【作者】 李学恕；

【导师】 赵玉芬；

【作者基本信息】 清华大学 ， 化学， 2006， 博士

【摘要】 蛋白酪氨酸磷酸酯酶与蛋白酪氨酸激酶一起承担信号传导作用,如果蛋白酪氨酸磷酸酯酶功能异常,就会导致一些疾病,如糖尿病、癌症和免疫缺陷症。因此开展对蛋白酪氨酸磷酸酯酶抑制剂的研究,并探索抑制剂与酶作用机理,这不仅对于研究生物体中的信号传导具有重要理论价值,也有利于开发出新型抗糖尿病、癌症和免疫缺陷症等疾病的新药。本论文旨在设计、合成并筛选出高效且细胞膜通透性好的蛋白酪氨酸磷酸酯酶抑制剂。主要研究成果如下:建立了合成新型磷杂环化合物的新方法。论文首先合成了磷杂苯并二氢吡喃-2,4-二酮和4-羟基磷杂香豆素,通过乙酰化、磺酰化、磷酰化以及烷基化反应构建了各种类型的磷杂香豆素,并利用O-(磷杂香豆素-4-基)对甲苯磺酸酯与有机锌试剂、苯硼酸和端炔的交叉偶联反应,合成了一系列4-芳基、烷基和炔基取代的磷杂香豆素。筛选出了细胞膜通透性好且抑制活性较高的磷杂香豆素蛋白酪氨酸磷酸酯酶抑制剂。论文对所合成的各种磷杂香豆素进行了蛋白酪氨酸磷酸酯酶(PTP1B和SHP-1)抑制活性测试,实验表明,大多数化合物具有中等或较好的抑制活性,其中化合物61b对蛋白酪氨酸磷酸酯酶SHP-1的抑制常数(k_i)为10μM。设计、合成并筛选出了活性较好的二酮类蛋白酪氨酸磷酸酯酶(PTP1B和SHP-1)抑制剂,并通过Sybyl工作站将筛选出来的二酮类抑制剂与酶PTP1B进行了分子对接,发现抑制剂与酶能较好地结合。建立了一种将慢抑制剂转变成快抑制剂的新方法。论文对慢抑制剂反-β-硝基苯乙烯类和香豆素类抑制剂进行O-磷酸化,这些被修饰的化合物同时具有蛋白酪氨酸磷酸酯酶底物和抑制剂结构(前抑制剂),由于底物与酶结合很快,随之在酶作用下去磷酸化,在酶活性口袋中释放出抑制剂而实现对酶的抑制。这种方法不但提高了抑制剂与酶的结合速度,而且提高了抑制剂的活性,为酶抑制剂的设计和改造提供了一种新的思路。研究并总结出了磷杂香豆素的电喷雾质谱裂解规律。论文采用电喷雾质谱并结合多级质谱技术系统研究和总结了磷杂香豆素的质谱裂解规律,这为其它含磷杂环化合物的结构表征提供了有用的方法。更多还原

【Abstract】 Protein tyrosine phosphatases (PTPs), in conjunction with protein tyrosine kinases (PTKs), serve as key regulatory components in signal transduction pathways. Defective or inappropriate regulation of PTP activity leads to aberrant tyrosine phosphorylation, which results in many human diseases, such as caners, diabetes and immune dysfunctions. It is very necessary to develop highly efficient PTP inhibitors and probe into their interaction mechanisms between the enzymes and the inhibitors, which contributes to the development of signal transduction in organisms and new drugs against diabetes, cancer and immune dysfunctions. In this dissertation, PTP inhibitors were designed, synthesized and screened, and some results were obtained as follows:A new method has been developed for the synthesis of heterocyclic compounds containing-phosphorus. The mixture of two isomers, phosphachroman-2,4-diones and 4-hydroxyphosphacoumarins, were synthesized. Various phoshpocouamarin derivatives were obtained by acetylation, sulfonylation, phophorylation and alkylation of the isomers, respectively. And a method for construction of new C-C bonds on the cores of phosphacoumarins has been developed through the cross-coupling reactions of 4-tosylphosphacoumarins with organic zincs, arylboronic acids and terminal alkynes, respectively, under palladium and/or copper-catalysts.Several phosphacoumarins with membrane permeability have shown good inhibition activity against SHP-1. These phosphacoumarins have been tested against PTP1B and SHP-1. The results showed that most of the phosphacoumarins had moderate to good inhibition activity against SHP-1, especially, compound 61b exhibited the highest inhibition activity against SHP-1 (K_i = 10μM).3-Benzylidenepentane-2,4-diones have been designed, synthesized and screened against SHP-1 and PTP1B. The bioassay showed that the kind of compounds had good inhibition activity against SHP-1 and PTP1B, and also displayed selectivity for PTP1B. The compounds with good inhibition were docked in the catalytic pockets of SHP-1 and PTP1B on the Sybyl workstation. The docking results are consistent to thebioassay results.A new strategy has been developed for the transition of slow-binding inhibitors to fast ones. Preinhibitors were designed and synthesized by phosphorylation of slow-binding inhibitors, derivatives of (E)-β-nitrostyrene and 7-hydroxycoumarin. The prepared preinhibitors contain parts of both PTP substrates and inhibitors. The preinhibitors could quickly bind with the catalytic site of PTPs, just like the natural substrates, phosphotyrosine. Once binding with catalytic site of PTPs, the preinhibitors were dephosphorylated under the catalysis of the enzyme, which led to the release of the inhibitors in the catalytic pocket. The released inhibitors could bind with the catalytic pocket of PTP and show the inhibition activity against PTPs. The strategy can not only improve the binding rate between inhibitors and enzymes, but also enhance the inhibition activity against enzymes, which brings out a new protocol for the design and modification of enzymatic inhibitors.Electrospray ionization mass spectrometry has been used as a useful tool for the structure determination of phosphacoumarin derivatives. In the dissertation, the positive ESI-MSn spectra of phosphacoumarins derivatives were investigated. The abundant characteristic fragment ions were observed, and the characteristic fragmentation pathways were analyzed, which are useful for the structural determination of other phosphacoumarin derivatives.更多还原