Exploring Ugi-Azide Four-Component Reaction Products for Broad-Spectrum Influenza Antivirals with a High Genetic Barrier to Drug Resistance
Influenza viruses are respiratory pathogens that are responsible for seasonal influenza and sporadic influenza pandemic. The therapeutic efficacy of current influenza vaccines and small molecule antiviral drugs is limited due to the emergence of multidrug-resistant influenza viruses. In response to the urgent need for the next generation of influenza antivirals, we utilized a fast-track drug discovery platform by exploring multi-component reaction products for antiviral drug candidates. Specifically, molecular docking was applied to screen a small molecule library derived from the Ugi-azide four-component reaction methodology for inhibitors that target the influenza polymerase PA C -PB1 N interactions. One hit compound 5 was confirmed to inhibit PA C -PB1 N interactions in an ELISA assay and had potent antiviral activity in an antiviral plaque assay. Subsequent structure-activity relationship studies led to the discovery of compound 12a, which had broad-spectrum antiviral activity and a higher in vitro genetic barrier to drug resistance than oseltamivir. Overall, the discovery of compound 12a as a broadspectrum influenza antiviral with a high in vitro genetic barrier to drug resistance is significant, as it offers a second line of defense to combat the next influenza epidemics and pandemics if vaccines and oseltamivir fail to confine the disease outbreak.
<!>Results and Discussion<!>Cellular antiviral mechanism of compound 12a.<!>Compound 12a has a higher in vitro genetic barrier to drug resistance than oseltamivir.<!>Docking model of compound 12a in PA c .<!>Conclusions<!>1-(1-{[1-(2,3-dihydro-1,4-benzodioxin-6-yl)-1 H-1,2,3,4-tetrazol-5-yl][4-(propan-2-yl)phenyl]methyl}piperidin-4-yl)-2,3-dihydro-1 H-1,3-benzodiazol-2-one (9j<!>Time-of-Addition Experiment.
<p>Influenza virus infection is responsible for both seasonal influenza as well as sporadic influenza pandemics 1 . In the annual influenza season, an estimated 10-20% of the human population is infected with the influenza virus. Despite the availability of influenza vaccines and small molecule antiviral drugs, the death toll of influenza virus-related illness surpasses that of breast cancer, which places the influenza virus among the top ten leading causes of death in the United States 2 . Moreover, the convenient transmission through airways, coupled with the high mortality rates associated with pandemic influenza viruses and highly pathogenic avian influenza (HPAI) viruses, renders the influenza virus a major public health concern 3 . For example, the CDC estimated the 2009 H1N1 influenza pandemic led to 284,000 deaths globally in the first 12 months of outbreak 4 . Over 400 cases of human infection by HPAI H7N9 were reported in the recent 2017 outbreak in China and the mortality rate was ~40% 5,6 . Therefore, next-generation vaccines and antiviral drugs are clearly needed with improved efficacy and antiviral spectrum to combat influenza virus infection.</p><p>Influenza vaccines remain the mainstay for the prophylaxis of influenza infection. They are normally effective in preventing seasonal influenza virus infection with an overall effectiveness of ~60% 7 . However, there is often a six-month delay from strain identification to batch production, which impedes its use at the beginning of an influenza outbreak 8 . Thus, small molecule antivirals are highly desired. They are not alternatives, but essential complements of influenza vaccines.</p><p>There are currently two classes of FDA-approved small molecule influenza antivirals: M2 channel blockers such as amantadine and rimantadine that inhibit the early stage of viral uncoating 9 , and neuraminidase inhibitors, such as oseltamivir, peramivir, and zanamivir that inhibit the last stage of viral egress 10 . The increasing incidences of drug-resistant viruses now call for the development of next generation influenza antivirals 11 . Indeed, amantadine and rimantadine are no longer recommended, due to the widespread M2-S31N mutant 12,13 . The 2008-2009 seasonal H1N1 strain circulating in the United States and Japan is completely resistant to the only orally bioavailable drug, oseltamivir, owing to H275Y mutation in the neuraminidase 14,15 . The emergence of drug-resistant viruses with acquired fitness of transmission is a timely reminder of the urgent need for new antivirals with a novel mechanism of action and a high genetic barrier to drug resistance.</p><p>In the search for novel influenza antivirals, we sought to utilize a fast-track drug discovery program by exploring multi-component reaction (MCR) products for inhibitors that target the influenza polymerase subunit PA C -PB1 N interactions. As drug discovery involves iterative cycles of design, synthesis, and pharmacological characterization, we envisioned that MCRs would greatly accelerate structure-activity relationship (SAR) studies during the drug discovery process, as final products can be conveniently synthesized in a one-pot one-step reaction, often containing 3 or more point of diversification [16][17][18] .</p><p>Influenza polymerase consists of three subunits PA, PB1, and PB2 (Fig. 1A) 19,20 . X-ray crystal structures show that the N-terminal tail of PB1 (PB1 N ) interacts extensively with the C-terminal domain of PA (PA C ) (Fig. 1B), and as such the concave shape of the PB1 N -binding site in PA C presents an attractive target for rational drug design 20,21 . Moreover, the PA C domain is highly conserved among different types and subtypes of influenza viruses, and compounds that inhibit PA C -PB1 N interactions have shown to have broad-spectrum antiviral activity [22][23][24] . Using the crystal structure of PA C bound to the PB1 N peptide (PDB: 3CM8) as a template, we screened two thousand compounds from an in-house library using the Schrödinger Glide standard precision docking program. The in-house library comprises a diverse set of compounds prepared by multicomponent reaction methodologies 25 . Top hits prioritized by in silico docking were tested in a PA C -PB1 N ELISA assay. Compound 5 was found to inhibit PA C -PB1 N interaction in a dose-dependent manner with an IC 50 of 4.3 ± 0.1 µM. The antiviral activity of compound 5 was confirmed by the plaque assay and it had single to submicromolar EC 50 values against several influenza A and B viruses, including both oseltamivir-sensitive and oseltamivir-resistant strains. Subsequent SAR led to the discovery of 12a with an improved selectivity index. Similarly, compound 12a had potent and broad-spectrum antiviral activity against several human clinical isolates of influenza A and B viruses. More importantly, no resistant virus was selected under drug selection pressure of compound 12a. Overall, we view the discovery of compound 12a -a broad-spectrum influenza antiviral -as proof-of-concept for the inherent advantage of our in-house fast-track MCR drug discovery platform, which has been recapitulated throughout the drug discovery arena.</p><!><p>Chemistry. Compound 5 was synthesized by the Ugi-azide 4-CR in methanol at room temperature in 72% yield (Fig. 2A). A focused library of compounds 9a-9u with diversity elements amine (R 1 ), aldehyde (R 2 ), and isocyanide (R 3 ) was synthesized according to the general procedure employed to afford compound 5 (Fig. 2B). Compounds 5 and 9a-9u were synthesized and tested as enantiomeric mixtures, whilst compounds 12a and 12b were prepared as diastereomeric mixtures (Fig. 2C), and separated by silica gel flash chromatography. The absolute stereochemistry of compound 12b was determined by X-ray crystallography (Fig. 2D).</p><p>PA C -PB1 N inhibition and antiviral activity of the initial hit compound 5. The initial hit 5 was confirmed as a potent inhibitor of the PA C -PB1 N polymerase subunit interactions in the ELISA assay with an IC 50 of 4.3 ± 0.1 µM (Fig. 3) and its antiviral activity was tested in an antiviral plaque assay. Compound 5 was found to inhibit multiple strains of influenza A and B viruses with single to submicromolar EC 50 values (Table 1). Its Structure-activity relationship studies of compound 5. Encouraged by the broad-spectrum antiviral activity of 5, we were thus interested in pursuing SAR studies to further optimize antiviral potency and its selectivity index. To this end, we synthesized a focused library of 21 compounds (9a-9u) using the expeditious Ugi-azide 4-CR. The library includes compounds that have points of diversity at the amine component R 1 (9a-9c), the aldehyde component R 2 (9d-9o), and the aldehyde and isocyanide components combined (R 2 & R 3 ) (9p-9u, and 12a-12b). All compounds were initially tested at 5 µM against the A/WSN/33 (H1N1) virus in the plaque assay to rule out compounds that have no cellular antiviral activity (Fig. 4). Nucleozin and oseltamivir carboxylate, two known influenza antivirals, were included as positive controls. It was found that the amine component, 4-(2-keto-1-benzimidazolinyl)piperidine, is essential for the antiviral activity, as compounds with amine modifications 9a-9c had no antiviral activity (Fig. 4 and Table 2). Amongst compounds with modifications of the aldehyde input R 2 (9d-9o), compounds 9i and 9k-9o had significantly improved antiviral activity compared with compound 5, and infected cells had less than 30% plaque formation when treated with 5 µM of compound. Compounds 9d, 9e, 9 g, and 9 h had moderate antiviral activity, while compounds 9 f and 9j were not active. These results suggest that hydrophobic aromatic groups such as benzene (9 g, 9i-9l), thiophene (9 m, 9o), and furan (9n) are preferred at the R 2 position. When R 2 substitution was benzene, a methoxyl group was tolerated at the ortho-, meta-, and para-positions (9k and 9 l). A small alkyl group such as ethyl (9 g) was also tolerated at the para-position; however, branched alkyl group such as isopropyl (9j) and a bulky substitution such as benzene (9 f) were not tolerated. For compounds with simultaneous modifications at both the aldehyde component R 2 and the isocyanide component R 3 (9p-9u), it was found that benzyl is preferred at R 3 (9p, 9 u versus 9q, 9r, 9 s and 9t), and compound 9p had similar antiviral activity as 9o (12.3% versus 19.5% plaque formation at 5 µM). Compound 9t was not active (73.0% plaque formation at 5 µM), possibly due to the presence of the bulky naphthalene substitution at the R 2 position. Compound 9 u had antiviral activity similar to compound 5 (22.7% vs 16.5% plaque formation at 5 µM) as it combines favorable substitutions from both the aldehyde component ((o-trifluoromethoxy)phenyl) and the isocyanide component (benzyl).   One feature of Ugi-azide 4-CR is that it produces a new chiral center during the reaction, and hence the product is a mixture of enantiomers. Although the enantiomers could be separated by chiral HPLC or other methods, chiral separation is generally time consuming and expensive, which presents a challenge for future development. Indeed, this somewhat compromises the advantages of exploring these specific chemotypes in the drug discovery arena. With grams of material required for possible downstream pharmacokinetic and in vivo animal studies, we therefore sought to develop a convenient synthesis and separation strategy to bypass chiral separation. Hence, we employed a chiral isocyanide, (S)-(−)-α-methylbenzyl isocyanide in Ugi-azide 4-CR, which we felt would be well-tolerated based on prior SAR results of 9a-9u. With this chiral isocyanide strategy, a mixture of diastereomers was thus produced (Fig. 2C) that were conveniently separated by silica gel flash column chromatography. The absolute stereochemistry of compound 12b was determined by X-ray crystallography as (R, S). It was found that the (S, S) diastereomer 12a had potent antiviral activity (1.9% plaque formation at 5 µM), while the (R, S) diastereomer 12b was not active (80.6% plaque formation at 5 µM) (Fig. 4).</p><p>Selected compounds were also tested for their inhibition of PA C -PB1 N interactions in the ELISA assay (Table 2). For compounds 9i, 9k, 9 m, 9o, and 12a, which had potent antiviral activity (less than 30% plaque formation at 5 µM), the IC 50 values in the ELISA assay ranged from 7.6 µM to 15.1 µM. For compounds 9c, 9 f, and 12b which had no antiviral activity (greater than 90% plaque formation at 5 µM), the IC 50 values were above 26.9 µM. Overall, there is in general a positive correlation between the compounds' PA C -PB1 N inhibition and their antiviral efficacy. The lack of a straight linear correlation was expected as the antiviral efficacy is a combined effect of PA C -PB1 N inhibition, cellular permeability, and potential off-target interactions.</p><p>Subsequently, the cellular cytotoxicity and broad-spectrum antiviral activity of one of the most potent compounds 12a was further profiled (Table 3). Compound 12a was not cytotoxic to either MDCK or A549 cells and the CC 50 values were greater than 150 µM and 98.1 ± 2.5 µM, respectively. When tested against a panel of human clinical isolates of influenza A and B viruses, compound 12a showed broad-spectrum antiviral activity, with EC 50 values ranging from 0.6 µM to 2.7 µM. It is noteworthy that compound 12a had no cross-resistance with the FDA-approved influenza antivirals amantadine and oseltamivir, as shown by the fact that compound 12a had potent antiviral activity against viruses that are resistant to amantadine, oseltamivir, or both (Table 3).</p><!><p>To gain insights into the cellular antiviral mechanism of 12a, we performed time-of-addition and RT-qPCR. As 12a was confirmed to inhibit influenza polymerase PA-PB1 interactions, it is expected to inhibit the intermediate stage of viral replication post viral fusion in the time-of-addition experiment. Furthermore, when the viral polymerase is inhibited, viral RNA transcription and replication should also be reduced. To confirm which stage of viral replication was affected by 12a, we performed a time-of-addition experiment by adding 12a at different time points during viral replication (Fig. 5A and B). Thus, it was found that the efficacy of 12a gradually decreased when it was added at later stages of viral replication (Fig. 5B). Pre-treatment of cells with 12a has little to no effect on viral replication, indicating that 12a might not target the host factors (Fig. 5B). As a control, oseltamivir carboxylate retained potent antiviral activity even when it was added 8 h post viral infection (Fig. 5A), which is consistent with its known mechanism of inhibiting viral egress. Next, to test whether 12a inhibits viral RNA transcription and replication, we performed RT-qPCR assay. Quantification of the viral nucleoprotein (NP) RNA expression levels by RT-qPCR revealed that the levels of vRNA, cRNA, and mRNA were all inhibited in a dose-dependent manner by 12a (Fig. 5C). The IC 50 values are consistent with the antiviral efficacy EC 50 values of compound 12a. Collectively, the results from the time-of-addition experiment and RT-qPCR are consistent with the antiviral mechanism of 12a by inhibiting the viral polymerase PA-PB1 interactions.</p><!><p>Drug-induced resistance is one of the major obstacles facing antiviral drugs 10 . We therefore designed serial viral passage experiments to characterize the genetic barrier to drug resistance of compound 12a (Fig. 6) [28][29][30][31] . In this experiment, the A/WSN/33 (H1N1) virus was amplified in the presence of increasing concentrations of compound 12a and the drug sensitivity of the resulting viruses at different passages was assayed against compound 12a using a plaque assay. Oseltamivir carboxylate was included as a control. Gratifyingly, viruses at passage 10 remained sensitive to compound 12a, and no increase in EC 50 value was observed (Fig. 6B and Table 4). In contrast, the EC 50 for oseltamivir carboxylate increased 10-fold at passage six and onwards, which is consistent with previous reports 32,33 . These results suggest that compound 12a targets a vital viral replication component such as the viral polymerase that is less prone to mutate. Overall, compound 12a demonstrated a high in vitro genetic barrier to drug resistance, rendering it a desirable drug candidate for further development.</p><!><p>To gain insights on how compound 12a binds to the PB1 N -binding pocket in PA C , we performed molecular docking using Schrödinger Glide software. In the docking model of 12a in PA, 12a snuggly fits in the PB1-binding pocket in PA (Fig. 7A), forming extensive hydrophobic interactions and multiple π-π interactions (Fig. 7B). For example, the phenyl ring from the isocyanide input (R 3 ) in 12a interacts with F707 and K643 through π-π and cation-π interactions, respectively. The thiophene ring from 12a forms π-π interactions with F710. In addition, the carbonyl from the benzimidazol-2-one in 12a forms a hydrogen bond with the E623 backbone amide NH, while the benzene ring of the benzimidazol-2-one fits in the hydrophobic pocket formed by F411 and I621.</p><!><p>Drug discovery is an expensive and time-consuming endeavor. On average, it costs billions of dollars and 10-15 years to advance a drug to the market 34 . Development of antiviral drugs comes with an even greater challenge because of the intrinsic issue of drug resistance, which could render years of research efforts futile 14 . To shorten the length of antiviral drug development, we are interested in further building our fast-track drug discovery platform by exploiting MCR products to identify hits with broad-spectrum antiviral activity and a high genetic barrier to drug resistance. Exploring MCRs for antiviral drug discovery offers the key advantage of expeditious synthesis that could feasibly enable timely development of newer generations of antivirals if resistance to earlier generations were to emerge. Toward this goal, we launched an in silico virtual screening campaign of an in-house, MCR-derived small-molecule library 25,35,36 against the highly conserved PA C subunit of the influenza polymerase.</p><p>The rationale for choosing this library were: (1) compounds in this library were synthesized by one-pot MCR reactions, which will ensure expeditious turnover of subsequent structure-activity relationship (SAR) studies; (2) the synthesis methodologies for compounds in this library are established and optimized, which ensures expeditious re-synthesis of interesting hits in gram quantities for subsequent mechanistic studies and in vivo animal studies and (3) the tetrazole ring embedded in all the molecules are cis-amide bond isosteres and as such are attractive constrained peptidomimetics 37 . As a proof-of-concept, our efforts have already yielded several hits. For example, compound 5 (Table 1) is active against multiple influenza A and B strains. Subsequent SAR by the expeditious Ugi-azide 4-CR led to the discovery of compound 12a that:   Importantly, by introducing a chiral isocyanide as the building block in the Ugi-azide 4-CR, we circumvent the problem of chiral separation and the diastereomeric product mixture was conveniently separated by silica gel flash chromatography. In summary, the promising in vitro antiviral efficacy of compound 12a warrants its further development as a next-generation influenza antiviral.</p><p>Experimental Section. Molecular Docking. The X-ray crystal structure of PAc-PB1 N (PDB: 3CM8) was used as the protein template for the molecular docking. Docking was performed using Schrödinger Glide. In house small molecule library was processed in Ligprep and all possible enantiomers and diasteromers were generated and docked individually. The grid box center was set as the centroid of workspace ligand (PB1 N ). The grid box size was set to dock ligands similar in size to the workspace ligand (PB1 N ). All compounds were initially docked using standard precision algorithm, and top 10% of the hits were further docking using extra precision algorithm. Hits were ranked by Glide score.</p><p>Chemistry. All commercially available chemicals were used without further purification. All final compounds were purified by flash column chromatography. Compound 5 and 9a-9u were synthesized and tested as a mixture of enantiomers. Compounds 12a and 12b were separated as pure diastereomers and tested individually. 1 H and 13 C NMR spectra were recorded on a Bruker-400 NMR spectrometer. Chemical shifts are reported in parts per million referenced with respect to residual solvent (DMSO-d6) 2.50 ppm and (Chloroform-d) 7.26 ppm or from internal standard tetramethylsilane (TMS) 0.00 ppm. The following abbreviations were used in reporting spectra: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublets. HPLC-grade solvents were used for all reactions. Flash column chromatography was performed using silica gel (230-400 mesh, Merck). Low-resolution mass spectra were obtained using an ESI technique on a 3200 Q Trap LC/MS/MS system (Applied Biosystems). High-resolution mass spectra were obtained using the positive ESI method for all the compounds, obtained in an Ion Cyclotron Resonance (ICR) spectrometer. The purity was   assessed by using a Shimadzu LC-MS with a Waters XTerra MS C-18 column (part #186000538), 50 × 2.1 mm, at a flow rate of 0.3 mL/min; λ = 250 and 220 nm; mobile phase A, 0.1% formic acid in H 2 O, and mobile phase B' , 0.1% formic in 60% isopropanol, 30% CH 3 CN and 9.9% H 2 O. All compounds submitted for testing in the ELISA and plaque assays were confirmed to be >95.0% purity by LC-MS traces. All compounds were characterized by proton and carbon NMR and MS.</p><p>Synthesis Procedures. Amine (1.0 equiv) and aldehyde (1.0 equiv) were mixed in methanol (5 ml) and stirred at room temperature for 15 minutes. Then TMS-azide (1.0 equiv) and isocyanide (1.0 equiv) were added sequentially and the resulting mixture was stirred at room temperature overnight. After that, the solvent was removed under reduced pressure and the crude product was purified with flash silica gel chromatography (Ethyl acetate in hexane 20-70%). (5). White solid. Yield: 65%. 1</p><!><p>one (9n). White solid. Yield: 68%. 1 H NMR (400 MHz, CDCl 3 ) δ 9.69 (s, 1 H), 7.66-7.39 (m, 2 H), 7.25-7.15 (m, 2 H), 7.14-7.00 (m, 5 H), 6.67 (dd, J = 1.9, 0.9 Hz, 1 H), 5.05 (s, 1 H), 4.41-4.30 (m, 4 H), 4.30-4.18 (m, 1 H), 3.04 (dd, J = 40.0, 11.3 Hz, 2 H), 2.71-2.04 (m, 4 H), 1.92-1.72 (m, 2 H). 13</p><p>White solid. Yield: 76%. 1 H NMR (400 MHz, CDCl 3 ) δ 9.82 (s, 1 H), 7.42-7.26 (m, 3 H), 7.25-7.15 (m, 1 H), 7.16-7.00 (m, 5 H), 6.93 (dd, J = 8.6, 2.5 Hz, 1 H), 5.17 (d, J = 0.5 Hz, 1 H), 4.42-4.32 (m, 4 H), 4.25 (tt, J = 12.2, 4.2 Hz, 1 H), 3.52 (d, J = 4.9 Hz, 1 H), 3.18-3.10 (m, 1 H), 3.04-2.96 (m, 1 H), 2.62-2.34 (m, 3 H), 2.23-2.12 (m, 1 H), 1.86-1.78 (m, 2 H). 13  Plaque assay. The plaque reduction assay was performed as previously reported 38 , except MDCK cells expressing ST6Gal I were used instead of regular MDCK cells 39 . Briefly, the confluent monolayers of ST6Gal MDCK cells were incubated with ~100 pfu virus samples in DMEM with 0.5% BSA for 1 h at 4 °C, then 37 °C for 1 h. The inoculums were removed, and the cells were washed with phosphate buffered saline (PBS). The cells were then overlaid with DMEM containing 1.2% Avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA) and NAT (2.0 µg/mL). To examine the effect of the compounds on plaque formation, the overlay media was supplemented with compounds at testing concentrations. At two days after infection, the monolayers were fixed and stained with crystal violet dye solution (0.2% crystal violet, 20% methanol). Influenza A virus A/ WSN/33 (H1N1) was obtained from Dr. Robert Lamb at the Northwestern University. Cytotoxicity assay. Evaluation of the cytotoxicity of compounds was carried out using the neutral red uptake assay 40 . Briefly, 80,000 cells/mL of MDCK or A549 cells in DMEM medium supplemented with 10% FBS and 100 U/mL Penicillin-Streptomycin were dispensed into 96-well cell culture plates at 100 µL/well. Twenty-four hours later, the growth medium was removed and washed with 100 µL PBS buffer; then for the cytotoxicity assay, 200 µL fresh DMEM (no FBS) medium containing serial diluted compounds was added to each well. After incubating for 48 h at 37 °C with 5% CO 2 in a CO 2 incubator, the medium was removed and replaced with 100 µL DMEM medium containing 40 µg/mL neutral red for four hours at 37 °C. The amount of neutral red uptake was determined at absorbance 540 nm using a Multiskan FC Microplate Photometer (Fisher Scientific). The CC 50 values were calculated from best-fit dose response curves with variable slope in GraphPad Prism version 5.</p><p>ELISA assay. To test the inhibitory activity of compound on PA C -PB1 N interaction, ELISA was performed 22 . Briefly, microtiter plates were coated with 400 ng of His-tagged PA 239-716 (PA C ) for 3 h at 37 °C, followed by blocking with 2% (wt/vol) BSA in phosphate buffer saline (PBS) for 1 h. After washing with PBS containing 0.3% Tween 20, plates were incubated with 200 ng of GST-tagged PB1 1-25 (PB1 N ) protein and compounds overnight at room temperature. Then the PA C -PB1 N interaction was detected using a horseradish peroxidase (HRP)-conjugated anti-GST monoclonal antibody and its chromogenic substrate TMB. The absorbance at 450 nm was read on a plate reader. The IC 50 values were calculated from best-fit dose response curves with variable slope in GraphPad Prism version 5.</p><!><p>A time-of-addition experiment was performed according to the procedure described earlier 29,41,42 . Briefly, MDCK cells were seeded at 6 cm 2 dishes at a 2 × 10 5 cells/dish cell density. After incubating for 24 h to allow cells to attach, the cells were infected with A/WSN/33 (H1N1) virus at a MOI of 0.01 at −2 h time point. Antiviral compounds, such as oseltamivir carboxylate (1 µM) or 12a (10 µM) was added at different time points before, during, or after viral infection as illustrated in Fig. 4. Viruses were harvested from the cell culture supernatant at 12 h post infection. The virus titers were quantified by plaque assay.</p><p>RNA Extraction and Real-Time PCR. Total RNA was extracted from influenza A/WSN/33 (H1N1) infected cells using Trizol reagents (Thermo Fisher Scientific). After removing genomic DNA by RQ1 RNase-Free DNase (Promega), the first strand of cDNA was synthesized using 1.2 μg of total RNA and AMV Reverse Transcriptase (Promega). vRNA specific primer (5′-AGCAAAAGCAGG-3′), cRNA specific primer (5′-AGTAGAAACAAGG-3′) or oligo (dT)18 was used for detecting influenza vRNA, cRNA or mRNA, respectively. Real-time PCR was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) using FastStart Universal SYBR Green Master (Rox) (Roche, Basel, Switzerland) and following influenza NP-specific primers: NP-F: 5′-AGGGTCAGTTGCTCACAAGTCC-3′; NP-R: 5′-TTTGAAGCAGTCTGAAAGGGTCTA-3′. GAPDH was also amplified to serve as a control using human GAPDH-specific primers (GAPDH-F: 5′-ACACCCACTCCTCCACCTTTG-3′ and GAPDH-R: 5′-CACCACCCTGTTGCTGTAGCC-3′). The amplification conditions were: 95 °C for 10 min; 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Melting curve analysis was performed to verify the specificity of each amplification. All experiments were repeated three times independently.</p><p>Serial viral passage experiments. Serial drug passage experiments were performed accordingly to previously published protocol 29,30,42 . Briefly, MDCK cells were infected with the A/WSN/33 (H1N1) virus at MOI 0.001 for 1 h. Then the inoculum was removed and MDCK cells were incubated with 1 µM compound 12a in the first passage and the concentration of 12a was gradually increased 2-fold in passages 2-7 and kept constant at 64 µM in passages 7-10. In each passage, the viruses were harvested when a significant cytopathic effect was observed, which usually takes 2-3 days after virus infection. The titers of harvested viruses were determined by plaque assay. The drug sensitivity after passages 3, 6, and 10 was determined via plaque assay as described previously 43 . Oseltamivir carboxylate was included as a control and similar fold of drug selection pressure was applied. The drug sensitivity of oseltamivir at passages 3, 6, and 10 was determined via plaque assay.</p>