| Influenza is an acute respiratory disease caused by influenza virus, resulting in millions of deaths every year, which is a major threat to human life and health. In recent years, due to the continuous variation of the influenza virus, cases of human infected highly pathogenic avian influenza viruses, such as H5N1 and H7N9, frequently emerged, which caused great panic in society. Neuraminidase (NA) is an important glycoprotein located on the surface of the influenza virus. It can cleave the glycosidic bonds of sialic acids from glycoproteins of target cells to promote the release of progeny virus during viral infection and can prevent progeny virus from enriched at the same time. NA can also hydrolysis the sialic acids in the respiratory mucosa to promote the spread of the virus in the respiratory tract. NA plays an important role in the virus replication and infection. Although the subtypes of NA are numerous, but a sequence containing more than ten amino acids is highly conserved in all influenza A and B viruses, which is an important theoretical basis for NA inhibitor design.Since zanamivir and oseltamivir were approved, NA inhibitors have become an important measure against influenza virus. Peramivir and laninamivir were approved in several countries later. NA has also become an important target for anti-influenza virus drug design, and a variety of structural types of NA inhibitors were designed and synthesized. Besides, there are many natural product molecules also have been reported to inhibit NA.In recent years, due to the NA-inhibiting drugs were widely used in clinical, drug-resistant virus strains have appeared. Because oseltamivir is an oral formulation, the most widely application leads to the most serious viral resistance. Although zanamivir and peramivir are administered by inhalation or intravenous injection, but resistant strains still begin to appear. Therefore, the development of novel, efficient and safe anti-influenza virus NA inhibitors is still the hot area of research.Hepatitis C is a liver disease caused by hepatitis C virus (HCV), may evolve to severe liver disease such as chronic hepatitis, cirrhosis and liver cancer. Approximately 180 million people have infected with HCV worldwide, causing hundreds of thousands of deaths each year, which has became another major threat to human health. There is no vaccine can effectively prevent HCV infection. NS3/4A is an important function protein of HCV possessing serine-like protease activity, and can cleave polyprotein precursors of HCV to form mature NS3-NS5B protein, which plays an important role in the replication and infection cycle of virus. NS3/4A is an important target for anti-HCV drug design. Since the triple therapy of boceprevir/telaprevir and interferon in combination with ribavirin is approved in the US for clinical application, more structurally diverse NS3/4A inhibitors have been reported, some of which have already in clinical studies. Given the side effects and drug resistance of the first generation inhibitors, development of safe, effective novel NS3/4A protease inhibitors are urgent.Methods:This study consists of two parts:Part 1 influenza virus NA inhibitors:We designed two series inhibitors, including chalcone-like non-classic NA inhibitors and phenyl acetic acid NA inhibitors. The first series:A lot of natural products have been reported to inhibit NA. On the basis of flavonoids NA inhibitors pharmacophore model pre-established by our group, we designed a series of compounds containing 2,4-aminophenol fragments by a ligand-based drug design method. Firstly, we introduced the aromatic-substituted β-acrylic acid side chain at C-4 position through amide bond. Secondly, in the C-2 position, alkyl or aromatic group were introduced to generate non-classical NA inhibitors with a chalcone-like skeleton.The second series:The crystal structure and its main amino acid in the active site of influenza virus NA has been elucidated, based on this, we designed and synthesized a series of phenyl acetic acid NA inhibitors using structure-based drug design method. The rigid structure of the benzene ring is considered to be the main factor that limits the activity, so we use phenyl acetic acid skeleton to increase flexibility. Taking account of the structure and position of the side chain in the benzoic acid NA inhibitors, we took 2-(3,4-diamino phenyl) acetic acid as a basic structural fragment. In order to increase the selectivity of the inhibitors, we modified amino-group at C-3 position to introduce a hydrophobic side chain.Part 2 HCV NS3/4A protease inhibitors:Through literature research, we chose linear peptide as backbone, acyl sulfonamide group as the basic pharmacophore to design novel inhibitors targeting NS3/4A protease, which were S-configuration triazole or amide bond at P2 position.Results:We assayed the activity of the two series of influenza virus NA inhibitors and one series of HCV NS3/4A protease inhibitor in vitro. The activity results of NA inhibitors showed that the activity of chalcone analogues NA inhibitors was poor, but the two compounds (A5) and (A13) with the best activity can still be further optimization as a lead compound. Phenyl acetic acid inhibitors NA activity results showed that the activity of this series was much better than A series, and compound (B9) had the best activity. Several compounds with alkylated amino group showed moderate NA subtype selectivity, among which (B9) displayed the best. Its activity against H5N1 was more than 10 times better than H9N2. NS3/4A protease inhibitor activity results showed that all the compounds had moderate inhibiting activity, and compound (C1) and (E3) were the best. In addition, we use SYBYLX-2.0 software to explore structure-activity relationship and the mode of the inhibitors action with enzyme active site.Conclusion:This study designed and synthesized several series of compounds targeting influenza neuraminidase and HCV NS3/4A protease, and examined the primary biological activity in vitro. Although the activity of the chalcone analogues is not good enough, the further study on structure-activity relationship on this type of inhibitors had important guiding significance. Phenyl acetic acid skeleton is novel for NA inhibitor design, and the activities of these compounds were potent. More importantly, they displayed selectivity for different NA subtypes, which provided a new direction to develop benzene ring NA inhibitors. Given the grim current anti-influenza virus situation, our research has important practical significance, r Although the activityNS3/4A protease inhibitors in this pape is not superior to the control telaprevir, the study of structure-activity relationship on chiral center provided a new way for the further development of such inhibitors. |
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