新型凝血因子Xa抑制剂的设计、合成及生物活性研究

发布时间：2018-05-12 22:22

本文选题：血栓栓塞性疾病 + 血栓　；参考：《山东大学》2017年硕士论文

【摘要】：心脑血管疾病是一类由于心脏或血管病变导致的循环系统功能紊乱的疾病的统称,是世界范围内尤其是亚洲地区的首要死亡原因,对人类的生命健康构成了严重威胁。其中血栓栓塞性疾病,如心肌梗死、脑卒中、深部静脉血栓,已成为全世界致残率和致死率最高的疾病之一,因此血栓的形成是引起心脑血管疾病的最主要因素。然而目前临床上常用的抗凝血药物如华法林等仍存在着诸多缺陷,例如给药不方便、不可预测的抗凝效果、治疗窗窄、能够与许多食物和药物发生相互作用等。因此,亟需开发更安全有效的新型抗凝血药物。Xa因子(FXa)位于人体内源性凝血途径和外源性凝血途径的交汇处,如果能够抑制FXa就可以同时阻断两条凝血途径,因此,FXa目前已成为抗凝药物研发中的一个十分具有吸引力的靶点。目前已经有一些口服的小分子FXa抑制剂在市场上销售,例如利伐沙班(rivaroxaban)、阿哌沙班(apixaban)以及依度沙班(edoxaban)。虽然新型FXa抑制剂克服了传统抗凝药物的缺点,但是,这些药物仍然存在一些缺陷,如药物的适应症狭窄、损害肝肾功能、以及发生出血时缺乏有效的解毒剂等。鉴于抑制剂本身的有效性和安全性问题,因此,亟需开发更安全有效的新型FXa抑制剂供临床应用。本论文以利伐沙班为先导化合物,通过研究分析"利伐沙班-FXa复合体"的晶体结构,发现FXa拥有两个最关键的结合口袋,分别是:特异性结合口袋S1和较大的疏水性口袋S4,同时两口袋之间存在着大约80°的夹角;因此,利伐沙班分子也相应的被分为三部分:P1区、P4区和中间的Linker区。FXa的特异性结合口袋S1为较小的疏水性口袋,正好可以容纳利伐沙班的P1区5-氯噻吩基团,同时5-氯噻吩基团与S1 口袋底部的Tyr228形成了 Cl-π作用力;而较大的疏水性口袋S4的氨基酸残基与利伐沙班P4区的苯环形成了 π-π堆积作用;同时,利伐沙班的Linker区与FXa的氨基酸Gly219形成了两个氢键。经过对先导化合物利伐沙班与FX a的结合模式的分析,我们基于电子等排、优势分子片段杂合等原理,将利伐沙班的P4区和Linker区进行了结构改造,如改变P4区的体积、疏水性和增加Linker区与FXa结合位点的作用力等,设计了吡咯烷酮类、噻二嗪类和吲哚环类三个系列FXa抑制剂,以期得到具有较高活性的新型骨架Xa因子抑制剂。同时,我们运用计算机辅助药物设计软件Sybyl X 1.3对所设计的三个系列化合物进行了分子模拟对接研究,研究结果表明:通过结构改造所设计的新化合物具有理论上的合理性。通过成环反应、酰化反应、缩合反应等实验步骤,对三个系列的目标化合物进行了定向合成,共合成了吡咯烷酮类、噻二嗪类和吲哚环类三个系列37个Xa因子抑制剂,并经光谱确证结构。首先测试了所合成FXa抑制剂的抗凝血活性,由化合物对血浆凝血酶原时间的影响来评价。通过使用凝血酶原时间测定仪,测定了在化合物不同浓度下贫血小板血浆的凝血酶原时间,然后计算出"使凝血时间延长一倍的化合物浓度"(PTCT2)。个别化合物具有一定的抗凝活性,与先导化合物利伐沙班(PTCT2 = 1.3μM)相比,仍存在差距。本论文进而对抗凝活性较好的化合物进行了 FXa抑制活性的测定。FXa能够促使发色底物S-2222裂解成多肽和对硝基苯胺,对硝基苯胺在405 nm处有紫外吸收。同样选取了rivaroxaban作为阳性对照药,由酶标仪测定出目标化合物在不同浓度下对吸光度的影响,并通过软件计算出所设计化合物对FXa的抑制率及IC50值。测试结果显示:个别化合物的活性较好,例如化合物H2、H14和H15,其IC50值分别为0.82、0.55和0.52μM,但与阳性对照药利伐沙班(IC50=5nM)尚存在差距。总之,基于靶标的合理药物设计及分子模拟,设计并通过定向合成得到了吡咯烷酮类、噻二嗉类和吲哚环类三个系列具有新型骨架的FXa抑制剂,尽管抗凝实验和酶活实验结果显示所设计的目标化合物活性未达到先导化合物利rivaroxaban的水平,但是通过研究分析构效关系,对我们更深入的研究和设计FXa抑制剂具有指导意义。
[Abstract]:Cardiovascular and cerebrovascular disease is a general name for a disorder of the circulatory system caused by heart or vascular lesions. It is the leading cause of death worldwide, especially in Asia, and poses a serious threat to human life and health. Thromboembolic diseases such as myocardial infarction, stroke, and deep venous thrombosis have become a whole. The formation of thrombus is one of the most important diseases in the world, so the formation of thrombus is the most important factor causing cardiovascular and cerebrovascular diseases. However, there are still many defects such as Hua Falin, such as inconvenient, unpredictable anticoagulant fruit, narrow treatment window, and many foods and drugs. Therefore, it is urgent to develop a more safe and effective anticoagulant drug.Xa factor (FXa), which is located at the intersection of endogenous coagulation pathways and exogenous coagulation pathways. If it can inhibit FXa, two clotting pathways can be blocked simultaneously. Therefore, FXa has become an attractive attraction in the development of anticoagulant drugs. Target. At present, some oral small molecule FXa inhibitors have been sold on the market, such as Lev Shaaban (rivaroxaban), opito Shaaban (apixaban) and idegree Shaaban (edoxaban). Although the new FXa inhibitors overcome the shortcomings of traditional anticoagulants, these drugs still have some defects, such as the stenosis of drug indications, and the loss of the drugs. The function of liver and kidney and the lack of effective antidote when bleeding. In view of the effectiveness and safety of the inhibitor itself, it is urgent to develop a more safe and effective new FXa inhibitor for clinical application. In this paper, the crystal structure of Lev Shaaban -FXa complex was studied and analyzed by the study of the crystal structure of the lefore Shaaban complex, and the discovery of F Xa has two most critical combined pockets, which are specific binding pocket S1 and larger hydrophobic pocket S4, and there are about 80 degrees between the two pockets; therefore, the lev Shaaban molecule is correspondingly divided into three parts: the P1 region, the P4 region and the Linker region.FXa in the P4 region, the specific binding pocket S1 is a smaller hydrophobic pocket, The 5- chlorthiophene group in P1 region of lefal Shaaban was accommodated, and the 5- chlorthiophene group formed a Cl- PI force with the Tyr228 at the bottom of the S1 pocket; the larger hydrophobic pocket S4 residues formed a pion pion accumulation with the benzene ring of the lev Shaaban P4 region; meanwhile, the Linker region of Lev Shaaban and the Gly219 of the FXa were formed. Two hydrogen bonds. Through the analysis of the binding mode of lefore Shaaban and FX a, based on the principle of electron equal row and hetero heterozygosity, the structure of the P4 and Linker regions of Lev Shaaban was reconstructed, such as changing the volume, hydrophobicity of the P4 region, and increasing the force of the Linker region with the FXa binding site, and designed pyrrole. Three series FXa inhibitors of alkanones, thiazoles and indole rings are used to obtain a new type of Xa factor inhibitor with high activity. At the same time, we used the computer aided drug design software Sybyl X 1.3 to study the molecular simulation of the designed series of compounds. The new compound has theoretical rationality. Through the cyclic reaction, acylation reaction, condensation reaction and other experimental steps, three series of target compounds were synthesized. A total of three series of 37 Xa factor inhibitors, pyrroliones, thiazoles and indole rings, were synthesized and confirmed by spectral identification. The anticoagulant activity of the synthetic FXa inhibitor was evaluated by the effect of the compound on the plasma prothrombin time. By using the prothrombin time analyzer, the prothrombin time of the blood poor plasma in different concentrations of the compound was measured, and the "PTCT2" was calculated. There is a certain anticoagulant activity, compared with the pilot compound leavin Shaaban (PTCT2 = 1.3 M), there is still a gap. In this paper, the determination of the FXa inhibitory activity of the compounds with better anticoagulant activity.FXa can cause the chromophore S-2222 to break into polypeptides and p-nitroaniline, and the p-nitroaniline has UV absorption at 405 nm. Rivaroxaban was used as a positive control drug. The effect of the target compound on the absorbance at different concentrations was measured by the enzyme scale, and the inhibition rate and IC50 value of the designed compound to FXa were calculated by software. The test results showed that the activities of individual compounds were better, such as H2, H14 and H15, and their IC50 values were 0.82,0.55 and 0.52 u M, respectively. But there is still a gap with the positive control drug Lev Shaaban (IC50=5nM). In a word, based on the target's rational drug design and molecular simulation, three series of pyrroliones, thiazoles and indole rings, which have a new skeleton of FXa inhibitors, are designed and carried out by directional synthesis, although the anticoagulant experiment and the results of enzyme activity experiments show the design of the IC50=5nM. The activity of the target compound does not reach the level of the lead compound, rivaroxaban, but by studying the structure-activity relationship, it has a guiding significance for us to further study and design the FXa inhibitors.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914;R96

【参考文献】