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

Partâ… : Advances in influenza virus NA and NA inhibitorsInfluenza is an acute viral infection of the upper respiratory tract that can affect millions of people every year. H5N1 is a type of virulent influenza in birds that is a pandemic threat causing viral disease in many species including humans. Despite these facts, effective and safe anti-influenza therapeutics are lacking, making anti-influenza therapy a high-priority and attractive area of drug discovery.In recent years, virology studies have resulted in an improved understanding of the replication mechanism of the virus. Several molecular targets have been identified for drug intervention, including hemagglutinin (HA), neuraminidase (NA), M2 protein and endonuclease. Vaccines against influenza virus are often ineffective due to the rapid emergence of mutant viral antigens. The antiviral drugs amantadine and rimantadine, which block the M2 protein ion channel, are effective only against type A influenza and produce undesirable side effects, and resistant mutants are rapidly generated. Treatments were limited before the development of influenza NA inhibitors (NAIs), which lead to a major breakthrough in the control of influenza. It is an attractive target for antiviral intervention because its active site is antigenically conserved among all clinically relevant strains.Neuraminidasem (NA), also called sialidase, is one of the two major surface glyproteins expressed by both type A and type B influenza virus, along with hemagglutinin. NA catalyses the cleavage of sialic acid (SA) residues from glycoproteins, glycolipids and oligosaccharides via the oxocarbonium intermediate. This catalytic activity is essential for influenza virus replication and infectivity since it liberates viruses from infected cells and prevents virus entrapment by respiratory secretions and virus self-aggregation. The cleavage of SA by NA is required for elution of newly synthesized virons from infected cells, whereas binding of HA to SA-containing receptors on the surface of host cells is essential for the fusion of influenza virus into the host cells. In addition, NA also facilitates the movement of the virus through the mucus of the respiratory tract. The catalytic activity of NA is essential for influenza virus replication and infectivity. It has been considered a suitable target for designing agents against influenza viruses.The high resolution X-ray crystal structures of NA complexed with a variety of inhibitors have been reported. These structures provided a detailed understanding of molecular interactions involved in the binding of various inhibitors to NA and formed the foundation for several successful rationale drug design programs, which lead to a variety of highly potent NA inhibitors (NAIs). Furthermore, the active site of NA was found to be highly conserved across all influenza A and B virus strains, rendering broad spectrum anti-influenza agents possible.The NAIs have currently emerged as promising therapeutics for the treatment of influenza. The approval of two such inhibitors of NA, Relenza (Zanamivir, by Glaxo Wellcome/Biota) and Tamiflu (Oseltamivir, by Roche/Gilead), further underscores the importance of NA as a valid anti-influenza drug target. Another two inhibitors, RWJ-270201 and A-315675 are undergoing phase III trials in North America and Europe.Partâ…¡: General thought in compound designWe firstly screened all the pyrrolidine derivatives in our compound library we synthesized before. The pharmacological result showed that N-acetyl-L-hydroxyproline exhibited modest activity against influenza virus A (H3N2) neuraminidase (IC₅₀= 48.73μM) and could be used as lead compound in further.In order to improve the affinity of the lead compound, we optimized the structure of N-acetyl-L-hydroxyproline with following chemical modifications: (i) N-acyl group in pyrrolidine ring was changed to other Boc protected or free amino acid residues; (ii) 2-carboxylic acid can be kept or converted to other derivatives such as methyl ester or hydroxymate; (iii) keep hydroxyl in 4-position or converted to free amino group.In our ongoing work, we wanted to use benzene ring to replace pyrrolidine ring and studied the substituted benzoic acid derivatives. And the pharmacological result showed that p-aminosalicylic acid (PAS), one antibacterial agent, exhibited modest activity against influenza virus A (H3N2) NA (IC₅₀= 10.27μM) and could be used as lead compound in future.Considering the SAR of lead compound (BANA 113, 4-acetamido-3-guanidine benzoic acid), we designed and synthesized several novel aromatic inhibitors of NA from commercially available PAS. In order to improve the affinity of lead compounds, we optimized the structure of PAS with the following chemical modifications: (i) C-1 carboxylic acid was kept or converted to other derivatives such as methyl ester or hydroxymate; (ii) C-2 hydroxy group in aromatic ring was changed to various phenolic ether in order to increase the hydrophobic interaction with NA; (iii) hydrogen at C-3 position was kept or replaced with amino group; (iv) amino group at C-4 position was acetylated; and (v) hydrogen at C-5 position was converted to free nitro group or amino group or guanido group.Partâ…¢: Synthesis of neuraminidase inhibitorsThe pyrrolidine derivatives possessing NA inhibitory activities were prepared from trans-4-hydroxy-L-proline. The starting materials, Boc-protected amino acids and the 4-hydroxy-L-proline methyl ester hydrochloride were prepared according to literature. The Boc-amino acids were activated with DCC and HOBt and then coupling with 4-hydroxy-L-proline methyl ester hydrochloride to yield A1-F1. The methylester A1-F1 was then hydrolyzed with NaOH/H₂O or treated with NH₂OK to generate obtain relative carboxylic acids A2-F2 or hydroximic acids A3-F3.4-Amino-pyrrolidine derivatives were prepared from intermediate A1-F1. Firstly, the hydroxyl group of A1-F1 were converted to mesyl group by methanesulfonate, and then reacted with sodium azide to generate configuration inversed azide. Amino derivatives A4-F4 can be synthesized by hydrogenation using Pd-CaCO₃. The methyl ester A4-F4 can be transformed to corresponding carboxylic acids A5-F5 and hydroximic acids A6-F6 as the methods mentioned before.The Boc-protected group can easily be removed with 3 N HC1 in ethyl acetate to give hydrochloride salts of pyrrolidine derivatives G1-G6. Finally, the amino group was acetylated with acetic anhydride to yield H1-H6.The salicylic acid derivatives possessing NA inhibitory activities were prepared from p-aminosalicylic acid (PAS). Benzoic acids J5-Q5 were synthesized from commercially available PAS. The methyl ester was prepared to avoid side-reactions of the carboxylate group. Then methyl ester underwent selective acetylation on the 4-amino group using acetic anhydride to provide amide. Selective alkylation of hydroxyl group with various alkylogen in the presence of K₂CO₃ or NaH gave intermediate J1-Q1 respectively. The compound, on nitration with fuming HNO3 and glacial acetic acid at 0℃gave nitro derivatives J2-Q2. Reduction of the nitro groups of J2-Q2 proceeded without problem using transfer hydrogenation, following the procedure described by Singh et al. Meanwhile the synthesis of J4-Q4 was accomplished in 60% yield by reaction of J3-Q3 with cyanamide and HC1. Hydrolysis of the methyl esters J4-Q4 with NaOH/H₂O yielded the target compounds J5-Q5.The structures of target compounds were identified by IR, ESI-MS and ¹H-NMR spectra.Partâ…£: Study on the inhibitory activities against NAAll the pyrrolidine compounds (54 compounds) were tested for their ability to inhibitory neuraminidase. Preliminary result showed that all the compounds displayed inhibitory activities with IC50 value from 1.56 to 90.79μM. Compound 35-F5 with NH2 group and methionine as hydrophobic side chain showed the best inhibitory activity (IC₅₀=1.56μM). Three compounds containing isoleucine (9-B3, 11-B5,12-B6) exhibited good activities (2.10～2.71μM). Two compounds (51-I3 and 54-I6) had lower IC₅₀ value (8.54μM and 3.57μM), but they possibly had anti-inflammatory activity. Generally, the compounds with NH₂ and -CONHOH group showed better activities than that with OH and -COOH group.And the preliminary result showed that 40 salicylic acid derivatives displayed inhibitory activities with IC50 value from 0.032μM to 9.26μM. Compound 59-J5 with two guanidine group at C-3 and C-5 and ethyl as hydrophobic side chain showed the best inhibitory activity (IC₅₀=0.032μM). The other five compounds containing guanidine(58-J4,59-J5,64-K5,79-N5,84-05 and 89-P5) exhibited good activities (0.036μM～0.049μM). Generally, the compounds with guanidine at C-5 and carboxyl group at C-1 showed better activities.In conclusion, we have described the synthesis and properties of a series of pyrrolidine and p-aminosalicylic acid derivatives as influenza NA inhibitors. All of the compounds were shown to possess potent influenza NA inhibitory activity, and the most potent compound of the series is compound 35-F5 (IC₅₀=1.56μM), which in addition to good enzyme inhibitory activity, displays potent anti-viral activity in vitro. We reported a more convenient and economical method of the synthesis of pyrrolidine and p-aminosalicylic acid NA inhibitors. Compared to the other research, 4-hydroxyproline and p-aminosalicylic acid we used appeared to be an ideal starting material because of its low cost and commercial abundance. 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. The compounds we have got all showed potent NA inhibitory activity, and this finding could further be used to design influenza NA inhibitors.