Ⅰ.苯并噁嗪衍生物液相平行组合合成及其生物活性研究 Ⅱ.SARS相关冠状病毒（SARS-CoV）融合抑制剂及SARS活性多肽与载体蛋白共轭物免疫性研究—两种SARS治疗策略的初步探索

【作者】 苑芸芸；

【导师】 刘刚；

【作者基本信息】 中国协和医科大学 ， 药物化学， 2007， 博士

【摘要】 第一部分以1,5-二氟-2,4-二硝基苯（DFDNB,1）为原料构建3,4-二氢-1,4-苯并噁嗪-3-酮化合物的设计与液相平行合成组合化学是近二十年来逐渐发展并成熟完善起来的一个新领域,其概念和技术已被广泛地应用于医药科学研究的各个方面。组合化学与高通量筛选技术相结合,促成了新药开发领域的一次飞跃,并且已经成为创新药物发现和优化环节中不可缺少的一项技术。组合化学起源于固相多肽合成化学。固相技术操作简便、纯化快速、反应完全及可实现自动化操作等优点,使固相组合化学得到了飞速发展。然而固相合成化学也有许多不足,诸如:某些在液相中进行的很好的反应,在固相上不是很理想,甚至不发生反应;需要设计并合成合适的连结分子;难以用常规波谱测试手段跟踪和检测反应过程;较难进行大量合成等等。液相合成可弥补上述固相反应的缺点,且结合固相试剂、清洁树脂及氟相技术的开发与应用,日益引起研究者们的兴趣。其中液相平行单分子合成更是备受关注。平行单分子合成是指同时合成一系列的单个分子,是单一产物的合成,可应用于单一产物的生物活性评价。这种方法已被广泛地用于合成各种小型化学库,特别适用于先导化合物的优化。1,5-二氟-2,4-二硝基苯（DFDNB,1）是一种蛋白交联剂,有四个潜在反应位点,通过引入不同的取代基团,可构建多样性的苯并杂环骨架。本论文以DFDNB（1）为起始原料,运用液相平行合成以及清洁树脂、微波辅助等方法,经定量亲核取代、芳香间二硝基还原、环合、苯胺或苯二胺衍生等步骤,探索和优化了“优势结构”（privileged structure）3,4-二氢-1,4-苯并噁嗪-3-酮的制备条件,合成了七类111个3,4-二氢-1,4-苯并噁嗪-3-酮衍生物及五类共65个基于3,4-二氢-1,4-苯并噁嗪-3-酮骨架的新三元杂环化合物,并合成了一个具有1040化合物的化学库。1) 3,4-二氢-1,4-苯并噁嗪-3-酮类似物的合成3,4-二氢-1,4-苯并噁嗪-3-酮衍生物具有多种生物活性,诸如抗炎、抗溃疡、抗高血压、抗真菌等。本论文以DFDNB（1）为起始原料,依次经由仲胺、酚、硫醇和羟基酸酯的亲核取代形成1,5-双取代-2,4-二硝基苯,在Pd-C/HCOONH₄或Na₂S₂O₄/K₂CO₃作用下将其间二硝基还原,还原中间体自动环合为6-氨基-3,4-二氢-1,4-苯并噁嗪-3-酮。还原产物中游离芳香氨基除与酸酐、（磺）酰氯、异（硫）氰酸酯、醛、双氰胺等化学试剂反应外,还可参与Ugi-4CC反应,从而引入诸如（磺）酰胺、（硫）脲、双胍等活性基团。部分氨基衍生反应在微波辅助下短时间内可得到高收率高纯度的目标产物。所探索的大部分反应具有反应条件温和、速度快、副产物少的特点,整个合成路线经四步反应引入两到五个多样性位点,适于构建化学库。2)基于3,4-二氢-1,4-苯并噁嗪-3-酮骨架的三环杂环化合物合成药物设计中经常会用到“整合”（intergrating）药效团（pharmacophore）或优势结构（privileged structure）的理念,以期获得双重药效。2-喹喔啉醇、苯并咪唑、苯并三氮唑、苯并羰基咪唑、2,3-喹喔啉-二酮五类杂环骨架具有广泛的生物活性,可谓是药物的“优势结构”,将其与“优势结构”1,4-苯并噁嗪-3-酮整合在一个分子里,除增加合成化合物的多样性外,还有可能提高发现具有特定生物活性的化合物的机率。本论文以DFDNB（1）为起始原料,依次经由伯胺、氨基酸酯和羟基酸酯的亲核取代,并采用Pd-C/HCOONH₄或Na₂S₂O₄/K₂CO₃将所得双取代的芳香间二硝基还原,得到自动环合为7-羟基-3,4,8,9-四氢-2H-[1,4]噁嗪[2,3-g]喹喔啉-3-酮或6,7-二氨基-3,4-二氢-1,4-苯并噁嗪-3-酮两类中间体。所得中间体再经过氧化脱氢或进一步与醛、亚硝酸钠、二硫化碳、三光气、草酰氯等试剂反应可得到五类新骨架:7-羟基-3,4-二氢-2H-[1,4]噁嗪[2,3-g]喹喔啉-3-酮（Ⅶ）,3,6,7,8-四氢咪唑[4’,5’:4,5]苯并[b][1,4]噁嗪-7-酮（Ⅷ,Ⅹ）,3,6,7,8-四氢[1,2,3]三唑[4’,5’:4,5]苯并[b][1,4]噁嗪-7-酮（Ⅸ）,1,2,3,6,7,8-六氢咪唑[4’,5’:4,5]苯并[b][1,4]噁嗪-2,7-二酮（Ⅺ）,3,4,6,7,8,9-六氢-2H-[1,4]噁嗪[2,3-g]喹喔啉-3,7,8-三酮（Ⅻ）。此外,所得目标产物中酰胺氮氢（Ⅸ,Ⅺ）及巯基（Ⅹ）还可进一步用卤代烷衍生。实验路线中大部分反应可在室温下进行,整个合成路线经四或五步反应,可引入三到四个多样性位点。3)构建3,6,7,8-四氢咪唑[4’,5’:4,5]苯并[b][1,4]噁嗪-7-酮（Ⅷ）化学库经过反应条件优化,并遵从构建化学库所必须满足的化合物取代及结构最大多样性及易操作性等规则,选用了不同类型的16种伯胺、1种羟基酸酯和72种不同取代的脂肪、芳香或杂环醛,以清洁树脂辅助的液相平行方式构建了含1040个3,6,7,8-四氢咪唑[4’,5’:4,5]苯并[b][1,4]噁嗪-7-酮（Ⅷ）的化学库。所有最终化合物经LC-MS及LC-ELSD分析表明:80.1%的化合物纯度大于80%。未清除完全的醛是造成所得部分化合物纯度低的主要原因。整个化学库合成历时两个月,经两步反应,引入两个多样性位点,体现了组合化学快速、高效的特点。第二部分SARS相关冠状病毒融合抑制剂及SARS活性多肽与载体蛋白共轭物免疫性研究严重急性呼吸综合症（severe acute respiratory syndrome,SARS）是2002年底开始流行的一种人类呼吸道传染病,始于我国广东省,并迅速蔓延至香港、加拿大等全球五大洲三十多个国家和地区。该病发病急、传染快、病死率高,严重威胁了全球公共卫生和社会经济的稳定。SARS的病原体是一种新的冠状病毒,称作SARS相关冠状病毒（SARS-CoV）,为正义单链RNA病毒。白其基因组测序完成后,人类对这一病毒逐渐有了深入的了解,包括其组成、受体、入侵机制、起源与进化等。T-20作为HIV-1融合抑制剂已应用于临床治疗艾滋病。本论文尝试合成了源于SARS-CoV S蛋白HR1及HR2的四条多肽（Pep 1D,1E,2B及HR2）,并由合作者研究了它们各自与T-20的相互作用。实验结果表明,T-20仅与一条源于HR1的多肽（Pep 1D）有中等相互作用,不足以提供临床治疗效果。本课题组在2003年运用“交叉重叠”的方法设计并合成了含十个氨基酸残基的多肽化学库,经血清抗原一抗体反应和进一步优化,筛选出了五个具有较好B细胞抗原性的多肽。本论文即以此为基础,选取其中三条多肽,将其与mcKLH、HBsAg共轭后免疫新西兰大白兔,以期为SAR5多肽疫苗的研制提供一些参考（其中,选用HBsAg作为载体蛋白还有可能开发多价疫苗）。目前一些相关数据仍在采集测试中。更多还原

【Abstract】 PartⅠ.Design and parallel synthesis of benzo[1,4]oxazin-3-one library in solution-phaseCombinatorial chemistry is a subject that has been developed and grown up quickly in the past twenty years.Its concepts and techniques have been widely accepted and used in various aspects of scientific research and industries,for example drug discovery and optimization,material science and catalysts.Quick development along with high-throughput or high content screening technologies,combinatorial chemistry has caused a big leap in new drug development in particularly and become an indispensable technique in the process of drug discovery and optimization. Combinatorial chemistry originates from solid-phase peptide synthesis.Nowadays, however,combinatorial generation of small heterocyclic molecules has heavily attracted attention for both organic and medicinal chemists due to their high diversities and drug-like properties.Solution-phase combinatorial synthesis,benefited from the development of solidified reagents,scavenging resins and fluorous techniques,has been widely practiced,among which solution-phase parallel single molecule synthesis was especially regarded.1,5-difluoro-2,4-dinitrobenzene（DFDNB,1） is a protein cross-linker,which has four potential reactive sites.Therefore,diversified benzofused heterocyclic skeletons are potentially able to be synthesized through the asymmetric introduction of different substitution groups.This thesis focused on synthesis of ’privileged structure’ of benzo[1,4]xazin-3-one scaffolds through four reaction steps in a parallel solution manner including quantitative neucleophilic substitution,reduction of meta-dinitro groups,autocyclization reaction and acylation.In all,one hundred and eleven benzoxazine analogs were synthesized.Sixty five compounds of five new skeletons based on banzoxazine were also generated.A chemical library with 1040 members was prepared.1) Synthesis of benzo[1,4]oxazin-3-one analogs Benzo[1,4]oxazin-3-ones have shown various biological activities,such as being anti-inflammatory,antiulcer,antipyretic,antihypertensive,and antifungal.The autocyclization of 6-amino-3,4-dihydro-benzo[1,4]oxazin-3-ones herein was discussed,following the neucleophilic substitutions of two fluorine atoms of DFDNB （1） with second amines/phenols/thiophenols,hydroxyacetates,and reduction of meta-dinitro groups.The remaining free aromatic amino group of 6-amino-3,4-dihydro-benzo[1,4]oxazin-3-ones was diversified through the acylation by anhydrides,sulfonyl chlorides,isocyanates,isothiocyanates,aldehydes, dicyanoamine.Particularly,Ugi-4CC reaction was successfully carried out at 6-amino group which greatly increased the substitution diversity.Several microwave assisted reactions were well practiced in this thesis to improve the conditions with shorter reaction time and higher purity.The optimized reaction conditions were compatible with chemical library synthesis which allows three or five diversity points.2) Synthesis of tri-heterocycles based on benzo[1,4]oxazin-3-one skeletonThere is always a considerable effort placed on the integration of privileged structures into one molecule for drug design.Quinoxalin-2-ol,benzoimidazole,benzotriazole, benzocarbonylimidazole,quinoxalin-2,3-dione are recognized as the privileged structures.Integrations of these scaffolds with benzo[1,4]oxazin-3-one will not only increased the compounds diversity,but also provide the new possibility to discover novel hits and lead compounds.After the neucleophilic substitution of two fluorine atoms of DFDNB（1） with aminoacetates/amines and hydroxyacetates subsequently, the meta-dinitro groups were reduced either by Pd/C-HCOONH₄ or Na₂S₂O₄/K₂CO₃ to yield 7-hydroxy-3,4,8,9-tetrahydro-2H-[1,4]oxazino[2,3-g]quinoxalin-3-one or 6,7-diamino-7-3,4-dihydro-2H-1,4-benzoxazin-3-one,respectively.The former was further oxidized in air to offer a new tricycle of 6-hydroxy-4H-1-oxa-4,5,8 -triazaanthracen-3-one（Ⅶ）.The latter reacted with aldehydes,sodium nitrite,carbon disulfide,triphosgene,oxalyl chloride to generate additional four new tricycles as: 3,8-dihydro-5-oxa-1,3,8-triazacyclopenta[b]naphthalene-7-one（Ⅷ,Ⅹ）,3,8-dihydro -5-oxa-1,2,3,8-tetraazacyclopenta[b]naphthalene-7-one（Ⅸ）,3,8-dihydro-1H-5-oxa -1,3,8-triazacyclopenta[b]naphthalene-2,7-dione（Ⅺ）,and 6-hydroxy-4,8-dihydro-1 -oxa-4,5,8-triazaanthracen-3,7-dione（Ⅻ）.The whole reaction routes were involved in four or five reaction steps,and thus three or four diversity points were introduced to the benzo[1,4]oxazin-3-one skeleton.3) Preparation of chemical library ofⅧConsidering the maximum molecular diversity,high quality（purity） and easy operation,sixteen primary amines,one hydroxyacetate and seventy-two aliphatic, aromatic or hetero aldehydes were selected to generate the library in a parallel manner. It was also referenced the reactivities of various reagents through our reaction optimization.The library synthesis was carried out in solution-phase in the aid of scavenging resin for purification.All final compounds were analyzed by LC-MS and LC-ELSD.The results indicated that 80.1%of the library members were over 80%in purity.The whole library synthesis took up two months to generate 1040 compounds individually by one person.This showed obviously the high performance and speediness of combinatorial chemistry.PartⅡ.Studies on SARS-CoV fusion inhibitors and conjugation of SARS immunogenic peptides with carrier proteins（mcKLH,HBsAg）Severe acute respiratory syndrome（SARS） is a human respiratory infection disease which began to prevail in the end of 2002.It first occurred in Guangdong Province, China,but spread to Hongkong and Canada shortly.SARS had caused an infection of more than thirty countries and regions across five continents.Due to its high infectivity and high mortality,SARS damaged greatly the public sanitation and the ecnomic stability of the whole world.SARS was caused by a new coronavirus,called SARS coronavirus（SARS-CoV）, which is a single chain（+） sense RNA virus.After decoding its gene sequence, knowkedge to this virus’s structure,receptor,invasion mechanism,origin,evolution and so on were understood step by step.Although some of these findings need more solid proofs,they still provide useful information for the precaution and therapy of SARS.T-20 is a clinical fusion inhibitor for HIV-1.Wondering if this reagent is also effective to SARS-CoV,we synthesized four peptides derived from the HR1 and HR2 domains of SARS-CoV S protein and studied the interaction between these peptides with T-20,respectively.The significant but moderate interaction between T-20 and an SARS-CoV HR1-derived peptide（Pep 1D） was observed,which is not enough to be able to provide therapeutic efficiency.Our group identified five B-cell immunoantigens of SARS-CoV through the synthsis and screening an ’overlapping’ peptide library.To explore the possibility of potential peptide vaccine for a clinical purpose,this dissertation primarily investigated （1） the conjugation between three peptides and mcKLH,HBsAg;（2） the immunity of the conjugates.更多还原