Design, synthesis, and biological evaluation of novel 7-deazapurine nucleoside derivatives as potential anti-dengue virus agents

Accepted Manuscript Design, synthesis, and biological evaluation of novel 7-deazapurine nucleoside derivatives as potential anti-dengue virus agents Cai Lin, Jianchen Yu, Muzammal Hussain, Yiqian Zhou, Anna Duan, Weiqi Pan, Jie Yuan, Jiancun Zhang PII:

S0166-3542(17)30317-0

DOI:

10.1016/j.antiviral.2017.11.005

Reference:

AVR 4186

To appear in:

Antiviral Research

Received Date: 24 April 2017 Revised Date:

1 October 2017

Accepted Date: 5 November 2017

Please cite this article as: Lin, C., Yu, J., Hussain, M., Zhou, Y., Duan, A., Pan, W., Yuan, J., Zhang, J., Design, synthesis, and biological evaluation of novel 7-deazapurine nucleoside derivatives as potential anti-dengue virus agents, Antiviral Research (2017), doi: 10.1016/j.antiviral.2017.11.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Design, synthesis, and biological evaluation of novel 7-deazapurine nucleoside derivatives as potential anti-dengue virus agents

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Cai Lin a, c, †, Jianchen Yu b, d, e, †, Muzammal Hussain a, c, Yiqian Zhou a, Anna Duan a, Weiqi Panf, Jie Yuan b, d, e*, Jiancun Zhang a, f*

Guangzhou Institutes of Biomedicine and Heath, Chinese Academy of Sciences, 190 Kaiyuan

Road, Guangzhou, 510530, PR China

Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education,

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b

Guangzhou, China

University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing, 100049, PR China

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c

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Guangdong Province Key Laboratory of Functional Molecules in Oceanic Microorganism (Sun Yat-sen University), Bureau of Education, Guangzhou, China

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Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

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State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Disease, Guangzhou Medical University, Guangzhou, PR China

Address correspondence and reprint request to:

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Jiancun Zhang, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou, 510530, PR China，Tel.: +862032015323; email address: [email protected]

†

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Jie Yuan, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou, Guangdong 510080, China, email address: [email protected]

These authors equally contributed to this work.

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Abstract

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Dengue fever, caused by four distinct serotypes of dengue virus (DENV-1 to -4), has become the fastest spreading human infectious disease in recent years. Despite extensive efforts, there is no specific antiviral treatment approved for dengue until now. Nucleoside inhibitors represent an actively pursued area to develop small-molecule anti-dengue virus agents. In this study, we

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designed and synthesized a series of 7-deazapurine nucleoside derivatives and evaluated their

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anti-DENV activity. Our design strategy and structure activity relationship studies revealed 6e as the most potent inhibitor (EC50 = 2.081±1.102 µM) of DENV replication. 6e suppressed RNA levels and DENV E protein expression, without causing any apparent cytotoxicity in A549 and HepG2 cells (CC50 = 150.06±11.42 µM, SI = 72.11 in A549 cells, and CC50 = 146.47±11.05 µM and SI = 63.7 in HepG2 cells). In addition, 6e showed similar inhibition potency against four

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serotypes of DENV, suggesting that it restrains some evolutionarily conserved targets essential for DENV replication. We conceive that 6e may serve as a promising lead compound for anti-

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DENV drug development.

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Key words: Dengue fever, Anti-DENV, Nucleoside Analogs

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1. Introduction

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Dengue fever, an acute systemic infection caused by dengue virus (DENV), has become one of the most burdensome and fastest spreading human infectious diseases (Shepard et al., 2016). DENV comprises four distinct serotypes (DENV-1 to -4), and represents the most prevalent mosquito-borne viral pathogen in humans. The global public health impact of dengue has rapidly

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increased in recent years, affecting over 2.5 billion people worldwide with an estimated annual

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epidemics of 390 million human infections, of which, 96 million manifest clinically (Bhatt et al., 2013). Over the past 50 years, the global incidence of dengue has grown dramatically and DENV is now endemic in more than 100 tropical and subtropical countries of the world. The year 2015 was particularly marked with worst dengue outbreaks worldwide, as compared to the previous year 2014. Sharp increases in dengue occurrence rates were reported in countries like Philippines

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(169, 000 cases), Malaysia (111, 000 cases), Brazil (1.5 million cases), and India (15, 000 cases only in Delhi), and many other countries continued to record cases until 2016 (WHO, 2016). Different factors, including unplanned rapid urbanization, climate changes and migration, have

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created a perfect storm for dengue expansion (Gubler, 2002; Simmons et al., 2012). According to

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World Health Organization (WHO) reports of 2015-2016, about half of the world’s population is now at risk of dengue infection (WHO, 2016). Therapeutically, there is no specific antiviral treatment approved for tackling rapidly increasing dengue outbreaks, except a recently introduced (in late 2015) first dengue vaccine Dengvaxia® by Sanofi Pasteur (Vannice et al., 2016; World Health, 2017). Dengvaxia has been registered now for use in individuals 9-45 years living in endemic countries. There are also some other vaccine candidates (based on subunit, DNA and purified inactivated virus platforms) at earlier 3

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stages of clinical development (For some recent reviews, see references (Martin and Hermida, 2016; Rothman and Ennis, 2016; Vannice et al., 2016; Wilder-Smith and Yoon, 2016)), which indicates that significant progress has been, and being, made in anti-DENV vaccine

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development. Looking from the perspective of developing anti-DENV antivirals, multiple

attempts have been conducted by both academia and industry over the past decade to identify DENV-specific inhibitors (Lim et al., 2013; Low et al., 2017; Schul et al., 2007). Majority of

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such efforts employed both target-based and cell-based approaches, and have led to the discovery of diverse classes of anti-DENV small-molecule inhibitors with different mechanisms of action,

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including: viral entry inhibitors (Wang et al., 2009; Zhou et al., 2008), capsid inhibitors (Byrd et al., 2013; Scaturro et al., 2014), inhibitors of DENV NS4B (van Cleef et al., 2013; Wang et al., 2015), inhibitors of DENV NS3 protease and helicase (Li et al., 2015; Yang et al., 2014), inhibitors of DENV NS5 methyltransferase (Benmansour et al., 2016; Xu et al., 2016),

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nucleoside and non-nucleoside inhibitors of RNA-dependent RNA-polymerase (RdRp) (Manvar et al., 2016; Yin et al., 2009; Yokokawa et al., 2016), and host target inhibitors (Wang et al., 2011). Nevertheless, no single compound has yet been generated as a clinical candidate, although

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the lessons learnt from all that efforts have provided a better rationale for the on-going anti-

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DENV drug discovery efforts (Lim et al., 2013). Nucleoside inhibitors represent the largest class of antiviral agents, and have been actively pursued for potential anti-DENV therapy in last few years. Many nucleoside inhibitors of DENV have originated from hepatitis C virus (HCV) drug discovery, as both viruses share some structural similarity because of belonging to the same Flaviviridae family (Chen et al., 2015). One particular example for this is the 2’-C-methyl substitution that was initially reported to inhibit HCV replication and then found to have anti-DENV activity as well (Migliaccio et al., 4

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2003). Nevertheless, some of these nucleosides are not sufficiently potent, and have no in vivo mouse activity (Chen et al., 2015).

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Structural modifications at 2’-position on the ribose ring of some nucleoside analogs have been demonstrated to achieve excellent potency against various viruses without interfering with the function of host cell polymerases (Chen et al., 2015; Migliaccio et al., 2003; Olsen et al., 2004).

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For instance, Sofosbuvir® (Fig. 1A) that is a sugar-modified 2’-deoxy-2’-fluoro-2’-C-methyl ribofuranosyl moiety has shown considerable antiviral activity against HCV. Binding of

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diphosphate metabolite of Sofosbuvir (2’-F/2’-CH3-UDP) in the HCV polymerase proved a disruption in the normal hydrogen bonding pattern observed for natural nucleotide substrates and 2’-OH/2’-CH3-containing analogs, which also provides a high barrier to the development of drug resistance (Appleby et al., 2015). But, it was limited to exert antiviral potency as broadly as 2’C-methyl ribofuranosyl moiety, and studies ought to be endorsed in other areas of RNA viruses.

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Similarly, 2’-deoxy-β-fluoro ribonucleosides (Fig. 1B) have been reported to have anti-HCV potency (Nauš et al., 2012; Smith et al., 2009), nevertheless, the data remains elusive for other RNA viruses. On the other hand, 7-deaza-adenine ribonucleosides (such as compounds C and D

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in Fig. 1) have been demonstrated to possess good inhibitory activity against HCV and DENV

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(Di Francesco et al., 2012; Latour et al., 2010). However, NITD008 (Fig. 1C) failed in the preclinical in vivo toxicity studies and was abandoned for further drug development (Chen et al., 2015). Likewise, bioisosteric replacement of the amino group at the 6-position of 7-deazapurine with a methyl group (Fig. 1D) was identified as a potent inhibitor of DENV (Wu et al., 2010), but this compound is nonselective and too toxic for clinical use as an antiviral agent. Based on these preliminary findings from literature, and inspired by our recently reported design strategy (Lin et al., 2016), we combined the two moieties: 2’-deoxy-2’-fluoro-2’-C-2-methyl ribose 5

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sugars and 7-deazapurine bases, together in a single scaffold and then performed structure activity relationship (SAR) studies to reveal a novel class of anti-DENV nucleoside inhibitors. The aim was to design and synthesize nucleoside analogs with improved potency and having less

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cytotoxicity. The first SAR studies prompted us to three novel scaffolds of 7-deazapurine

analogs possessing anti-DENV activity (Fig. 2). Herein, we describe the syntheses and biological

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evaluation of a novel series of anti-DENV nucleoside inhibitors with a reasonable SAR revealed. 2. Materials and Methods

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2.1 Biological materials 2.1.1. Cell lines

The A549, HepG2, and Vero cell lines, purchased from American Type Culture Collection (ATCC), were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10 % fetal bovine serum (FBS) (Hyclone, Logan, UT), 2 mM

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L-glutamine, 100 mg/ml streptomycin and 100 units/ml penicillin (Invitrogen, Carlsbad, CA), at 37 °C and 5% CO2. C6/36 Aedes albopictus cells (ATCC, CRL-1660) 18 were maintained at

2.1.2. Viruses

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28 °C and 5% CO2 in DMEM supplemented with 10% FBS.

The DENV1 (Hawaii Strain), DENV2-NGC (New Guinea C strain), DENV3 (Philippine H87

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Strain), and DENV4 (GZ/9809/2012) were kindly provided by Guangzhou Centers for Disease Control and Prevention and were propagated in C6/36 cells. A monolayer of C6/36 cells was inoculated with DENV at a multiplicity of infection (MOI) of 1, and incubated at 28 °C for about 4 days (in case of DENV2) and 7 days (in case of DENV1, DENV3, and DENV4). The supernatants were collected and clarified by centrifugation at 4 °C, 2000× g for 5 min, and were filtrated with 0.45 µM filters. Virus stocks were titrated by Flow Cytometry-Based Assay in

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C6/36. 2.1.3. Experimental chemistry Reagents were purchased from commercial suppliers and used without further purification unless

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otherwise noted. All synthetic compounds were always dissolved in DMSO (Sigma, China) and prepared for the biological evaluation.

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2.2 Biological experiments

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2.2.1. Cytotoxicity assay

MTT assay was used to determine cytotoxicity of each compound. A549 cells were seeded in 96well flat-bottom plate at a density of 1×104. After 24 h incubation, cells were treated with different concentrations of the corresponding compound in triplicates, and were further incubated

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for 48 h at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS. At the end of the incubation, each well was added by 28 µl of 5 mg/ml MTT solution (Sangon Biotech, Shanghai, China), and then the whole plate was kept at 37 °C for 4 h. Finally, the MTT solution was

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carefully replaced with 200 µl DMSO (Sigma, China), and optical density of the wells was measured at 490 nm Synergy 2 Multi-Detection Microplate Readers (BioTek, U.S.). The

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cytotoxic concentration of compounds that reduced cells survival by 50% (CC50) was determined by GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA). For each value, results were obtained from average of three experiments. 2.2.2. Real-Time Quantitative PCR (RT-qPCR) analysis DENV RNA copy number was measured with the method of RT-qPCR. A549 cells were seeded in 24-well flat-bottom plate at a density of 1×105 cells/well at 37 °C and 5% CO2 in DMEM supplemented with 2% FBS. After 24 h, cells were incubated in the medium consisting of DENV 7

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(MOI of 1) and different concentrations of each compound for 2 h at 37 °C. After incubation, the cells were washed with sterile phosphate buffer saline (PBS) three times and incubated with the medium mixed with different concentrations of each compound, respectively, for further 48 h.

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DENV RNA of each well was extracted from the cell culture supernatant using LogPure Viral DNA/RNA Kit (Magen, Guangzhou, China) by 50 µl RNA-free water. 10 µl of the total RNA extraction was reverse translated with Thermo Scientific RevertAid First Strand cDNA Synthesis

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Kit (Thermo Fisher Scientific, Guangzhou, China) in 20 µl volume system. The real-time RTPCR assay was performed by adding 2 µl of extracted DENV-2 RNA to 18 µl reaction system

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which contained 10 µl Light Cycler® 480 Probes Master (Roche, China), 5 µl ddH2O, 1 µl Forward Primer in 10 µM (DENV1 5’–CAAAAGGAAGTCGYGCAATA-3’, DENV2 5’CAGGCTATGGCACYGTCACGAT-3’, DENV3 5’-GGACTGGACACACGCACTCA-3’, DENV4 5’-TTGTCCTAATGATGCTGGTCG-3’), 1 µl Reverse Primer in 10 µM (DENV1 5’-

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CTGAGTGAATTCTCTCTACTGAAC-3’, DENV2 5’-CCATCTGCAGCAACACCATCTC-3’, DENV3 5’-CATGTCTCTACCTTCTCGACTTGTCT-3’, DENV4 5’TCCACCTGAGACTCCTTCCA-3’), and 1 µl Probe in 10 µM (DENV1 5’-

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CATGTGGTTGGGAGCACGC-3’, DENV2 5’-CTCTCCGAGAACAGGCCTCGACTTCAA-3’, DENV3 5’-ACCTGGATGTCGGCTGAAGGAGCTTG-3’, DENV4 5’-

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TTCCTACTCCTACGCATCGCATTCCG-3’) (Santiago et al., 2013). The amplification was performed using the DNA Engine Option system (MJResearch/Bio-Rad, Hercules, CA) with the following thermal cycling conditions: initial denaturation at 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 sec, 55 °C for 60 sec. The copy numbers of viral RNA in the samples were measured with a standard curve that was generated with a 10-fold serially diluted viral RNA extracted from DENV inoculums of known titer. The standard curve consisted of 6 points of

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concentration ranging from 10-8 to 10-3 ratio of virus inoculums. Each concentration was assayed in triplicate. For each value, results were obtained from average of three experiments.

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2.2.3. Western blot analysis A549 cells were seeded in 6-well flat-bottom plate at a density of 3×105 cells/well at 37 °C and 5% CO2 in DMEM supplemented with 2% FBS. After 24 h, cells were incubated in the medium

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consisting of DENV-2 (MOI of 1) and different concentrations of each compound for 2 h at 37 °C. After the incubation, cells were washed with sterile PBS three times and incubated with

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the medium mixed with the corresponding concentrations of each compound, respectively, for further 48 h. Finally, cells were harvested and lysed in 1× sampling buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM PMSF, 10% glycerol, 6% SDS, 5% mercaptoethanol and 0.1% bromophenol blue before sonication. The protein concentration of the lysate was determined by

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using the Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA) according to its protocol, with BSA as the standard. Proteins were separated by 12% sodium dodecyl sulfate polyacrylmide gel electrophoresis (SDS-PAGE), transferred onto PVDF

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membranes, blocked with 5% non-fat milk in Tris-buffered saline (TBS) (20 mM), Tris-HCl (pH 7.6), 135 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature, and then reacted with

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antibody against dengue virus PrM protein (GTX128092, GeneTex, Irvine, USA) or α-tubulin (Sigma) at 4 °C overnight. Bound antibodies were revealed by horseradish peroxidase-labeled secondary antibodies and enhanced chemiluminescence using a commercial kit (Thermo Fisher Scientific, Rockford, IL) according to the protocol of the manufacturer. 2.2.4. Immunofluorescence staining assay The A549 cells were seeded in 24 well-plate which already had coverslips at a density of 2×104

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cells/well, at 37 °C and 5% CO2 in DMEM supplemented with 2% FBS. After 24h, the cells were transfected with DENV2 (MOI of 1) or complexed with respective concentration of the compound for 2h at 37°C. After the incubation, cells were washed with sterile PBS three times

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and incubated with the medium mixed with the corresponding concentrations of the compound for further 48 h. The cells were fixed with cold 4% PFA (Paraformaldehyde, Sigma) for 20 min at -20 °C, and were then blocked with 10% BSA (Albumin from bovine serum) for 1 h. Finally,

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the cells were incubated with anti-E protein mouse monoclonal antibody (sc-65659, Santa Cruz Biotechnology) and detected using Rhodamine labeled anti-mouse antibody (Jackson

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Immunolab). 3. Results 3.1. Chemistry

The synthesis of the compounds is described in detail in the supplemental material. The 6-chloro

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intermediate 1 was prepared as described in our previously reported route (Lin et al., 2016). The synthesis of three designed scaffolds (shown in Fig. 2) and their analogs has been shown in Schemes 1 to 4 in the supplemental material. Halogenation was employed for 1 with N-

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Iodosuccinimide (NIS), N-Bromosuccinimide (NBS) and N-Chlorosuccinimide (NCS) to yield

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the halogenated nucleosides 2a-c in modest yields, respectively. Amination and deprotection of 1 and 2a-c with NH4OH and 1,4-dioxane led to the simply substituted analogs 3a-d in high yields. The 6-chloro-7-deazapurine 1 was transformed to 6-methyl derivative 4 by using AlMe3 in the presence of Pd(PPh3)4. The same conditions of halogenation reactions were employed to 4 affording 5a-c in good yields. The subsequent cleavage of benzyl protecting groups gave analogs 6a-d (with yields 55%-65%) in two steps (Scheme 1 in the supplemental material).

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The 7-iodo-7-deazapurine nucleosides 3b and 6b served as key intermediates for the preparation of the target nucleosides via various palladium-catalyzed reactions (Scheme 2 in supplemental material). Cross-coupling of iodide 3b with zinc cyanide using Pd(PPh3)4 as a catalyst, afforded

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the nitrile derivative 3e in 13% yield. The 3e was then oxidized by NH4OH and H2O2 in 1,4dioxane to afford formamido derivative 3f in 50% yield.

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The vinyl derivative 6e and the thienyl nucleosides 3g and 6f were obtained in 48-65% yields when 3b and 6b were subjected to Stille reaction, via treatment with tributylvinyltin and 2-

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tributylstannylthiophene, respectively. A series of 7-heteroaryl 7-deazapurine analogs 3h-3j were accessed through replacement with required boronic acids under Suzuki conditions. The Sonogashira coupling was utilized between iodinated nucleosides 3b, 6b and triethylsilylacetylene and arylacetylene to yield the corresponding modifications (3k-i and 6g) in

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7-position of nucleobases (Scheme 2 in the supplemental material).

The 1-α-bromo sugar 8 was produced as the major isomer, after treatment of the lactol with HBr in acetic acid as a bromo-generating reagent, in high yield without a need for purification. SN2-

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type coupling of 1-α-bromo sugar 8 with 6-chloro purine 9 was achieved to provide the desired 6-chloro purine analog 10 as described in our previously reported route (Lin et al., 2016). The

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following methylation of 10 gave 2’-deoxy-2’-fluoro-6-methyl nucleoside 11. Then, all selected 7-substituted analogs 13a-13g in the context of the 2’-deoxy-2’-fluoro ribose series were obtained with the similar synthetic protocols to those already described for the corresponding ribose analogs (Schemes 3 and 4 in the supplemental material). 3.2. Evaluation of anti-DENV2 activity and SAR analysis

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We evaluated the anti-DENV2 activity of all the synthesized compounds (Tables 1-3) using a in vitro cell-based assay, while NITD008 and Ribavirin were used as the positive controls (Tseng et al., 2014). .To explore the SAR trend and further establish the effect of alternative modifications

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introduced in the 7-position of the 7-deaza-adenine base for anti-DENV2 potency, we

synthesized a panel of 12 selected compounds with regular substituents varying from small

groups to bulky ones. For the scaffold 3, four compounds showed modest anti-DENV2 activity

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with EC50 values less than 25 µM in the in vitro cell-based assay (summarized in Table 1);

nevertheless, the unsubstituted compound 3a exhibited no inhibitory activity against DENV2,

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suggesting that the electron-withdrawing alternatives boost anti- DENV2 potency at 7-position. The iodo (3b) and thienyl (3g) derivatives demonstrated almost similar anti-DENV2 potencies (EC50 values of 14.000±4.223 µM and 13.630±3.572 µM, respectively) as compared to that of Ribavirin (EC50 = 11.821±1.583 µM), while 3c with bromo substitution showed slightly less

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potency (EC50=18.600±3.278 µM). The compound 3k with 7-ethynyl substituent demonstrated an EC50 value of 21.690±7.378 µM that was likely attributed to its intrinsic toxicity (CC50> 50 µM). The 7-cyano and 7-formamido derivatives (3e and 3f, respectively) were not active against

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DENV2. Similarly, the bulky group derivatives (3h, 3i, 3j, 3l) also failed to inhibit DENV2 replicons which indicated that bulky groups at 7-position could be unfit in the binding site,

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although 7-thienyl-7-deazapurine nucleoside showed a low micromolar anti-DENV2 effect (EC50 = 13.630±3.572 µM). Moreover, the previously published 7-vinyl-7-deazapurine nucleoside analog was not tested because of its high cytotoxicity (Shi et al., 2011). The 7-deazapurine ribonucleosides have been reported to show promising activity against HCV and DENV when their H-bond-donating amino group at position 6 was replaced by H-bond acceptor groups or even by an isosteric but nonpolar methyl group (Naus et al., 2014). Based on 12

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these observations and inspired by the SAR analysis of scaffold 3, we introduced seven selected small groups at the 7-position of the scaffold 6. The results of these modifications are shown in Table 2. Unexpectedly, only the vinyl analog 6e displayed improved intrinsic potency (EC50 =

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2.081±1.102 µM) without any apparent cytotoxicity in A549 cells. 6e was more potent and active than the reference compounds, NITD008 (EC50 = 4.84±1.61 µM) and Ribavirin (EC50 =

11.821±1.583 µM). These findings revealed that the methyl group at 6-position was more

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beneficial in terms of inhibitory activity than the amino group when an appropriate substitution

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was endorsed at 7-position.

As reported previously (Nauš et al., 2012), 2’-deoxy-2’-β-fluoro modification in the ribose combined with 7-deazapurine has demonstrated potent antiviral activity against HCV. Whether such kinds of 2’-position modifications enhance anti-DENV potency of nucleoside analogs still remains elusive. Again the SAR analysis of scaffold 3 prompted us to explore this aspect, and we

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further investigated different modifications in the 2-position of ribose (shown in Table 3). Remarkably, the inhibitory potency was improved against DENV2, as exemplified by the iodo (13b), bromo (13c), chloro (13d) and ethynyl (13g) derivatives, with EC50 values of 0.650±0.231

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µM, 0.183±0.112 µM, 1.870±1.792 µM and 5.100±1.210 µM, respectively. In particular,

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compound 13c demonstrated most significant inhibitory activity. However, despite possessing promising biological potencies, compounds 13b, 13d, 13e and 13g had narrow therapeutic windows because of possessing micromolar cytotoxicity values (Table 3). 3.3. Cytotoxicity assay and selectivity index (SI)

We determined the cellular toxicity of all the synthesized compounds in A549 cells by using MTT assay. As depicted in Tables 1 and 2, nineteen compounds having 2’-deoxy-2’-fluoro-2’-

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C-methyl ribofuranosyl moiety on the ribose ring did not show inhibitory effect on the growth of A549 cells. The underlying reason might be the presence of methyl group at 2’-position that may likely boost the selectivity in the host cells. However and surprisingly, A549 cells were more

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sensitive to the scaffold 13. The CC50 values of compounds 13b, 13c, 13d, 13e and 13g in A549 cells were ranging from 3 µM to 30 µM. Although having CC50 value of just 4.690±1.990 µM against A549 cells, compound 13c demonstrated a two folds better selectivity index (SI, SI =

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26.06) than that of Ribarivin (SI = 12.05) (Table 4). To exactly reflect the intrinsic cytotoxicity of compound 13c, a concentration required to inhibit A549 cells growth by 15% (CC15) was

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detected as the maximal non-toxic dose (MNTD). The results showed that the value of CC15 (0.18±0.07 µM) of compound 13c was almost same to one of its EC50 (0.183±0.112 µM), indicating that the survival of the host cells decreased apparently along with progressive inhibition of DENV-2 growth. More importantly, compound 6e proved to have strong inhibitory

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activity without any cytotoxic effects against A549 cells. 6e possessed a SI value (72.11) that is much better in comparison to that of Ribavirin (SI = 12.05), and similar to that of NITD008 (SI = 71.41). The CC50 value of 6e was up to 150 µM, and its MNTD was found to be substantially

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safe (CC15 = 47.36±5.13 µM). Overall, the SAR analysis combined with anti-DENV activity and SI studies revealed that the two compounds 6e and 13c are the potent DENV inhibitors which

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were selected for subsequent biological/cytotoxic evaluation. 3.4. Dose−response antiviral potency and cell viability in curves We performed real-time RT-qPCR analysis with specific primers to determine the DENV RNA expression with the inhibitory activity curves of compounds 6e and 13c, while Ribavirin and NITD008 served as the positive controls. As shown in Fig. 3, compounds 6e and 13c consistently reduced DENV replication in the A549 cells at various concentrations after 48 h 14

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treatment. Results indicated that the DENV-2 inhibition by 6e and 13c was in dose-responsive manner. The results of MTT cell viability assay revealed that compound 13c was harmful for the A549 cells, but no significant cell cytotoxicity was detected for the compound 6e. We further

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verified the cellular toxicities of 6e and 13c in HepG2 and Vero cells. As is evident from the Fig. 4a & 4b and Supplementary Table S1, 13c demonstrated a certain level of cytotoxicity against the tested cell lines (Fig. 4b), whereas, 6e did not possess any cytotoxic potential against HepG2

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(CC50 = 146.47±11.05 µM and SI = 63.7) and Vero (CC50 = 79.17±12.50 µM and SI = 58.6) cells. Albeit, Vero cells appeared to be more sensitive to 6e. In our previous work, 6e was also

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reported non-toxic against MDCK cells (Lin et al., 2016). Considering the cytotoxic potential, we conceived that 6e may be a promising lead in managing DENV infection. 3.5. Western blot and Immunofluorescence staining assay

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The DENV prM (membrane) protein and E (envelope) protein are both important in the formation and maturation of the viral particle. To further confirm that 6e inhibits DENV replication, we determined its inhibitory effects on DENV prM protein expression by using

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Western blotting with anti-PrM and anti-α-tubulin antibodies and also performed side-by-side comparison through immunofluorescence staining by detection of DENV E protein. The results

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(shown in Fig. 5a and 5b) demonstrated that compound 6e was more potent than the two positive controls, Ribavirin and NITD008. Compound 6e reduced DENV replication in A549 cells at low concentrations that were consistent with values that of EC50 and dose-response curves. Moreover, 6e reduced DENV replication in A549, HepG2 and Vero cell lines at the concentrations of 1 and 5 µM (Fig. 5c). In addition, 6e efficiently reduced DENV E protein expression at lower concentration without any cellular toxicity (Fig. 6). The inhibition of DENV replicon by 6e was in dose-responsive manner ranging from 1 to 25 µM. 15

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3.6. Inhibitory potency across the four serotypes of dengue virus We next examined the antiviral potency/activity of compound 6e across the four DENV

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serotypes. As shown in Table 5, 6e was active against the four serotypes and exhibited a broad range of potencies at concentration of 5 µM, which suggested that 6e might interact with an evolutionarily conserved target that is essential for virus replication.

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4. Discussion

The design and synthesis of nucleoside analog inhibitors represent an attractive approach for

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anti-DENV drug discovery (Chen et al., 2010a; Chen et al., 2010b; Chen et al., 2015). Especially, testing nucleoside inhibitors targeting other viruses (in particular HCV) in DENV is an actively pursued area for DENV antiviral development (Chen et al., 2015). Some of the nucleoside inhibitors targeting HCV have already been shown to be active in DENV (Chen et al.,

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2015). In this study, we successfully used our previously reported strategy (Lin et al., 2016) and combined the two moieties: 2’-deoxy-2’-fluoro-2’-C-2-methyl ribose sugars and 7-deazapurine bases, together in a single scaffold and then performed SAR studies at the 7-position of the base.

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Our chemistry efforts led to the discovery of a potent anti-DENV nucleoside inhibitor 6e.

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The combination of 2’-deoxy-2’-fluoro-2’-C-methyl ribose sugars and 7-deazapurine bases produced some potent anti-DENV nucleosides. The exploration of SAR at 7-position revealed that some nonpolar and electron-withdrawing alternatives with small volume are required in the active pocket. The initial SAR prompted us to further optimize chemical structures, even in the absence of target identification. In addition, 6-methyl substitution on bases, non-polar H-bondaccepting groups, replaced successfully normal amino groups, thus guiding a novel chemical optimization. 16

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Some 7-deazapurine nucleosides without any modifications in sugars, such as tubercidin and 6methyl-7-deaza-adenosine, have been found very toxic (Wu et al., 2010). Therefore, the main focus of our study was to explore some novel 7-deazapurine analogs having less cytotoxicity and

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improved selectivity towards the viral target. The cytotoxicity of 2’-deoxy-2’-fluoro-2’-C-methyl derivatives was found negligible by MTT assay, nevertheless, 2’-deoxy-2’-β-fluoro analogs showed apparent cellular toxicity which further hinders there potential to be developed as anti-

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DENV therapies. It might be due to the lack of the methyl group at 2’-position, which likely led to interference with the enzyme of the host cells. Nine out of the twenty-six compounds showed

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inhibitory activity for DENV replication, amongst which 6e and 13c displayed significant potency with SIs at least 6-fold and 2-fold, respectively, higher than that of Ribavirin. Especially, compound 6e demonstrated no detectable cytotoxicity in the replicon assay when raised up to a concentration of 47 µM for 48 h. Furthermore, exploration of the cytotoxic potential of 6e in

6e (Fig. 4).

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three different cell lines revealed a close trend, with Vero cells appearing to be more sensitive to

Our results indicated that 6e demonstrates anti-DENV activity by inhibiting the DENV

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replication in a dose-dependent manner. This conclusion was supported by two lines of evidence.

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First, by using real-time RT-qPCR analysis, we showed that compound 6e suppressed viral RNA levels. Second, by using Western blot and immunofluorescence staining assays, we demonstrated that 6e reduced the DENV prM protein and E protein expression in dose-dependent manner. Moreover, comparative immunofluorescence staining also indicated a striking difference in the inhibitory effects of 6e in normal cells and DENV replicon at concentrations ranging from 1 to 25 µM (Fig. 6). Further screening against four serotypes of DENV showed that 6e demonstrates a similar level of activity against DENV1-4 (Table 5), which indicates that the enzymatic 17

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target(s) of 6e is likely to be highly conserved and regulates the process of virus replication. Whether 6e suppresses the replication of other flaviviruses, such as HCV and yellow fever virus

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(YFV), remains an open question. Two major issues remain to be addressed for further development of compound 6e. One issue may be to confirm whether 6e targets viral polymerase after conversion into its corresponding

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triphosphate nucleotide inside the cell. It will also be useful to determine the intrinsic potency of 6e against viral polymerase by using more than one clinically relevant cell lines. Another issue

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may be to confirm the in vivo efficacy of 6e in dengue mouse models and explore its pharmacokinetic properties and safety profile.

In summary, we have discovered 6e as a promising candidate for anti-DENV drug development. The combination of the highly specific chain-terminating 2’-deoxy-2’-fluoro-2’-C-methyl-ribose

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modification and the chemically stable 6-methyl-7-deazapurine has resulted a direct anti-DENV compound with promising therapeutic potential. Further studies ought to be endorsed to

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of 6e.

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determine the pharmacokinetic properties and safety profiles as well as the mechanism of action

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Acknowledgments The research was supported by Guangzhou Science and Technology Project (201707020046),

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Guangzhou health care collaborative innovation key project (201704020227), and an innovation grant form the State Key Laboratory of Respiratory Disease (2016). M.H is sponsored by CASTWAS President fellowship for international PhD students. The authors would also like to

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express our deep gratitude to Prof. Dr. Zheng Yin of Tsinghua University and Prof. Dr. Luqing

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Shang of Nankai University for providing a sample of NITD008.

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Appleby, T. C., Perry, J. K., Murakami, E., Barauskas, O., Feng, J., Cho, A., Fox, D., Wetmore, D. R., McGrath, M. E., Ray, A. S., 2015. Structural basis for RNA replication by the hepatitis C virus polymerase. Science 347, 771-775. Benmansour, F., Eydoux, C., Querat, G., de Lamballerie, X., Canard, B., Alvarez, K., Guillemot, J. C., Barral, K., 2016. Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and 3-phenyl-5-[(E)-2(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole derivatives as dengue virus inhibitors targeting NS5 polymerase. Eur J Med Chem 109, 146-156. Bhatt, S., Gething, P. W., Brady, O. J., Messina, J. P., Farlow, A. W., Moyes, C. L., Drake, J. M., Brownstein, J. S., Hoen, A. G., Sankoh, O., et al., 2013. The global distribution and burden of dengue. Nature 496, 504-507. Byrd, C. M., Grosenbach, D. W., Berhanu, A., Dai, D., Jones, K. F., Cardwell, K. B., Schneider, C., Yang, G., Tyavanagimatt, S., Harver, C., et al., 2013. Novel benzoxazole inhibitor of dengue virus replication that targets the NS3 helicase. Antimicrob Agents Chemother 57, 1902-1912. Chen, Y.-L., Yin, Z., Duraiswamy, J., Schul, W., Lim, C. C., Liu, B., Xu, H. Y., Qing, M., Yip, A., Wang, G., 2010a. Inhibition of dengue virus RNA synthesis by an adenosine nucleoside. Antimicrobial agents and chemotherapy 54, 2932-2939. Chen, Y.-L., Yin, Z., Lakshminarayana, S. B., Qing, M., Schul, W., Duraiswamy, J., Kondreddi, R. R., Goh, A., Xu, H. Y., Yip, A., 2010b. Inhibition of dengue virus by an ester prodrug of an adenosine analog. Antimicrobial agents and chemotherapy 54, 3255-3261. Chen, Y.-L., Yokokawa, F., Shi, P.-Y., 2015. The search for nucleoside/nucleotide analog inhibitors of dengue virus. Antiviral research 122, 12-19. Di Francesco, M. E., Avolio, S., Pompei, M., Pesci, S., Monteagudo, E., Pucci, V., Giuliano, C., Fiore, F., Rowley, M., Summa, V., 2012. Synthesis and antiviral properties of novel 7-heterocyclic substituted 7deaza-adenine nucleoside inhibitors of Hepatitis C NS5B polymerase. Bioorganic & medicinal chemistry 20, 4801-4811. Gubler, D. J., 2002. Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends in microbiology 10, 100-103. Latour, D. R., Jekle, A., Javanbakht, H., Henningsen, R., Gee, P., Lee, I., Tran, P., Ren, S., Kutach, A. K., Harris, S. F., 2010. Biochemical characterization of the inhibition of the dengue virus RNA polymerase by beta-d-2′-ethynyl-7-deaza-adenosine triphosphate. Antiviral research 87, 213-222. Li, L., Basavannacharya, C., Chan, K. W., Shang, L., Vasudevan, S. G., Yin, Z., 2015. Structure-guided Discovery of a Novel Non-peptide Inhibitor of Dengue Virus NS2B-NS3 Protease. Chem Biol Drug Des 86, 255-264. Lim, S. P., Wang, Q. Y., Noble, C. G., Chen, Y. L., Dong, H., Zou, B., Yokokawa, F., Nilar, S., Smith, P., Beer, D., et al., 2013. Ten years of dengue drug discovery: progress and prospects. Antiviral Res 100, 500-519. Lin, C., Sun, C., Liu, X., Zhou, Y., Hussain, M., Wan, J., Li, M., Li, X., Jin, R., Tu, Z., 2016. Design, synthesis, and in vitro biological evaluation of novel 6-methyl-7-substituted-7-deaza purine nucleoside analogs as anti-influenza A agents. Antiviral Research. Low, J. G., Ooi, E. E., Vasudevan, S. G., 2017. Current Status of Dengue Therapeutics Research and Development. J Infect Dis 215, S96-S102. 20

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Manvar, D., Kucukguzel, I., Erensoy, G., Tatar, E., Deryabasogullari, G., Reddy, H., Talele, T. T., Cevik, O., Kaushik-Basu, N., 2016. Discovery of conjugated thiazolidinone-thiadiazole scaffold as anti-dengue virus polymerase inhibitors. Biochem Biophys Res Commun 469, 743-747. Martin, J., Hermida, L., 2016. Dengue vaccine: an update on recombinant subunit strategies. Acta Virol 60, 3-14. Migliaccio, G., Tomassini, J. E., Carroll, S. S., Tomei, L., Altamura, S., Bhat, B., Bartholomew, L., Bosserman, M. R., Ceccacci, A., Colwell, L. F., 2003. Characterization of resistance to non-obligate chainterminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro. Journal of Biological Chemistry 278, 49164-49170. Naus, P., Caletkova, O., Konecny, P., Dzubak, P., Bogdanova, K., Kolar, M., Vrbkova, J., Slavetinska, L., Tloust'ova, E., Perlikova, P., et al., 2014. Synthesis, cytostatic, antimicrobial, and anti-HCV activity of 6substituted 7-(het)aryl-7-deazapurine ribonucleosides. J Med Chem 57, 1097-1110. Nauš, P., Perlíková, P., Bourderioux, A., Pohl, R., Slavětínská, L., Votruba, I., Bahador, G., Birkuš, G., Cihlář, T., Hocek, M., 2012. Sugar-modified derivatives of cytostatic 7-(het) aryl-7-deazaadenosines: 2′C-methylribonucleosides, 2′-deoxy-2′-fluoroarabinonucleosides, arabinonucleosides and 2′deoxyribonucleosides. Bioorganic & medicinal chemistry 20, 5202-5214. Olsen, D. B., Eldrup, A. B., Bartholomew, L., Bhat, B., Bosserman, M. R., Ceccacci, A., Colwell, L. F., Fay, J. F., Flores, O. A., Getty, K. L., 2004. A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties. Antimicrobial agents and chemotherapy 48, 3944-3953. Rothman, A. L., Ennis, F. A., 2016. Dengue Vaccine: The Need, the Challenges, and Progress. J Infect Dis. Santiago, G. A., Vergne, E., Quiles, Y., Cosme, J., Vazquez, J., Medina, J. F., Medina, F., Colon, C., Margolis, H., Munoz-Jordan, J. L., 2013. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus. PLoS Negl Trop Dis 7, e2311. Scaturro, P., Trist, I. M., Paul, D., Kumar, A., Acosta, E. G., Byrd, C. M., Jordan, R., Brancale, A., Bartenschlager, R., 2014. Characterization of the mode of action of a potent dengue virus capsid inhibitor. J Virol 88, 11540-11555. Schul, W., Liu, W., Xu, H. Y., Flamand, M., Vasudevan, S. G., 2007. A dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs. J Infect Dis 195, 665-674. Shepard, D. S., Undurraga, E. A., Halasa, Y. A., Stanaway, J. D., 2016. The global economic burden of dengue: a systematic analysis. Lancet Infect Dis. Shi, J., Zhou, L., Zhang, H., McBrayer, T. R., Detorio, M. A., Johns, M., Bassit, L., Powdrill, M. H., Whitaker, T., Coats, S. J., 2011. Synthesis and antiviral activity of 2′-deoxy-2′-fluoro-2′-C-methyl-7deazapurine nucleosides, their phosphoramidate prodrugs and 5′-triphosphates. Bioorganic & medicinal chemistry letters 21, 7094-7098. Simmons, C. P., Farrar, J. J., Nguyen v, V., Wills, B., 2012. Dengue. N Engl J Med 366, 1423-1432. Smith, D. B., Kalayanov, G., Sund, C., Winqvist, A., Maltseva, T., Leveque, V. J. P., Rajyaguru, S., Pogam, S. L., Najera, I., Benkestock, K., 2009. The design, synthesis, and antiviral activity of monofluoro and difluoro analogues of 4′-azidocytidine against hepatitis C virus replication: the discovery of 4′-azido-2 ′-deoxy-2′-fluorocytidine and 4′-azido-2′-dideoxy-2′, 2′-difluorocytidine. Journal of medicinal chemistry 52, 2971-2978. Tseng, C.-H., Lin, C.-K., Chen, Y.-L., Hsu, C.-Y., Wu, H.-N., Tseng, C.-K., Lee, J.-C., 2014. Synthesis, antiproliferative and anti-dengue virus evaluations of 2-aroyl-3-arylquinoline derivatives. European journal of medicinal chemistry 79, 66-76.

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van Cleef, K. W., Overheul, G. J., Thomassen, M. C., Kaptein, S. J., Davidson, A. D., Jacobs, M., Neyts, J., van Kuppeveld, F. J., van Rij, R. P., 2013. Identification of a new dengue virus inhibitor that targets the viral NS4B protein and restricts genomic RNA replication. Antiviral Res 99, 165-171. Vannice, K. S., Durbin, A., Hombach, J., 2016. Status of vaccine research and development of vaccines for dengue. Vaccine. Wang, Q.-Y., Patel, S. J., Vangrevelinghe, E., Xu, H. Y., Rao, R., Jaber, D., Schul, W., Gu, F., Heudi, O., Ma, N. L., 2009. A small-molecule dengue virus entry inhibitor. Antimicrobial agents and chemotherapy 53, 1823-1831. Wang, Q. Y., Bushell, S., Qing, M., Xu, H. Y., Bonavia, A., Nunes, S., Zhou, J., Poh, M. K., Florez de Sessions, P., Niyomrattanakit, P., et al., 2011. Inhibition of dengue virus through suppression of host pyrimidine biosynthesis. J Virol 85, 6548-6556. Wang, Q. Y., Dong, H., Zou, B., Karuna, R., Wan, K. F., Zou, J., Susila, A., Yip, A., Shan, C., Yeo, K. L., et al., 2015. Discovery of Dengue Virus NS4B Inhibitors. J Virol 89, 8233-8244. WHO, 2016. World Health Organization. 2016. Dengue and severe dengue: Fact sheet. 2016 update (http://www.who.int/mediacentre/factsheets/fs117/en/; accessed 09 May 2016). Wilder-Smith, A., Yoon, I. K., 2016. Edging closer towards the goal of a dengue vaccine. Expert Rev Vaccines 15, 433-435. World Health, O., 2017. Dengue vaccine: WHO position paper, July 2016 - recommendations. Vaccine 35, 1200-1201. Wu, R., Smidansky, E. D., Oh, H. S., Takhampunya, R., Padmanabhan, R., Cameron, C. E., Peterson, B. R., 2010. Synthesis of a 6-methyl-7-deaza analogue of adenosine that potently inhibits replication of polio and dengue viruses. Journal of medicinal chemistry 53, 7958-7966. Xu, H. T., Colby-Germinario, S. P., Hassounah, S., Quashie, P. K., Han, Y., Oliveira, M., Stranix, B. R., Wainberg, M. A., 2016. Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase. Antimicrob Agents Chemother 60, 600-608. Yang, C. C., Hu, H. S., Wu, R. H., Wu, S. H., Lee, S. J., Jiaang, W. T., Chern, J. H., Huang, Z. S., Wu, H. N., Chang, C. M., Yueh, A., 2014. A novel dengue virus inhibitor, BP13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral NS2B/NS3 protease. Antimicrob Agents Chemother 58, 110-119. Yin, Z., Chen, Y.-L., Schul, W., Wang, Q.-Y., Gu, F., Duraiswamy, J., Kondreddi, R. R., Niyomrattanakit, P., Lakshminarayana, S. B., Goh, A., 2009. An adenosine nucleoside inhibitor of dengue virus. Proceedings of the National Academy of Sciences 106, 20435-20439. Yokokawa, F., Nilar, S., Noble, C. G., Lim, S. P., Rao, R., Tania, S., Wang, G., Lee, G., Hunziker, J., Karuna, R., et al., 2016. Discovery of Potent Non-Nucleoside Inhibitors of Dengue Viral RNA-Dependent RNA Polymerase from a Fragment Hit Using Structure-Based Drug Design. J Med Chem 59, 3935-3952. Zhou, Z., Khaliq, M., Suk, J. E., Patkar, C., Li, L., Kuhn, R. J., Post, C. B., 2008. Antiviral compounds discovered by virtual screening of small-molecule libraries against dengue virus E protein. ACS Chem Biol 3, 765-775.

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Figure legends Fig. 1: Compounds with potent antiviral activity.

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Fig. 2: Three designed scaffolds as inhibitors of DENV.

Fig. 3: Dose-response curves of compounds 6e and 13c in DENV assay and A549 cells. (a) Inhibitory effects of compounds 6e and 13c, and the two positive controls Ribavirin and

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NITD008 on DENV 2 RNA yield. Real time PCR assays were performed using compounds 6e, 13c, NITD008, and Ribavirin, respectively. The antiviral activity is presented as the percent

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inhibition. 6e and 13c demonstrated more potent suppression of RNA levels as compared to Ribavirin and NITD008. (b) Concentration-dependent anti-DENV 2 activity of 6e in cell viability assay. (c) Concentration-dependent anti-DENV 2 activity of 13c in cell viability assay. (d) Concentration-dependent anti-DENV 2 activity of NITD008 in cell viability assay (e)

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Concentration-dependent anti-DENV 2 activity of Ribavirin in cell viability assay. Fig. 4: Cell viability curves after treatment with 6e and 13c in three different cell lines (A549, HepG2 and Vero cells). The cell lines were incubated with 6e or 13c at various concentrations

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for 48 h. Cell viability was then measured using an MTT assay and presented as a percentage of 490 nm absorbance derived from the compound-treated cells. (a) Concentration-dependent

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decrease in cell viability of three cell lines after treatment with 6e. (b) Concentration-dependent decrease in cell viability of three cell lines after treatment with 13c. (c) Concentration-dependent decrease in cell viability of three cell lines after treatment with NITD008. Overall, 13c appeared to be more cytotoxic against the three cell lines as compared to 6e. Fig. 5: (a) Inhibition of DENV2 prM protein expression in A549 cells by compound 6e and NITD008. (b) Inhibition of DENV2 prM protein expression in A549 cells by compound 6e and 23

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Ribavirin. A549 cells were incubated with tested compound 6e and the positive controls NITD008 and Ribavirin at the indicated concentrations. After 48 h of incubation, cell lysates were extracted and analyzed by Western blotting with anti-PrM protein and anti-α-tubulin

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antibody. (c) Inhibition of DENV2 prM protein expression in A549, HepG2 and Vero cells by compound 6e.

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Fig. 6: (a) Side-by-side comparison of anti-DENV activity in immunofluorescence staining. A549 cells were infected and treated with compound 6e. Red color represents the detection of

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DENV E protein, blue for cell stain. (b) Standard yield reduction assay was performed using indicated concentration of compound 6e. DENV titers were determined and expressed as % of control. DMSO (0.1%) treatment served as the mock controls. DENV2 (MOI of 1) treatment

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served as the controls. Error bars represent the SD from three experiments.

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DENV-2 R1

1

3a

H

2

3b

I

3

3c

Br

4

3d

Cl

5

3e

6

3f

7

EC50(µM)b

A549 cell

CC50(µM)c

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Compound

>100

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Table1.Inhibitory effect of 6-amino nucleoside derivatives 3a-l in DENV-2 RNA yield and the cytotoxic evaluation in A549 cellsa.

>100 >50

18.600±3.278

>50

>100

>100

CN

>100

>100

CONH2

>100

>100

3g

13.630±3.572

>100

8

3h

>100

>50

9

3i

>100

>100

10

3j

>100

>50

11

3k

21.690±7.378

>50

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14.000±4.223

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13

NITD008

14

Ribavirin

>100

>100

-

4.84±1.61

345.63±22.11

-

11.821±1.583

142.480±12.560

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12

a

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The data are the mean values±standard deviation from three independent experiments. b Concentration required to inhibit DENV-2 virus growth by 50%.The EC50s were determined by real time PCR assay (see Materials and Methods for details). c Cytotoxic concentration required to inhibit A549 cells growth by 50%. The CC50s were determined by MTT assay (see Materials and Methods for details).

6a

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1

Compound

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Entry

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Table2.Inhibitory effect of 6-methyl nucleoside derivatives 6a-g in DENV-2 RNA yield and the cytotoxic evaluation in A549cellsa.

DENV-2

A549 cell

EC50(µM)b

CC50(µM)c

H

>100

>100

R2

2

6b

I

>100

>100

3

6c

Br

>100

>100

4

6d

Cl

>100

>100

5

6e

2.081±1.102

150.06±11.42

6

6f

>100

>100

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6g

8

NITD008

9

Ribavirin

>100

>100

-

4.84±1.61

345.63±22.11

-

11.821±1.583

142.480±12.560

a

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The data are the mean values±standard deviation from three independent experiments. b Concentration required to inhibit DENV-2 virus growth by 50%.The EC50s were determined by real time PCR assay (see Materials and Methods for details). c Cytotoxic concentration required to inhibit A549 cells growth by 50%. The CC50s were determined by MTT assay (see Materials and Methods for details).

1

R3

DENV-2

A549 cell

EC50(µM)b

CC50(µM)c

13a

H

>100

>100

13b

I

0.650±0.231

3.470±1.540

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2

Compound

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Table 3.Inhibitory effect of 6-methyl nucleoside derivatives 13a-g in DENV-2 RNA yield and the cytotoxic evaluation in A549cellsa.

3

13c

Br

0.183±0.112

4.690±1.990

4

13d

Cl

1.870±1.792

18.000±2.240

5

13e

25.200

30.400±3.710

6

13f

>100

>100

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8

NITD008

9

Ribavirin

5.100±1.210

19.300±3.140

-

4.84±1.61

345.63±22.11

-

11.821±1.583

142.480±12.560

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7

a

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The data are the mean values±standard deviation from three independent experiments. b Concentration required to inhibit DENV-2 virus growth by 50%.The EC50s were determined by real time PCR assay (see Materials and Methods for details). c Cytotoxic concentration required to inhibit A549 cells growth by 50%. The CC50s were determined by MTT assay (see Materials and Methods for details).

Table 4. Anti-DENVselectivity index (SI) of the selected compounds. CC50(µM)

6e

150.06±11.42

13c

4.69±1.99

Ribavirin a

EP 345.63±22.11

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NITD008

CC15(µM)a

EC50(µM)

SIb

47.36±5.13

2.081±1.102

72.11

0.18±0.07

0.183±0.112

26.06

108.23±11.31

4.84±1.61

71.41

28.32±2.75

11.821±1.583

12.05

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Compound

142.48±12.56

Concentration required to inhibit A549 cells growth by 15%. The data are the mean values±standard deviation from three independent experiments. CC15s were detected as the maximal non-toxic dose (MNTD). b Selectivity index, SI = CC50/EC50.

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DENV-3

DENV-4

79.2±4.2

72.2±5.4

76.2±6.2

68.5±4.8

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Inhibitiona (%)

DENV-1

a

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Cells were infected with the indicated viruses at an MOI of 1 and treated with6e at the concentration of 5 µM. The data are mean values±standard deviation from three independent experiments.

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Fig 1. NH2

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Fig 2. NH2

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HO

HO Me F

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HO

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HO

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Highlights Dengue fever has become the fastest spreading human infectious disease in recent years

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Currently, there is no approved therapy for dengue virus infection

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Designed and synthesized a series of 7-deazapurine nucleoside derivatives as dengue

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virus inhibitors

6e emerged as the most potent inhibitor of viral replication against four serotypes of

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dengue virus

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6e may serve as a promising lead compound for anti-dengue virus drug development

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•