究结果表明5f抑制NF-κB和MAPK通路的磷酸化。本研究为苯酞类化合物的应用改造以及开发新的抗炎药提供理论和实践基础。更多还原

【Abstract】 Inflammation is the first alarm signal of the body’s response to the invasion of microorganisms and pathogens,leading to the occurrence of a variety of clinical chronic diseases.Unfortunately,the anti-inflammatory drugs currently in clinical use generally have poor selectivity and obvious side effects in clinical applications,which limit the clinical application of traditional anti-inflammatory drugs and require the development of new anti-inflammatory drugs.Ligustilide is a traditional Chinese medicine extract with strong anti-inflammatory and analgesic properties isolated from traditional Umbelliferae Chinese medicine Angelica sinensis.However,due to the unstable chemical properties of ligustilide,its C-3 linkage The butenyl group is easily oxidized or degraded by light,heat and enzymes in the environment,and the druggability is poor.In this study,the structure of ligustilide was designed and synthesized to obtain a structurally stable derivative,and the anti-inflammatory activity of the obtained product was evaluated,which provided a scientific basis for the follow-up structure-activity relationship and anti-inflammatory mechanism research.Derivative synthesis:In this paper,phthalide was used as raw material to obtain ligustilide through nucleophilic substitution,Birch reduction reaction and elimination reaction,and then a series of derivatives were synthesized by nucleophilic substitution reaction between amine and ligustilide.According to the activity results,a series of the most active compound 4j was screened out,and the benzene ring structure of the compound 4j was modified to obtain a series two.The series of derivatives were further explored to connect different groups on the benzene ring and the groups in different positions of the benzene ring.Effects on anti-inflammatory activity.A total of 36compounds were obtained,and the structures of all target compounds were verified by¹HNMR,¹³CNMR and HRMS.Evaluation of anti-inflammatory activity in vitro:.First,the Griess reagent method was used to detect the inhibitory effect of all compounds on LPS-induced NO production in RAW264.7 cells,and the preliminary activity screening was carried out.According to the experimental results,it was concluded that:1.When the group connected to N of a series of compounds is a six-membered ring,it can increase the partial The anti-inflammatory activity of the compound,extending the carbon chain between N and the benzene ring found that the activity was the best when one C was extended,and the compound 4j with better activity was obtained in series one;2.When a strong electron-donating group was connected to the benzene ring in series two The activity was obviously weakened,but when the strong electron withdrawing group trifluoromethyl（-CF3）was connected to the benzene ring,the compound 5f with the best activity was obtained,and its IC₅₀ for inhibiting NO was 1.61±0.11μM,which was much lower than the two positive controls.The IC₅₀ of indomethacin（2.81±0.06μM）and the IC₅₀ of dexamethasone（2.22±0.02μM）.In this paper,compound 5f with the best inhibitory rate of NO was used as a candidate compound,and its effects on inflammatory factors IL-1β,TNF-αand IL-6 were tested.Compound 5f inhibited the IC₅₀ of IL-1β,TNF-αand IL-6.18.22±0.12 n M,6.2±0.02μM and 2.61±0.31μM were higher than the positive drug dexamethasone(IC₅₀ were 566.09±0.32 n M,65.34±0.17μM and 15.03±0.36μM,respectively).Finally,the anti-inflammatory mechanism of these compounds was studied by Western Blot and other methods.Compound 5f inhibited the expression of NLRP3,COX-2 and i NOS proteins.The effect of all compounds on the cell proliferation activity of RAW264.7 cells,African green monkey kidney cells（Vero cells）and human normal hepatocytes（LO2 cells）was also measured by MTT and the compounds were not cytotoxic in the effective concentration range.Conclusion:In this paper,ligustilide derivatives were obtained by transforming the lactone ring structure of ligustilide.The anti-inflammatory activity of this series of compounds was tested in vitro,and the compound 5f with the best activity was screened out.Further summarizing the structure-activity relationship of the compounds,it was found that the lactone ring of ligustilide was changed to a lactam ring and the butenyl group at the C-3 position was added,the chemical stability was significantly improved,and the N atom of the lactam ring was connected to a benzyl group.best active.The benzene ring linking group on the benzyl group is more reactive than other positions at the para position.The results of inflammatory mechanism studies showed that 5f inhibited the expression of NLRP3,COX-2 and i NOS proteins.This study provides a theoretical and practical basis for the application and transformation of phthalide compounds and the development of new anti-inflammatory drugs.更多还原