【摘要】：本論文由兩個部分組成：第一部分是以丙型肝炎病毒(Hepatitis C Virus, HCV)非結(jié)構(gòu)蛋白NS5A抑制劑GL100953為先導(dǎo)化合物，設(shè)計合成了新型苯并噻唑類抗HCV化合物，并對其生物活性進行了測試和評價；第二部分是發(fā)現(xiàn)了一種新穎的多取代苯類化合物合成方法，即以噻唑烷二酮或羅丹寧構(gòu)建的螺環(huán)化合物為底物，經(jīng)氫氧化鉀處理一步構(gòu)建得到多取代苯類化合物。 一、新型苯并噻唑類HCV NS5A抑制劑的設(shè)計、合成和抗HCV活性研究 全球目前約有1.7億人感染了HCV病毒，且感染發(fā)病率還在逐年遞增，已經(jīng)成為一種受到高度重視的健康危害。如得不到有效治療，HCV感染還會導(dǎo)向發(fā)展為肝硬化和肝癌。目前主要使用利巴韋林和聚乙二醇干擾素聯(lián)合治療HCV感染，但是該治療方案能發(fā)揮效用的基因型有限，效果不佳，并容易引起耐藥。非結(jié)構(gòu)蛋白NS5A是HCV RNA復(fù)制必不可少的關(guān)鍵病毒蛋白，基于NS5A研發(fā)抗HCV新藥是目前的熱點領(lǐng)域。 本研究課題以HCV NS5A抑制劑GL100953為先導(dǎo)化合物，開展了苯并噻唑類新型抑制劑的設(shè)計、合成和抗HCV活性研究。首先，對先導(dǎo)化合物GL100953的苯基噻唑母核進行了骨架躍遷研究，發(fā)現(xiàn)了苯并噻唑類化合物GL110509的抗HCV抑制活性優(yōu)于先導(dǎo)化合物GL100953。其次，對化合物GL110509的酰胺部分進行構(gòu)效關(guān)系研究，設(shè)計合成了酰胺類(A01-A13)、胺類(B01-B02)、磺酰胺類(C01-C03)和雙磺酰胺類(D01)四類化合物。其中，化合物B02、C03和D01的抗HCV抑制活性最為突出，IC50值分別為1.46mol·L-1、5.76mol·L-1和2.99mol·L-1。在此基礎(chǔ)上，對雙磺酰胺衍生物D01進行結(jié)構(gòu)優(yōu)化，發(fā)現(xiàn)2-氟衍生物D02具有最佳的抗HCV抑制活性和選擇性，其抑制活性達到納摩爾級(IC50值為486.2nmol·L-1)，并且細胞毒性低(CC5050μmol·L-1)。在2-氟衍生物D02的基礎(chǔ)上，進一步優(yōu)化另一側(cè)脯氨酸上取代基。發(fā)現(xiàn)去除芐氧羰基后所得化合物D15抑制活性(IC50=0.4mol·L-1)與D02(IC50=0.49mol·L-1)基本相當，說明芐氧羰基對化合物的抑制活性貢獻不大。但在脯氨酸上引入3-苯丙�；�，，化合物D19的活性比D02提高了約2.5倍，IC50值達到0.19μmol·L-1是所有目標化合物中抗HCV抑制活性最強的。此外，D19的細胞毒性非常低，CC50值大于50μmol·L-1，選擇性指數(shù)SI大于357，是一個比較有前景的先導(dǎo)化合物，值得進一步結(jié)構(gòu)優(yōu)化。由于目前還沒有NS5A相關(guān)分子水平篩選模型的報道，本課題組初步建立了基于表面等離子共振(SPR)的檢測方法，可用于評價化合物與NS5A的相互作用。 二、五取代苯類化合物的合成新方法研究 多取代苯類化合物是常見且應(yīng)用廣泛的功能分子，常用作化工產(chǎn)業(yè)中重要的中間體和有機材料，并且普遍存在于藥物中。但是現(xiàn)有多取代苯類化合物的合成方法存在諸多缺陷，例如反應(yīng)條件苛刻，需要使用過渡金屬或重金屬催化劑參與反應(yīng)，相對合成成本較高，多采用甲苯、乙腈等有毒溶劑等。 本課題組前期基于噻唑烷二酮或羅丹寧構(gòu)建了螺環(huán)化合物。進一步研究發(fā)現(xiàn)以氫氧化鉀為堿，EtOH/H2O混合液(1:1)為溶劑，可經(jīng)一步反應(yīng)合成五取代苯類化合物。該方法具有較高的收率(50.26%~73.26%)，并且具有良好的底物適用范圍，使用的氫氧化鉀廉價易得，使用的溶劑較為綠色環(huán)保。該方法不僅提供了一種全新的、經(jīng)濟的、綠色環(huán)保的多取代苯類化合物合成方法；而且新合成的多取代苯類化合物同時具有醛基、酚羥基和酰胺基三種活潑性的反應(yīng)基團，可進行進一步衍生化，因此也可作為重要的醫(yī)藥和染料化工原料進行開發(fā)。 綜上所述，本研究共設(shè)計合成得到64個以苯并噻唑為母核的抗HCV新化合物，并完成了一項多取代苯合成新方法學研究。本論文的創(chuàng)新點在于：(1)設(shè)計合成得到了新型苯并噻唑類NS5A抑制劑，發(fā)現(xiàn)多個化合物的抗HCV活性達到了nM級。尤其是化合物D19具有高效低毒的特點，為開發(fā)抗HCV創(chuàng)新藥物提供了高質(zhì)量的先導(dǎo)結(jié)構(gòu)，為NS5A相關(guān)的化學生物學研究提供了新穎的分子探針；(2)發(fā)展了一項多取代苯合成新方法，具有反應(yīng)效率高，綠色環(huán)保，易衍生化等優(yōu)點。
[Abstract]:This paper consists of two parts: the first part is a novel benzothiazole anti-HCV compound designed and synthesized with GL100953, a non-structural protein NS5A inhibitor of hepatitis C virus (HCV), as the lead compound, and its biological activity was tested and evaluated; the second part is the discovery of a novel polysubstituted benzene. Polysubstituted benzenes were synthesized by one-step reaction with thiazolidinedione or rhodanine as substrates.
Design, synthesis and anti HCV activity of a new benzothiazole HCV NS5A inhibitor
There are about 170 million people infected with HCV worldwide, and the incidence of infection is increasing year by year, which has become a highly valued health hazard. Without effective treatment, HCV infection will also lead to cirrhosis and liver cancer. Non-structural protein NS5A is a key viral protein essential for replication of HCV RNA. The development of new anti-HCV drugs based on NS5A is a hot area at present.
In this study, a novel benzothiazole inhibitor GL100953 was designed, synthesized and its anti-HCV activity was studied. Firstly, the skeleton transition of the parent nucleus of benzothiazole GL100953 was studied. It was found that the anti-HCV inhibitory activity of benzothiazole GL110509 was better than that of the precursor GL110509. Compound GL100953. Secondly, the amides of GL110509 were studied by structure-activity relationship. Four compounds were designed and synthesized, including amides (A01-A13), amines (B01-B02), sulfonamides (C01-C03) and disulfonamides (D01). On this basis, the structure of disulfonamide derivative D01 was optimized. It was found that the 2-fluoride derivative D02 had the best anti-HCV inhibitory activity and selectivity. Its inhibitory activity reached nanomolar level (IC50 value 486.2 nmol L-1), and its cytotoxicity was low (CC5050_ 50_ micromol L-1). On the basis of the 2-fluoride derivative D02, the other derivative D02 was further optimized. It was found that the inhibitory activity of D15 (IC50 = 0.4mol L 1) was similar to that of D02 (IC50 = 0.49mol L 1), indicating that benzyloxycarbonyl had little contribution to the inhibitory activity of the compounds. However, when 3-phenylpropionyl was added to proline, the inhibitory activity of D19 was about 2.5 times higher than that of D02, and the IC50 value was 0. In addition, the cytotoxicity of D19 is very low, the CC50 value is greater than 50 micromol. L-1 and the selectivity index SI is greater than 357. It is a promising lead compound and deserves further structural optimization. A method based on surface plasmon resonance (SPR) was developed to evaluate the interaction between compounds and NS5A.
A new method for the synthesis of two, five substituted benzene compounds
Polysubstituted benzenes are common and widely used functional molecules, often used as important intermediates and organic materials in the chemical industry, and are ubiquitous in drugs. However, the existing synthesis methods of polysubstituted benzenes have many drawbacks, such as harsh reaction conditions, need to use transition metals or heavy metal catalysts. The reaction costs higher relative synthesis cost, such as toluene, acetonitrile and other toxic solvents.
The spirocyclic compounds were synthesized from thiazolidinedione or rhodanine in the previous stage. Further studies showed that PENTASUBSTITUTED benzenes could be synthesized by one-step reaction using potassium hydroxide as the base and EtOH/H2O mixture (1:1) as the solvent. The method had high yield (50.26%~73.26%) and good substrate application. This method not only provides a new, economical and environmentally friendly synthesis method of polysubstituted benzenes, but also has three active reaction groups, aldehyde group, phenolic hydroxyl group and amide group, which can be further derived. Therefore, it can also be used as an important pharmaceutical and Dyestuff Chemical raw material.
In summary, 64 new anti-HCV compounds with benzothiazole as the parent nucleus were designed and synthesized, and a new method of synthesizing polysubstituted benzenes was completed. Compound D19 has the characteristics of high efficiency and low toxicity, providing a high-quality lead structure for the development of innovative anti-HCV drugs, and providing a novel molecular probe for NS5A-related chemical and biological research; (2) A new synthetic method of multi-substituted benzene has been developed, which has the advantages of high reaction efficiency, green environmental protection and easy derivation.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R91;R914.5

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