Design, Synthesis And Preliminary Activity Assay Of Novel Neuraminidase Inhibitors Of Influenza Virus

ObjectivesInfluenza is an acute viral infection of the upper respiratory tract that can affect millions of people each year. Recently, the global outbreak of H5N1 (high pathology avian flu) and H1N1 (swine flu) has heightened pandemic attention, thus the anti-influenza therapy became an attractive area of drug discovery.The development of influenza neuraminidase inhibitors (NAIs) is the major breakthrough in the control of influenza. Several potent and specific NA inhibitors have been developed, among which zanamivir (Relenza, by Glaxo Wellcome/Biota) and oseltamivir (Tamiflu, by Roche/Gilead) have been approved for the treatment and prophylaxis of human influenza virus infection. Beside, peramivir, which was developed by Biocryst, has been authorized for Emergency Use in 2009 H1N1 influenza, allowing the use of the drug in intravenous form for hospitalized patient only in case where the other available treantment are ineffective or unavailable.Neuraminidase (NA, sialidase) is one major surface glyprotein expressed by both type A and type B influenza virus. Its major function is catalyzing the cleavage of sialic acid (SA) residues from glycoprotein, glycollipid and oligosaccharide via the oxocarbonium intermediate. It is thought to promote virus entry, and thereby enhances infection efficiency, and rationally designed NA inhibitors (NAIs) that prevent release of progeny virions away from infected cells are effective for the treatment of influenza. Additionally, the structure of the active site is highly conserved across all nine influenza A NA subtypes and influenza B subtypes, which makes NA to be a high-priority and promising target for anti-influenza agents.In our study, two series of novel neuraminidase inhibitors were designed, synthesized and evaluated for their preliminary enzyme inhibition in vitro, aiming to discover novel and potent NA inhibitors as lead compounds.MethodsIn our initial screening, we found that the 2-amino-4-thiazole-acetic ester and 2-phenyl-thiazolidine-4-carboxylic acid exhibited substantial NA inhibitory activity. Considering that there haven't been any thiazoles or thiazolidines reported as neuraminidase inhibitors, we chose them as the lead compounds for further modification and optimization.To contain different substituents to interact with the four binding pockets of the NA active sites, we performed the following chemical modifcations to 2-amino-4-thiazole-acetic ester:(1) amino group at C-2 position was coupled with a few kinds of Boc-protected amino acids with different hydrophobic side chains; Boc-protected amino group was kept or be converted to free amino group; (2) carboxyl ethyl ester at C-4 position was kept or converted to carboxylic group.For 2-phenyl-thiazolidine-4-carboxylic acid, the following chemical modifications were performed:(1) benzaldehydes with different sustituents and 2-furaldehyde were used to couple with L-cysteine hydrochloride; (2) The thiazolidines were alkylated with chloroacetyl chloride and phenylacetyl chloride; (3) The chlorides were converted to amines.ResultsAll the target compounds were identified by 1H-NMR, and HR-MS spectra, and tested for their ability to inhibit NA. Preliminary result showed that most of the compounds displayed enhanced inhibitory activities compared to the lead compounds, although not so good as the positive control (oseltamivir carboxylate). Thiazole derivatives L4d, L5d, L8d and thiazolidine derivatives Y4b, Y4c, Y4f exhibited relatively potent activities and could be used as lead compounds for further optimization.We also conducted structure-activity research of the target compounds via computer assisted software. The quantitive structure-activity relationship (QSAR) model of thiazolidine derivatives was construtcted by means of the comparative molecular field analysis (CoMFA) method. The QSAR model we built may guide the structure optimization in the future.ConclusionIn summary, two series of novel neuraminidase inhibitors were designed, synthesized and evaluated for their preliminary NA inhibitory activity. Most of the target compounds were shown to possess increased influenza NA inhibitory activities compared to the lead compounds. The most potent compound is Y4f, which is about 7-fold less potent than oseltamivir carboxylate. These result showed that thiazole and thiazolidine derivatives could be developed as novel NA inhibitors. Moreover, the compounds that showed good NA inhibitory activities could be further modified as lead compounds to develop more potent and novel influenza NA inhibitors.We also established a consistent 3D-QSAR model which was critical to predictive structure-based drug design and potent compounds discovery, that would potentially be useful for further study.