Boronic Acid Modifications Enhance the Anti-Influenza A Virus

†Key Laboratory of Marine Drugs, Ministry of Education, Ocean University of ... Shandong Provincial Key Laboratory of Glycoscience and Glycotechnolo...
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Boronic Acid Modifications Enhance the Anti-Influenza A Virus Activities of Novel Quindoline Derivatives Wei Wang,†,‡,⊥ Ruijuan Yin,†,‡,⊥ Meng Zhang,†,⊥ Rilei Yu,†,‡ Cui Hao,§ Lijuan Zhang,*,§ and Tao Jiang*,†,‡,∥ †

Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, Ocean University of China, Qingdao 266003, P. R. China ‡ Marine Biomedical Research Institute of Qingdao, Qingdao 266003, P. R. China § Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College, Qingdao, 266003, P. R. China ∥ Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, P. R. China S Supporting Information *

ABSTRACT: The unique glycan-binding ability of chemically synthesized boronic acid derivatives makes them emerging candidates for developing anti-influenza A virus (IAV) drugs. Herein we report the synthesis and the anti-IAV activities of three series of novel boronic acid-modified quindoline derivatives both in vitro and in vivo. Boronic acid-modified compounds 6a and 7a effectively prevented the entry of virus RNP into the nucleus, reduced virus titers in IAV infected cells, and also inhibited the activity of viral neuraminidase. Compound 7a possessed broad antiviral spectrum and was able to inhibit cellular NF-κB and MAPK signaling pathways to block IAV infection. More importantly, IAV infected mice treated with compound 7a showed better survival rates than mice treated with oseltamivir, a popular anti-IAV drug. Thus, our study provides not only an antiviral preclinical candidate but also useful information for further research and development of boronic acid-modified anti-IAV drugs.

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INTRODUCTION Influenza pandemics cause significant morbidity and mortality, especially in high-risk populations such as the elderly, the very young, and those suffering from chronic illness.1,2 Given the magnitude of influenza pandemics as a threat to the global population, it is crucial to have as many prevention and treatment options as possible. The glycan receptor binding and specificity of influenza A virus (IAV) hemagglutinin as well as the IAV neuraminidase that removes terminal sialic acid of glycans are critical for virus infection and transmission in humans.3,4 The two most popular anti-IAV drugs, zanamivir and oseltamivir, are directed against the viral neuraminidase.5 Despite these successes, concerns regarding drug resistance, toxicity, and cost still remain.6 Hence, the development of novel anti-IAV agents with high efficiency and low toxicity is of high importance. Chemically synthesized boronic acid derivatives7,8 have been developed into drugs9,10 or biomarker identification reagents11,12 that work either inside cells or at the cell surface due to unique glycan interactions13,14 and Lewis acidity of the boronic acid moiety under physiological conditions. Especially GSK5852 (1 in Figure 1a), a boronic acid derived inhibitor of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, © 2017 American Chemical Society

entered clinical trials in 2011 and is being developed for the treatment of infection with HCV. The boronic acid moiety was found to be a critical pharmacophore for enhanced inhibition of HCV NS5B polymerase.15 Quindoline (2 in Figure 1a) belongs to the indoloquinoline alkaloid family, which has been reported to possess antiplasmodial,16 anti-inflammatory,17 and antitumor activities.18,19 The indoloquinoline moiety can penetrate cells, interact with DNAs, and kill cancer cells by three established mechanisms.18,19 Our previous work has shown that boronic acid modifications could reduce the cytotoxicity of some quindoline derivatives (3 and 3a in Figure 1a) mainly through decreasing the cell penetration and nuclear localization in cancer cells.20 Moreover, some viruses such as IAV and human papillomavirus (HPV) mainly use cell surface glycans as their receptors for endocytosis, and these viral particles also contain many glycan chains that interact with host cells.21−23 Since boronic acid derivatives possess good glycan-binding ability13,14 and reduce the nuclear localization of quindoline,20 we hypothesized that the introduction of boronic acid to Received: December 1, 2016 Published: March 7, 2017 2840

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phenylboronic acid-modified quindoline derivatives (6a−c and 7a−c) were obtained by amidation of 11-amino-10H-indolo[3,2-b]quinoline (5a−c) with 4-carboxyphenylboronic acid, or 3-carboxyphenylboronic acid, respectively, as in Scheme 1 (Figure 1b). The structure of compounds was characterized by nuclear magnetic resonance spectroscopy (NMR) and high resolution mass spectrometry (HRMS) analysis (see Supporting Information). Boronic Acid Modified Quindoline Derivatives Delayed the Nuclear Import of Virus RNP with Lower Cytotoxicity. Our previous work showed that boronic acid modifications could reduce the cytotoxicity of some quindoline derivatives through decrease of the nuclear localization in cancer cells.24 Thus, we first tested the cytotoxicity of these quindoline derivatives (5a−c, 6a−c, 7a−c) in A549 and H1299 cells. The results indicated that after 48 h exposure at a concentration of 20 μM, boronic acid modified quindoline derivatives (6a−c, 7a−c) were less toxic than the boronic acidfree quindoline derivatives (5a−c), and these tendencies were consistently seen in both A549 and H1299 cells (Figure 2a). Moreover, the structure−activity relationship (SAR) studies showed that compounds 6a and 7a had superior anti-IAV effects to other compounds (5a−c, 6b, 6c, 7b, 7c), and the selectivity index (SI = CC50/IC50) of 7a (SI = 27.9) was comparable to that of oseltamivir (SI = 36.2) (Supporting Information Table S1), which suggested that the modification and location of boronic acid group was crucial for the anti-IAV activities of quindoline derivatives. Furthermore, quindoline has intrinsic fluorescence, so after incubation with compounds 5a, 6a, and 7a for 4 h at 37 °C, the intrinsic fluorescence was detected by confocal microscopy. As shown in Figure 2b, the green fluorescence of 5a treated A549 cells was found both in and outside the cell nucleus. However, the A549 cells treated with 6a or 7a exhibited less fluorescence inside their nuclei than outside, and the higher level of fluorescence was mostly in cytoplasm and close to cell membranes (Figure 2b). The results indicated that the boronic acid-modified quindoline derivatives reduced nuclear entry to below quantifiable levels. Since IAV replication occurs in the nucleus, we then explored whether these compounds could prevent IAV from the nucleus entry by immunofluorescence assay of virus NP protein. In brief, influenza virus (A/Puerto Rico/8/34 [H1N1]; PR/8) (multiplicity of infection (MOI) = 1.0) was pretreated with 10 μM 5a, 6a, or 7a for 1 h at 37 °C before infection, and then after virus adsorption, the infecting media containing 10 μM of these three compounds were added to A549 cells, respectively. At 2 h postinfection (p.i.), high level of red fluorescence was detected in the non-drug-treated IAV infected cells, and the fluorescence was found in both the cytoplasm and the nucleus (Figure 2c). However, after the treatment with 5a, 6a, or 7a, the fluorescence was mainly observed in the cell cytoplasm and barely in the nucleus (Figure 2c), suggesting these compounds might interfere with the import of virus RNP into the nucleus. Quantitation of data of the fluorescence intensity in the nucleus showed that compounds 6a and 7a significantly prevented IAV from entering the nucleus in A549 cells (Figure 2d). Compound 7a showed most obvious colocalization with virus NP in cytoplasm, suggesting that 7a may exert the strongest inhibition of the nuclear import of virus RNP (Figure 2e). Moreover, some fluorescence could be detected in the nuclei of cells treated with each of the three compounds at 4 h p.i. (Figure 2c), which suggested that 6a and 7a might be able to delay the entry of virus RNP into nucleus.

Figure 1. Structures of compounds 1, 2, quindoine derivatives, and the synthetic strategy. (a) Structures of compounds 1, 2, and quindoine derivatives with boronic acid modification (3−3a). (b) Synthesis of boronic acid modified quindoine derivatives: (I) aromatic diamine, 2ethoxyethanol, 100 °C, 2 h (5a, 71.3%; 5b, 61.2%; 5c, 63.0%); (II) 4carboxyphenylboronic acid or 3-carboxyphenylboronic acid, DMTMM, 2-ethoxyethanol, 25 °C, 17 h (6a, 53.1%; 6b, 68.1%; 6c, 83.0%; 7a, 59.5%; 7b, 72.3%; 7c, 57.4%).

quindoline derivatives might interfere with virus binding and entering the cell due to increased interactions with the viruses or the cell surface based on boronolectin studies. Therefore, we successfully designed and synthesized three series of novel quindoline derivatives without (5a−c) or with boronic acid modifications (6a−c and 7a−c) as shown in Figure 1. We explored the anti-IAV activities and mechanisms of these three series of novel boronic acid-modified quindoline derivatives both in vitro and in vivo. Boronic acid-modified compounds 6a and 7a effectively prevented the entry of virus RNP into the nucleus and reduced the virus titer in IAVinfected cells. More importantly, IAV-infected mice treated with compound 7a had better survival rates than mice treated with oseltamivir, suggesting that this compound has the potential to be developed into novel anti-IAV agents in the future.

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RESULTS Synthesis of Novel Quindoline Derivatives. To synthesize the quindoline derivatives, the key intermediate of 11-chloroquindoline 4 (Figure 1b) was prepared following the procedure reported by Bierer.24 The substitution reaction of compound 4 with the commercial aromatic diamines then produced quindoline derivatives containing three different types of arylamine side chains at the C-11 position (5a−c). The 2841

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Figure 2. Boronic acid modified quindoline derivatives delayed the nuclear import of virus RNP with low cytotoxicity. (a) After a 48 h exposure to the quindoline derivatives (20 μM), the cell viability of H1299 and A549 cells was measured by resazurin assay. Values are the mean ± SD (n = 3). (b) A549 cells were incubated with the derivative 5a, 6a, or 7a (20 μM) for 4 h at 37 °C. After that, the cells were fixed and stained with DAPI before confocal imaging analysis. Scale bar represents 10 μm. (c) PR8 virus (MOI = 1.0) was pretreated with 5a, 6a, or 7a for 1 h at 37 °C before infection, and then after adsorption at 4 °C for 1 h, the media containing 10 μM of each compound were added to cells, respectively. At 2 or 4 h postinfection (p.i.), the localization of virus NP protein was evaluated by immunofluorescence assay. Scale bar represents 20 μm. (d) The average fluorescence intensity of NP proteins in cell nucleus at 2 h p.i. was measured by ImageJ (NIH) version 1.33u (USA) to calculate the average intensity per unit area of cell nucleus of different images (n = 30). Significance: (∗) P < 0.05 vs virus control group (PR8). (e) The colocalization of virus NP protein and 5a, 6a, or 7a at 2 h p.i. was observed by confocal imaging. Scale bar represents 20 μm.

Boronic Acid Modification Enhanced the Inhibitory Effects of Quindoline Derivatives against Different Influenza Viruses. If the boronic acid-modified quindoline derivatives prevented the IAV from entering the nuclei as shown in Figure 2c, they should in principle inhibit IAV replication. To test this, compounds 5a, 6a, and 7a were assayed for their ability to inhibit IAV multiplication in MDCK cells using a plaque assay.25 In brief, PR8 virus was preincubated with or without compounds 5a, 6a, 7a or oseltamivir (10 μM) for 1 h at 37 °C before infection. Then after incubation at 37 °C for 24 h, the virus yields were evaluated by plaque assay. As shown in Figure 3a, preincubation of PR8 with 6a or 7a at the concentration of 10 μM significantly reduced the virus titers of IAV (P < 0.05), suggesting that compounds 6a and 7a may be able to inactivate viral particles directly. Compound 7a showed the best inhibitory effects on viral titer (P < 0.01), while 5a did reduce virus titer to some extent but without significance (Figure 3a). Oseltamivir (10 μM) also significantly reduced the virus titer (P

< 0.05) but not as well as 7a (Figure 3a). Moreover, boronic acid-modified compounds 6a and 7a showed lower cytotoxicity in MDCK cells compared to that of 5a (Figure 3b). The 50% cytotoxic concentrations (CC50) of compounds 5a, 6a, and 7a were about 37.9 ± 1.6, 78.6 ± 5.7, and 142.1 ± 1.3 μM, respectively (Table S1). Thus, boronic acid modification may enhance the anti-IAV effects of quindoline derivatives in vitro with lower toxicity. To further evaluate the inhibitory effects of compound 7a on IAV infection, the virus yield reduction assay was performed with different concentrations of 7a. Briefly, PR8 virus was preincubated with or without 7a (2.5, 5, 10 μM) for 1 h at 37 °C before infection. At 24 h p.i., the virus yields were determined by plaque assay. As shown in Figure 3c, preincubation of PR8 with 7a at the concentrations of 2.5− 10 μM reduced the viral titers in a dose-dependent manner. 7a significantly decreased the virus titer when used at >5 μM (p < 0.05) (Figure 3c). Moreover, the 50% inhibitory concentration (IC50 value) of compound 7a for virus yield reduction was 2842

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Figure 3. Boronic acid modification enhanced the inhibitory effects of quindoline derivatives against different influenza viruses. (a) PR8 (MOI = 1.0) was pretreated with 5a, 6a, 7a, or oseltamivir (10 μM) for 1 h at 37 °C before infection, and then after adsorption, the media containing 10 μM of each compound were added to cells, respectively. At 24 h p.i., the virus titer in cell supernatants was determined by plaque assay. Values are the mean ± SD (n = 3): (∗) P < 0.05, (∗∗) P < 0.01 vs virus control group. (b) After 48 h exposure to 5a, 6a, or 7a at the indicated concentrations, the cell viability of MDCK cells was measured by resazurin assay. Values are the mean ± SD (n = 3). (c) MDCK cells were infected with 7a (2.5, 5, 10 μM) pretreated PR8 and incubated at 37 °C for 24 h. Then the virus yields were determined by plaque assay. Values are the mean ± SD (n = 3): (∗) P < 0.05, (∗∗) P < 0.01 vs virus control group. (d) HA titers from single-cycle high-MOI (MOI = 3.0) assays performed on MDCK cells infected with PR8, Minnesota, and Cal09 and treated with the indicated concentrations of 7a. Mean percentage HA titers were calculated as a percentage of HA titers from untreated cells for each group. Values are the mean ± SD (n = 4). (e) Approximately 50−100 PFU/well of PR8, cal09, or Minnesota virus was preincubated with 7a (0, 2.5, 5, 10 μM) for 1 h at 37 °C before infection, respectively. Then the virus−7a mixture was transferred to MDCK cells, incubated at 4 °C for 1 h, and subjected to plaque reduction assay. (f) Plaque number from plaque reduction assays performed on MDCK cells infected with the three viruses and treated with the indicated concentrations of 7a. Values are the mean ± SD (n = 4).

about 2.2 ± 0.1 μM, and the selectivity index (CC50/IC50) for 7a was about 64.6 in MDCK cells. Thus, compound 7a possesses inhibitory effects against IAV replication in vitro. To explore whether compound 7a exerts broad antiviral spectrum, the hemagglutination (HA) assay and plaque reduction assay were used to evaluate the inhibition effects of 7a against PR8 virus and two other clinical isolates of influenza viruses, H1N1 strain (A/California/04/2009) (Cal09) and H3N2 strain (A/swine/Minnesota/02719/2009) (Minnesota). Briefly, IAV (MOI = 3.0) infected MDCK cells were treated with 7a for 24 h, and then the HA titers in cell supernatants were determined. As shown in Figure 3d, for all three viruses tested, the reduction of virus HA titer with increasing

concentrations of 7a was dose-dependent. Compound 7a was effective against all strains tested including PR8, Cal09, and Minnesota, with IC50 values at 2.5 ± 0.4, 4.3 ± 0.7, and 3.0 ± 0.4 μM, respectively, superior to the effects of 5a, 6a, and sseltamivir (Table S2). Moreover, the multicycle replication assay showed that 7a also inhibited the plaque formation in PR8, Cal09, and Minnesota-infected cells when used at >2.5 μM, with IC50 values at 3.2 ± 0.5, 3.7 ± 0.6, and 2.3 ± 0.9 μM, respectively (Figure 3e, Figure 3f, and Table S2). Furthermore, both the single-cycle and multicycle replication assay all showed that the IC50 values of 5a were much higher than that of 6a and 7a (Table S2), further establishing boronic acid incorporation enhances the anti-IAV effects of quindoline derivatives. In 2843

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Figure 4. Influence of different treatment conditions of compound 7a on IAV infection. (a) MDCK cells were infected with PR8 (MOI = 1.0) by four different treatment conditions. (i) Previrus: PR8 was pretreated with 7a (10 μM) at 37 °C for 1 h before infection. (ii) Precell: MDCK cells were pretreated with 10 μM 7a before infection. (iii) Adsorption: MDCK cells were infected in media containing 7a (10 μM) at 4 °C for 1 h. (iv) After adsorption: After removal of unabsorbed virus, 7a (10 μM) was added to cells. At 24 h p.i., the virus yields were determined by plaque assay. Values are the mean ± SD (n = 3): (∗) P < 0.05 vs virus control group. (b) The inhibition effects of 7a and anti-HA antibody on IAV-induced aggregation of chicken erythrocytes were evaluated by hemagglutination inhibition (HI) assay. (c) Inactivated PR8 virus was incubated with 5a, 6a, or 7a (10 μM) or zanamivir (10 μM) for 30 min at 37 °C, and then NA enzymatic activity was determined by a fluorescent assay. Values are the mean ± SD (n = 3). (d) Vero cells expressing A/PR8 HA were first treated with trypsin and then incubated for 15 min in the presence of 7a. Then, the pH was lowered to 5.0 and the cells were incubated for 10 min at 37 °C in the presence of 7a. Following syncytium formation for 2 h at 37 °C, the cells were stained with Hema3Stat Pak and examined by microscopy.

interact with MDCK cell surface directly. Addition of 7a after adsorption could also reduce virus titers but without significance (Figure 4a). Thus, 7a might be able to inactivate virus particles directly before infection to block the adsorption process of IAV. Since compound 7a may interact directly with virus particles, we then explored whether 7a had interaction with virus surface hemagglutination (HA) protein by using the hemagglutination inhibition (HI) assay. The results showed that the anti-HA antibodies significantly inhibited the PR8 virus-induced aggregation of chicken erythrocytes at the concentrations of 0.625−5 μg/mL (Figure 4b). However, 7a could not inhibit virus-induced aggregation of chicken erythrocytes even at a concentration of 10 μM (Figure 4b), suggesting that 7a may have no direct interaction with virus HA protein. Moreover, the potential effect of 7a on the acid-induced membrane fusion was also investigated by a HA syncytia assay.26,27 Briefly, HAexpressing Vero cells were pretreated with compound and then

summary, boronic acid modified 7a can inhibit both H1N1 and H3N2 viruses in vitro. Influence of Different Treatment Conditions of Compound 7a on IAV Infection. The time-of-addition assay was performed to determine the stage(s) at which 7a exerted its inhibition actions in vitro. In brief, compound 7a was added to MDCK cells at four distinct time points: pretreatment of viruses, pretreatment of cells, during virus adsorption, and after virus adsorption. Subsequently, the antiviral activity was determined by plaque assay.25 As shown in Figure 4a, pretreatment of PR8 virus with 10 μM 7a for 1 h before infection significantly reduced the virus titer of IAV, compared to the nondrug treated virus control group (P < 0.05), suggesting that 7a may directly interact with IAV particles. Addition of 7a simultaneously with virus adsorption also possessed good inhibition on the virus titers (P < 0.05) (Figure 4a). However, pretreatment of cells did not decrease virus titers in vitro (Figure 4a), suggesting that 7a may not 2844

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Figure 5. Involvement of MAPK and NF-κB signaling pathways in the anti-IAV actions of compound 7a. (a−d) IAV (MOI = 1.0) infected cells were treated with compound 7a (5, 10 μM) or ribavirin (50 μg/mL) for 4 h, and then the phosphorylation of ERK1/2 (a), p38 (b), NF-κB (c), and Akt (d) proteins was evaluated by Western blot. Blots were also probed for β-actin and GAPDH proteins as loading controls. The result shown is a representative of three separate experiments. (e−h) Quantification of immunoblot for the ratio of p-ERK1/2 (e), p-p38 (f), p-NF-κB (g), and p-Akt (h) protein to β-actin or GAPDH. The ratio for noninfected cells (M) was assigned values of 1.0, and the data are presented as the mean ± SD (n = 3). Significance: (#) P < 0.05, (##) P < 0.01 vs normal control group (Mock); (∗) P < 0.05, (∗∗) P < 0.01 vs virus control group (PR8).

6a and 7a (Figure 4c). These results suggested that boronic acid modifications might be able to enhance the interaction between virus NA protein and quindoline derivatives to block virus infection. Indeed, molecular docking of 5a, 6a, and 7a to NA showed that the boronic acid group was positioned to the binding site directly forming H-bonds with nearby residues (Supporting Information Figure S1), which might partially explain the better anti-NA activity of 6a and 7a than that of 5a. In addition, the boronic acid of compounds 6a and 7a could interconvert from the trigonal, planar sp2 form to anionic, tetrahedral sp 3 complexes by coordination of various nucleophiles28 of the binding site. The covalent coordination binding of the boronic acid to the nucleophile group of a residue of the binding site, very likely S98, might be responsible for the stronger NA inhibition activity of 6a and 7a. Involvement of MAPK and NF-κB Signal Pathways in the Anti-IAV Actions of Compound 7a. The results in Figure 2 showed that compound 7a can access the cell cytoplasm and delay the transport of virus RNP complex, so we further explored if 7a could influence the cellular signaling pathways required for virus replication. The MAPK signaling

briefly exposed to pH 5.0 to trigger refolding of HA, resulting in fusion of plasma membranes and syncytium formation. As shown in Figure 4d, overexpression of HA protein in Vero cells leads to significant syncytium formation upon lowering the pH from 7.0 to 5.0 without compound treatment. No inhibition of HA syncytium formation was seen with 7a, whether added during the preacid stage or acid stage (Figure 4d), suggesting that the virus HA protein may not be the target of compound 7a. We next asked if the decreased virus titer was due to direct inhibition of the viral neuraminidase (NA) activity by performing an NA inhibition assay. In brief, NA proteins in viral suspensions of PR8 were incubated with 10 μM 5a, 6a, or 7a at 37 °C for 30 min, and then the enzymatic activity was determined by a fluorescent assay as described previously.25 Zanamivir was employed as a positive control (10 μM). As shown in Figure 4c, 6a and 7a had decent NA inhibition activity (inhibition by 56% and 72%) at a concentration of 10 μM whereas compound 5a without boronic acid modifications had almost no NA inhibition activity (Figure 4c). Zanamivir had superior NA inhibition activity (>85%) when compared to 2845

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Figure 6. Anti-IAV effects of quindoline derivatives in vivo. (a) Viral titers in lungs. After treatment with 5a, 6a, or 7a (10 mg kg−1 day−1) for 4 days, the pulmonary viral titers were evaluated by plaque assay. Values are the mean ± SD (n = 3). Significance: (∗) P < 0.05 vs virus control group. (b) Survival rate. IAV infected mice received oral therapy with 5a, 6a, or 7a (10 mg kg−1 day−1) for the entire experiment. Results are expressed as percentage of survival, evaluated daily for 14 days. Significance: (∗) P < 0.05 vs virus control group (placebo). (c−h) Histopathologic analyses of lung tissues on day 4 p.i. by HE staining (×10). The representative micrographs from each group are shown. Mock: noninfected lungs. PR8: IAV infected lungs without drugs. PR8 + oseltamivir-20: IAV infected lungs with oseltamivir (20 mg kg−1 day−1) treatment. PR8 + 5a-10: IAV infected lungs with 5a (10 mg kg−1 day−1) treatment. PR8 + 6a-10: IAV infected lungs with 6a (10 mg kg−1 day−1) treatment. PR8 + 7a-10: IAV infected lungs with 7a (10 mg kg−1 day−1) treatment. The red arrows indicate the presence of inflammatory cells in the alveolar walls and serocellular exudates in the lumen.

pathway was reported to be required for efficient vRNP export from nucleus, and the inhibitors of MAPK pathway could reduce IAV replication and inflammatory symptoms.29−31 In this study, ERK1/2 protein was significantly activated in virus control group to approximately 1.4-fold higher than normal control group at 4 h p.i. (p < 0.01) (Figure 5a and Figure 5e). However, after treatment with 7a (5 and 10 μM) for 4 h, the expression level of phosphorylated ERK1/2 protein significantly decreased from about 1.4 to about 0.7 and 0.6-fold of normal control group, respectively (p < 0.01) (Figure 5a and Figure 5e). Moreover, treatment with 7a (5 and 10 μM) for 4 h significantly reduced the expression level of phosphorylated p38 protein from about 5.1-fold to about 3.3- and 2.5-fold of normal control group, respectively (p < 0.05) (Figure 5b and Figure 5f). These data suggested that 7a may inhibit MAPK pathways to interfere with IAV replication.

Furthermore, the influence of compound 7a on the NF-κB signaling pathway, which is responsible for efficient virus vRNA synthesis, was also evaluated. As shown in Figure 5c and Figure 5g, phosphorylated NF-κB significantly increased to 1.8-fold higher than the normal control group after IAV infection for 4 h. However, treatment with 7a (5 and 10 μM) for 4 h significantly inhibited the activation of NF-κB from 1.8 to about 0.9, and 0.4-fold of normal control group, respectively (p < 0.05) (Figure 5g). Moreover, the activation of Akt protein which is associated with NF-κB pathway and IAV endocytosis was also evaluated (Figure 5d and Figure 5h). The results indicated that treatment with 7a (5 and 10 μM) for 4 h could significantly decrease the expression level of phosphorylated Akt protein from 1.4 to about 1.2, and 0.8-fold of normal control group, respectively (p < 0.05) (Figure 5h). Therefore, cellular NF-κB pathway may also be involved in the anti-IAV actions of 7a in vitro. 2846

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Boronic Acid Modification Also Enhanced the AntiIAV Effects of Quindoline Derivatives in Vivo. The data shown so far indicated the boronic acid modified quindoline derivatives prevented the entry of virus RNP into the nucleus and reduced the virus titer in IAV infected cells. However, the most important question that needed to be addressed was whether the novel compounds had anti-IAV activity in vivo. We explored such activity using the mouse pneumonia model as described previously.32 In brief, IAV-infected mice received compound 5a, 6a, or 7a orally (10 mg kg−1 day−1) or placebo once daily for the entire experiment and then sacrificed at 4 days postinfection. Subsequently, the pulmonary viral titers were determined by plaque assay.25 As shown in Figure 6a, after treatment of 6a or 7a (10 mg kg−1 day−1) for 4 days, the pulmonary viral titers were significantly decreased compared to that of the virus control group (p < 0.05), suggesting that oral therapy with boronic acid-modified quindoline derivatives could effectively inhibit IAV multiplication in mice. 5a could also reduce the pulmonary viral titers to some extent but without significance (P > 0.05) (Figure 6a). Oseltamivir (20 mg kg−1 day−1) treatment also showed significant reduction of virus titers in mice lungs (p < 0.05) (Figure 6a). Moreover, the survival experiments were also performed to evaluate the effect of quindoline derivatives on the survival of IAV infected mice. As shown in Figure 6b, oral administration with 6a or 7a (10 mg kg−1 day−1) increased survival rates significantly as compared to the placebo-treated control group (P < 0.05). In the particular experiment shown in Figure 6b, by day 14 after infection, only 30% of the mice in the placebo group survived whereas 90% of 7a-treated mice survived, which was superior to that of oseltamivir (20 mg kg−1 day−1) treated mice (80%). In summary, the data in Figure 6b showed that the boronic acid-modified compound 7a had superior anti-IAV activities in vivo in the mouse model tested. To further evaluate the effects of quindoline derivatives on viral pneumonia in mice, histopathology analysis was also performed. As shown in Figure 6c−h, lung tissues in the virus control group showed marked infiltration of inflammatory cells in the alveolar walls and the presence of massive serocellular exudates in the lumen (Figure 6d). However, mice treated with boronic acid-modified compounds 6a and 7a (10 mg kg−1 day−1) following infection had intact columnar epithelium in the bronchiole without inflammatory cell infiltration (Figure 6g and Figure 6h). The compound 5a treated lungs also had intact columnar epithelium in the bronchiole but with some serocellular exudates in the lumen (Figure 6f). Moreover, the lung tissues with oseltamivir (20 mg kg−1 day−1) treatment also showed intact columnar epithelium without infiltration of inflammatory cells (Figure 6e). Therefore, the boronic acid modification may also enhance the anti-IAV effects of quindoline derivatives in vivo.

also be involved in the anti-IAV actions of boronic acidmodified quindoline derivatives. As a result of the combined molecular targets and mechanisms, these novel quindoline derivatives showed better anti-IAV activities in vivo compared to that of the established neuraminidase inhibitor oseltamivir. Since boronic acid derivatives possess good glycan-binding ability13,14 and reduce the nuclear localization of quindoline,20 we hypothesized that the introduction of boronic acid to quindoline derivatives might interfere with virus binding and entering the cell due to increased interactions with the viruses or the cell surface. Herein, our data demonstrated that the largely cytoplasm-localized boronic acid-modified quindoline derivatives were less toxic to cancer cells than the nucleuslocalized quinoline derivatives. Boronic acid-modified compounds 6a and 7a delayed the entry process of virus RNP into nucleus to interfere with IAV replication. Boronic acid modification enhanced the inhibitory effects of quindoline derivatives against influenza virus in vitro. The compound 7a possessed better anti-IAV effects than 6a and the nonmodified compound 5a (Figure 3a and Table S2), which suggested that the boronic acid modification and the spatial structure are important for the anti-IAV actions of quindoline derivatives. Moreover, compound 7a was able to inhibit both H1N1 and H3N2 virus strains in vitro, suggesting that 7a possessed broad antiviral spectrum. The time-of-addition assay showed that pretreatment of PR8 virus before infection or treatment of virus during adsorption all significantly inhibited the virus titer of IAV, suggesting that 7a may have direct interaction with IAV particles. Combined with the results that pretreatment of PR8 could delay the import of virus RNP into nucleus and inhibit the activity of virus neuraminidase, we posit that 7a may be able to inactivate virus particles directly before infection to block the adsorption and internalization of IAV. Moreover, 7a could not inhibit IAVinduced aggregation of chicken erythrocytes and did not influence HA syncytia formation, suggesting that the virus HA protein may not be the target of 7a. The IAV NA protein, as a glycoprotein, was reported to be able to promote IAV attachment and entry into target cells during the initial stage of virus infection, in addition to promoting viral release.33−35 The results of NA inhibition assay and molecular docking indicated that the covalent coordination binding of the boronic acid to the nucleophile group of a residue of the binding site, very likely S98, might be responsible for the stronger NA inhibition activity of compounds 6a and 7a than that of 5a (Figure 4c and Figure S1). In addition, the molecular docking of NA with 7a and 7a′, an analogue of 7a obtained by replacement of boronic acid with carboxyl group, showed that 7a may form three hydrogen bonds with D69 and E37 respectively, whereas 7a′ only forms one hydrogen bond with D69 (Figure S2), suggesting that the boronic acid group may be essential to the binding affinity of 7a to NA. Therefore, boronic acid modifications may be able to enhance the interaction between NA protein and quindoline derivatives to attenuate the activity of NA, which interferes with IAV entry process. The fact that compound 7a can enter into cell cytoplasm and delay the nuclear import of virus RNP prompted us to explore the influence of 7a on cellular signaling pathways required for virus replication. Cellular MAPK and NF-κB signaling pathways are reported to be required for efficient virus vRNA synthesis and vRNP export from nucleus, and the inhibitors of these pathways can reduce IAV replication and inflammatory

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DISCUSSION AND CONCLUSIONS The glycan receptor binding specificity of influenza A virus (IAV) hemagglutinin (HA) and IAV neuraminidase (NA) that removes terminal sialic acid of glycans are not only used to define the specific type of IAVs but also critical for virus infection and transmission in humans. To take advantage of the unique glycan-binding property of chemically synthesized boronic acid derivatives, we showed that three series of newly synthesized quindoline derivatives inhibited the entry of the virus RNP into nucleus as well as the viral neuraminidase activity. Cellular NF-κB and MAPK signaling pathways may 2847

DOI: 10.1021/acs.jmedchem.6b00326 J. Med. Chem. 2017, 60, 2840−2852

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symptoms.29−31 The PI3K/Akt signaling pathway was reported to be required for efficient IAV endocytosis.36,37 In this study, we found that compound 7a could significantly inhibit the activation of cellular ERK1/2, p38, and NF-κB proteins in IAVinfected cells (Figure 5), which suggested that 7a may interfere with the activation of MAPK and NF-κB signaling pathways. Moreover, 7a also inhibited the activation of Akt protein which is associated with NF-κB pathway and IAV endocytosis. Thus, the cellular NF-κB and MAPK signaling pathways may be involved in the anti-IAV actions of compound 7a in vitro. It was reported that compounds that bind to NA proteins may be able to interfere with the activation of EGFR pathway, thus inhibiting the endocytosis of IAV.38 7a also inhibited the activation of downstream signals of EGFR such as Akt and NFκB (Figure 5), suggesting that 7a may also block IAV entry through inhibiting the activation of EGFR pathway. The in vitro antiviral effects of boronic acid modified quindoline derivatives were mirrored in a murine pneumonia model of influenza. Oral treatment of PR8-infected mice with 6a or 7a markedly improved the survival rate, decreased pulmonary viral titers, and attenuated the inflammatory symptoms (Figure 6). The survival benefit of 7a (10 mg kg−1 day−1) observed in our study was superior to that of oseltamivir (20 mg kg−1 day−1), and the boronic acid modified 6a and 7a showed better anti-IAV effects in vivo than the nonmodified compound 5a. Thus, boronic acid modification could also enhance the anti-IAV effects of quindoline derivatives in vivo. Considering that 7a is a small molecule that can enter the cell cytoplasm and that oral therapy of 7a had comparable anti-IAV effects to those of the positive control drug oseltamivir, the boronic acid-modified compound 7a may potentially be used for prevention and treatment of IAV by oral administration in the future. In conclusion, boronic acid modifications not only reduced quindoline’s cytotoxicity but also increased the anti-IAV effects of the quindoline both in vitro and in vivo. The boronic acidmodified quindoline derivatives could inhibit both H1N1 and H3N2 virus replication probably through interfering with the entry process of virus RNP into the nucleus as well as the NA activity inhibition. Most importantly, the compound 7a treated IAV infected mice had better survival rate compared to the mice treated with established anti-IAV drug oseltamivir. Thus, this study provides valuable information about the influence of boronic acid modifications on the anti-IAV activities of the quindoline derivatives, which has important implications in research and development of boronic acid-modified anti-IAV drugs.

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b]quinoline 4 (5 mmol), aromatic diamine (25 mmol), and 1 drop of concentrated HCl in 2-ethoxyethanol (30 mL) was heated at 100 °C for 2 h. Then the mixture was cooled, poured into ethyl acetate (100 mL), and the resulting precipitate was filtered. The filter cake dissolved into methanol (20 mL) and then poured into saturated sodium bicarbonate solution (200 mL). The resulting precipitate was filtered, washed with neutral water (3 × 15 mL), and then dried to give the crude product 5a−c, which was purified by recrystallization in methanol. General Procedure for the Synthesis of 11-(4Carbonylphenylboronic acid side chain)-10H-indolo[3,2-b]quinoline (6a−c). DMT-MM was synthesized by using a published procedure.39 A mixture of 5 (1.0 mmol), 4-carboxyphenylboronic acid, (0.22 g, 1.3 mmol), and DMT-MM (0.36 g, 1.3 mmol) was stirred at room temperature in 2-ethoxyethanol (10 mL) for 18 h. The mixture was poured into water (60 mL), the resulting precipitate was filtered, washed with neutral water (3 × 15 mL), and then dried to give the crude product 6, which was purified by flash chromatography on silica gel (chloroform/methanol, 8:1 and then 1:1). General Procedure for the Synthesis of 11-(3Carbonylphenylboronic acid side chain)-10H-indolo[3,2-b]quinoline (7a−c). A mixture of 5 (1.0 mmol), 3-carboxyphenylboronic acid, (0.22 g, 1.3 mmol), and DMT-MM (0.36 g, 1.3 mmol) was stirred at room temperature in 2-ethoxyethanol (10 mL) for 18 h. The mixture was poured into water (60 mL), the resulting precipitate was filtered, washed with neutral water (3 × 15 mL), and then dried to give the crude product 7, which was purified by flash chromatography on silica gel (chloroform/methanol, 8:1 and then 1:1). General Procedure for Purity Determination by HPLC. An Agilent 1260 HPLC system (Agilent Technologies, Palo Alto, CA, USA) comprised a quaternary solvent delivery system, an online degasser, an autosampler, a column temperature controller, and DAD detector coupled with an analytical workstation. The column configuration was an COSMOSIL C8 reserved phase column (5 μm, 250 mm × 4.6 mm). The compounds were dissolved in methanol, and injection volume was 10 μL. Detection wavelength was set at 254 nm, the flow rate was 1.5 mL min−1, and the column temperature was maintained at 25 °C. The mobile phase was elution solution which was mixed with solvent A (ammonium acetate, 50 mM, pH 4.3) and B (methanol). For compounds 5a−c, the mobile phase was as follows: A:B = 1:1. For compounds 6a−c and 7a−c, the mobile phase was gradient elution program which was as follows: 0−15 min, A 45 35%, B 5565%. The above gradient program would result in the retention time between 4 and 10 min. Results indicated that all the compounds used in our anti-influenza virus test had a purity of more than 95% (Supporting Information Table S3). Compound Characterization: 5a−c, 6a−c, 7a−c. 11-(2Aminophenyl)amino-10H-indolo[3,2-b]quinoline (5a). Yield, 71.3%; mp 170−175 °C; 1H NMR (500 MHz, DMSO-d6) δ (ppm) 10.38 (s, 1H, NH), 8.33−8.35 (d, J = 7.35 Hz, 1H), 8.13−8.14 (d, J = 8.45 Hz, 1H), 8.06−8.08 (d, J = 8.50 Hz, 1H), 7.91 (s, 1H, NH), 7.61−7.64 (t, J = 7.30 Hz, 1H), 7.52−7.56 (m, 2H), 7.38−7.40 (t, J = 7.50 Hz, 1H), 7.23−7.27 (m, 1H), 6.82−6.86 (m, 2H), 6.40−6.43 (m, 2H), 5.20 (brs, 2H, NH2); 13C NMR (126 MHz, DMSO-d6) δ (ppm) 144.5 (C), 144.3 (C), 143.1 (C), 141.1 (C), 131.5 (CH), 129.8 (CH), 129.2 (CH), 128.1 (CH), 127.4 (CH), 124.2 (CH), 123.8 (C), 123.7 (CH), 121.7 (CH), 121.0 (C), 120.4 (C), 120.0 (C), 119.9 (CH), 117.0 (CH), 115.8 (CH), 12.5 (CH); HRMS (ESI) m/z calcd for C21H17N4, 325.1453; found, 325.1441. 11-(3-Aminophenyl)amino-10H-indolo[3,2-b]quinoline (5b). Yield, 61.2%; mp 173−179 °C; 1H NMR (500 MHz, DMSO-d6) δ (ppm) 10.65 (s, 1H, NH), 8.54 (s, 1H, NH), 8.33−8.35 (d, J = 7.65 Hz, 1H), 8.18−8.22 (m, 2H), 7.64−7.67 (t, J = 7.35 Hz, 1H), 7.55− 7.60 (m, 2H), 7.48−7.51 (t, J = 7.60 Hz, 1H), 7.25−7.28 (t, J = 6.35 Hz, 1H), 6.87−6.90 (t, J = 7.90 Hz, 1H), 6.09−6.13 (t, J = 8.85 Hz, 1H), 6.01 (s, 1H), 5.76 (s, 1H), 5.19 (brs, 2H, NH2); 13C NMR (126 MHz, DMSO-d6) δ (ppm) 149.9 (C), 145.8 (C), 145.4 (C), 144.7 (C), 145.4 (C), 143.7 (CH), 129.8 (CH), 128.8 (C), 128.4 (C), 126.9 (CH), 126.1 (CH), 124.1 (CH), 123.7 (CH), 122.3 (C), 121.6 (CH), 121.5 (C), 119.7 (CH), 112.6 (CH), 107.0 (CH), 105.2 (CH), 102.0

EXPERIMENTAL SECTION

Materials and General Methods. All starting materials and solvents were obtained from commercial sources and used without further purification. Thin-layer chromatography (TLC) was performed on precoated silica-gel 60 F254 plates (E. Merck). Column chromatography was performed on silica gel (200−300 mesh; Qingdao Marine Chemical Company, Qingdao, China). Melting points were determined on a Mitamura-Riken micro-hot stage and not corrected. 1H NMR and 13C NMR spectra were obtained on a Pro pulse 500 MHz spectrometer (Agilent) with tetramethylsilane (Me4Si) as the internal standard, and chemical shifts were recorded in δ values. Mass spectra were recorded on a Q-TOF Global mass spectrometer. The important intermediate 11-chloroquindoline (4) was synthesized as reported.24 General Procedure for the Synthesis of 11-Amino-10Hindolo[3,2-b]quinoline (5a−c). A mixture of 11-Cl-10H-indolo[3,22848

DOI: 10.1021/acs.jmedchem.6b00326 J. Med. Chem. 2017, 60, 2840−2852

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(CH); HRMS (ESI) m/z calcd for C21H17N4, 325.1453; found, 325.1436. 11-(4-Aminophenyl)amino-10H-indolo[3,2-b]quinoline (5c). Yield, 63.0%; mp 175−181 °C; 1H NMR (600 MHz, DMSO-d6) δ (ppm) 9.99 (s, NH), 8.33 (s, NH), 8.28−8.29 (d, J = 7.74 Hz, 1H), 8.22−8.23 (d, J = 8.22 Hz, 1H), 8.11−8.12 (d, J = 8.28 Hz, 1H), 7.587.61 (m, 1H), 7.50−7.55 (m, 2H), 7.38−7.41 (m, 1H), 7.21−7.24 (m, 1H), 6.71−6.73 (d, J = 8.76 Hz, 2H), 6.55−6.57 (d, J = 8.70, 2H), 4.87 (s, 2H); 13C NMR (151 MHz, DMSO-d6) δ (ppm) 146.2 (C), 145.7 (C), 144.2 (C), 143.3 (C), 133.2 (C), 130.2 (C), 129.5 (C), 129.4 (C), 126.7 (CH), 123.6 (CH), 123.3 (CH, C), 122.2 (CH), 121.5 (CH), 120.7 (2CH), 120.4(CH), 119.6(CH), 115.3 (2CH), 112.5(CH); HRMS (ESI) m/z calcd for C21H17N4, 325.1453; found, 325.1446. (4-((2-((10H-Indolo[3,2-b]quinolin-11-yl)amino)phenyl)carbamoyl)phenyl)boronic Acid (6a). Yield, 53.1%; mp 240−248 °C; 1H NMR (600 MHz, DMSO-d6) δ (ppm) 11.11 (s, 1H, NH), 10.44 (s, 1H, NH), 8.56 (s, 1H), 8.22−32 (m, 4H), 7.51−8.00 (m, 9H), 7.10−7.30 (m, 3H), 6.81 (s, 1H); 13C NMR (151 MHz, DMSOd6) δ (ppm) 168.7 (C), 147.0 (C), 145.7 (C), 145.4 (C), 144.0 (CH), 140.8 (C), 129.9 (CH), 129.7 (2CH), 127.0 (CH), 126.7 (2CH), 124.5 (CH), 123.4 (CH), 123.0 (CH), 122.1 (CH), 121.7 (CH), 119.8 (CH), 112.56 (C), 110.9 (CH), 110.6 (CH), 106.2 (CH); HRMS (ESI) m/z calcd for C28H22BN4O3, 473.1785; found, 473.1769. (4-((3-((10H-Indolo[3,2-b]quinolin-11-yl)amino)phenyl)carbamoyl)phenyl)boronic Acid (6b). Yield, 68.1%; mp 236−243 °C; 1H NMR (600 MHz, DMSO-d6) δ (ppm) 10.82 (s, 1H), 10.10 (s, 1H), 8.82 (s, 1H), 8.35−8.36 (d, J = 7.8 Hz, 2H), 8.22−8.23 (d, J = 8.22 Hz, 2H), 7.87−7.89 (d, J = 8.28 Hz, 2H), 7.82−7.83 (d, J = 8.22 Hz, 1H), 7.51−7.67 (m, 5H), 7.26−7.38 (m, 3H), 7.16−7.19 (t, J = 7.8 Hz, 1H), 6.51−6.56 (m, 1H); 13C NMR (151 MHz, DMSO-d6) δ (ppm) 166.1 (C), 144.4 (CH), 143.8 (CH), 140.6 (CH), 138.2 (CH), 136.7 (CH), 134.4 (2CH), 130.3 (CH), 129.6 (CH), 127.8 (CH), 127.0 (2CH), 124.6 (CH), 123.7 (CH), 121.9 (CH), 120.1 (CH), 112.8 (C); HRMS (ESI) m/z calcd for C28H22BN4O3, 473.1785; found, 473.1798. (4-((4-((10H-Indolo[3,2-b]quinolin-11-yl)amino)phenyl)carbamoyl)phenyl)boronic Acid (6c). Yield, 83.0%; mp 242−250 °C; 1H NMR (600 MHz, DMSO-d6) δ (ppm) 10.59 (s, 1H, NH), 10.17 (s, 1H, NH), 8.81 (s, 1H, NH), 8.35−8.37 (m, 3H), 8.24−8.26 (d, J = 7.32 Hz, 1H), 8.20−8−22 (d, J = 8.76 Hz, 1H), 7.88−7.94 (m, 4H), 7.65−7.67 (m, 3H), 7.56−7.58 (m, 2H), 7.50−7.52 (t, J = 6.20 Hz, 1H), 7.25−7−29 (m, 1H), 6.81−6.83 (dd, J = 8.70 Hz, 2.76 Hz, 2H); 13C NMR (151 MHz, DMSO-d6) δ (ppm) 165.87 (C), 146.8 (C), 145.4 (C), 143.8 (CH), 140.8 (C), 136.9 (C), 134.6 (2CH), 132.2 (C), 129.9 (CH), 129.6 (C), 128.9 (C), 128.1 (C), 127.4 (C), 127.0 (2CH), 126.9 (CH), 125.7 (CH), 124.3 (CH), 123.4 (CH), 122.3 (CH), 122.1 (CH), 121.8 (CH), 119.9 (CH), 116.5 (CH), 112.6 (CH); HRMS (ESI) m/z calcd for C28H22BN4O3, 473.1785; found, 473.1762. (3-((2-((10H-Indolo[3,2-b]quinolin-11-yl)amino)phenyl)carbamoyl)phenyl)boronic Acid (7a). Yield, 51.1%; mp 232−239 °C; 1H NMR (600 MHz, DMSO-d6) δ (ppm) 10.81 (s, 1H, NH), 10.41 (s, 1H, NH), 9.25 (brs, 1H, NH), 8.38−8.46 (m, 3H), 8.20− 8.27 (m, 3H), 7.96−8.04 (m, 2H), 7.72−7.79 (m, 2H), 7.57−7.61 (m, 3H), 7.45−7.48 (t, J = 8.10 Hz, 1H), 7.30−7.32 (t, J = 8.10 Hz, 1H), 7.12−7.18 (m, 2H), 6.80 (s, 1H); 13C NMR (151 MHz, DMSO-d6) δ (ppm) 167.2 (C), 143.4 (CH), 138.9 (CH), 137.8 (CH), 135.6 (C), 134.9 (CH), 134.4 (CH), 134.0 (CH), 131.3 (C), 130.8 (CH), 130.0 (CH), 127.8 (CH), 126.6 (CH), 126.5 (CH), 124.6 (CH), 124.0 (CH), 123.9 (CH), 122.4 (CH), 120.4 (CH), 113.1 (C); HRMS (ESI) m/z calcd for C28H22BN4O3, 473.1785; found, 473.1793. (3-((3-((10H-Indolo[3,2-b]quinolin-11-yl)amino)phenyl)carbamoyl)phenyl)boronic Acid (7b). Yield, 72.3%; mp 235−241 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.92 (s, 1H, NH), 10.62 (s, 1H, NH), 10.41 (s, 1H, NH), 8.61−8.63 (d, J = 8.10 Hz, 1H), 8.54− 8.56 (d, J = 8.10 Hz, 1H), 8.27−8.35 (m, 4H), 7.91−7.96 (m, 3H), 7.62−7.70 (m, 3H), 7.45−7.48 (t, J = 7.60 Hz, 1H), 7.33−7.40 (m, 2H), 6.95−6.97 (d, J = 7.95 Hz, 1H); 13C NMR (126 MHz, DMSOd6) δ 167.3 (C), 143.2 (CH), 141.0 (CH), 140.6 (C), 138.7 (C), 138.0

(CH), 137.06 (C), 136.3 (C), 135.1 (C), 134.7 (CH), 134.2 (CH), 132.4 (CH), 132.1 (CH), 130.4 (C), 130.2 (CH), 130.0 (CH), 128.3 (CH), 125.4 (C), 125.0 (CH), 123.4 (CH), 121.4 (CH), 120.9 (C), 120.8 (C), 117.6 (CH), 117.4 (CH), 115.3 (C), 114.1 (C), 114.0 (CH); HRMS (ESI) m/z calcd for C28H22BN4O3, 473.1785; found, 473.1790. (3-((4-((10H-Indolo[3,2-b]quinolin-11-yl)amino)phenyl)carbamoyl)phenyl)boronic Acid (7c). Yield, 57.4%; mp 237−245 °C; 1H NMR (600 MHz, DMSO-d6) δ (ppm) 10.63 (s, 1H, NH), 10.20 (s, 1H, NH), 8.99 (s, 1H, NH), 8.40 (s, 1H), 8.36−8.38 (d, J = 6.50 Hz, 1H), 8.28−8.30 (d, J = 7.05 Hz, 3H), 8.21−8.23 (d, J = 7.10 Hz, 1H), 7.97−7.99 (t, J = 5.60 Hz, 2H), 7.67−7.71 (m, 3H), 7.57− 7.58 (d, J = 3.25 Hz, 2H), 7.46−7.53 (m, 2H), 7.25−7.30 (m, 1H), 6.87−6.89 (d, J = 8.70 Hz, 2H); 13C NMR (151 MHz, DMSO-d6) δ (ppm) 166.3 (C), 143.8 (CH), 140.1 (C), 137.4 (CH), 135.0 (CH), 134.0 (CH), 132.9 (CH), 130.0 (CH), 129.7 (CH), 128.0 (2CH), 127.3 (C), 125.1 (C), 124.3 (CH), 123.6 (CH), 122.0 (2CH), 121.8 (CH), 120.0 (CH), 117.1 (CH), 112.7 (C); HRMS (ESI) m/z calcd for C28H22BN4O3, 473.1785; found, 473.1804. Cell Culture and Virus Infection. Human lung cancer cell A549 and H1299 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and grown in Ham’s F-12 medium supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Madin−Darby canine kidney (MDCK) cells were grown in RPM1640 medium supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Vero cells were grown in DMEM medium supplemented with 10% FBS, 100 U/ mL of penicillin, and 100 μg/mL of streptomycin. Influenza virus (A/ Puerto Rico/8/34 [H1N1]; PR/8) was propagated in 9-day-old embryonated eggs for 3 days at 36.5 °C. Influenza H1N1 virus A/ California/04/2009 (Cal09) and H3N2 strain A/swine/Minnesota/ 02719/2009 (Minnesota) were propagated in MDCK cells for 3 days at 37 °C. For virus infection, virus propagation solution was diluted in PBS containing 0.2% BSA and was added to cells at the indicated multiplicity of infection (MOI). Virus was allowed to adsorb for 1 h at 4 °C. After removal of the virus inoculum, cells were maintained in infecting media (RPM1640, 4 μg/mL trypsin) at 37 °C in 5% CO2. Cytopathic Effect (CPE) Inhibition Assay. The cytopathic effect (CPE) inhibition assay was performed as described previously.40 MDCK cells in 96-well plates were first infected with IAV (MOI = 0.1) and then treated with different compounds in triplicate after removal of the virus inoculum. After 48 h of incubation, the cells were fixed with 4% formaldehyde for 20 min at room temperature (rt). After removal of the formaldehyde, the cells were stained with 0.1% crystal violet for 30 min. The plates were then washed and dried followed by solubilization of the dye with methanol, and the intensity of crystal violet staining for each well was measured at 570 nm. The concentration required for a test compound to reduce the CPE of IAV by 50% (IC50) was determined. Cytotoxicity Assay. The cytotoxicity of compounds was measured by the resazurin (Sigma-Aldrich, USA) assay as described previously.41 Confluent MDCK, A549, and H1299 cell cultures in 96-well plates were exposed to different concentrations of compounds in triplicate and then incubated at 37 °C for 48 h. After that, 20 μL of resazurin was added to each well at a final concentration of 0.2 mg/mL and incubated for 16 h at 37 °C. Then fluorescence of resazurin was measured using a SpectraMax M5 plate reader (Molecular Devices, USA) with excitation and emission wavelengths of 544 and 595 nm, respectively. Cell viability was expressed as a percentage of nontreated control. Indirect Immunofluorescence Assay. The immunofluorescence assay was performed according to W. Wang.40 Influenza A virus (PR8) (MOI = 1.0) was pretreated with 10 μM 5a, 6a, or 7a for 1 h at 37 °C before infection, and then after virus adsorption at 4 °C for 1 h, the infecting media containing 10 μM of these three compounds were added to A549 cells, respectively. At 2 or 4 h postinfection, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. Then cells were permeabilized using 0.5% (v/v) Triton X-100 in PBS for 5 min before being incubated with 2% BSA/PBS for 1 h at 37 °C. Cells were washed and incubated with anti-influenza virus NP 2849

DOI: 10.1021/acs.jmedchem.6b00326 J. Med. Chem. 2017, 60, 2840−2852

Journal of Medicinal Chemistry

Article

antibody (Santa Cruz, USA) overnight at 4 °C. After washing, the cells were incubated with FITC-labeled secondary antibody (Boster, China) for 50 min at 37 °C. Finally, cells were washed and observed using a laser scanning confocal microscope (Zeiss LSM 510, Jena, Germany). Plaque Assay. Confluent cell monolayers in 6-well plates were incubated with 10-fold serial dilutions of influenza A virus at 4 °C for 1 h. The inoculum was removed; cells were washed with PBS and overlaid with maintenance DMEM medium containing 1.5% agarose, 0.02% DEAE-dextran, 1 mM L-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 μg/mL TPCK-treated trypsin. After incubation for 3 days at 37 °C in a humidified atmosphere of 5% CO2, cells were fixed with 0.05% glutaraldehyde, followed by staining with 1% crystal violet in 20% ethanol for plaque counting. Hemagglutination (HA) Assay. The hemagglutination (HA) assay was performed as previously reported.42 Standardized chicken red blood cell (cRBC) solutions were prepared according to the WHO manual. Virus propagation solutions were serially diluted 2-fold in round bottomed 96-well plate, and 1% cRBCs were then added at an equal volume. After 60 min of incubation at 4 °C, RBCs in negative wells sedimented and formed red buttons, whereas positive wells had an opaque appearance with no sedimentation. HA titers are given as hemagglutination units/mL (HAU/mL). HA Syncytium Assay. The HA syncytium assay was performed as previously described with some modification.26,27 In brief, Vero cells were transfected with the PR8 HA plasmid (2 μg per well) using Lipofectamine and Plus reagent (Invitrogen). At 16 h posttransfection, the HA was first cleaved by incubation with 5 μg/mL TPCK-trypsin (Sigma) for 15 min at 37 °C. After preincubation with test compound during 15 min at 37 °C, the cells were incubated with a pH 5.0 buffer containing test compound. After exactly 10 min of incubation at 37 °C, the cells were rinsed, and medium containing 10% FBS was added, followed by 2 h of incubation at 37 °C. Finally, the cells were stained with the Hema3Stat Pak (Fisher Scientific, USA) according to the manufacturer’s instructions. Syncytia were visualized and photographed using a Zeiss Axio Observer inverted microscope with an attached digital camera. Neuraminidase Inhibition Assay. The influenza neuraminidase inhibitor detection kit (Beyotime, China) was used to measure the inhibition of NA activity as described previously.25 Briefly, inactivated PR8 virus supernatants were added to a 96-well plate and then mixed with different compounds (diluted in 33 mM MES buffer (pH 3.5), 4 mM CaCl2) at 37 °C for 30 min. Then MUNANA (20 μM) was added as the substrate and incubated at 37 °C for 30 min. The reaction was stopped by the addition of stop solution (25% ethanol, 0.1 M glycine, pH 10.7). Fluorescence was measured using a SpectraMax M5 plate reader with excitation and emission wavelengths of 360 and 440 nm, respectively. Computational Modeling. The initial structures of compound 5a and carbon derivatives of compounds 6a and 7a were drawn using ChemD, and their 3D structures were minimized in AMBER 12 using gaff force field.43 The crystal structure of neuraminidase A/PR/8/34 is unavailable, and its homology model was built using the crystal structure of neuraminidase of A/Brevig Mission/1/1918 H1N1 in complex with zanamivir28 as the template in Modeller 9.12.44 Details on homology modeling processes were described in a previous study.45 The high sequence identity (93%) between neuraminidase A/Brevig Mission/1/1918 H1N1 and neuraminidase A/PR/8/34 ensures the accuracy of the homology model. Ligand docking of compound 5a and carbon derivatives of compounds 6a and 7a to the homology model was performed using MOE using AMBER12:EHT force field.46 The induced fit docking approach was applied for consideration of the flexibility of the side chains of the residues at the binding site. The produced conformation of with the best score was selected for the analysis. Western Blot Assay. PR8 virus (MOI = 1.0) was pretreated with or without compound 7a for 1 h at 37 °C before infection, then after virus adsorption, the infecting media containing 7a at different concentrations (5, 10 μM) were added to MDCK cells. After incubation for 4 h, the cell lysate was separated by SDS−PAGE and

transferred to a nitrocellulose membrane. After being blocked in Trisbuffered saline (TBS) containing 0.1% Tween 20 (v/v) and 5% nonfat milk (w/v) at 4 °C overnight, the membranes were rinsed and incubated at room temperature (rt) for 2 h with antiphosphorylated NF-κB, ERK1/2, p38, Akt antibody, or anti-β-actin and GAPDH antibodies (Cell Signaling Technology, Danvers, USA) as control. The membranes were washed and incubated with AP-labeled secondary antibody (1:2000 dilutions) at rt for 2 h. The protein bands were then visualized by incubating with the developing solution (p-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine (BCIP)) at rt for 30 min. The relative densities of proteins were all determined by using ImageJ (NIH) version 1.33u (USA). In Vivo Experiments. Four-week-old female Kunming mice (average weight, 14.0 ± 2.0 g) were housed and studied under protocols approved by the Animal Care and Use Committee of Ocean University of China. Mice were inoculated intranasal with PR8 (4 HAU/mouse) diluted in 40 μL of 1× PBS and randomly divided into experimental groups. Two hours after inoculation, mice received lavage administration of compounds 5a, 6a, 7a (10 mg kg−1 day−1, 0.2 mL/10 g) or placebo, and the treatments were repeated once daily for the entire experiment. Mice were weighed and euthanized on day 4 after inoculation by spinal dislocation method, and lungs were removed and weighed. The lung specimens were homogenized in 1× PBS for determination of viral titers by plaque assay.25 Histopathological analysis was performed using H&E staining on samples collected on 4 days postinfection (dpi) as described previously.47 In the survival experiments, 10 mice per group were intranasally infected with PR/8 virus (8 HAU/mouse) at day 0. Oseltamivir (20 mg kg−1 day−1) was used as positive control drug as described previously.48 The drug administration was repeated once daily for 7 days. Mice were monitored daily for weight loss and clinical signs. If a mouse lost body weight over 25% of its preinfection weight, it was defined as dead and humanely euthanized immediately; the rest of the mice were sacrificed at the end of experiment on 14 dpi. Ethics Statement. All animal experiments were approved by the Institutional Animal Care and Use Committee at Ocean University of China (Grant OUCYY-2016002). All methods were performed in accordance with the animal ethics guidelines of the Chinese National Health and Medical Research Council (NHMRC). Statistical Analysis. All data are representative of at least three independent experiments. Data are presented as the mean ± SD. Statistical significance was calculated by SPSS 10.0 software using oneway ANOVA, with P values of