摘要
肿瘤目前已是严重威胁人类的生命健康的常见病、多发病。全球每年新发病人数已超过1000万,而死亡人数也上升到了700万以上。而且近年来由于环境污染以及不良的生活习惯,使得肿瘤尤其是恶性肿瘤的发病率不仅逐年上升,同时还逐渐呈现年轻化的趋势。因此,对肿瘤的防治工作已经非常迫切。
抗肿瘤药物是目前恶性肿瘤的主要治疗手段。但由于目前临床应用的抗肿瘤药物存在诸多问题(水溶性差、毒副作用严重以及易产生获得性耐药性),不能满足临床治疗的需要。因此,新型的抗肿瘤药物的研究开发成为药物研究的重点领域之一,其中微管蛋白抑制剂又是目前抗肿瘤药物研究的热点方向。
微管(microtubulel是真核细胞的重要组分,也是重要的抗肿瘤药物作用靶标。现有研究表明,微管蛋白(tubulin)中存在三个主要的药物结合位点:紫杉醇结合位点(Taxol site)、长春碱结合位点(Vinblastine site)以及秋水仙碱结合位点(Colchicinesite)。但在这三个位点中,秋水仙碱位点由于自身结合腔的体积较小,有利于小分子抗肿瘤抑制剂的研究。
本课题以微管蛋白中的秋水仙碱结合位点为靶标,分析研究结合位点的结构性质,并模拟其与抑制剂的结合,确定各亚区域的性质、关键作用残基以及潜在的结合部位;从抑制剂结构中的共有基团入手,逐步扩充共有基团的性质,从而假设了抑制剂的结构模板。再从活性构象出发并结合已有的构效关系研究,提出抑制剂的三维药效团模型以及影响活性的部分结构因素。在此基础上,开展新型微管抑制剂的设计、合成、结构改造以及体外活性研究。
一.微管蛋白中秋水仙碱药物结合位点的研究
1.结合位点的性质
采用多拷贝同时搜寻(MCSS)方法,以不同性质的官能团为探针对结合位点进行分析。根据官能团的空间分布位置,将结合位点分为三个疏水口袋(Ⅰ,Ⅱ和Ⅲ)以及三个极性区域(Ⅳ,Ⅴ和Ⅵ)。疏水口袋Ⅰ和Ⅱ为主要的疏水基团分布区,但是在Ⅱ中芳香性基团的分布在能量上占优且优势构象为垂直的插入其中。Ⅲ位于结合位点的底部,受其空间所限,只能接受体积较小的脂肪族基团且能量较高。极性基团主要分布在极性区域Ⅳ和Ⅴ中,前者中氢键受体、供体基团无明显差别,但后者中氢键受体基团分布的能量与数量更有优势。Ⅵ则位于Ⅰ和Ⅲ之间,极性基团有分布但能量较高。此外,β-Cys241的-SH因为带有微弱的极性,因而附近可以分布两种不同的基团,如芳香性和氢键受体基团。属于极性区域Ⅳ中α-Ser178的骨架O、α-Thr181的骨架-NH,以及位于Ⅴ中的β-Ala250～Leu255间的骨架-NH的周围均富集了不少极性基团的分布,提示了这些残基的重要性。
对结合位点的静电势研究表明,整个结合位点主要表现为电中性,但是在Ⅰ的底部与Ⅵ具有负电特性,而在极性区域Ⅳ和Ⅴ却为正电特性。这一分析,与MCSS的研究结果相符,且也同晶体结构中Colchicine的构象较为匹配。而呈负电的和正电的区域,却没有被已有抑制剂占据,因而可以作为潜在的部位参与结合。
2.蛋白与抑制剂的结合模式研究
采用AutoDock软件,模拟了蛋白与抑制剂的结合。按照结合能量以及出现的次数,确定抑制剂的结合构象。结果表明,抑制剂主要通过与疏水口袋Ⅰ和Ⅱ间的疏水作用以及与极性区域Ⅳ间的氢键作用而与蛋白结合。抑制剂结构中体积较大且非平面的疏水部分(如三甲氧基苯环)位于疏水口袋Ⅰ中,其中的氢键受体原子(如O)或者苯环与β-Cys241的-SH存在极性作用(氢键或者阳离子-π作用)。平面的疏水部分则垂直的姿态插入Ⅱ中,其中的疏水侧链伸向Ⅱ的内部,而氢键供体原子或者受体原子,分别与α-Ser178或者α-Thr181形成氢键。此外,抑制剂中位于Ⅰ和Ⅱ二者之间的结构部分上的O原子(如=O)的空间位置比较相似,均指向loop区的β-Ala250的骨架-NH。
对AutoDock对接得到的抑制剂结合构象又进行了Affinity的柔性对接方法考察结合位点中残基的构象改变情况。结果表明:主要的结合残基β-Cys241、β-Leu248、α-Thr179、α-Val181以及β-Asn352侧链的构象改变较大,可能是因为随着周围配体中的基团位置的改变而产生相应的变化。但是结合位点中的其余残基的构象均未发生明显改变。
3.确定关键作用残基以及潜在药物结合部位
根据MCSS中官能团的分布位置与能量,提示了极性区域Ⅰ中的β-Cys241、Ⅳ中的α-Scr178和α-Thr181,以及Ⅴ中的β-Ala250～Leu255等残基可能具有一定的重要性。在抑制剂的结合构象中,β-Cys241、Ⅳ中α-Ser178和α-Thr181均以氢键的形式参与了结合。同时,参考已有的构效关系研究,失去与β-Cys241或者α-Ser178以及α-Thr181间的极性作用,对活性具有一定的负面影响,因而明确的指出它们为关键的结合残基。
由于Ⅴ中的β-Ala250～Leu255之间的骨架-NH位置集中且又有开阔的空间,而且MCSS计算中氢键受体官能团的能量较低,因而是一个比较理想的氢键供体区域。然而,对接研究表明,现有的抑制剂均未与该区域结合。此外,现有抑制剂一个共同问题是较低的脂水分配系数,因此,该区域可作为潜在的药物部位,通过引入新的极性基团改善化合物的水溶性及增加氢键作用而增强与蛋白的结合。疏水口袋Ⅲ和极性区域Ⅵ也并没有参与结合,但是因为它们直接位于重要的疏水口袋Ⅰ的下方,因而也可以作为新的结合部位为改造Ⅰ中的基团提供指导。
二.秋水仙碱位点抑制剂的结构特征研究
1.二维结构特征与结构模板
从经典的抑制剂结构中的共有基团受到启发,假设了其结构特征。随后按照性质特点,对共有基团进行扩展,进而此特征逐步扩大,即:由1～4个原子作为桥连连接起来的2个疏水环或基团(分别以A和B表示),其一与TMP类似,另一与邻甲氧基酚环或者1,2-亚甲二氧基苯环类似。最终提出了二维的结构模板,并结合已有的结构,更进一步指出了具体的结构类型。
2.抑制剂的三维药效团模型模型的构建
按照对接模拟得到的抑制剂结合构象中官能团的空间位置,并参考关键的结合残基的性质,提出了由以下六个元素组成的药效团模型,即:2个疏水中心(H1,H2)、疏水侧链H3、2个氢键受体(A1,A2)和极性原子(P)。H1和A1位于疏水口袋Ⅰ中,A1与β-Cys241存在氢键作用;H2和H3位于疏水口袋Ⅱ中;P位于极性区域Ⅳ中,与α-Ser178或α-Thr181形成氢键;A2靠近loop区的β-Ala250的骨架-NH。同时由于极性区域Ⅴ可以作为潜在的结合部位,因而在A2和loop之间,还假设存在另一个氢键受体点A3以同loop区中的骨架-NH形成氢键。其中H1和A1组成了平面P1,H2、H3以及P构成平面P2,但是平面P1-P2之间存在42～60的夹角。而结合已提出了结构特点,药效团元素中的H1和A1组成了模板的A部分位于疏水口袋Ⅰ中,H2、H3以及P构成B部分位于疏水口袋Ⅱ和极性区域Ⅳ中,A2或A3则属于桥连部分以连接A和B部分。
3.影响活性的其它结构因素
在模拟了不同结构改造的现有抑制剂与蛋白的结合时发现,改变H1部分疏水基团的体积,改变H2部分的平面性,以及改变结构模板中的AB部分位于桥连的空间位置时,对活性具有重大影响。因而参考已有的结构改造研究,指出了影响活性其它的结构因素为:除了必须的疏水性外,H1部分的体积大小、H2部分的平面性以及桥连部分是否能保证将AB位于其同侧。
三.新型小分子抑制剂的计算机辅助设计
以上述研究为基础开展先导化合物的设计工作。首先根据三维药效团模型中各个药效元素的性质与特点选择不同的官能团;随后按照结合位点各个区域的性质,采用LUDI挑选不同的连接片段以手工方法将上述官能团连接起来,得到了多种不同的先导模板。最后通过接模拟它们与蛋白的结合模式,并综合考虑其与蛋白的结合能量、分子间相互作用能、LUDI打分结果、形成的氢键个数以及酯水分配系数,最终确定了咪唑酮环为桥连的结构作为先导化合物。在设计的时候,还考虑了以下几个因素:遵循提出的药效团模型,保证结构中存在六个药效点;符合提出了结构特征与结构模板,结构中仍然存在A、B以及桥连这三个部分,且连接片段必须保证A、B部分位于桥连的同侧;匹配结合位点的性质,按照疏水口袋Ⅰ和Ⅱ,以及Ⅳ的特征选择合适的官能团;利用新发现的潜在结合区域,引入新的亲水基团;合成的难度以及结构改造的空间。
四.合成与结构修饰研究
参考相关文献,提出了一条反应条件温和,后处理简便且易于结构改造的合成路线。即:以三甲氧基苯甲酸为原料,经酰氯化后与乙酰乙酸乙酯发生酰基化反应,随后脱除乙酰基后再与NBS反应得到的溴代产物,在碱性条件下和脲直接环合得到关键中间体(Ⅵ)。此后,中间体(Ⅵ)按照桥连部分改造的需要,又分别与不同的芳胺、酚等反应;或者还原为醇或醛后,得到反酯、烯胺或者乙烯结构。
结构改造中,还对B部分进行结构修饰,考察了B部分中取代基的体积大小、供电子/吸电子、取代基的位置以及个数等因素对活性的影响。最终得到了20个化合物,经数据库检索所有结构均为首次报道。
同时对中间体咪唑酮的合成方法进行了改进,提出了先溴代再与脲环合的2步反应路线,以乙酸乙酯:石油醚(v:v=1:3)重结晶即可得到产物,总收率达到55.4%。与已有文献中的先亚硝化、经还原后再与异氰酸反应的合成方法相比,新路线的反应步骤简短、处理方便,而且提高了收率。
五.抑制剂的活性测试研究
肿瘤细胞生长抑制实验实验表明,所有化合物对六种不同的肿瘤细胞都具有一定的生长抑制作用,体现了广谱的抗肿瘤活性。其中对LOVO(人结肠癌细胞)、CEM(人白血病细胞)、MDA-MB-435(人乳腺癌细胞)抑制作用明显。化合物CON-2、CON-3、CON-8、CON-10、CON-11以及CON-13对这三个细胞的活性低于5μM,其中CON-7、CON-14最高活性分别达到了176nM和330nM。此外,CON-7、CON-14对除SKOV-3(人卵巢癌细胞)外的其它五种瘤细胞具有明显的生长抑制作用,活性范围主要集中在0.17nM～5μM之间。
抗血管实验表明,大部分的化合物都能较好的抑制人脐静脉内皮细胞(ECV-304)的生长,9个化合物的活性低于16μM。同时,此实验结果与抑瘤实验活性存在一定的相关性。
初步的构效关系分析表明:B部分的芳环中有供电子基团或者氢键供体基团的存在对活性有增强作用,而间位取代的活性又强于对位和邻位取代。
生物活性数据不仅验证了最初的设计思想,更为进一步的研究设计新结构类型的微管蛋白抑制剂提供了重要参考。
Malignant tumors represent one of the most common human diseases that seriously threaten the life of the people.Internationally,there are more than 10 million new cancer cases and more than 7 million cancer-related deaths reported each year,making it urgent to take efficacious measures to prevent,detect and treat tumor.
Antitumor agents have been one of the major methods to combat against this fatal disease,prolonging the life time and improving the life quality of the patients.But searching for the effective drugs with simple structure and proper physiochemical features is always the pursuing goal of many researchers.
As an important cellular component in all the eukaryotic cells,microtubules have many key biological functions,including intracellular transport,morphogenesis,motility and cell division.Structurally,microtubule is composed ofα-βtubulin heterodimers.Targeting at microtubules by inhibiting or inducing the assembly form its subunits(α-βtubulin) by several agents will result in the apoptosis of the cell.Tumor cells acquire unlimited replicative potential and extremely dependent upon microtubule and are vulnerable to the drugs that bind at microtubules.Additionally,proliferating endothelial cells that form neovasculature in the tumor are also sensitive to tubulin-binding agents.These findings suggest that microtubules will continue to be among the most promising cancer targets for the development of new anticancer drugs.
According to the different binding areas,the microtubule binding agents can be mainly divided into three classes:the taxol-site agents,the vinblastin-site agents and the colchicine-site agents.But in general,the molecular structures of the last class compounds are much simpler than those binding bind in the other two domains.The simplicity of these molecules offers promise for the rational design of antitubulin agents.Hitherto,many diverse colchicine-site inhibitors(CSI) have been discovered(i.e.,Indonacin,CA-4,E7010 and Curacin A),which have proven or potential utility as antitumor drugs.
Although large number of compounds has been reported,the research method on the inhibitors still focuses on the traditional protocols,e.g.the random screening or the structural modification.The research based on the structures of the protein or the inhibitors was rarely revealed.Furthermore,despite of bearing high activity,the further application of these found agents was interfered by the poor water solubility,severe site effects and acquired drug resistance.
For the above reasons,the colchicine site on the microtubule was selected as the target to find novel antitumor agents in this study.By analyzing the structure of the binding site and simulating the binding modes with its agents,the properties of the sub-regions of the site,the key binding residues and potential binding areas were determined.Derived from the active conformations and the available structural-activity relationship of the reported inhibitors,the structural model of the agents was built,and the other structural factors affecting the binding affinity were also proposed.Based on the above results,a systemic research platform from the views of the receptor and the ligand was constructed.Directed by this platform,several novel scaffolds were designed and then selected on the principle of synthesis easiness and binding energies.Next,proper synthesis routes and modification were selected.Finally,in vitro biological test was conducted on all the compounds.
Ⅰ.The research of the colchicine binding site on the microtubules
1.The properties of the subregions of the binding site
The multiple copy simultaneous search(MCSS) methodology was used to explore the binding site.The results showed that the binding site could be divided into three hydrophobic pockets(Ⅰ,ⅡandⅢ) and three polar regions(Ⅳ,ⅤandⅥ).
The pocketⅠandⅡare the main distribution areas for the hydrophobic probes,but inⅡthe aromatic groups are superior to others,and preferred the vertical conformations. PocketⅢlocated in the deep bottom of the binding site,only the groups with smaller size were accepted but with higher energy.The regionⅣandⅤwere responsible for the almost the polar groups,while the latter favorite the hydrogen bond acceptor groups. Several polar probes were found inⅥ,but with higher energies.
The electrostatic potential of the binding site was revealed by Delphi,which showed that nearly all the site was nearly neutral,while the bottom of theⅠandⅥwere exhibited as negative charge,the regionⅣandⅤwere represented as positive charge.This result was in accordance to the finding of MCSS,and to the conformation of the colchicines in the crystal structure.
2.The binding modes between the tubulin and the inhibitors
Simulated in the docking software AutoDock and Affinity,the binding modes were studied.The active conformations showed the agents mainly depended on the hydrophobic interaction with the pocketⅠandⅡ,and the hydrogen bond with the regionⅣin binding with tubulin.The larger hydrophobic part of the agent located in the pocketⅠ,with the hydrogen bond acceptor(O) or the aromatic ring polar bonding;with the -SH of theβ-Cys241.The planar hydrophobic part inserted in the pocketⅡ,hydrogen bonding with theα-Ser178 andα-Thr181.
The soft docking study was further applied in the Affinity moduler on the conformations obtained from AutoDock,to investigate the movement of the residues lineing the site.Only the conformations of the side chains of the major binding residuesβ-Cys241,β-Leu248,α-Thr179,α-Val181andβ-Asn352 had changed much,due to the motion of neighbouring groups of the ligands.
3.The key binding residues in the binding site and the potential binding area
According to binding energy and the number of copies of the distributed groups in the MCSS calculation,theβ-Cys241 in the pocketⅠand theα-Ser178 andα-Thr181 in the regionⅣmight be necessary for binding.By docking simulation,tire above three residues were almost involved in the binding with the agents.So they were identified as the key residues.
Though not involved in binding,the polar regionⅤwas suggested as a potential binding area by the distribution of hydrogen bond acceptor groups in the MCSS.While the cluster of the backbone-NH in V and the enclosed broad space also present its influential role in binding.
The other two unoccupied pocketsⅢandⅥwere just under the important hydrophobic pocketⅠ.They might provide some new strategy for modification on the groups locating in the pocketⅠ.
Ⅱ.The structural features of the colchicine site inhibitors
1.Proposition of the two dimensional structural features and the structural scaffold
Hinted by the common groups in some of the inhibitors,a coarse structural feature was proposed.According to the properties,the common groups were expanded to the other groups.Then the structural feature was modified,which is:two hydrophobic groups (presented as part A and B) were connected by a bridge part that is composed of 1-4 atoms.One group may be similar to TMP,the other should similar to 2-methoxyphenol. The structural scaffold was also supposed,furthermore three detained scaffolds were provided.
2.Construction of the three dimensional pharmacophore
According to their binding conformations,the structures of the inhibitors were divided into three parts,namely A,B and the bridge between them.The pharmacophore of the inhibitor(the hydrophobic centers H1 and H2,the hydrophobic group H3,the hydrogen-bond acceptors A1 and A2 and the polar atom P) was built.In the pharmacophore,H1 and A1 located in the hydrophobic pocketⅠ,polar bond could be found between A1 andβ-Cys241;H2 and H3 was buried in pocketⅡ;P was in polar regionⅣby hydrogen bonding withα-Ser178 orα-Thr181;A2 was in the vicinity ofβ-Ala250 in the regionⅤ,but no obvious interaction was identified between them.Another hydrogen bond acceptor A3 was proposed between A2 and regionⅤ.
3.The other structural factors affected the binding affinity of the agents
By combining the reported modification results and computer simulation,the structural factors affected the activity were enclosed.The factors included the volume size of H1,and the planarity of H2,while the bridge part should be in rigid form to maintain parts A and B to be in the same side of the bridge(in cis-conformation).A potential hydrogen-bond acceptor A3 was proposed between A2 and the loop area forming the regionⅤ.
Ⅲ.Design of lead compounds
Based on the above results,the lead compounds were designed.Firstly,according to the properties of the structural elements of the pharmacophore,the corresponding proper groups were selected.But their positions in the site were adjusted in MCSS.Then diverse linkage parts were designed to link the elements by LUDI screening.By optimization and docking,the binding conformations of diverse lead scaffolds were simulated.At last,the final structure was determined by the binding conformation,the binding energy,the LUDI score points and the numbers of hydrogen bonds.During design,the following factors were taken in account:complied with the pharmacophore that composed of six elements; complied with the structural model that the scaffold should contain the A,B and the bridge part;the linkage part should keep the groups in theⅠandⅡin the cis-conformation;the hydrophilic groups was introduced to interacted with the key residues,especial the residues in potential binding areaⅤ;the synthetic accessibility and the modification margin.Finally, the imidazolones were selected as the lead compound.
Ⅳ.Synthesis and structural modification of the lead compound
Based on the idea that the structural model was composed of three parts(A,B and the bridge),the lead compound could be synthesized by these three parts sequentially.That is, the bridge part was firstly added to the part A,and then the part B was added.In planning the synthetic route,the following factors were considered:the reaction condition should be as mild as possible;the reagent should be available;the structural modification should be easily managed.
Modification was conducted on part B.The position,the number,the volume size and the electronic acceptor/donor of the substituents on the aromatic were all studied.
Finally,more than 20 compounds were synthesized,which were also revealed to be firstly reported by searching the database.Furthermore,the synthesized mthods of the key intermediate was modified.
Ⅴ.Biological assay
Cytotoxicity activity,antivascular activity and Tubulin polymerization assays were conducted to explore the effect of diverse substituents on activity with MTT method.
The results showed that nearly all the compounds could inhibit the growth the six tumor cell types effectively.The electron donor groups and the hydrogen bond donor groups could enhance the activity.Also the substituents in the m-position were advantageous than the p-position.
引文
[1]Nogales E.Structural insights into microtubule function.Annu.Rev.Biophys.Biomol.Struct.2001,30:397-420.
[2]Downing K.Nogales E.Tubulin and microtubule structure.Curr.Opin.Cell Biol.1998,10(1):16-22.
[3]Burns R.G.Alpha- beta- and gamma-tubulins:sequence comparisons and structural constraints.Cell Motil.Cytoskeleton 1991,20(3):181-189.
[4]Jordan A.,Hadfield J.A.,Lawrence N.J.et al.Tubulin as a target for anticancer drugs:agents which interact with the mitotic spindle.Med.Res.Rev.1998,18(4):259-296.
[5]Rodionov V.I.,Borisy G.G.Microtubule treadmilling in vivo.Science 1997,275(5297):215-218.
[6]罗深秋.医用细胞生物学.上海:第二军医大学出版社,2004,140-143.
[7]Philip E.T.Vascular Targeting Agents as Cancer Therapeutics.Clinical Cancer Research 2004,10(2):415-427.
[8]凌诒萍.细胞生物学.北京:人民卫生出版社,2005,119-121.
[9]雷远成.三种甘蓝型油菜小孢子发生过程中微管骨架的变化研究.成都:四川大学,2003.
[10]陈诗书.汤雪明.医学细胞与分子生物学.上海:上海医科大学出版社,2000,289
[11]Wittmann T.,Hyman A.,Desai A.The spindle:a dynamic assembly of microtubules and motors.Nat.Cell Biol.2001,3(1):28-34.
[12]Gundersen G.G.,Cook T.Microtubules and signal transduction.Curr.Opin.Cell Biol.1999,11(1):81-94.
[13]Jordan M.A.,Wilson L.Microtubules and actin filaments:dynamic targets for cancer chemotherapy.Curr.Opin.Cell Biol.1998,10(1):123-130.
[14]Wilson L.,Panda D.,Jordan M.A.Modulation of mierotubule dynamics by drugs:a paradigm for the actions of cellular regulators.Cell Struct.Funct.1999,24(5):329-335.
[15]Michael M.,Iwao O.Chemistry and Chemical Biology of Taxane Anticancer Agents.The Chemical Record 2001,1(3):195-211.
[16]Nicolaou K.C.,Roschangar F.,Vourloumis D.Chemical Biology of Epothilones.Angew.Chem.Int.Ed.1998,37(15):2014-2045.
[17]He L.,Orr G.A.,Horwitz S.B.Novel molecules that interact with microtubules and have functional activity similar to Taxol.Drug Discovery Today 2001,6(22):1153-1164.
[18]Richard J.K.,Paraskevi G.,Sarath P.G.et al.The Microtubule-Stabilizing Agent Discodermolide Competitively Inhibits the Binding of Paclitaxel(Taxol) to Tubulin Polymers Enhances Tubulin Nucleation Reactions More Potently than Paclitaxel and Inhibits the Growth of Paclitaxel-Resistant Cells.Molecular Pharmacology 1997,52:613-622.
[19]Nettles J.H.,Li H.,Cornett B.et al.The binding mode of epothilone A on alpha,beta-tubulin by electron crystallography.Science 2004,305(5685):866-869.
[20]Lowe J.,Li H.,Downing,K.H.et al.Refined structure of alpha beta-tubulin at 3.5 A resolution.J.Mol.Biol.2003,313(5):1045-1057.
[21]Nogales E.,Wolf S.G,Downing K.H.Structure of the alpha beta tubulin dimer by electron crystallography.Nature 1998,391(6681):199-203.
[22]Alain D.,Anna K.,Barret J.M.Novel Aspects of natural and modified vinea alkaloids.Curr.Med.Chem.Anti-Cancer Agents 2002,2(1):55-70.
[23]Bai R.,Durso N.A.,Sackett D.L.,Hamel E.Interactions of the sponge-derived antimitotic tripeptide hemiasterlin with tubulin:comparison with dolastatin 10 and cryptophycin 1.Biochemistry 1999,38(43):14302-14310.
[24]Poncet J.The dolastatins.Curr.Pharm.Des.1999,5(3):139-62..
[25]Brossi A.Bioactive alkaloids.4.Results of recent investigations with colchicine and physostigmine.J.Med.Chem.1990,33(9):2311-2319.
[26]Stephanie D.,Sylviane G.R.Drugs that inhibit tubulin polymerization:the particular case of podophyllotoxin and analogues.Curr.Med.Chem.Anti-Cancer Agents 2002,2(1):71-90.
[27]Gian C.T.,Tracey P.,Giovanni S.et.al.Medicinal Chemistry of combretastatin a4:present and future directions.J.Med.Chem.2006,49(11):3033-3044.
[28]Owa T.,Yoshino H.,Okauchi T.et.al.Discovery of novel sulfonamides targeting G1 phase of the cell cycle.J.Med.Chem.1999,42:3789.
[29]Ravelli R.B.,Gigant B.,Curmi P.A.,Jourdain I.,Lachkar S.,Sobel,A.,Knossow M.Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain.Nature 2004,428(6979):198-202.
[30]Miranker A.,Karplus M.Functionality maps of binding sites:a multiple copy simultaneous search method.Proteins 1991,11(1):29-34.
[31]Ravelli R.B.,Gigant B.,Curmi P.A.,Jourdain I.,Lachkar S.,Sobel,A.,Knossow M.Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain.Nature 2004,428(6979):198-202.
[32]InsightII Computer Program release version 2000,Accelrys Inc.San Diego California.
[33]SYBYL Computer Frogram release version 6.9,Tripos Associates Inc.St.Louis Missouri.
[34]Morris G.M.,Goodsell D.S.,Halliday R.S.Automated docking using a lamarckian genetic algorithm and an empirical binding free energy function.J.Comput.Chem.1998,19(14):1639-1662.
[35]Luty Z.R.,Wasserman P.F.W.,Stouten C.N.et.al.A molecular mechanics/grid method for the Evaluation of Ligand-Receptor Interactions.J.Comp.Chem.1995,16(4):454-464.
[36]Joseph-McCarthy D.,Hogle J.M.,Karplus M.Use of the multiple copy simultaneous search(mcss)method to design a new class of picornavirus capsid binding drugs.FROTEINS:Structure Function and Genetics 1997,29(1):32-58.
[37]Caflisch A.,Miranker A.,Karplus M..Multiple copy simultaneous search and construction of ligands in binding sites:application to inhibitors of hiv-1 aspartic proteinase.J.Med.Chem.1993,36(15):2142-2167.
[38]Cushman M.,Nagaratlmam D.,Gopal D.et al.synthesis and evaluation of stilbene and dihydrostilbene derivateives as potential anticancer agents that inhibit tubulin polymerization J.Med.Chem.1991 34 2579.
[39]Kong Y.L.,Grembecka J.,Edler M.C.et al.Structure based discovery of boronic acid bioisotere of combretastatin A-4.Chem.Biol.2005,12(9):1007-1014.
[40]Bei S.B.,Medina J.C.,Santha D.et.al.Selective covalent modification of b-tubulin residue Cys-239 by T138067 an antitumor agent with in vivo efficacy against multidrug-resistant tumors.Proc.Natl.Acad.Sci.USA 1999,96(10):5686-5691.
[41]Petitclerc E.,Deschesnes R.G.,Cote M.F.Antiangiogenic and antitumoral activity of phenyl-3-(2-chloroethyl)ureas:a class of soft alkylating agents disrupting microtubules that are unaffected by cell adhesion-mediated drug resistance.Cancer research 2004,64(14):4654-4563.
[42]Hna X.H.,Genini D.,Gussio R.et al.Biochemical genetic analysis of indanocine resistance in human leukemia.Cancer Res.2001,61(19):7248-7254.
[43]Kim D.Y.,Kim K.H.,Kim N.D.et.al.Design and biological evaluation of novel tubulin inhibitors as antimitotic agents using a pharmacophore binding model with tubulin.J.Med.Chem.2006,49(19):5664-5670.
[44]Li Y.W.,Zhou Y.J.,Zhu J.et al.Study on the binding modes of the colchicine-site Inhibitors and construction of their structural model.Acta chim.Sinica.2008,in press.
[45]Li Y.W.,Zhou Y.J.,Zhu J.et al.Study on the structural features of colchicine-site Inhibitors.Journal of pharmaceutical practice 2007,25:372.
[46]Gaukroger K.,Hadfield J.A.,Lawrence N.J.et al.Structural requirements for the interaction of combretastatins with tubulin:how important is the trimethoxy unit.Org.Biomol.Chem.2003,1(6):3033-3037.
[47]Ducki S.,Mackenzie G.,Lawrence N.J.et al.Quantitative structure-activity relationship (5D-QSAR) study of combretastatin-like analogues as inhibitors of tubulin assembly.J.Med.Chem.2005,48(2):457-465.
[48]Pettit G.R.,Rhodes M.R.,Herald D.L.et al.Synthesis and evaluation of structural modifications of(Z)- and(E)-Combretastatin A-4.J.Med.Chem.2005,48(12):4087-4099.
[49]Cushman M.,Nagarathnam D.,Gopal D.,Chakraborti A.K.,Lin C.M.,Hamel E.Synthesis and evaluation of stilbene and dihydrostilbene derivatives as potential anticancer agents that inhibit tubulin polymerization.J.Med.Chem.1991,34(8):2579-2588.
[50]Zelleka G.,Leonard J.,Ping S.C.,et al.Synthesis of alkoxy-substituted diaryl compounds and correlation offing separation with inhibition of tubulin polymerization:differential enhancement of inhibitory effects under suboptimal polymerization reaction conditions.J.Med.Chem.1992,35(6):1058-1067.
[51]LeBlanc R.,Dickson J.,Brown T.Stewart M.et al.Synthesis and cytotoxicity of epoxide and pyrazole analogs of the combrestatins.Bioorg.Med.Chem.2005,13(21):6025-6034.
[52]Banwell M.G.,Hamel E.,Hocldess et al.4,5-Diaryl-1H-pyrrole-2-carboxylates as combretastatin A-4/lamellarin T hybrids:Synthesis and evaluation as anti-mitotic and cytotoxic agents.Bioorg.Med.Chem.2006,14(13):4208-4638.
[53]Bellina,F.,Cauteruccio S.,Monti S.,Rossi R.Novel imidazole-based combretastatin A-4 analogues:Evaluation of their in vitro antitumor activity and molecular modeling study of their binding to the colchicine site of tubulin.Bioorg.Med.Chem.Lett.2006,16(22):5757-5762.
[54]Li L.,Wang H.K.,Kuo S.C.et al.synthesis and biological evaluation of 3',6,7-substituted 2-phenyl-4-quinolones as antimicrotubule agents.J.Med.Chem.1994,37(20):3400-3407.
[55]Liou J.P.,Chang Y.L.,Kuo F.M.et al.Concise synthesis and structure-activity relationships of combretastatin A-4 analogues 1-aroylindoles and 3-aroylindoles as novel classes of potent antitubulin agents.J.Med.Chem.2004,47(17):4247-4257.
[56]Luty B.A.,Wasserman Z.R.,Stouten P.F.W.et al.A molecular mechanics/grid method for evaluation of ligand-receptor interactions.J.Comp.Chem.1995,16(4):454-464.
[57]Bohm H.J.The computer program LUDI:a new method for the de novo design of enzyme inhibitors.J.Comput.-Aided Mol.Des.1992,6(1):61-78.
[58]LeBlanc R.,Dickson J.,Brown T.Stewart M.et al.Synthesis and cytotoxicity of epoxide and pyrazole analogs of the combrestatins.Bioorg.Med.Chem.2005,13(21):6025-6034.
[59]Banwell M.G.,Hamel E.,Hockless et al.4,5-Diaryl-1H-pyrrole-2-carboxylates as combretastatin A-4/lamellarin T hybrids:Synthesis and evaluation as anti-mitotic and cytotoxic agents.Bioorg.Med.Chem.2006,14(13):4028-4038.
[60]Bellina,F.,Cauteruccio S.,Monti S.,Rossi R.Novel imidazole-based combretastatin A-4 analogues:Evaluation of their in vitro antitumor activity and molecular modeling study of their binding to the colchicine site of tubulin.Bioorg.Med.Chem.Lett.2006,16(22):5757-5762.
[61]Li L.,Wang H.K.,Kuo S.C.et al.synthesis and biological evaluation of 3',6,7-substituted 2-phenyl-4-quinolones as antimicrotubule agents.J.Med.Chem.1994,37(20):3400-3407.
[62]Beckers T.,Reissmann T.,Schmidt M.et al.2-Aroylindoles a novel class of potent orally active small molecule tubulin inhibitors.Cancer Res.2002,62(11):3113-3119.
[63]Duan J.X.,Cai X.H.,Meng F.Y.et al.Potent antitubulin tumor cell cytotoxins based on 3-aroyl indazoles.J.Me& Chem.2007,50(5):1001-1006.
[64]Kuo C.C.,Hsieh S.P.,Pan W.Y.et al.BPROL075 a novel synthetic indole compound with antimitotic activity in human cancer ceils exerts effective antitumoral activity in vivo.Cancer Res.2004,64(13):4621-4628.
[65]Martino G.D.,Regina G.L.,Coluecia A.et al.Arylthioindoles potent inhibitors of tubulin polymerization.J.Med.Chem.2004,47(25):6120-6123.
[66]Martino G.D.,Edler M.C.,Regina G.L.et al.New arylthioindoles:potent inhibitors of tubulin polymerization.2.structure-activity relationships and molecular modeling studies.J.Med.Chem.2006,49(3):947-954.
[67]Kiyoshi Tanemura;Tsuneo Suzuki;Yoko Nishida,et.al.A mild and efficient procedure for a-bromination of ketones using N-bromosuccinimide catalysed by ammonium acetate.Chem.Commun.2004,4:470-471.
[68]Robert Dnschinsky;L.Allend Olan.A Simple synthesis ofdl-desthiobiotin and related substances.J.Ame.Chem.Soc.1945,67(12):2079-2084.
[69]Robert Dnschinsky;L.Allend Olan.Studies in the Imidazolone Series.The synthesis of a lower and a higher homolog of desthiobiotin and of related substances.J.Ame.Chem.Soc.1946,68(11):2350-2355.
[70]Mosmann T..Rapid colorimetric assay for cell growth and survival:application to proliferation and cytotoxicity assays.J.Immunol.Methods 1983,65(I-2):55-63.
[71]Charmantray F.,Demeunynck M.,Carrez D.et al.4-hydroxymethyl-3-aminoacridine derivatives as a new family of anticancer agents.J.Med.Chem.2003,46(6):967-977.
[72]Robley C.,Willamc J.R.,Jammes C.et al.Preparation of tubulin from brain.Methods in enzymology 1982,85(B):376-385.