【作者】 张仰明；

【导师】 吕伟； 汤杰；

【作者基本信息】 华东师范大学 ， 有机化学， 2009， 博士

【摘要】 先导化合物的发现是药物发现阶段的关键步骤，是目前药物化学研究中最活跃的一个领域。基于优势结构单元构建小分子库的理念吸引了药物化学家越来越多的注意，并已成为药物化学的研究热点之一。本论文工作主要围绕一些嘧啶并环类化合物的合成方法学研究和多样性导向的化合物库设计与合成及其相关生物活性的评价来展开。第一部分：含有嘧啶并环结构单元的活性化合物数目众多，许多已上市及临床阶段的药物分子中都含该结构单元，是优势结构之一。我们在合成广谱抗真菌药物伏立康唑中间体6-乙基-5-氟-4-羟基嘧啶过程中，发现Pd-C/H₂/NaHCO₃体系能有效脱除嘧啶环上的氯原子。深入研究发现该体系能区域选择性地优先氢解诸多2，4-二氯嘧啶类化合物中的4-位氯原子，得到2-氯嘧啶类产物。初步的方法学研究表明Pd-C/H₂/NaHCO₃条件下区域选择性氢解2，4-二氯嘧啶类化合物是现有的2-氯嘧啶类化合物制备方法的有效补充。在上述方法学研究的基础上，我们设计并合成了三个具有2-3个不同反应活性位点的嘧啶并环砌块A1-3，作为构建该类小分子库的起始原料。通过亲核取代反应和Suzuki偶联反应，砌块A1依次与芳胺砌块B1-5、仲胺砌块C1-6和硼酸砌块D1-10作用，基于尽可能拓展化学空间的多样性导向原则，我们初步构建了一个含80个化合物的嘧啶并环类小分子库Library 1，并在蛋白激酶c-Met上对化合物的抑制活性进行了评价。结果表明Library 1中的绝大部分化合物不是很好的c-Met抑制剂，其中LXY-78是活性最好的化合物，在浓度为10μg/mL时，对c-Met的抑制率为47．8%。Library 1在腺苷受体上的评价工作仍在进行之中。第二部分：S-腺苷同型半胱氨酸水解酶（SAHH）是一种在细胞内广泛存在的酶，它催化腺苷同型半胱氨酸（AdoHcy）水解生成腺苷（Ado）和同型半胱氨酸（Hcy）。抑制SAHH将导致细胞内AdoHcy的蓄积，从而对转甲基反应产生反馈性抑制作用。与机体其它细胞相比，淋巴细胞的活化更依赖于这种由SAHH调控的甲基化作用，因而抑制SAHH的活性，就有可能实现免疫抑制。与早期的SAHH不可逆抑制剂相比，DZ2002是近年来研究比较多的一种Ⅲ型可逆抑制剂。研究发现DZ2002在有效降低不可逆抑制剂所固有的细胞毒性的同时，仍然保持了良好的酶抑制活性。DZ2002在一系列炎症和自身免疫性疾病的动物模型上都具有良好的预防和治疗作用。DZ2002具有较高的治疗指数，在毒性剂量和治疗剂量间有较宽的距离，这些都提示DZ2002有可能发展成为高效低毒的新型免疫抑制剂。基于DZ2002的良好的应用前景，有必要对它的合成工艺进行研究。我们以价廉易得的大宗化商品L-苹果酸为手性原料，丙酮叉保护，硼烷选择性还原4-位羧基到醇。所得醇转化为对甲苯磺酸酯后，与商品化原料腺嘌呤偶联，所得偶联产物于甲醇中用乙酰氯处理，在脱去丙酮叉保护的同时实现甲酯化反应，得到目标分子DZ2002。以L-苹果酸计，经5步反应，重结晶后总收率为6．54%，所得样品经测试满足临床前研究的各项质量指标。基于上述合成路线，我们首次报道了DZ2002的比旋光值和ee值，为今后进一步的工艺优化奠定了基础。DZ2002小试工艺合成路线简洁，所用原料成本低，反应条件温和，后处理方便，易于放大。为了进一步考察手性与DZ2002生物活性的关系，我们设计并合成了DZ2002的三个相关异构体。活性测试结果显示，四个化合物中DZ2002的酶抑制活性最强(IC₅₀=21．2．μM，Table 3-1)，SAHH抑制活性顺序依次为：（S）-DZ2002＞（Rac）-DZ2002＞（R）-DZ2002＞（Ketone）-DZ2002。四个化合物的体外免疫抑制活性与它们对SAHH的抑制活性相一致，DZ2002的体外免疫抑制活性最强。这些结果表明DZ2002分子中的手性中心采取（S）绝对构型最有利于活性的保持。通过计算模拟DZ2002与SAHH的结合情况，发现DZ2002中手性的不同会导致化合物与酶结合时其边链的空间伸展方向的不同，并导致与SAHH残基形成氢键个数的不同，这在一定程度上解释了DZ2002分子中的手性影响其活性的作用机制。更多还原

【Abstract】 With a DOS （diversity-oriented synthesis） approach,the installation of suitablefunctionalities at suitable places of certain privileged structure may lead to a higherscreening hit rate later.This strategy of building libraries of small molecules basedon some privileged structures has attracted many medicinal chemists,representingone trend in lead discovery in recent years.1)Many bio-active compounds contain pyrimidine-fused cyclic structures,which also occur in many drugs on market or under clinical stage.During oursynthesis of 6-ethyl-7-fluoro-4-hydroxy uracil,the key intermediate of Voriconazole,we found Pd-C/H₂/NaHCO₃ system can replace the chlorine atom in the pyrimidinering with a hydrogen atom efficiently.Further application study showed that many2-chloropyrimidines can be obtained regioselectively from readily available2,4-dichloropyrimidines using above system in good to excellent yields,constitutingan useful alternative synthesis of 2-chloropyrimidines.Based on above synthetic methodology study,we designed and prepared threepyrimidine building blocks A1-3 with 2-3 different reacting points.Employingsubstitution and Suzuki coupling reaction,aryl amine blocks B1-5,secondary amineblocks C1-6 and boric acid blocks D1-10 were installed to block A1 smoothly,leading to the construction of Library 1 containing 80 compounds.Library 1 wasevaluated on protein kinase c-Met and Adenosine Receptors.LXY-78,the mostpotent compound on c-Met in Library 1,displayed a 47.8% enzyme inhibition inconcentration of 10 mg/mL.Evaluations on Adenosine Receptors are currently underprogress.2)Within recent years,4-（6-Amino-purin-9-yl）-2（S）-hydroxy-butyric acidmethyl ester （DZ2002） has been recognized as a potent typeⅢreversible inhibitor ofSAHH.DZ2002 is able to reversibly bind to the open form of the enzyme, maintaining a similar potency with much reduced toxicity which hampers furtherclinical applications of irreversible SAHH inhibitors.Biological study indicatesDZ2002 is a promising noval therapeutic agent for immune-related diseases.Due to its promising prospect,there is a need to develop a practical syntheticprocess to meet the sample need for related pre-clinical study.Our synthesis startedwith cheap and commercially available optical malic acid.In brief,（S）-malic acid wasfirst transformed to acetonide.Acetonide was reduced to alcohol,which wasimmediately converted to its tosylate.Coupling of tosylate with adenine gave desiredkey intermediate in a yield of 32% after silica-gel chromatograph.The keyintermediate was readily transformed to DZ2002 upon spontaneous deprotection andesterification in methanol solution of HCl.Because of the rigid conformation ofacetonide during reaction sequences,2-chiral center was highly reserved and potentialracemization was avoided.Following this process,target DZ202 was obtained in atotal yield of 6.54% after 5 steps reaction and recrystallization.According to thisprocess,about 120 grams of DZ2002 was synthesized and used for related biologicalstudy.We also designed and synthesized DZ2002 related steroisomers so as toinvestigate the necessaty of chiral center.Later biological study showed that that（S）-DZ2002 was the strongest inhibitor among four compounds (IC₅₀=21.2μM).The order of enzyme inhibition potency was （S）-Z2002＞（Rac）-DZ2002＞（R）-DZ2002＞（Ketone）-DZ2002.Respective suppressive potency appeared to matchthe inhibitory potency of the SAHH.These results indicated that maintenance of2-chiral center in the molecule as （S）-configuration is important for binding withSAHH.Simplified docking results showed that different numbers of hydrogen bondsinteracted with SAHH was formed providing useful information to understand theirefficacy difference.更多还原