A Novel Potent and Highly Specific Inhibitor against Influenza Viral N1

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A Novel Potent and Highly Specific Inhibitor against Influenza Viral N1N9 Neuraminidases: Insight into Neuraminidase-inhibitor Interactions Nongluk Sriwilaijaroen, Magesh Sadagopan, Akihiro Imamura, Hiromune Ando, Hideharu Ishida, Miho Sakai, Erika Ishitsubo, Takanori Hori, Setsuko Moriya, Takeshi Ishikawa, Kazuo Kuwata, Takato Odagiri, Masato Tashiro, Hiroaki Hiramatsu, Kenji Tsukamoto, Taeko Miyagi, Hiroaki Tokiwa, Makoto Kiso, and Yasuo Suzuki J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01863 • Publication Date (Web): 20 Apr 2016 Downloaded from http://pubs.acs.org on April 21, 2016

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Journal of Medicinal Chemistry

A Novel Potent and Highly Specific Inhibitor against Influenza Viral N1N9 Neuraminidases: Insight into Neuraminidase-inhibitor Interactions

Nongluk Sriwilaijaroen,*,†,∞,∆ Sadagopan Magesh,‡,◊,∆ Akihiro Imamura,‡ Hiromune Ando,‡,§ Hideharu Ishida,‡ Miho Sakai,∥ Erika Ishitsubo,∥ Takanori Hori,∥ Setsuko Moriya,⊥ Takeshi Ishikawa,#,◊ Kazuo Kuwata,∇ Takato Odagiri,○ Masato Tashiro,○ Hiroaki Hiramatsu,∞ Kenji Tsukamoto,¶,◊ Taeko Miyagi,⊥ Hiroaki Tokiwa,*,∥,× Makoto Kiso,*,‡,§ and Yasuo Suzuki*,∞

†

Department of Preclinical Sciences, Faculty of Medicine, Thammasat University,

Pathumthani 12120, Thailand, ‡

Department of Applied Bioorganic Chemistry, #Division of Prion Research, Center for

Emerging Infectious Disease, and ∇United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan, §

Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-

8501, Japan, ∥Department

of Chemistry, and ×Research Center for Smart Molecules, Rikkyo University,

Tokyo 171-8501, Japan, ⊥Division

of Cancer Glycosylation Research, Institute of Molecular Biomembrane and

Glycobiology, Tohoku Pharmaceutical University, Sendai 981-8558, Japan, ○

Influenza Virus Research Center, National Institute of Infectious Diseases, Tokyo 208-0011,

Japan, ∞

Health Science Hills, College of Life and Health Sciences, Chubu University, Aichi 487-

8501, Japan, ¶

Research Team for Zoonotic Diseases, National Institute of Animal Health, Ibaraki 305-

0856, Japan,

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ABSTRACT People throughout the world continue to be at risk for death from influenza A virus, which is always creating a new variant. Here we present a new effective and specific anti-influenza viral neuraminidase (viNA) inhibitor, 9-cyclopropanecarbonylamino-4-guanidinoNeu5Ac2en (cPro-GUN). Like zanamivir, it is highly effective against N1-N9 avian and N1N2 human viNAs, including H274Y oseltamivir-resistant N1 viNA, due to its C-6 portion still being anchored in the active site, different from the disruption of oseltamivir’s C-6 anchoring by H274Y mutation. Unlike zanamivir, no sialidase inhibitory activity has been observed for cPro-GUN against huNeu1-huNeu4 enzymes. Broad efficacy of cPro-GUN against avian and human influenza viruses in cell cultures comparable to its sialidase inhibitory activities makes cPro-GUN ideal for further development for safe therapeutic or prophylactic use against both seasonal and pandemic influenza.

Keywords: influenza; viral neuraminidase; 9-pentanoylamino-4-guanidino-Neu5Ac2en; 9cyclopropanecarbonylamino-4-guanidino-Neu5Ac2en; drug resistance; human neuraminidase


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INTRODUCTION Influenza A viruses efficiently infect cells with balance assistance of hemagglutinin (HA) that binds to a specific sialyl surface receptor, which is essential for initiating viral entry into the host cell, and neuraminidase (viNA) that catalyzes sialic acid removal, which is critical for viral spread to the next host. All of the 16 identified HA and 9 identified NA subtypes have been isolated from avian hosts except for new subtypes H17, H18, N10 and N11 from H17N10 and H18N11 viruses found only in bats, being clearly different from all known influenza viruses.1,2 Only H1N1, H2N2 and H3N2 subtypes of all 198 possible subtypes have emerged and adapted to human hosts; however, only H1N1pdm09 and H3N2 have continued to be detected in humans.3-5 While influenza epidemics continue to threaten human health annually, outbreaks of avian influenza viruses, especially subtypes H5 and H7, in humans have been occasionally reported and could spark a pandemic leading to serious illness and a high death rate followed by social disruption and economic loss.5-7 Control of this worrisome zoonotic virus requires integration of an understanding of influenza virus properties and drugs/vaccines that would effectively eliminate all strains of influenza viruses. An anti-influenza virus drug is important for prevention and treatment of influenza due to the limitation of influenza vaccine potential. Emergence of adamantane-resistant mutants (mostly having S31N substitution in the M2 protein) among avian and human influenza A viruses together with central nervous system side effects limit the use of adamantanes.8,9 There has been extensive development of drugs targeting viNA, resulting in several effective derivatives as shown in Figure 1: (i) substrate-like αNeu5Ac (1)-based (dihydropyran) derivatives including Neu5Ac2en (DANA; 2),10 4-guanidino-Neu5Ac2en (zanamivir; GUN; 3),11,12 9-O-octanoyl-7-O-methyl-4-guanidino-Neu5Ac2en (prodrug laninamivir octanoate; 4), 7-O-methyl-4-guanidino-Neu5Ac2en (active metabolite laninamivir; 5),13 9-pentanoylamino-4-guanidino-Neu5Ac2en (Pen-GUN; 6 in this study) and 9-cyclopropanecarbonylamino-4-guanidino-Neu5Ac2en (cPro-GUN; 7 in this study); (ii) ACS Paragon Plus Environment


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cyclohexene derivatives including ethyl ester prodrug oseltamivir phosphate (OP; 8) and active metabolite oseltamivir carboxylate (OC; 9);14 and (iii) a cyclopentane derivative, peramivir15 (10). Three of these viNA-targeting drugs have been approved by the U.S. Food and Drug Adminstration (FDA), and inhaled zanamivir, oral oseltamivir phosphate and intravenous peramivir have become the main drugs recommended for influenza treatment and prophylaxis.16 Another viNA-targeting drug undergoing clinical trials for FDA approval17 is inhaled laninamivir octanoate with long-lasting antiviral activity. Laninamivir octanoate18 was approved in Japan for influenza treatment in 201019 and for prophylaxis in 2013.20 Inhaled zanamivir is preferred over oral oseltamivir for some patients such as patients with poor gastrointestinal absorption, and intravenous zanamivir (unapproved but available if requested to the GlaxoSmithKline manufacturer for compassionate use individually) is preferred for patients with impending respiratory failure.21 Seasonal influenza A (H1N1) virus strains developed resistance to oseltamivir, and one strain with an H274Y mutation in viNA (Amino acid numbering of viNA is based on N2 numbering throughout this article.) that is transmissible among untreated people became predominant globally in 2007-2008.22 In early 2009, a novel influenza A (H1N1) pandemic virus emerged, and oseltamivir-resistant H274Y H1N1pdm09 variants have been increasingly detected.3,23 H274Y substitution in viNA of H5N1 viruses has also been detected in humans who died from H5N1 virus infection.24 H1N1pdm09 virus with H274Y substitution in 2 immunocompromised patients rapidly emerged during a short course of oseltamivir therapy and was reported to lead to clinical failure of intravenous peramivir for a reduction in viral shedding in one of these cases.25 These H1N1, H1N1pdm09 and H5N1 variants are still susceptible to zanamivir.16,25,26 Variants resistant to zanamivir, such as H3N2 harboring a Q136K mutation in viNA,27 have rarely been detected. Neuropsychiatric adverse reactions in patients treated with oseltamivir have been reported.28 Inhibitory activities against human neuraminidases (huNeus) by zanamivir have been reported.29,30 The huNeus play pivotal roles in several biological processes:31 lysosomal


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Neu1 plays a role in lysosomal function,32 cytosolic Neu2 plays a role in cytoskeletal functions,33 integral membrane protein Neu3 plays a role in cell transformation,34 differentiation35 and cell contact,36 and inner membrane-associated Neu4 plays a role in mitochondrial apoptosis and lysosomal function.37,38 There is therefore a need for new specific and effective anti-influenza drugs that are less prone to resistance. Differences in human host-virus neuraminidase active sites provide us with ample opportunities for rational development of DANA to novel specific viNA inhibitors (NAIs), Pen-GUN and cPro-GUN (Scheme 1). cPro-GUN has shown strong anti-sialidase and antiinfluenza activities against all N1-N9 avian influenza virus subtypes recently isolated from ducks or a mallard (N10 subtype from bat influenza virus lacks sialidase activity.39) and against N1, N1pdm09 and N2 influenza virus clinical isolates including amantadine-resistant S31N and oseltamivir-resistant H274Y mutants. The structural basis of cPro-GUN that disfavors binding to huNeus and favors binding to both wild-type and mutant forms of viNA has been revealed.

RESULTS Active-site sequences and structures of neuraminidases of influenza A virus and humans: implications for selective antiviral drug design. The highly conserved amino acids in the viNA active site are compared with active-site huNeus in Figure 2a (full sequence alignment of viNA and huNeus shown in Figure S1). Only 8 viNA residues exhibit significant homology with the counterparts of huNeus: 5 homologous amino acids (R118, R292, R371, D151 and Y406 in viN2 numbering) in catalytic sites that are important for catalytic function and the other 3 homologous amino acids (R156, E277 and E425 in viN2 numbering) in framework sites generating the appropriate geometry for enzyme catalysis. Structural comparison of viN2 NA (PDB: 1ivf)40 with huNeu2 (PDB: 1vcu)41 active sites in complex with DANA in Figure 2b indicates two regions with highly variable amino acids that could ACS Paragon Plus Environment


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distinguish binding preference between viNA and huNeu, which are aligned with the C-5 and C-6 positions of the bound DANA. In agreement with amino acid differences, there are striking differences in charge distribution between viNA and huNeu2 in the C-5 and C-6 binding regions as shown in Figure 2c: more positive potential at the mouth of the viNA crevice than that of the huNeu2 crevice coordinates C-5 and C-6 portions of DANA. The C-5 acetamido group and the opposing C-1 carboxyl group anchor DANA into the active site and were maintained in all reported potent sialidase inhibitors.42 Focusing on the C-6 glycerol side chain composed of the C-7, C-8 and C-9 hydroxyl groups of the DANA template, the C-9 position is promising for modification to generate a derivative for inhibition of viNAs but not huNeus, since there is a striking difference between viNA (Arg, Ile, Glu and Asn) and huNeu (Tyr, Leu and Gln) binding pockets. Our previous studies43,44 showed that modifications at the C-9 position on the C-6 portion of DANA can provide inhibitors with higher selectivity for viNAs over huNeus than their reference DANA. We replaced the C-9 hydroxyl group with a carbonylamino group to maintain interactions of the C-9 position with Glu or Asn in viNAs but reduce interactions with Gln or Tyr in huNeus. Based on the capacity of the C-6 binding pocket of viNAs mainly via Ile or Pro to accommodate hydrophobic interactions,14 the carbonylamino group was linked with a range of hydrophobic groups that are different in size and geometry (linear, branched, and cyclic) including C1 methyl, C3 propyl, isopropyl and cyclopropyl, C4 2-methylpropyl, n-butyl and tert-butyl, C5 2-ethylpropyl and cyclopentyl and C6 phenyl groups. Of this carbonylamino linked hydrophobic series, 9cyclopropanecarbonylamino-Neu5Ac2en displayed the most potent inhibition selectively against all tested N1, N2 and N3 viNAs with IC50 values ranging from 10 to 100 µM against viNAs, from 700 to 800 µM against huNeus1 and 4, and more than 1,000 µM against huNeus2 and 3, whereas the reference DANA produced IC50 values ranging from 1 to 10 µM against the viNAs and from 50 to 150 µM against huNeus1, 2, 3 and 4. Although connection of the n-butyl group to the carbonylamino group giving 9-pentanoylamino-4-guanidino-


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Neu5Ac2en derivative showed less potency than 9-cyclopropanecarbonylamino-Neu5Ac2en against the viNAs, its inhibitory activities were not observed at the highest tested concentration of 1,000 µM against huNeus2, 3 and 4. Replacement of the C-4 hydroxyl group of DANA with the bulky basic guanidinyl group, giving 4-guanidino-Neu5Ac2en (GUN, which has the generic name zanamivir), is known to improve inhibitory activity against viNAs.12 Here the bulky basic guanidinyl group was introduced into the C-4 position of S8 and S9 (Scheme 1) since it could be stabilized in negative charges contributed to by Asp and Glu in this C-4 viNA binding pocket and it would be unfavorable to be buried in the huNeu corresponding binding pocket with Arg and Met or Ile (Figure 2). Taken together, the two factors of (i) different environments including amino acid residues and charge distributions around the C-4 and C-6 binding pockets in the viNA and huNeu enzymes and (ii) greater capacity of the viNA binding pockets than that of the huNeu equivalent pockets to accommodate different inhibitors provide us with an opportunity to logically develop new DANA-based inhibitors holding the C-4 guanidinyl group in combination with the C-9 carbonylamino linked to an n-butyl or to cyclopropyl group, named Pen-GUN and cPro-GUN, respectively (Scheme 1), that exhibit interesting features of broad-spectrum inhibition against all viNAs but not their human counterparts, different from zanamivir (details below), thereby being effective and safe for human application. Inhibition of human neuraminidases. Inhibitory activities of zanamivir and GUN derivatives against four recombinant huNeu1-huNeu4 are shown in Figure 3a. Zanamivir showed significant inhibitory activities against huNeu2, huNeu3 and huNeu4 with IC50 values in the micromolar range: mean IC50 values of 15.33 µM for huNeu2, 8.17 µM for huNeu3, and 392.00 and 1043 µM for huNeu4 when 2′-(4-methylumbelliferyl)-α-D-5-Nacetylneuraminic acid (4MU-Neu5Ac) and Neu5Acα2-3Galβ1-4Glcβ1-1'Cer (GM3) were used as substrates, respectively. Neither of the GUN derivatives displayed inhibitory activity against any huNeu even at a concentration of 1 mM with either 4MU-Neu5Ac or GM3 as a



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substrate, suggesting that they cannot cause the adverse effect of huNeu-associated host toxicity in treated humans. Structural analysis of inhibitor binding to huNeus. Similar to interactions with Neu2, which is in agreement with a previous report,30 zanamivir forms salt bridges with Arg and Glu, forms hydrogen-bond networks with Arg, Glu, Tyr and Asp, and undergoes other interactions including electrostatic interactions and van der Waals interactions with active-site residues of huNeu3 and huNeu4 (Figure 3b). The huNeu1-zanamivir complex revealed steric interference between nonpolar side chains of Leu139 and Ile79 and positively charged side chain of Arg97 with the C-4 bulky basic guanidinyl group of zanamivir. Docking analysis (Figure 3b) demonstrated binding of cPro-GUN to huNeus was limited by steric hindrance between Leu139, Ile79 and Arg97 in huNeu1, and Arg41 and Met85 in huNeu2, Ile26 and Arg45 in huNeu3, and Arg43 in huNeu4 with the C-4 basic guanidinyl side chain of cProGUN and between Asn261 in huNeu1, Tyr181 and Gln270 in huNeu2, Arg114 in huNeu3 with the carbonyl cyclopropane part of the C-6 side chain of cPro-GUN. This, together with the energy requirement to generate negatively charged and neutral regions for proper binding of the bulky basic C-4 side chain and the hydrophobic part of the C-6 side chain, respectively, cooperatively make huNeus resistant (reduce binding) to cPro-GUN compound. This study should pave the way for designing anti-viNA agents that can bypass their binding to human enzyme counterparts. Inhibition of influenza virus neuraminidases. The sialidase inhibitory activities of GUN derivatives were investigated in parallel with those of zanamivir and OC. Figure 4a shows curve comparison of sialidase inhibitory activities of each compound against each viNA subtype principally divided into two phylogenic groups: group-1 with an open 150-cavity (V149): N1, N4, N5 and N8; group-1 lacking a 150-cavity (I149): N1pdm09; and group-2 with a closed 150-cavity (I149 with a D147-H150 salt bridge): N2, N3, N6, N7 and N9. The IC50 values obtained after nonlinear curve fitting are shown in Figure 4b. Zanamivir appeared


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to be more effective than OC against both group-1 viNAs (mean IC50 values in the ranges of 0.47-5.53 nM for zanamivir and 2.01-9.20 nM for OC). In contrast, OC is more effective against group-2 viNAs (mean IC50 values in the ranges of 1.60-11.51 nM for zanamivir and 0.35-6.94 nM for OC). GUN derivatives are similarly effective against both groups of viNAs; the sialidase inhibitory activities of cPro-GUN were comparable to those of zanamivir against group-2 viNAs and to those of OC against group-1 and group-1/pdm09 viNAs with mean IC50 values ranging from 2.37 to 46.16 nM, approximately 15-243-times lower than those of PenGUN having mean IC50 values ranging from 109.20 to 5,253.00 nM. cPro-GUN, which showed strong inhibitory activities against all N1-N9 NA subtypes, could be used not only against epidemic/pandemic influenza but also for prevention of the next pandemic. Structural analysis of inhibitor binding to N1 through N9 viNAs. Interaction energy between residues in the viNA active site and each inhibitor (Table S1) showed that most of the active site residues of N1 to N9 NAs have more favorable binding interactions with cProGUN than with Pen-GUN. Analysis of all viNA-inhibitor binding energies confirmed that binding of cPro-GUN has stronger affinity than that of Pen-GUN to all viNA subtypes, in agreement with their anti-sialidase activities (Figure 4c). An overview of structures of cProGUN binding to N1-N9 active sites shown in Figure 4d indicated that cPro-GUN forms variation in shape related to the shape of the active site pocket of each NA subtype, suggesting that cPro-GUN and each viNA active site are flexible enough to be reshaped on the basis of their interactions until there is a perfect fit. Simple diagrams of hydrophobic, hydrophilic and H-bond interactions of cPro-GUN within the N1-N9 active sites are provided in Figure 4d. Different interactions of cPro-GUN with N1-N9 NA subtypes could be due to variability in the viNA sequences between subtypes directly or indirectly affecting reorientation of active site residues to obtain optimal architecture for favorable accommodation of the inhibitor. Interactions between Pen-GUN and N1-N9 NAs are shown in Figure S2.



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Inhibition of influenza virus wild-type and mutant neuraminidases. One of the major problems of combating influenza is rapid generation by the causative influenza RNA virus of variants not only to escape host immunity but also to compromise anti-influenza drugs. In accordance with a previous report,45 the mean IC50 value in µM (4.48 µM) obtained for OC against A/Kitakyushu/10/2006 (H1N1) with H274Y NA mutation was more than 1,000-fold higher than that in nM (2.77 nM) against A/Yamaguchi/20/2006 (H1N1) carrying wild-type NA, whereas zanamivir was equally effective in nM against both viNAs (2.50 nM for A/Kitakyushu/10/2006 and 1.19 nM for A/Yamaguchi/20/2006) (Figure 4b). The GUN derivatives were found to remain highly active against H274Y-N1 NA. The mean IC50 values were 670.30 nM for Pen-GUN and 93.82 nM for cPro-GUN compared to those of 213.30 nM for Pen-GUN and 13.83 nM for cPro-GUN against A/Kitakyushu/10/2006 and A/Yamaguchi/20/2006, respectively. Therefore, cPro-GUN might be useful as an alternative to oseltamivir for treating H274Y-N1 NA-carrying influenza viruses. Structural analysis of inhibitor binding to wild-type and mutant neuraminidases. Approximately 240-times greater decrease in anti-sialidase activity of OC than that of compared to cPro-GUN against wild-type (A/Yamaguchi/10/06) to H274Y (A/Kitakyushu/20/06) N1 NAs (Figure 5a) could be deduced from inhibitor-NA interactions as follows. In agreement with previous reports,26,46 structural interactions between OC and wild-type/H274Y-N1 NAs shown in a static 2D image in Figure 5b and diagrammatically detailed in Figure 5c revealed that the bulkier Y274 substituent makes E276 with the charged carboxyl group move about 1.32 Å inward (red arrow in Figure 5b) into the NA active site, causing disruption of the hydrophobic environment normally formed in the wild-type N1 NA for accommodation of the C-6 hydrophobic pentyloxy side chain of OC. cPro-GUN bound to wild-type and H274Y-N1 NAs (Figure 5b and 5c) revealed that the C-8 hydroxyl group of cPro-GUN retains a hydrogen bond with the charged carboxyl group of E276 with closer distance in the H274Y-N1 variant. Although the H274Y mutant pushes S246 about 0.78 Å


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further (blue arrow in Figure 5b) from the binding site, resulting in loss of S246 bonding with the C-8 hydroxyl group on cPro-GUN, the carbonyl group on cPro-GUN can provide compensation by forming hydrogen bonds with R152. The cyclopropane group of cPro-GUN appears to make contact with a network of hydrophobic faces of S246 and I222 residues in the active sites of both wild-type and H274Y-N1 NAs. In the case of Pen-GUN, its detailed interactions with the active sites of wild-type and H274Y-N1 NAs differ from those of cProGUN; notably, its hydrophobic n-butyl part is compensated by reorientation of D347, and H274Y makes the carboxyl group of D151 move 1.40 Å inward and react with the C-7 hydroxyl group of Pen-GUN, resulting in loss of bonding between the amino group of R292 and the carbonyl group of Pen-GUN (Figure S3). However, all of the C-2, C-4, C-5 and C-6 side chains of GUN derivatives retain interactions contributing to anchor them into the H274Y-N1 active site. Figure 5d shows that anti-influenza activities of OC and cPro-GUN against A/Yamaguchi/10/06 and A/Kitakyushu/20/06 viruses are in good agreement with antisialidase activities (Figure 5a). Inhibition of replication of avian and human influenza viruses in cell culture. By using a cell counting kit-8, GUN concentrations in the range of 0.064 to 1,000 µM were shown to have no deleterious effects on MDCK and modified MDCK cells with a high level of α2,6linked sialic acids on the cell surface (AX4 cells). By using a combination of quantitative fluorescence-based and qualitative chromogenic-based virus growth inhibition methods, it was shown that anti-avian influenza virus activity was produced in MDCK cells by Pen-GUN with IC50 values in the micromolar range (IC50 = 0.45 to 47.68 µM), by cPro-GUN with IC50 values in the submicromolar range (IC50 = 11.85 to 395.70 nM), and by OC and zanamivir with IC50 values in the nanomolar range (IC50 = 0.85 to 28.22 nM for OC and IC50 = 2.73 to 65.00 nM for zanamivir) against avian influenza viruses of all N1-N9 NA subtypes (Table 1). In agreement with results for MDCK cells, in AX4 cells, cPro-GUN exhibited higher antihuman influenza virus activity than that of Pen-GUN (IC50 values: 24.70-389.60 nM for Pen-



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GUN versus 1.27-27.65 nM for cPro-GUN) but lower activity than that of zanamivir (IC50 values of 0.61-4.37 nM) and OC (IC50 values of 0.04-1.71 nM) against H1N1/2009, H1N1/2006 and H3N2/2008 viruses carrying NA wild types (Table 1). OC showed remarkably elevated anti-influenza activity in comparison with its anti-sialidase activity against H3N2/2008 viruses (mean H3N2 IC50 values: 4.35 nM in enzymatic assay versus 0.06 nM in cell-based assay), suggesting that its other possible effect(s) producing synergistic inhibition with its anti-sialidase effect may be involved in its anti-influenza virus (H3N2) activity. In agreement with results of the enzymatic assay, OC showed more than 1,000-fold reduction in its anti-influenza virus activity against A/kitakyushu/20/2006 (H1N1) carrying H274Y NA (IC50 = 3.91 µM) compared to that against A/Yamaguchi/10/2006 (H1N1) with NA wild type (IC50 = 1.71 nM). Pen-GUN and cPro-GUN displayed anti-influenza activities with IC50 values of 389.60 and 27.65 nM, respectively, against the NA wild-type virus and with IC50 values of 337.35 and 106.02 nM, respectively, against the H274Y-N1 virus. The efficacy of cPro-GUN to reduce virus titer and reduce virus distribution to neighboring cells warrants it as a good compound of choice for further development to treat influenza virus infection.

DISCUSSION Safety and broad efficacy to counter all highly mutable influenza RNA viruses are key goals of anti-influenza drug development. Remarkable differences in amino acid sequences and structures of the active sites of viNAs and endogenous huNeus including shape and electrostatic potential distribution of/in their cavities allow us to successfully discover GUN derivatives with specific binding to viNAs. Important structural characteristics of GUN derivatives disfavored by huNeu binding include the large basic C-4 guanidinyl side chain and the bulky C-6 hydrophilic plus hydrophobic side chain causing steric hindrance. Whereas the active sites of huNeus may be not flexible enough to rearrange the conformation to fit the ACS Paragon Plus Environment

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GUN derivatives, the active sites of viNAs can adapt their conformations to bind to the GUN derivatives. Like OC and zanamivir, they showed potency against sialidase activities of all N1 to N9 viNAs of duck/mallard influenza virus isolates and all N1, N1pdm09 and N2 viNAs of human influenza virus isolates. Comparison of their inhibitory activities against different viNA subtypes divided into group-1 viNAs (N1, N1pdm09, N4, N5 and N8) and group-2 viNAs (N2, N3, N6, N7 and N9) showed that i) zanamivir is more potent than OC against group-1 viNAs but less potent than OC against group-2 viNAs, in agreement with results of a previous study.47 This could be explained by the notable structural difference between two NA groups at the 150-cavity (residues 147-152) adjacent to the active site. Group-1 viNAs with an open cavity containing a Val149 hydrophobic side chain pointing away from the cavity would be more preferable for entry of zanamivir with the bulky basic C-4 guanidino group and the hydrophilic C-6 glycerol side chain before undergoing an open-to-closed conformational change. On the other hand, group-2 NAs without the cavity carry a D147-H150 salt bridge and Ile149 with the hydrophobic side chain pointing toward the cavity could scaffold more favorable binding of OC with the small basic C-4 amino group and the hydrophobic C-6 pentyl ether group. N1pdm09 NA contains Ile149 but no D147-H150 salt bridge48 and thus, the 150 loop of the N1pdm09 NA is sufficiently flexible to accommodate zanamivir access. ii) cPro-GUN showed potency nearly equivalent to that of OC against group-1 viNAs and potency approximately equal to that of zanamivir against group-2 viNAs, which could be explained by its structure. As in the case of OC and zanamivir, GUN derivatives retain the C-1 carboxylate group and the opposing C-5 acetamido group. Like zanamivir, they bear a C-4 guanidinyl group. Their C-6 side chain retains hydrophilicity with hydroxyl groups at C-7 and C-8 positions but has modification of the hydroxyl group at the C-9 position with a carbonylamino group. Like OC, the C-6 side chain has an additional hydrophobic group with an n-butyl group or a cyclopropyl group next to the carbonylamino group.



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It is notable that cPro-GUN, different from Pen-GUN only in that a butyl group in Pen-GUN is substituted with a cyclopropyl group, showed more than 100-times high sialidase inhibitory activity, in agreement with results of docking studies indicating that viNA binding by cPro-GUN has higher negative energy (high affinity) than binding by Pen-GUN. Possible explanations are: (i) the linear alkyl n-butyl group poorly fits in the viNA active site because of the size of linear alkyl chain, (ii) unlike the small alkyl ring, the cyclopropane group, which is a rigid planar molecule that should be entropically favorable, resulting in increase in binding affinity, the linear alkyl chain, n-butyl group, is more flexible and thus suffers a higher loss in entropy upon binding to the viNA binding pocket, and (iii) rearrangement of the side chains of the charged amino acid residues located in the NA active sites for the highly lipophilic structure of the n-butyl group should pose geometric constraints. The development of resistance in influenza viruses reduces drug efficacy and limits the use of drugs. New anti-influenza drugs that are effective against the currently increasing H274Y-N1 viruses and the sustained S31N-M2 viruses49 and less prone to resistance are needed for controlling influenza. GUN derivatives were found to inhibit sialidase activity of A/Kitakyushu/10/2006 (H1N1) harboring the H274Y viNA mutant without a significant difference in its inhibitory activity, whereas OC showed more than 1,000-fold decrease in its inhibitory activity in comparison to its activities against A/Yamaguchi/20/06 (sensitive strain) and A/Narita/1/2009 (amantadine-resistant strain). This is in accordance with the results of a previous study showing that H274Y mutation in N1 viNA, but not in N2 viNA, exhibits a great decrease in susceptibility to OC.50 Exploring structural interactions of GUN derivatives/OC with the H274Y-N1 viNA variant and with the corresponding wild-type N1 viNA revealed that the bulkier Y274 in N1 viNA forces the carboxylate side of the E276 face near to the hydrophobic 3-pentyl ether on the C-6 side chain of OC, and thus OC could not bind effectively to the H274Y N1 viNA, in agreement with previous findings.14,26 GUN derivatives carrying the corresponding C-6 side chain with hydroxyl, hydroxyl and


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carbonylamino groups at C-7, C-8 and C-9, respectively, can interact with hydrophilic groups in the H274Y N1 viNA active site. Connected to the carbonylamino group of the GUN derivatives, the hydrophobic butyl/cyclopropyl group is accommodated in the hydrophobic patch formed by reorientation of S246 and I222 within the N1 viNA of either A/Yamaguchi/20/06 or A/Kitakyushu/10/2006. If a mutation that disrupts this hydrophobic patch occurs, the hydrophilic part of the C6 side chain of GUN could still anchor to the active site and thus GUN derivatives may be less prone to aggressive resistance. The GUN derivatives were proved to have broad efficacy against replication of all subtypes of influenza viruses isolated from avian and human origins in MDCK and AX4 cells, respectively, without cytotoxicity. Less anti-influenza virus activities of those compounds against the avian influenza virus isolates tested in MDCK cells than their anti-sialidase activities tested in the viNA inhibition assay, in agreement with results of previous studies51,52 could be explained by host range restriction of influenza virus replication in MDCK cells. The key viral factors affecting efficient virus replication include balanced functions of receptorbinding HA and receptor-destroying viNA; poor binding of HA to an inappropriate host cell surface results in less requirement of cleaving activity of viNA, leading to less susceptibility of influenza virus to NAIs.53 Using AX4 cells with a large amount of α-2,6 sialic acid receptors matching well with epithelial cells of the human respiratory trachea, virus yield reduction by GUN derivatives, OC and zanamivir showed a good correlation with their viNA specific inhibition as indicated by comparable IC50 values, suggesting that anti-influenza virus activity of these compounds is mainly due to their sialidase inhibitory activity. Of note, OC anti-influenza virus activities were clearly elevated compared to its anti-viNA activities against A/H3N2 viruses isolated in 2008. The viNA activity is naturally influenced by HA binding activity and vice versa, and thus not only viNA structure but also HA structure including amino acid residues forming the HA receptor binding site and the number, location and structure of glycan moieties on the HA molecule could contribute to NAI anti-influenza



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virus activity. Other targets of action of NAI, such as fusion mediated by HA,54 cannot be ruled out. A better understanding of the biological complexity of influenza virus replication and mode of action of NAI may lead to the development of more powerful NAI. Interestingly, it has recently been shown that an HA inhibitor can enhance antiviral activity of an NAI and vice versa, offering a new powerful approach for efficient influenza therapy.55 In conclusion, not only the efficacy of anti-influenza drugs against all influenza virus strains but also safety in treated patients remains challenges. GUN derivatives can efficiently inhibit sialidase activities of wild-type N1-N9 and oseltamivir-resistant viNAs, with cProGUN showing potency similar to that of OC and zanamivir currently in clinical use, but not that of human orthologous enzymes. Moreover, GUN derivatives with the C-6 side chain holding hydrophilic and hydrophobic moieties seem to be less susceptible to resistance. In cell cultures, cPro-GUN has been proved to strongly inhibit viral replication and halt the spread of influenza viruses to neighboring cells. Thus, cPro-GUN is an attractive candidate as a first line of defense in people at high risk for infection and as a safe therapeutic drug.

EXPERIMENTAL SECTION Synthesis of Pen-GUN and cPro-GUN compounds. Summary of synthesis procedures. To synthesize Pen-GUN (6) and cPro-GUN (7), the key compound S1 (5-acetamido-4-azido7,8,9-tri-O-acetyl-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic acid methyl ester) was synthesized using the procedure described previously.56 Then compound S1 underwent multi-step conversion to the target structures (6 and 7) as depicted in the synthetic route (Scheme 1). Hydrogenolysis of the azido compound S1 in the presence of a Lindlar catalyst gave an amine derivative that was directly protected with Boc anhydride to afford compound S2. In order to selectively introduce the required functionality at the C-9 position, O-acetate protecting groups of compound S2 were removed with sodium methoxide


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(CH3ONa) in methanol to give triol S3. O-9 of compound S3 was then selectively protected with p-toluenesulfonyl chloride (TsCl) to afford S4, which was successively converted to an azido group with sodium azide (NaN3) to give compound S5. Staudinger type reduction of compound S5 with trimethyl phosphine (PMe3) gave the intermediate amine, which was successively converted to compound S6 or S7 using respective NHS esters. The t-Boc group of compound S6 or S7 was removed with trifluoroacetic acid (TFA) and subsequently guanylated with N,N'-bis-(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (bis-BocPCH) to give N,N'-di(Boc)-protected quanidino compound S8 or S9. Finally, the target compounds 6 and 7 were obtained by de-protection of methyl ester and t-Boc groups with LiOH and TFA, respectively. NMR and mass spectroscopic data of all synthesized compounds were in agreement with their structures. General procedures. 1H NMR spectra were recorded with a JEOL JNM-ECA600 (600 MHz) spectrometer and 13C NMR spectra were recorded with a Bruker Advance III 500 (125 MHz) spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) and are relative to the central peak of the solvent, which was CDCl3, CD3OD or D2O. In order to confirm assignments, 2D NMR experiments were performed using COSY for proton-proton interactions and using HMQC and HMBC for proton–carbon interactions. High resolution mass spectral (HR-MS) data were obtained in positive or negative ion mode on a Bruker Daltonics microTOF-II with an electrospray ionization (ESI) source. Purity of the targeted compounds was quantitatively determined by HPLC on a Shodex Asahipak NH2P-50 4D column (4.6 mm i.d. x 150 mm) at 40 °C with UV detection at 210 nm. Each compound (5 µg) was eluted with 65% (v/v) CH3CN in 15 mM potassium phosphate (pH 5.2) with a flow rate of 0.5 ml/min. Both target compounds, 6 and 7, had 97.5% purity. Details of synthesis procedures. According to the synthetic route shown in Scheme 1, each compound at each step of the procedure was synthesized to the target compounds (compounds 6 and 7) as detailed below.



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5-Acetamido-7,8,9-tri-O-acetyl-4-(N-tert-butoxycarbonyl) amino-2,3,4,5-tetradeoxy-Dglycero-D-galacto-non-2-enopyranosonic acid methyl ester (S2). After 7.95 g (15 mmol) of compound S1 had been dissolved in 250 mL of ethyl acetate, 4 g of a Lindlar catalyst was added and then Boc anhydride (20 mL) was added to the reaction system. The reaction mixture was stirred under a hydrogen atmosphere of 1 atm for 2 days at 50 °C. After completion of the reaction, the reaction mixture was filtered through Celite, and the filtered product was washed with methanol. The filtrate and washings were combined and distilled off under reduced pressure. The residue thus obtained was purified by silica gel column chromatography (eluant, 100% ethyl acetate) to obtain 9.1 g (98%) of S2 as white foam. 1H NMR (CDCl3) δ 1.41 (9H, s, NHCOC(CH3)3), 1.92, 2.05, 2.07, 2.10 (12H, 4 × s, 3 × OCOCH3, 1 ×NHCOCH3), 3.78 (3H, s, COOCH3 ), 4.13 to 4.23 (3H, m, H-5, H-6, H-9a), 4.46 (1H, td, J 4,NH 9.6 , J 4,3 2.1, J 4,5 9.6, H-4), 4.66 to 4.70 (2H, m, NH4, H-9b), 5.30 (1H, ddd, J 8,7 7.5, J 8,9a 5.5, J 8,9b 2.8, H-8), 5.46 (1H, dd, J 7,8 4.8, J 7,6 1.3, H-7), 5.54 (1H, d, J NH,5

8.9, NH5), 6.90 (1H, d, J 3,4 2.1, H-3); HR-MS (Positive): Calcd. for C23H34N2O12

(M+Na)+, 553.2009, Found 553.2012. 5-Acetamido-4-(N-tert-butoxycarbonyl)amino-2,3,4,5-tetradeoxy-D-glycero-D-galactonon-2-enopyranosonic acid methyl ester (S3). After 5.3 g (10 mmol) of compound S2 had been dissolved in 50 mL of methanol, a catalytic amount of sodium methoxide was added to the reaction mixture at room temperature and the mixture was stirred overnight. After completion of the reaction, the reaction system mixture was adjusted to pH 7 with Dowex50×8 (H+). The reaction mixture was filtered and evaporated to yield a white solid, which was purified by silica gel column chromatography (eluant, 5% methanol in chloroform) to yield 3.03 g (75%) of S3 as a white amorphous solid. 1H NMR (CD3OD) δ 1.44 (9H, s, NHCOC(CH3)3), 1.98 (3H, s, NHCOCH3), 3.57 to 3.67 (2H, m, H-7, H-9a), 3.76 (3H, s, COOCH3 ), 3.81 (1H, dd, J 9b,9a 11.2, J 9b,8 3.1, H-9b), 3.87 (1H, ddd, J 8,7 11.2, J 8,9a 4.5, J 8,9b

2.2, H-8), 4.05 (1H, dd, J 5,4 8.7, J 5,6 9.8, H-5), 4.22 (1H, dd, J 6,5 9.8, J 6,7

A Novel Potent and Highly Specific Inhibitor against Influenza Viral N1

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