【作者】 王静；

【导师】 叶光明；

【作者基本信息】 安徽医科大学 ， 药学（专业学位）， 2020， 硕士

【摘要】 类风湿性关节炎（RA）是一种慢性的全身性的自身免疫性疾病,女性较男性更易患病,在老年人群体中也更易发现。主要影响患者关节滑膜,临床表现为对称的关节受累,包括关节痛、肿胀、炎症,并伴随着增生性滑膜,软骨损害,骨侵蚀和全身性后果,严重者甚至限制患者的功能性活动,导致残疾,对患者本身和社会都造成经济负担。尽早发现和治疗对病人有利。目前临床治疗RA的药物主要包括小分子药物:非甾体抗炎药（non-steroidal anti-inflammatory drugs,NSAIDs）、糖皮质激素和化学合成类抗风湿性药物（disease modifying antirheumatic drugs,DMARDS）等,但这些药物有较多的不良反应。大分子生物制剂,即针对RA的发病机理,对疾病多环节的靶点进行特异性的抗体研制,包括TNF抑制剂、白介素受体抑制剂和以T、B细胞为靶点的生物抑制剂,但是这些生物制剂价格昂贵,疗效也不确切,还需要注射给药,有时有严重的感染等不良反应,因此使用较为谨慎。因此对寻找多靶点多途径的治疗RA的新药物十分必要和迫切。林兵等人前期发现从中药豆豉姜中的二氯甲烷活性部位提取出一个二苄基丁烷型木脂素类化合物LCA,经实验研究,提示有明显的抗炎活性。目的:完成从豆豉姜中提取出来的具有抗炎活性的化合物LCA的全合成,同时为了寻找活性更高的具有抗炎活性的新化合物;还合成了五个系列的新的LCA衍生物,并探讨其构效关系。方法:通过LCA结构的分析,对其进行反合成的逆向拆分,设计了两条不同的合成路线,其中一条是不对成的合成路线,经过酯化、氢化、苄基保护、水解、取代等反应成功合成了目标化合物。此外通过不对称全合成路线衍生而来的中间体W₁和对称合成路线的中间体W₂,在羧基上引入了不同的脂肪链伯仲胺、取代哌嗪、取代苄胺和取代苯胺等侧链基团,合成了A、B、C、D、E、五类LCA衍生物,并对合成的五类化合物中27个化合物进行细胞毒性实验和LPS刺激下的RAW.264.7细胞生成NO含量抑制实验。结果:通过两种不同的合成路线,完成了LCA的全合成,并进行了结构确认。并以LCA为先导化合物,重点设计合成了33个新的化合物,并对其中的27个化合物进行初步的细胞毒性试验和LPS刺激下RAW264.7细胞NO含量抑制实验。细胞毒性结果显示除了B类化合物中的w-2-17、D类化合物中的w-1-57和w-2-21、E类化合物中的w-4-2和w-4-14分别在浓度低于3μM,3μM,15μM,3μM和15μM时对细胞的增殖无显著性影响,即不影响细胞的生长,对RAW264.7细胞无毒性,其余所测化合物在所测浓度范围内均无细胞毒性。LPS刺激下RAW264.7细胞NO含量抑制实验结果显示,A类化合物w-1-34、D类化合物w-1-61在30μM浓度对细胞NO含量有抑制作用,B类化合物w-2-17、E类w-4-18在3μM浓度对细胞NO含量有抑制作用,而E类w-4-14提示在1.5μM浓度就对NO有抑制作用,且呈剂量依赖性。同时计算27个化合物的IC₅₀值。结论:综上所述,根据计算出来的化合物IC₅₀值,5类LCA衍生物中,共有5个LCA衍生物具有显著抗炎活性,结果显示,A类化合物中引入4-OCH₃苯基哌嗪的w-1-34(IC₅₀=45.49μM)抗炎活性高,B类化合物中引入4-OCH₃苄胺的w-2-17(IC₅₀=1.44μM)抗炎活性最优,D类化合物中活性最好的为引入2-OCH₃苯胺的环酰亚胺化合物w-1-61(IC₅₀=17.10μM),E类化合物中活性较高的为引入4-OCH₃苄胺的化合物w-4-14(IC₅₀=8.20μM)和引入4-CH₃苄胺的化合物w-4-18(IC₅₀=29.51μM),此次LCA衍生物抗炎活性实验为以后设计更多具有抗炎活性衍生物提供基础。更多还原

【Abstract】 Rheumatoid arthritis（RA）is a chronic systemic autoimmune disease that arises more frequently in females than males and frequently observed in the elderly.It mainly affects the patient’s joint synovium.The clinical manifestations are symmetrical joint involvement,including joint pain,swelling,inflammation,accompanied by proliferative synovium,cartilage damage,bone erosion,and systemic consequences.In severe cases,even the patient’s functional activities are restricted.Leading to disability,causing financial burden to patients themselves and society.Early detection,early diagnosis,and early treatment are currently more effective treatments.At present,the drugs for clinical treatment of RA mainly include small-molecule drugs:non-steroidal anti-inflammatory drugs（NSAIDs）,glucocorticoids and chemically modified antirheumatic drugs（DMARDS）,etc.However,these drugs have more adverse reactions.Macromolecular biologics,including TNF inhibitors,interleukin receptor inhibitors,and bioinhibitors targeting T and B cells which against the pathogenesis of RA.However,injection is the only way to use these biologicals.Therefore,there are adverse reactions such as severe infections and expensive,so they are used with caution.Therefore,it is necessary and urgent to find new drugs for multi-target and multi-pathway treatment of RA.Researches by Lin Bin and others found that LCA was extracted from the active site of dichloromethane in traditional Chinese medicine from Litsea cubeba（Lour.）Pers root.After experimental research,it has been shown that there is obvious anti-inflammatory activity from LCA.Purpose:To complete the synthesis of the compound with anti-inflammatory activity LCA extracted from Litsea cubeba（Lour.）Pers root,and to find new compounds with higher activity with anti-inflammatory activity;we also synthesized five series of new LCA derivatives,and The structure-effect relationship is discussed.Method:Through the analysis of the LCA structure,the reverse separation of the reverse synthesis was carried out,and two different synthetic routes were designed,one of which was an incompatible synthetic route,after esterification,hydrogenation,benzyl protection,hydrolysis,substitution,etc.The reaction successfully synthesized the target compound.In addition,through the intermediate W₁ of the asymmetric synthesis route and the intermediate W₂ of the symmetric synthesis route,different side chain groups such as primary and secondary aliphatic chains,substituted piperazine,substituted benzylamine and substituted aniline were introduced into the carboxyl groups to synthesize A,B,C,D,E,five types of LCA derivatives,and 27 compounds of the five types of compounds were subjected to cytotoxicity test and LPS stimulated RAW.264.7 cells to produce NO content inhibition test.Results:The cytotoxicity tests showed that in addition to the w-2-17,w-1-57 and w-2-21 in class D compounds,and w-4-2,w-4-14 in class E compounds are at concentrations below 3μM,3μM,15μM,3μM and 15μM have no cytotoxicity,there is no significant effect on cell proliferation during another compound in the tested concentration range.NO inhibition experiment tests showed that the A compound w-1-34 and D compound w-1-61 at 30μM concentration can inhibit the NO content of cells,B compound w-2-17 E compound w-4-18 inhibited the NO content of cells at a concentration of 3μM,while E compound w-4-14 suggested that it inhibited NO at a concentration of 1.5μM in a dose-dependent manner.In summary,5 compounds have anti-inflammatory activity.Simultaneously calculate the IC₅₀ values of 27 compounds.Conclusion:A total of 5 LCA derivatives described above have anti-inflammatory activity.The test show that 4-OCH₃phenylpiperazine introduced w-1-34(IC₅₀=45.49μM)has high anti-inflammatory activity in Class A compounds,and in Class B compounds The anti-inflammatory activity of w-2-17(IC₅₀=1.44μM)introduced with 4-OCH₃ benzylamine was the best.highest activity among the class D compounds is the cyclic imide compound w-1-61(IC₅₀=17.10μM)introduced with 2-OCH₃ aniline.Among the E compounds,the higher activity was the compound w-4-14(IC₅₀=8.20μM)introduced with 4-OCH₃benzylamine and the compound w-4-18(IC₅₀=29.51μM)introduced with 4-CH₃benzylamine.The LCA derivative anti-inflammatory activity experiment provides the basis for designing more derivatives with anti-inflammatory activity in the future.更多还原