Design, Synthesis And Preliminary Activity Assay Of L-Hydroxyproline Derivatives As Neuraminidase Inhibitors

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:(â…°) more natural amino acids and their analogues were introduced to the nitrogen atom of the pyrrolidine ring, (â…±) N-Boc-protecting group was replaced by different N-acyl groups, especially acetyl group, which almost exists in all potent NA inhibitors, (â…²) 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:(â…°) the hydroxy group was transformed to thioketone to construct the 1,4-dithia-7-azaspiro [4.4]nonane scaffold, (â…±) various natural amino acids or their analogues were introduced to the nitrogen atom of the pyrrolidine ring, (â…²) 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.