【作者】 周宏钰；

【导师】 闫兵；

【作者基本信息】 山东大学 ， 药物化学， 2009， 博士

【摘要】 组合化学发展至今,其平行合成、分析及纯化技术均得到了较大的提高,随着高通量筛选技术的不断发展,组合化学在先导化合物的开发和优化过程中得到了广泛的应用。本课题致力于开发新的抗肿瘤药物,这种药物能同时杀死对药物敏感的肺癌细胞H460及多药耐药性的肺癌细胞H460/TaxR,而且对人正常成纤维细胞（NHFB）不表现毒性。在已知噻唑烷酮类先导化合物的前提下,我们用液相组合化学及氟化学方法合成了多个噻唑烷酮类化合物库来优化其抗肿瘤活性及选择性。在得到初步构效关系基础上我们设计了优化化合物库来进一步优化其抗肿瘤活性并利用P糖蛋白（P-gp）模型来深入研究其构效关系,我们还设计了活性化合物结构模型来对所得结果进行解释。活性小分子要成为药物需要具备很多条件,很多在细胞实验中有很好的活性的小分子因为较低的水溶性和渗透性,或者较高的体内毒性而被淘汰。小分子的物理化学性质可以通过结构修饰或是聚合物包被来改善,但总是受操作性或颗粒大小的限制,把小分子修饰于纳米材料表面是改善其性质的一种新的方法。随着纳米材料在生命体系中的广泛应用,纳米材料的生物相容性和毒性问题越来越受到大家的关注,解决纳米材料生物相容性的方法是表面化学修饰。本课题将组合化学技术运用于纳米材料的表面修饰,对多壁碳纳米管（MWNTs）表面进行共价组合化学修饰,通过多重生物实验来检测其生物相容性,在得到了生物相容性好的多壁碳纳米管的同时得到了初步的纳米材料表面化学修饰与生物相容性的构效关系。制约纳米材料表面修饰化学的一个主要因素为纳米材料的表面化学分析,在多壁碳纳米管化合物的合成过程中,我们在定性、定量碳纳米管表面官能团方面积累了一些经验,发展了诸如傅立叶变换红外光谱（FTIR）和魔角核磁技术（MAS NMR）在碳纳米管表面化学分析中的应用。为了验证并拓展MAS NMR在纳米表面化学分析中的应用,我们设计并合成了另外一种被广泛研究的纳米材料-金纳米颗粒（GNPs）,通过NMR条件的优化,得到了较高质量的GNPs的¹H及2D核磁谱图。在MAS ¹H NMR中,氢的信号强度与其距离金纳米表面的远近有显著的关系,距离越近,信号越弱;同时苯环及其附近的氢在魔角核磁谱图中总以宽峰形式出现。最后我们在这些经验基础上设计合成了系列双官能团修饰的金纳米颗粒化合物库,为其生物学实验提供了材料。本论文共分为两个部分,第一部分为噻唑烷酮类化合物库的合成、抗癌活性及构效关系研究,第二部分为生物相容性纳米材料的设计、合成及表征。第一部分噻唑烷酮类化合物库的合成、抗癌活性及构效关系研究肺癌是世界上死亡率最高的癌症之一,其中超过80%的肺癌为非小细胞肺癌（NSCLC）。但是癌症治疗的瓶颈并非人们没有发现能杀死癌细胞的药物,而是很少发现在能杀死癌细胞的同时对正常细胞无毒性作用的药物。另一方面,癌症治疗失败的原因往往因为高剂量给药而产生的多药耐药性（MDR）。因此剂量引发的毒性和耐药性的产生是癌症治疗失败的两个主要原因。MDR可由多种机制产生,其一为与耐药相关的一些药物转运蛋白的高表达。在MDR中起主要作用而且研究最为深入的是MDR/P-gp高表达。因此我们急需研发新一类非P-gp底物的抗癌药物,这类药物在能同时杀死药物敏感和多药耐药性癌细胞的同时对正常细胞不表现毒性。前期的细胞实验筛选得到两个噻唑烷酮类化合物,这两个化合物能杀死对紫杉醇敏感和已产生耐药性的NSCLC细胞H460和H460/TaxR,同时对作为对照的NHFB无明显毒性。基于此,我们设计了一系列化合物库来改善噻唑烷酮类化合物对NSCLC细胞的活性和选择性,同时研究此类化合物抗癌活性-化合物结构关系。在第一轮活性实验中,我们用液相组合化学方法合成了185个2-芳基亚胺基取代-4-噻唑烷酮类化合物和17个2-芳基亚胺基-3-取代-4-噻唑烷酮类化合物,用氟化学手段合成了45个2-芳基-4-噻唑烷酮类化合物。所有化合物在10μM浓度下对H460、H460/TaxR和NHFB进行活性筛选,能杀死两个NSCLC细胞系但对正常细胞无毒性的化合物被筛选出来。初期实验表明3-位胺基位置取代使噻唑烷酮类化合物失去了抗癌活性和选择性,同时2-位芳基亚氨基为保持其抗癌选择性必需基团。重复实验筛选得到11个最有活性的化合物。为了进一步改善其抗癌选择性并研究其构效关系,我们对活性化合物结构进行分析并在此基础上设计并合成了包含170个化合物的优化化合物库,化合物的平均纯度达到95%。经过10μM浓度下初步筛选得到40个活性化合物,用5μM浓度筛选得到11个较高活性化合物。对11个化合物的剂量依赖实验得到此类化合物对H460和H460/TaxR的最佳半数抑制浓度为300 nM,同时即使在最高作用浓度下（100μM）,绝大多数活性化合物对NHFB仍没有达到半数抑制,其抗癌选择性得到显著提高。选取13个能杀死H460但不能杀死耐药性H460/TaxR和11个能同时杀死H460和H460/TaxR的噻唑烷酮化合物进行P-gp底物模拟实验。第一组中有9个化合物符合P-gp底物药物模型（70%）,但第二组中仅有4个化合物符合（36%）。药物模型实验表明活性化合物极有可能不是P-gp底物。构效关系研究表明活性化合物在噻唑烷酮的2-和4-位取代芳基位置多为供电基团,2-位芳胺基中的取代基团可以在苯环的2-和4-位,但是在4-位芳亚甲基中的取代基团只能发生在4-位。噻唑烷酮中的胺基不能被取代。通过对11个活性化合物进行结构模型分析表明活性化合物共同特征为两个氢键受体和3个疏水性部位,结构模型的建立可以指导将来的此类化合物的设计。第二部分生物相容性纳米材料的设计、合成及表征每年有成千上万的活性小分子被筛选出来,而真正能成为药物的小分子少之又少,这是因为从小分子到药物需要具备很多条件,因此很多在细胞实验中活性很好的化合物因为低的水溶性或渗透性而被淘汰。改善小分子物理化学性质的两个常用方法为化学修饰和用高聚物进行包被,然而两种方法或者太费时费力,或者因为粒子半径太大而受到限制。与此同时,随着纳米技术的发展,将小分子连接于纳米材料表面来改善小分子在生命体系中的性质越来越受到大家的关注。MWNTs是被广泛应用于材料、电子及生命体系的纳米材料之一,但是我们有必要来寻找较高生物相容性的MWNTs来将其更好地用于生命体系,改善其生物相容性的方法之一即为纳米材料的表面修饰。我们设计了一系列实验来定性研究纳米材料的生物相容性,如蛋白结合、细胞毒性和免疫反应实验等。组合化学法修饰纳米材料来筛选生物相容性纳米材料的优势在于其不仅能通过筛选实验得到理想的纳米材料,还可以通过生物相容性与结构关系研究来指导纳米材料性质的优化。在没有前期经验的前提下,我们计划通过最大的结构多样性来修饰碳纳米管。化学修饰通过羧基化MWNTs与酪氨酸的反应来进行。酪氨酸选来作为连接体是因为它有三个活性基团,当一个反应基团用来连接MWNTs后,剩余基团仍可以仍可在两个反应位点进行多样性修饰,同时因为酪氨酸作为氨基酸具有生物相容性。8种有机胺和9种酰氯被选择来组建一个包含80种f-MWNTs的化合物库（其中8种为反应中间体）。LC/MS被用来监测洗涤过程的效率及最终纳米材料的纯度。FTIR用来表征功能化MWNTs（f-MWNTs）表面特征官能团,例如当原始多壁碳纳米管被氧化为羧基碳纳米管时,在1713cm^-1左右出现的IR吸收峰即为羧基中羰基的特征吸收。我们首次将装备nano probe的MAS NMR技术应用于f-MWNTs的表面化学分析中,以f-MWNTs 26、50、28和76为例,它们含有相似的苯磺酰基,所不同的是与f-MWNTs 26和50相比,f-MWNTs 28和76在苯环3-位有一硝基,硝基的出现降低了苯环上的电子云密度,所以比较而言f-MWNTs 28和76的苯环的MASNMR信号（7.64-8.71ppm）比f-MWNTs 26和50（7.29-7.94ppm）向低场移动。f-MWNTs表面官能团的含量由元素分析中氮和氢的含量来计算,对其中的部分中间体,表面官能团的含量由UV-Vis测定其裂解Fmoc含量的方法进行确证。为了筛选出合适的f-MWNTs来进行优化,我们对其进行了蛋白结合实验、细胞毒性实验和免疫反应实验研究,所有f-MWNTs按蛋白结合能力、细胞毒性和免疫反应由低到高进行排序,然后对每一个f-MWNTs的各项排序指标进行加和,所以较低的指标加和说明一个f-MWNTs具有较好的生物相容性。生物相容性与结构进行构效关系分析显示,前5名生物相容性好的f-MWNTs有3个包含AC005结构单元,同时所有包含AC005结构单元的f-MWNTs都位于前25名生物相容性好的f-MWNTs中。同样AC008、AC006、AC004、AM003和AM004为有益于增强生物相容性的结构单元。通过这种利用最大多样性设计f-MWNTs纳米材料化合物库来筛选生物相容性f-MWNTs的实验,我们证明了组合化学方法在纳米材料的表面修饰中的应用价值。受MAS NMR在f-MWNTs纳米材料化合物库的分析过程中应用的启发,我们设计并合成了4种化学修饰的GNPs,并成功地将MAS NMR技术应用于GNPs表面化学分析。与f-MWNTs相比,GNPs具有较好的生物相容性,简单的合成方法及统一的粒子半径等优点。在利用TEM和FTIR对纳米材料进行初步表征基础上,从溶剂、温度及转速等多个方面对MAS NMR进行优化。在优化条件下,得到了GNPs的较高分辨率的MAS ¹H和2D NMR谱图,包括COSY、TOCSY和HSQC。结果显示在MAS ¹H NMR谱图中,氢的信号强度与其所在位置距离GNPs表面的远近有显著关系,距离越小,信号强度越小。同时,GNPs表面的苯环官能团及基附近氢总以宽峰形式出现,推测是因为不同苯环的π-π共扼作用所致。通过对2D谱图信号的归属,我们可以确定GNPs表面的复杂结构。纳米材料的另一个优点是其多官能团修饰,即可以将具有不同功能的官能团同时修饰于一种纳米材料表面。在化学表征及生物筛选方法建立的基础上,我们设计并合成了一系列双官能团修饰的GNPs。FTIR结果及对其裂解产物进行LC/MS表征确定了两种官能团在纳米材料表面同时存在并且其相对含量的多少,同时纳米材料表面小分子的含量与反应过程中小分子的用量有对应关系,其绝对含量可由LC/CLND来计算（实验未完成）。当用荧光材料FITC和叶酸（FolicAcid,FA）来修饰GNPs时,荧光分析确定了GNPs表面FITC的存在。用FA受体高表达的KB细胞系和不表达FA受体的A594细胞进行细胞摄取实验,结果证实了GNPs表面FA的存在。应用上述实验建立的优化条件及分析方法,我们设计并合成了一个包含42种纳米材料的GNPs化合物库,关于这一个化合物库的生物筛选实验正在进行中。更多还原

【Abstract】 The developments of combinatorial chemistry,parallel synthesis,analysis, automatic purification,and high throughput screening techniques have been greatly improved in the past decades.The application of combinatorial chemistry has been widely applied in the discovery and optimization of lead compounds.This study is committed to the development of new anti-cancer drugs,which can kill both the drug-sensitive lung cancer cells H460 and paclitaxel-resistant lung cancer cells H460/TaxR,but do not show toxicity towards normal human fibroblast（NHFB）. Using thizaolidinones as lead compounds,solution-phase combinatorial chemistry and fluorous chemistry were adapted to synthesize a series of 4-thiazolidinone libraries.Based on the preliminary structure-activity relationship（SAR） exploration, a lead optimization library was designed and synthesized to further optimize the anti-tumor activity and cytoselective toxicity.A series of potent compounds with less toxicity and a much wider expected therapeutic window for their cytoselective toxicity for both multidrug-sensitive and -resistant cancer cells were identified.A separate pharmacophore for the most active compounds showed a common arrangement of two hydrogen bond acceptors and three hydrophobic regions.These most active compounds were less likely to be P-glycoprotein（P-gp） substrates.A number of conditions are required for an active small molecule to be a drug. Many active compounds in cell screening are abandoned due to low aqueous solubility,low permeability or high toxicity in vivo.The physicochemical properties of small molecules can be changed by chemical modification or encapsulation of small molecules with polymeric micelles as well as dendrimers.However,they are always subjected to operational restrictions,or particle sizes.An alternative new approach to improve the nature of small molecules is small-molecule surface modification on nanoparticles.With the application of nanoparticles in biological systems,the concern about the biocompatibility and toxicity is increasing.Surface chemical modification is one of the methods to address such problems.In this study, combinatorial chemistry technology was firstly applied to the surface modification of multi-walled carbon nanotubes（MWNTs）.Through multiple biological screening, such as protein binding assay,cyototoxicity and immune response,the MWNTs with better biocompatibility were identified as well as the structure-biocompatibility relationship.The surface characterization restricts the development of chemical modification of nanoparticles.In the synthesis of MWNT library,we developed several methods for qualitatively and quantitatively determination of surface functional groups on MWNTs,such as FTIR and magic angle spinning NMR（MAS NMR）.In order to verify and expand the usage of MAS NMR in the nano-surface chemical analysis,we designed and synthesized another widely studied nanoparticles,gold nanoparticles （GNPs）.Through the optimization of NMR conditions,the high quality of ¹H and 2D NMR spectra were obtained.In the ¹H MAS NMR spectrum,the signal density of hydrogen is highly dependent on the distance between the surface of GNPs and protons in the ligand molecule.The closer the distance,the weaker the signal density is.Furthermore,NMR spectra of aromatic protons seem to always have a broad base compared with aliphatic ligands,indicating some degree ofπ-πstacking effects.The final parts of this study focus on the design and synthesis of a series of bifunctional GNPs for biological experiments.PartⅠ:Design,Synthesis,Cytoselective Toxicity,and Structure Activity Relationships（SAR） of Thiazolidinone DerivativesLung cancer is the number one cause of cancer-related deaths worldwide.More than 80%of bronchogenic malignancy is from non-small cell lung cancer（NSCLC）. However the bottleneck to the development of effective anticancer drugs does not lie on an inability to identify chemicals that will kill cancer cells.Instead,the bottleneck lies on our inability to identify chemicals that will kill cancer cells at concentrations that do not harm patients.On the other hand,currently available therapies have been only temporarily successful for most cancer patients because they frequently lead to resistance.Thus,resistance to treatment and dose-limiting toxicity are two primary reasons for the failure of anticancer therapies.Resistance to treatment may arise through multidrug resistance（MDR）,which is a phenotype of cross-resistance to multiple drugs.One mechanism underpinning MDR is the overexpression of the MDR-1 gene that encodes the transmembrane,ATP-dependent,drug efflux transporter P-gp in response to chemotherapy.Therefore,it’s an important step for the success of cancer therapy to discover new compounds that are not substrates of P-gp and are affective against both drug-sensitive and drug-resistant cancer cells but spare normal human cells.Two thiazolidinone derivatives have been identified to inhibit the growth of paclitaxel-sensitive and -resistant NSCLC cell lines H460 and H460/TaxR.Both of them showed relatively low toxicity toward NHFB.To search more active compounds with an acceptable therapeutic window,thus helping to reveal structure-activity relationship for cytoselective anticancer activity,a series of thiazolidinone derivatives were designed.185 2-arylimino-4-thiazolidinones and 17 2-arylimino-3-substituted-4-thiazolidinones were successfully prepared using solution phase parallel synthesis method and 45 2-aryl-4-thiazolidinones were synthesized using fluorous chemistry method.In the primary screening,all compounds were tested against H460,H460/TaxR and NHFB at a concentration of 10μM.Compounds which exhibited toxicity to both cancer cells but not to normal cells were selected. Primary screening indicated that the nitrogen substitution blocked the cytoselective toxicity of the compounds and the imino group at 2-positon of thiazolidinones was necessary for keeping activities.The confirmation assays identified 11 compounds as potent agents for inducing cytoselective toxicity.To optimized the cytoselectivity and explore the structure-activity relationship of active compounds,the early lead optimization library was designed based on those potent compounds.We synthesized, purified and obtained 170 designed compounds with an average purity of 95%.The single-concentration（10μM） primary screening identified 40 hits.Using a lower concentration（5μM）,11 compounds were confirmed and selected for dose-response studies,the 50%inhibitory concentration(IC₅₀) of the 11 compounds for H460 and H460_taxR cells was as low as 300 nM.For most of active compounds,NHFBs did not reach 50%cell killing at the highest compound concentration used（100μM）.One of the most potent compounds possessed IC₅₀ against H460 at 500 nM and H460/taxR at 210 nM.Structures of 13 compounds that killed H460,but not drug-resistant H460/taxR, and 11 active compounds that killed both cancer cells,but not normal cells were selected for P-gp pharmacophore modeling studies.9 of 13 molecules（70%） were found to map to the P-gp substrate pharmacophore in group 1.In contrast,only 4 of 11 molecules（36%） in group 2 mapped to the P-gp substrate pharmacophore.This suggested that active compounds were less likely to be P-gp substrates.SAR analysis showed that substitution groups were dominantly electron-donating groups at the 2- or 4-position for both rings.Substitution at phenylimino position allowed groups such as -Me,-Cl at both the 2- and 4-positions.However,Substitution at benzylidene position was restricted to only the -NMe₂ group at the 4-position.Compounds with nitrogen substitutions on the 4-thiazolidinone ring B did not show any cytoselective toxicity. We,therefore,tentatively concluded that nitrogen substitution blocked such activity. Anti-cancer molecule pharmacophore was generated with 11 most active compounds. The results indicated that two hydrogen bond acceptors and three hydrophobic regions were common features for all active compounds.This anticancer compound pharmacophore may be useful in further database searching to scaffold-hop to find novel anticancer molecules.Further studies to determine the mechanism of these anticancer compounds,as well as to optimize anticancer activity within this series of compounds,will be desirable.PartⅡ:Design and synthesis of nanoparticles with better characterization and biocompatibilityHundreds of thousands of active compounds are screened for best drug candidates each year,however,only a few less of them can be drugs in the end.That’s because a lot of factors are needed for an active compound to become a drug,i.e.some active compounds in vitro experiment are restricted by bad aqueous solubility or poor permeability in vivo.Chemical modification and encapsulation of drug molecules with the core of polymeric micelles as well as dendrimers are two common methods to address the problems;however,the methods are time-consuming or restricted by the large sizes.Interestingly,the use of nanoparticulate pharmaceutical carries,which have small size from 1 to 100 nm,to enhance the efficiency of many drugs well were established over the past decades.MWNTs have attracted wide biological and medical interests besides the vast interests in their technological and engineering applications. For applications of MWNTs in biological systems,it is urgent to explore the MWNTs with better biocompatibility.Surface modification is an solution to enhance the biocompatibility of nanoparticles.Combinatorial modifications of MWNTs’ surface would enable us to map unknown chemical space more effectively and to rapidly discover biocpmpatible functional MWNTs（f-MWNTs） with reduced toxicity,and reveal structure-activity relationships at the same time.Nano-combinatorial chemistry will accomplish two missions:1) to search for an initial lead candidate through combinatorial synthesis and screening and 2) once a lead is available,to optimize the lead in order to meet a set of criteria for nanomedicine candidate.To discover biocompatible nanotubes without a prior knowledge of targets,we decided to expose multiple biological targets to the maximum surface structural diversity.Starting with carboxylated f-MWNT,a tyrosine molecule is first attached as a linker because of its biocompatibility as an amino acid and its two reacting points（amine and carboxyl groups） for further diversification in two dimensions.8 amines and 9 acylators were selected for library synthesis（80 members including 8 intermediates in library）.To confirm the purity of final f-MWNTs,LC/MS method was developed for monitoring the effectiveness of each purification process.FTIR was adapted for characterization of special functional groups on the surface of MWNTs,For example,when pristine-MWNT was oxidized, an FTIR band at 1713 cm^-1 appeared indicating the formation of carboxylic acid groups.We were the first to use MAS NMR with Nano probe to acquire ¹H NMR spectra of nanotube-bond molecules in this investigation.F-MWNTs 26,50,28,and 76 have a benzenesulfonyl group except that there was a nitro group at 3-position in 28 and 76.The existence of nitro group reduced the electron density of the phenyl ring and caused a downfield shift in 28 and 76（7.64-8.71 ppm） compared to 26 and 50（7.29-7.94 ppm）.The loading of selected f-MWNTs were calculated by elemental analysis and confirmed by quantification of released Fmoc group from intermediates with UV-Vis spectroscopy.In order to select suitable candidates for further optimization and in vivo study,we studied the protein binding properties,cytotoxicity and immune response of the f-MWNT library.The f-MWNTs with less protein binding,immune response,and cytotoxicity were ranked from 1 to 80.The sum of a f-MWNT’s ranking was named multi-assay score.Smaller multi-assay score is superior in term of overall biocompatibility.Structure-biocompatibility relationship analysis showed that three out of the top five lead candidates,contained the building block AC005 and all eight AC005 containing f-MWNTs were in the top 25 in term of multi-assay score.AC008, AC006,AC004,AM004 and AM003 also showed moderate correlation to the multi-assay score.By rapidly approaching to surface molecular diversity on MWNTs and accelerating discovery of biocompatible nanomedicine carriers,we demonstrated the general utility of nano combinatorial chemistry approach in nanomedicine discovery.Encouraged by the primary results of the usage of MAS NMR for surface characterization of MWNTs,we successfully expanded the method to GNPs for full structural characterization.Compared with MWNTs,GNPs had advantages of better biocompatibility,easier synthesis process and unique size distribution.We effectively optimized high resolution MAS NMR（HRMAS NMR） experimental conditions by selecting solvent,temperature,spinning speed and pulse sequence for GNPs.Using the optimized condition,we also successfully obtained the ¹H and 2D HRMAS NMR for GNPs,such as COSY,TOCSY and HSQC.We found that there are significant differences in detection sensitivity depending on the distance between the surface of GNP and protons in the ligand molecule,and the loss of sensitivity for protons closer to the nanoparticle surface is consistent with an early finding.Furthermore,NMR spectra of aromatic protons of ligands attached to GNP seem to have a broad base compared with aliphatic ligands,indicating some degree ofπ-πstacking effects. Chemical shift assignment was useful for characterization of more complex structure by 2D spectra.Our results demonstrate that ¹H HRMAS NMR holds great potential for structural elucidation of molecules bound to nanoparticle surface.Recognizing that nanoparticles had the advantages of multifunctionalization,we designed and synthesized double functional GNPs with different ratios of two free ligands.We tried different methods to determine the loading of each ligands, including FTIR and cleavage-LC/MS.The loading of certain ligand increased with the increased usage of the free ligand.The conclusion was confirmed by design and synthesis of another group of double functional GNPs.When FITC and folic acid（FA） were used to construct multifunctional GNPs,the existence of FITC was confirmed by fluorescence spectroscopy of the GNPs.Two cell lines,KB cells over expressing FA receptors and A495 with low content of FA receptors,were used for cell uptake experiments.The difference of Confocal images of two cell lines demonstrated the existence of FA on the surface of GNPs.Using the optimized conditions,a double-functional GNP nano-combinatorial library containing 42 members was successfully synthesized and characterized.The development of new quantitative method for GNPs and the bioassays for the GNP library are on going.更多还原