【作者】 贡建志；

【导师】 徐文方；

【作者基本信息】 山东大学 ， 药物化学， 2010， 博士

【摘要】 目的意义神经氨酸酶(Neuraminidase, NA)是甲型和乙型流感病毒侵染、复制、成熟和释放等过程中的关键酶,其作用至关重要。与血凝素(Hemagglutinin,HA)和基质蛋白M2相比,神经氨酸酶具有成为药物靶点的独特优势,其活性位点与唾液酸的结合口袋比血凝素的更深。通过设计高亲和力的小分子化合物,模拟其催化反应的过渡态,即可以实现对神经氨酸酶的强力抑制,而这种小分子的化合物在后期药物开发中更具吸引力。另外,因其活性位点在甲、乙型流感病毒中均高度保守,设计研发神经氨酸酶抑制剂类药物可以同时治疗甲、乙型流感。这都使得神经氨酸酶成为抗流感病毒药物研究中的理想靶点。1999年,FDA先后批准两个神经氨酸酶抑制剂(zanamivir和oseltamivir)用于流感的预防和治疗。Zanamivir由于口服生物利用度低,需要吸入给药,给儿童和老人用药造成很大的不便;oseltamivir口服给药,但因产能有限,其用药成本一直居高不下。且两者均已对部分病毒株产生耐药甚至交叉耐药。因此,研究高效、低毒的新型神经氨酸酶抑制剂是当前抗流感病毒药物研发的重要方向,具有重大的临床意义和广阔的市场前景。本研究以神经氨酸酶为靶点,通过分析底物或抑制剂与酶活性位点的相互作用,借助计算机技术,运用基于结构药物设计方法,结合“类肽”策略对前期合成的化合物进行优化并设计新结构骨架的化合物,经体外初步活性筛选,以期发现具有较好神经氨酸酶抑制活性的先导化合物。研究方法基于前期合成的小分子类肽吡咯烷类化合物具有一定程度的神经氨酸酶抑制活性,在对其进行构效关系研究的基础上,进行分子结构优化：①吡咯环氮原子上的侧链用更多类型的氨基酸及其类似物取代,构建类肽结构：②将侧链上结构不稳定的N-叔丁氧羰基替换为N-酰基,尤其是大多数神经氨酸酶抑制剂中都存在的N-乙酰基；③保留活性测试表现较好的C-2位羧基和C-4位S-氨基,设计了一系列新型的N-酰基吡咯烷类化合物。分析已上市的oseltamivir与神经氨酸酶活性位点的相互作用模式,结合课题组前期实验基础,发现了具有良好研究前景的硫杂螺环骨架。通过对L-羟脯氨酸进行合理修饰：①C-4位衍生为疏水性的硫杂螺环;②在吡咯环仲胺氮原子上引入多种类型的氨基酸及其类似物,构建类肽结构,期望氨基能与酶活性位点的负电中心发生相互作用；③C-2位衍生为甲酯、羧基、羟肟酸、酰肼等基团,期望能与酶活性位点的正电中心发生静电相互作用,设计了一系列新型的硫杂螺环类化合物。两个系列目标化合物的合成均以L-羟脯氨酸作为起始原料,该原料经济易得,实验过程中所涉及的路线科学合理,得到的两个系列64个新型吡咯烷及硫杂螺环类化合物,其结构经过核磁共振氢谱、高分辨质谱和红外光谱确证,均未见国内外报道。本研究使用商品化的神经氨酸酶抑制剂筛选试剂盒,以有荧光特性的化合物为酶促反应底物,建立了适合筛选小分子神经氨酸酶抑制剂的荧光分析法,对所合成的目标化合物进行了初步的体外活性筛选。研究结果体外抑酶活性试验显示,所合成的化合物对神经氨酸酶均具有一定程度的抑制活性。第一系列的吡咯烷类化合物表现出较好的活性,第二系列的硫杂螺环类化合物活性相对较差。其中,第一系列的化合物G10g、G101、G10m、第二系列的化合物M12f、M14f、M16f可以继续进行结构修饰和优化。以所合成的化合物为对象,利用Tripos/SYBYL软件包对其进行了构效关系研究。通过分子对接,探讨了两个系列化合物与神经氨酸酶活性区域的作用模式;采用比较分子力场分析(CoMFA)方法构建了硫杂螺环类化合物的定量构效关系(QSAR)模型,结合模型给出的等势线图对其构效关系进行了探讨。所构建的CoMFA模型具有较好的交叉验证系数和一定的预测能力。研究结论本研究以神经氨酸酶为靶点,在前期工作的基础上,设计、合成了两个系列具有全新结构的小分子类肽目标化合物,原料经济易得,技术路线合理可行。经体外抑酶活性筛选,发现了具有进一步研究价值的先导化合物。探讨了抑制剂分子与酶的作用位点及可能的作用模式,并建立了具有一定预测能力的CoMFA模型,对化合物的进一步结构改造具有指导意义,对神经氨酸酶抑制剂类抗流感病毒药物的研究进行了有益的探索。更多还原

【Abstract】 Objectives Neuraminidase (NA, EC 3.2.1.18) is one of two major surface glycoproteins on both type A and B influenza viruses and is essential for viral replication and infection in vitro. NA promotes virus entry into host cells during the initial stage and facilitates the release of the newly formed virions from the infected cells at the final stage of viral replication. The high conservation (up to 75%sequence variation) around the enzyme active site in influenza A and B viruses makes NA an attractive target on the rationale of structure-based drug design (SBDD) for the development of antiviral agents. Inhibition of NA by complexation with the active site could lead to potent molecules for having a broad-spectrum activity against various flu strains.Recently, several potent and specific NA inhibitors have been developed, among which zanamivir and oseltamivir phosphate have been approved for the treatment and even prophylaxis of human influenza virus infection. Zanamivir has low bioavailability and requires topical administration using disk inhaler technology, which can cause problems in patients with underlying respiratory disease. In contrast, oseltamivir phosphate is administered as capsules with good compliance and has very high bioavailability and long half-life. However, on the account of the emergence of drug-resistant variants and some undesirable side effects, and the high cost for clinical use, it is very necessary and urgent to develop novel NA inhibitors with different scaffolds than zanamivir and oseltamivir phosphate.Basd on the detailed understanding of the molecular interactions involved in the binding of substrate or various inhibitors to the NA active site, we optimized compounds previous synthesized and designed another series with novel scaffold, with the strategy of structure-based drug design, and "peptidomimetics". After the preliminary enzyme inhibition assay in vitro, it is expected to find promising lead with novel scaffold which possess potential NA inhibitory activity.Methods In our previous study, a series of Boc-amino acid-containing pyrrolidine derivatives with potent NA inhibitory activities in vitro was described. The structure-activity relationship (SAR) information for the variation of substituents on the pyrrolidine ring was also related with anti-influenza activity. These preliminary results can provide some guidelines for further lead modification and optimization of these pyrrolidine-based NA inhibitors.In order to improve the bioactivity and explore the SAR of exocyclic side chain, some novel pyrrolidine derivatives were designed by following these strategies:(ⅰ) more natural amino acids and their analogues were introduced to the nitrogen atom of the pyrrolidine ring, (ⅱ) N-Boc-protecting group was replaced by different N-acyl groups, especially acetyl group, which almost exists in all potent NA inhibitors, (ⅲ) the functional essential carboxyl group in position 2 and amino group in position 4 were still maintained.According to analyze the interaction of oseltamivir and the NA active site, combined to the former experimental basis, we found that 1,4-dithia-7-azaspiro[4.4] nonane maybe a promising scaffold for developing new type NA inhibitors. In order to improve its affinity, we optimized the structure of 1,4-dithia-7-azaspiro[4.4] nonane based on L-hydroxyproline with the following chemical modifications:(ⅰ) the hydroxy group was transformed to thioketone to construct the 1,4-dithia-7-azaspiro [4.4]nonane scaffold, (ⅱ) various natural amino acids or their analogues were introduced to the nitrogen atom of the pyrrolidine ring, (ⅲ) the carboxyl group was kept or converted to other derivatives such as methyl ester, hydroxymate or hydrazine.Using commercial available L-hydroxyproline as starting material, we synthesized two series of L-hydroxyproline derivatives. The structures of target compounds were identified by ~1H-NMR, HR-MS and IR spectra.Preliminary evaluation of the target compounds was determined by the commercial NA inhibitory screening kit. This fluorescence method is suitable for the high-throughput screening of NA inhibitors in vitro.Results The NA inhibition assay showed that the activity of pyrrolidine derivatives was improved by comparing with the previous compounds, but not better than the positive control, oseltamivir. The 1,4-dithia-7-azaspiro[4.4]nonane derivatives had relatively weaker activity than pyrrolidines, but there is still large space for this series to improve its potency.With the aid of Tripos/SYBYL package, we also studied the structure-activity relationship of the target compounds. The interaction model of the pyrrolidine derivatives with the NA active site were discussed by using molecular docking approach. The quantitive structure-activity relationship (QSAR) model of 1,4-dithia-7-azaspiro[4.4]nonane derivatives were construtcted by means of the comparative molecular field analysis (CoMFA) method. The CoMFA model had good cross-validated coefficient (q2) and predictive potency. The contour map (steric and electrostatic) derived from the CoMFA model could give some guidelines for further structral modifications.Conclusions In summary, we have described the synthesis and properties of two series of pyrrolidine and 1,4-dithia-7-azaspiro[4.4]nonane derivatives with novel chemical structures as influenza NA inhibitors. Some of the target compounds were shown to possess potent influenza NA inhibitory activity. These feasible schemes were more convenient and economical for the synthesis of potential NA inhibitors. We also established a consistent QSAR model which was critical to predictive structure-based drug design and discovering potent compounds that would potentially be useful for antiviral therapy.更多还原