【作者】 孙娟；

【导师】 朱海亮；

【作者基本信息】 南京大学 ， 生物学， 2015， 博士

【摘要】 1,4-苯并二噁烷(1,4-Benzodioxan,BZD)类衍生物具有抗菌、抗癌、抗炎等广泛的生物活性,它们在化学界、医学界及药物学界引起了广泛的关注。由于BZD基团的稳定性、低毒性及生物多样性,在有机分子中引入BZD基团往往能显著提高分子的生物活性,从而成为药物研究的一个热点。本论文以1,4-苯并二噁烷为基本骨架,基于已有的活性结构,利用计算机辅助药物设计手段,运用比较分子力场分析法结合Docking计算,建立具有良好相关性及预测能力的各类生物活性小分子。引入噁二唑、哌嗪环、噻唑烷二酮等药效团骨架,设计并合成了五个系列1,4-苯并二噁烷类新型衍生物,其中新化合物72个,所有化合物都利用元素分析、1H NMR、MS等手段进行了结构表征,测试了部分化合物的13CNMR谱,通过单晶X-射线衍射结构分析确定了 3个化合物的分子结构。进行了系统的活性筛选和构效关系研究,其中很多化合物具有良好的抗炎、抗癌和抗菌作用,简述如下。(1)设计并合成了 一系列17个含噁二唑骨架的新型1,4-苯并二噁烷类MetAP2抑制剂,并测试了它们的生物活性。部分化合物表现出高效低毒的特性,与阳性对照TNF-470(IC50=1.96μM for HUVEC)活性相当。尤其是化合物A1具有最强的抗人脐静脉内皮细胞增殖活性(IC50= 1.16μM)和MetAP2抑制活性(IC50 =2.08 μM)。此外进一步的细胞凋亡实验表明化合物A1能够抑制细胞内MetAP2活性从而诱发细胞凋亡。计算机分子模拟计算结果表明,化合物A1存在和靶蛋白MetAP2中Co2+形成络合物的配位功能基团,因此它可以深入其结合口袋发挥作用。同时,MetAP2活性中心与小分子抑制剂A1之间存在氢键及π-π相互作用力,这进一步为MetAP2抑制剂的研究提供了强有力的理论支持。(2)将双酰肼骨架引入到1,4-苯并二噁烷结构中,设计并合成了一系列19个1,4-苯并二噁烷双酰肼类PLK1抑制剂。模拟对接结果显示,双酰肼骨架的引入显著提高了化合物与PLK1活性中心氨基酸残基相互作用的能力,尤其在取代基为间甲氧基的情况下,化合物B11与PLK1的结合能为-37.7233 Kal/mol。这可能是因为吸电子基团的取代基降低了苯环的电子云密度,导致活性中心口袋变大,使苯环顺利进入PLK1的活性中心发挥作用。此外,化合物B11对宫颈癌细胞的抗肿瘤活性IC50值为0.17 μM,对PLK1的抑制活性IC50值达到0.03 μM,这个结果明显高于阳性对照Poloxin(IC50= 2.31μM)。(3)在保持1,4-苯并二噁烷噁二唑骨架不变的情况下,用活性基团哌嗪结构取代苯环,合成了一系列17个1,4-苯并二噁烷噁二唑哌嗪类FAK抑制剂,并且测试了它们对HepG2、Hela、SW1116以及BGC823四种肿瘤细胞的抗增殖生物活性。实验结果研究表明,这些化合物对FAK表现出潜在的抑制活性,其中最优化合物为C13(IC50= 0.78μM)。构效关系分析显示,苯环上的取代基为强吸电子基团的时候,能提高靶点蛋白FAK活性中心氨基酸残基的电子云密度,使其化合物难以进入活性空腔发挥作用,而邻位取代基被最优构象的化合物包裹在内部阻止了其增强电子云密度的功能,从而利于化合物进入活性空腔发挥抗癌活性。(4)运用药物拼合原理,引入具有抗炎活性的哌嗪药效团,设计出20个1,4-苯并二噁烷哌嗪类COX-2抑制剂。首先借助计算机辅助药物设计软件,根据能量打分情况,筛选并合成出活性较好的11个化合物,并培养出3个目标化合物的单晶(D1、D3和D7)。然后测试了它们的系列抗炎活性,通过角叉菜胶致大鼠足趾肿胀实验、镇痛活性实验以及胃溃疡实验,筛选出最优化合物D11,对足趾肿胀的抑制率在3 h时达到92%,且无任何胃损伤症状,是非常好的高效低毒类潜在药物。此外,体外实验进一步证明化合物D11是一类高度选择性COX-2抑制剂。(5)以FabH为药物靶点,首先通过计算机模拟药物设计软件计算模拟对接了80个不同的1,4-苯并二噁烷噻唑烷二酮哌嗪类衍生物,根据结合能的数值筛选出17个与FabH结合较稳定的化合物。然后对筛选出的17个小分子化合物进行ADMET模拟,选择并合成了具有较高药代动力学特征的6个潜在药物。随后对这6个化合物进行了抗菌活性和E.coli FabH抑制活性的评估研究。实验结果显示,这一系列化合物对革兰氏阴性菌的效果明显优于革兰氏阳性菌,IC50值普遍低于5μM,个别化合物的活性远远高于阳性对照药Penicillin(IC50 = 6.25 μM)。FabH的抑制活性实验结果与抗菌结果的关系进一步表明这个系列化合物的抗菌活性是通过抑制FabH的活性产生的。以E10(IC50 = 1.56 μM for E.coli and IC50 =0.06μM for FabH)为代表的系列化合物有望成为以FabH为靶点的新型抗菌先导药物。更多还原

【Abstract】 Compounds containing a 1,4-benzodioxan skeleton have received significant attention in chemical,medicinal and pharmaceutical research as this structural scaffold has broad biological activities,such as antibacterial,anticancer and anti-inflammatory activities.Since its stability,low toxicity,activity diversity and unexpected activities enhancement in an organic molecule,therefore,it has become a hotspot of pharmaceutical research.In this thesis,we focus on the identification and modification of all kinds of bioactive small molecules using 1,4-benzodioxan as a basic skeleton.Means of Computer Aided Drug Design was utilized,in which,combined with Docking calculation and comparative molecular field analysis.We reported in the present work the synthesis,active screening and structure-activity relationships of five series of 1,4-benzodioxan derivatives on the basis of introducing pharmacophore skeleton,such as oxadiazole,piperazine and thiazolidinediones,72 of which were firstly reported.All of the compounds were characterized by elemental analysis,1H NMR,and MS spectra,and some compounds were characterized by 13C NMR as well.Besides,3 compounds were structurally determined by single crystal X-ray structural analysis.Many of the designed compounds have good anti-inflammatory,anticancer and antibacterial activities.The results were concluded as follows:(1)A series of seventeen novel MetAP2 inhibitors containing 1,4-benzodioxan and oxadiazole skeletons has been synthesized and tested their biological activities.Some compounds exhibited excellent activities with low toxicity,comparable to the positive control TNF-470(IC50 = 1.96 μM for HUVEC).Compound A1 showed the best anti-proliferative activity(IC50＝1.16 μM)and inhibitory activity on MetAP2(IC50=2.08 μM).Further apoptosis experiment showed that compound A1 could inhibit MetAP2 activity in cells and induce apoptosis.Computer simulation results showed that compound A1 not only forming a complex ligand functional group with Co2+ of protein MetAP2,but also deeping into the binding pocket to exert its function.Meanwhile,the hydrogen bonding and π-π interactions between MetAP2 activity center and compound A1 provide a strong theoretical support for MetAP2 inhibitor studies.(2)A series of nineteen novel PLK1 inhibitors containing 1,4-benzodioxan and diacylhydrazine skeletons has been synthesized according to the introduction of diacylhydrazine skeleton.Docking simulation results exhibited that the interactions between the amino acid residues and the designed compounds were significantly improved through introduction of the diacylhydrazine backbone.Particularly,the binding energy between compound Bll(with m-methoxy substituent)and PLK1 was-37.7233 Kal/mol.This maybe because the electron-withdrawing groups can reduce the electron density of the benzene ring,making the active center larger and easier to be get in for the benzene ring.In addition,compound Bll showed the best inhibitory activities on cervical cancer cells(ICs50=0.17 μM)and PLKI(IC50= 0.03 μM),prior to the positive control Poloxin(IC50 = 2.31 μM).(3)Keeping 1,4-benzodioxan and oxadiazole skeleton unchanged,a series of seventeen novel FAK inhibitors has been synthesized with piperazine ring instead of benzene ring,and tested their antitumor activities on four cancer cell lines(HepG2,Hela,SW1116 and BGC823).The results showed that most of the compounds had potential FAK inhibitory activities,and compound C13 showed the best inhibitory activity on FAK(IC50= 0.78 μM).According to the results of active screening and structure-activity relationships,the electron-withdrawing groups can improve the electron density of FAK amino acid residues,making the compounds difficult to enter and play a role.While the ortho-substituent was wrapped by the optimal conformation compounds and the function was blocked,thereby it is beneficial for compounds to play the antitumor activities.(4)According to the combination principle,a series of twenty COX-2 inhibitors with 1,4-benzodioxan and piperazine as basic skeletons has been designed.First,using the computer-aided drug design software,eleven compounds with better activities were screened and synthesized according to the energy situation scoring.The crystal structure of compounds Dl,D3 and D7 were determined by single crystal X-ray diffraction analysis.And then tested a series of anti-inflammatory activities.According to the carrageenan-induced swelling experiments in rats foot-being,analgesic activities and ulcer experiments,compound Dll was performed the best anti-inflammatory(92%inhibition rate),and without ulcer side effect,which is a potential efficiency and low toxicity drug.In addition,the in vitro experiment showed that compound Dll was a highly selective COX-2 inhibitor.(5)A series of eighty thiazolidinedione derivatives with 1,4-benzodioxan as a basic skeleton has been designed with computer simulation technology.Seventeen compounds were chosen according to the bing energy.Then six potential drugs were selected and tested their antibacterial and FabH inhibitory activities by ADMET simulation.The experimental results showed that the activities against Gram-negative bacteria is much better than Gram-positive bacteria,and the IC50 values are generally less than 5 μM.Activities of individual compounds is much better than the positive control Penicillin(IC50 = 6.25 μM).The results of FabH inhibitory activities of the tested compounds were in agreement to the structure relationships of their antibacterial activities.This agreement suggested that antibacterial activities of the synthesized compounds may be derived from the inhibition of FabH enzymatic activities.The experiment results indicated that the compound E10(IC50 = 1.56 μM for E.coli and IC50 = 0.06 μM for FabH)was a potential FabH inhibitor.更多还原