【作者】 孙良鹏；

【导师】 朴虎日；

【作者基本信息】 延边大学 ， 药物化学， 2011， 博士

【摘要】 蛋白酪氨酸磷酸酯酶1B (PTPIB)是胰岛素和瘦素信号转导通路的负调节因子。小鼠PTP1B基因敲除及反义核苷酸治疗实验表明PTP1B是治疗糖尿病和肥胖症的潜在靶标。因此,小分子PTP1B抑制剂可能成为治疗Ⅱ型糖尿病以及肥胖症的新途径。近年来,运用基于PTP1B酶三维结构的设计方法,已经发现了大量的高效、高选择性的小分子抑制剂,很多化合物对PTP1B的抑制活性已经达到了纳摩尔级。然而,令人遗憾的是,这些化合物本身具有较高的负电荷,在生物体内主要以负离子的形式存在,不利于透过细胞膜,导致生物利用度差；有些抑制剂对不同的PTPase的选择性不好,不利于有效作用于特定的PTPase靶点。这些因素限制了它们被开发成为有治疗价值的药物。寻求有效性、特异性和药学性质之间的平衡,是研究和开发小分子PTP1B抑制剂作为口服抗糖尿病药物所面临的巨大挑战。本论文旨在设计、合成并筛选出高效且细胞膜通透性好的特异性PTP1B抑制剂。主要研究成果如下：(1)利用基于结构设计的方法,以底物酪氨酸磷酸酯为模板：以萘环替代苯环；以羧酸替代底物的磷酸酯部分,模拟其与酶的催化位点结合；同时,引入疏水性基团,使其与催化位点周边的区域通过疏水性作用结合,设计合成出了三个系列萘丙氨酸衍生物及类似物。活性研究表明,这些化合物是一类高效的、竞争性的PTP1B抑制剂,可以与底物共同竞争酶的结合位点；对于其他PTPase具有较好的选择性,比较PTP1B和TCPTP,77a、79h和810分别表现出3倍、6倍和7倍的选择性。同时,这些化合物对CDC25B具有较明显的抑制作用。通过计算机辅助软件Discovery Studio 3.0模拟化合物77a与酶PTP1B的结合,发现抑制剂与酶能较好地结合,而且,疏水性基团2-氟苄基可以延伸至非催化区域,并通过疏水性作用与氨基酸残基Ile219和Met258结合,增强了与酶的结合力。这一点与我们当初的设计思想不谋而合,也与实验数据相互符合。(2)基于文献报道,很多从天然物中分离得到得查尔酮类化合物对PTP1B具有较好的抑制作用,并且发现结构中的酚羟基对活性影响明显。因此,设计合成出了2,4-二羟基查尔酮、2,4,6-三羟基查尔酮和5,7-二羟基黄烷酮类化合物,考察其与PTPlB抑制活性间的构效关系。活性研究结果表明,大多数化合物对PTP1B具有较强的抑制活性,而且作为竞争性抑制剂,与底物共同竞争酶的结合位点；对于其他PTPase具有较好的选择性,其中化合物85d对PTP1B的抑制活性是TCPTP的9倍。另外,通过评价在CHO/hIR细胞上对胰岛素受体磷酸化水平的影响实验,发现5,7-二羟基黄烷酮类化合物87m能增强胰岛素的磷酸化水平,并呈现一定的化合物浓度梯度依赖性。(3)利用基于结构设计的方法,并结合查尔酮类化合物在PTPlB抑制活性方面的优势,以查尔酮为母体,在B环上引入酪氨酸磷酸酯模拟物,设计了两个系列化合物。活性研究表明,这些化合物均对PTP1B具有较强的抑制活性,ICso为0.56-16.3μM。进一步活性研究正在进行中。(4)基于杂环类酪氨酸磷酸酯模拟物在PTP1B抑制剂研发中的重要性,并结合查尔酮类化合物在PTP1B抑制活性方面的优势,以查尔酮为母体,在B环上分别引入2-芳基-5(4H)恶唑酮和N-(4-苯基-噻唑-2-基)芳醛腙结构,设计了两个系列化合物。活性研究表明,这些化合物均对PTP1B具有较强的抑制活性,IC50为1.16-15.31μM。进一步活性研究正在进行中。本文合成了共计八个系列190个目标化合物,目标化合物经1H NMR和MS确证了结构,部分化合物测试了13C NMR。根据生物活性测试结果,对化合物的构效关系进行了分析,总结出了一些规律,为此类化合物进一步的设计和结构优化提供了依据,同时,也为PTP1B抑制剂的研发提供了新的化合物模板。更多还原

【Abstract】 Protein tyrosine phosphatase 1B (PTP1B) has received much attention due to its pivotal role in type II diabetes and obesity as a negative regulator of the insulin and leptin-signaling pathway. Studies from two laboratories have shown that PTP1B knockout mice exhibit phenotypes of increased insulin sensitivity, improved glucose tolerance, and resistance to diet-induced obesity. In a more therapeutically relevant study, Zinker et al. have demonstrated that a PTP1B antisense oligonucleotide normalized blood glucose and improved insulin sensitivity in diabetic mice via a mechanism of lowering PTP1B protein expression. Thus, small molecule PTP1B inhibitors have considerable therapeutic potential for the treatment of TypeⅡdiabetes and obesity.Recent studies have provided important insights into the basic structural requirements for PTP IB-substrate and -inhibitor interactions and have suggested that identification of highly selective, catalytic site-directed PTP IB inhibitors is possible. Disappointingly, nearly all medicinal chemistry efforts to date have been severely hindered because of the lack of suitable druglike lead compounds for optimization to drug candidates. As is well documented in the literature, the majority of competitive inhibitors of PTP1B incorporate highly charged anions that mimic the pTyr substrateⅠ, such as difluoromethylphosphonates (DFMP), carboxymethylsalicyclic acids (CMS), and oxalylaminobenzoic acids (OBA). Compounds containing negatively-charged non-hydrolyzable pTyr mimetics have exhibited excellent potency (at nanomolar concentrations) in vitro, however, the low cell permeability and low bioavailability of these compounds have limited their application for the development of effective drugs. Therefore, we sought a new pTyr mimetic that would not only be a potent inhibitor of PTP1B, but one which would also avoid or minimize the polarity.In this dissertation, potent and specific PTP1B inhibitors were designed, synthesized and screened, and some results were obtained as follows:(1) Based on the structure of the pTyr substrate, we assumed that desired druglike lead compounds could be obtained if the monoacid based fragment was reserved and the phosphate group of pTyr was effectively replaced by hydrophobic groups, in which the carboxylic group mimics the interactions of the phosphate group of pTyr with residues in the PTP1B catalytic site and hydrophobic groups improve the stability of the enzyme/inhibitor complex through hydrophobic interactions with the active site and surrounding subpockets. As a starting point toward this goal, three series of 2-naphthol tyrosine analogues have been designed and synthesized using structure-based design programs. The bioassay showed that these compounds had good inhibition activity against PTP1B, and were identified as reversible and competitive PTP1B inhibitors. These compounds also displayed better selectivity for other PTPase,77a、79h and 81o possess the best selectivity of 3-,6-and 7-fold for PTP1B over TCPTP, respectively. The docking results showed that the -COOH group could well bind into the active site, and the lipophilic 2-fluorobenzyl group of 77a lies on top of a largely hydrophobic region of the protein, consisted of Ile219, Met258. These results are consistent to our original assumption and the bioassay results.(2) As is well documented in the literature, a number of naturally occurring chalcones have been reported to possess better inhibition activity against PTP1B and the position and the number of the hydroxyl group attached to the chalcone rings are of major importance in the improvement of potential inhibitory effects against PTP1B. Therefore, a series of hydroxyflavonoids were designed and synthesized considering the effect of different skeletons (2,4,6-trihydroxychalcones,2,4-dihydroxychalcones, and 5,7-dihydroxyflavanone). The bioassay showed that these compounds had good inhibition activity against PTP1B, and were identified as reversible and competitive PTP1B inhibitors. These compounds also displayed better selectivity for other PTPase,85d possesses the best selectivity of 9-fold for PTP1B over TCPTP. Further evaluation in cell models illustrated that 87m enhanced insulin receptor phosphorylation in CHO/hIR cells.(3) Considering the advantages of inhibition activity against PTP1B of chalcones, 4-(((5Z)-5-(4-((E)-3-phenyl-3-oxoprop-1-enyl)benzylidene)-2,4-dioxothiazoli din-3-yl)methyl)benzoic acid derivatives and (2E)-methyl 2-((5Z)-5-(4-((E)-3-(3-chlorophenyl)-3-oxoprop-1-enyl)benzylidene)-4-oxothiazolidin-2-ylidene-amino)thiazole-5-carboxylate derivatives were designed and synthesized using structure-based design programs. The bioassay showed that these compounds had good inhibition activity against PTP1B with IC50 values in a range of 0.56-16.3μM, and further bioassays are in progress.(4) Considering the advantages of inhibition activity against PTP1B of chalcones, (4Z)-4-(4-((E)-3-phenyl-3-oxoprop-1-enyl)benzylidene)-2-aryloxazol-5(4H)-one derivatives and N-(4-phenyl-thiazol-2-yl)-4-((E)-3-phenyl-3-oxoprop-l-en-1-yl)benzaldehyde hydrazone derivatives were designed and synthesized using heterocyclic pTyr mimetics. The bioassay showed that these compounds had good inhibition activity against PTP1B with IC50 values in a range of 1.16-15.31μM, and further bioassays are in progress.In this paper,190 compounds were designed and synthesized, and the structures of all synthesized compounds were characterized by 1H-NMR and MS. Part of the compounds were characterized by 13C-NMR. The structure-activity relationships were also discussed. And the results will provide experimental and theoretical clues for designing of novel potent and specific PTP1B inhibitors.更多还原