Inhibition of Enterovirus A71 by a Novel 2-Phenyl-Benzimidazole Derivative

Enterovirus A71 (EV-A71) infection has emerged as a significant public health concern at the global level. Epidemic events of EV-A71 have been reported worldwide, and this succession of outbreaks has heightened concern that EV-A71 may become a public health threat. In recent years, widespread A71 enterovirus also occurred in European countries. EV-A71 infection causes hand-foot-mouth disease (HFMD), herpangina, and fever. However, it can sometimes induce a variety of neurological complications, including encephalitis, aseptic meningitis, pulmonary edema, and acute flaccid paralysis. We identified new benzimidazole derivatives and described theirin vitrocytotoxicity and broad-spectrum anti-enterovirus activity. Among them, derivative 2b resulted in interesting activity against EV-A71, and therefore it was selected for further investigations. Compound 2b proved to be able to protect cell monolayers from EV-A71-induced cytopathogenicity, with an EC50 of 3 µM. Moreover, Vero-76 cells resulted in being significantly protected from necrosis and apoptosis when treated with 2b at 20 and 80 µM. Compound 2b reduced viral adsorption to Vero-76 cells, and when evaluated in a time-of-addition assay, the derivative had the highest effect when added during the infection period. Moreover, derivative 2b reduced viral penetration into host cells. Besides, 2b did not affect intestinal monolayers permeability, showing no toxic effects. A detailed insight into the efficacy of compound 2b against EV-A71 showed a dose-dependent reduction in the viral titer, also at low concentrations. Mechanism of action investigations suggested that our derivative can inhibit viral endocytosis by reducing viral attachment to and penetration into host cells. Pharmacokinetic and toxicity predictions validated compound 2b as a good candidate for furtherin vivoassays.


Introduction
Enterovirus A-71 (EV-A71) is a positive-strand RNA virus belonging to the Picornaviridae family, genus Enterovirus, commonly associated with mild diseases in children. EV-A71 clinical manifestations include hand-foot-mouth disease (HFMD), herpangina, and fever, but it also may involve the central nervous system (CNS). Neurological manifestations comprise meningitis, brainstem encephalitis, and acute flaccid paralysis. Epidemic events of EV-A71 have been reported in the last ten years in Australia, Japan, Malaysia, Taiwan, Vietnam, and China, and this succession of outbreaks has heightened concern that EV-A71 may become a public health threat [1,2]. In recent years, widespread A71 infections occurred also in European countries, such as the Netherlands, France, and Spain (https://www.eurosurveillance.org). HFMD occurrences constitute a perpetual menace in China, resulting in 7.2 million cases between 2008 and 2012 [3]. EV-A71 emerged as responsible for about 80% of occurred severe cases and 93% of the deaths during this time [4]. As a major neurotropic causative agent of HFMD, EV-A71 has replaced poliovirus as the most clinically important enterovirus following the global eradication of poliomyelitis [5,6]. HFMD is a benign and common childhood disease, easily transmitted via the fecal-oral routes and characterized by mucocutaneous ulcerative vesicles in the mouth as well as lesions on the hands and feet. While HFMD is caused by several members of the Enterovirus genus, HFMD caused by EV-A71 is considered to be the most pathogenic. Fatal neurological complications have been reported in children, with clinical symptoms such as aseptic meningitis, poliomyelitis-like acute flaccid paralysis, and brainstem encephalitis that may lead to cardiovascular collapse and pulmonary edema [7]. Seven EV A71 genogroups are known (A-G) [8]. Genogroup A contains the prototype strain BrCr, which was isolated in the United States from a 2-month-old male with aseptic meningitis, but it was also documented in China in 2008. Viruses belonging to genogroups B and C have been circulating more largely and contain six and five genotypes (B0-B5 and C1-C5). Genogroups D and G have been documented in India and genogroups E and F in Africa were recognized more recently. Several countries within the Asia-Pacific region have reported large outbreaks of EV-71 related to new genotypes B and C. Most of the EV-A71 strains circulating in Europe belong to genotype C [8].
Up until now, two inactivated vaccines have been developed and marketed exclusively in China [9]. With no established antiviral treatment for EV-A71 infection [10], management of severe EV-A71 infections is mainly aimed at alleviating disease symptoms using supportive treatments such as mechanical cardiopulmonary support systems and the administration of milrinone to prevent cardiopulmonary failure and improve clinical outcome in patients [11]. Other treatments include intravenous administration of immunoglobulin, interferon-alpha, and corticosteroids, all leading to favorable clinical outcomes in patients. With currently limited antivirals against EV-A71, it is fundamental to develop techniques and strategies to expand the drug discovery pipeline [12].
Among a long-lasting antiviral discovery project, we synthesized several small molecules that were subjected to a wide antiviral [13][14][15] and anti-infective screening [16,17]. Some of them turned out as active against representative Enteroviruses [18]. Previously reported benzotriazole derivatives which showed antiviral activity against Coxsackievirus B5 [18] and respiratory syncytial virus (RSV) [19] were used as prototypes for a scaffold simplification strategy and bioisostere substitutions. By the scaffold simplification strategy, the side chain on the main scaffold was reduced in size, choosing small substituted phenyl moieties. The benzotriazole scaffold was replaced by a bioisostere benzimidazole skeleton. They were selected as lead compounds and underwent classical chemical scaffold modification in order to improve potency and selectivity. In this study, we focused on identifying promising enteroviruses drug candidates and anew series of variously substituted 2-phenyl-benzimidazole derivatives (1a,b-5a,b) was designed, synthesized, and tested against a panel of relevant enteroviruses.

Synthesis
All starting materials were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Benzimidazole ring closure, to obtain derivatives 1a,b-5a,b, was carried out as described in the literature [20] by mixing o-phenylenediamines (1)(2)(3)(4)(5) and commercial aldehydes (a and b) in a ratio of 1:1,dissolvingthe mixture in a few mL of acetonitrile (CH 3 CN), and adding H 2 O 2 30% (ratio 1:7) and HCl 37% (ratio 1:3.5) at room temperature for a proper time, generally a few hours to a maximum of an overnight time period. The solid product obtained was filtered off, washed with CH 3 CN and then with water until neutral pH, and ultimately dried overnight in an oven. Products were obtained as pure solids by crystallization from ethanol and then characterized. Retention factors (R f ) were measured by thin-layer chromatography (TLC) using Merck F-254 commercial plates and a proper mixture of petroleum ether (PE) and ethyl acetate (EA) as eluent. Nuclear magnetic resonance (NMR) spectra were registered in solutions in deuterated DMSO or acetone and were recorded with a Bruker Avance III 400 NanoBay (400 MHz) instrument. 1 H-NMR chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane (TMS) used as internal standard, at 400 MHz. Chemical shift values are reported in ppm (δ) and coupling constants (J) are reported in Hertz (Hz). Signal multiplicities are represented as s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of doublets), t (triplet), q (quadruplet), and m (multiplet). 13 C-NMR chemical shifts are reported downfield from tetramethylsilane (TMS) used as internal standard, at 100 MHz.A suitable method among the APT (attached proton test) and jmod (J-modulated spin-echo for X-nuclei coupled to H-1 to determine the number of attached protons) was selected for each compound. The solutions for ESI-MS measurements were prepared by dissolving the solid compounds in HPLC acetonitrile to obtain a concentration of 1.0-2.0 ppm. Mass spectra in the positive-ion mode were obtained on a Q Exactive Plus Hybrid Quadrupole-Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA) mass spectrometer. The solutions were infused at a flow rate of 5.00 µL/min into the ESI chamber. The spectra were recorded in the m/z range 150-800 at a resolution of 140,000 and accumulated for at least 2 min in order to increase the signal-to-noise ratio. The instrumental conditions used for the measurements were as follows: spray voltage 2300 V, capillary temperature 250 • C, sheath gas 10 (arbitrary units), auxiliary gas 3 (arbitrary units), sweep gas 0 (arbitrary units), and probe heater temperature 50 • C. ESI-MS spectra were analyzed by using Thermo Xcalibur 3.0.63 software (Thermo Fisher Scientific), and the average deconvoluted monoisotopic masses were obtained through the Xtract tool integrated within the software.

Cytotoxicity Assays
HeLa and LLC-MK2 cells were seeded in 96-well plates at an initial density of 5 × 10 5 cells/mL, in Minimum Essential Medium with Earle's salts (MEM-E), L-glutamine, 1 mM sodium pyruvate, and 25 mg/L kanamycin, supplemented with 10% fetal bovine serum (FBS). Vero-76 cells were seeded in 96-well plates at an initial density of 5 × 10 5 cells/mL, in Dulbecco's modified Eagle medium (D-MEM), with L-glutamine and 25 mg/L kanamycin, supplemented with 10% FBS. Cell cultures were then incubated at 37 • C in a humidified, 5% CO 2 atmosphere, in the absence or presence of serial dilutions of test compounds. The test medium used for the cytotoxic assay as well as for the antiviral assay contained 1% of the appropriate serum. Cell viability was determined after 72-96 h at 37 • C by the MTT method for HeLa and Vero-76 as previously reported [22].

Antiviral Assays
Compounds' activity against EV-A71 and EV-D68 was based on inhibition of virusinduced cytopathogenicity in Vero-76 and HeLa cells, respectively, acutely infected with an m.o.i. of 0.01. Briefly, Vero-76 and HeLa cells were seeded in 96-well plates at a density of 2 × 10 4 and 3 × 10 4 cells/well, respectively, and were allowed to form confluent monolayers by incubating overnight in growth medium at 37 • C in a humidified CO 2 (5%) atmosphere. Cell monolayers were then infected with 200 and 300 PFU (50 µL of a proper virus dilution) in maintenance medium (D-MEM and MEM-Earl, with L-glutamine, 1 mM sodium pyruvate, and 0.025 g/L kanamycin, supplemented with 0.5% inactivated FBS, respectively) to give an m.o.i of 0.01. After 2 h, 50 µL of maintenance medium, without or with serial dilutions of test samples, wasadded. After a 3/4-day incubation at 37 • C, cell viability was determined by the MTT method [23]. Compounds' activity against CV-B3, CV-B4, CV-B5, and Echo 9 was determined by plaque reduction assays in infected cell monolayers, as previously reported [24]. Plc (Pleconaril), NM107 (2 -C-methylcytidine),and Rup (Rupintrivir) were used as references and internal controls.

Yield Reduction Assay
Vero-76 cells were inoculated with EV-A71 at an m.o.i. of 0.1 in maintenance medium and tested compounds at non-cytotoxic concentrations. Following the 2-h adsorption period at 37 • C and 5% CO 2 , the inoculum was removed and replaced with fresh medium containing the same concentration of compounds. After 96 h at 37 • C and 5% CO 2 , each sample was harvested and diluted with serial passages, starting from 10 −1 up to 10 −8 . The titer of the serial dilutions of the virus-containing supernatant was determined by a standard plaque assay, counting the number of obtained plaques in at least two different dilutions for each concentration. NM107 (2 -C-Methyl-Cytidine) was used as a reference compound.

Apoptosis Assay
To assess levels of apoptosis following 2b derivative treatment, a flow cytometric analysis, using the cell apoptosis kit Annexin V/Propidium Iodide (PI) double staining uptake (Invitrogen, Life Technologies, Italy), was used. Vero-76 cells, at the density of 2 × 10 5 cells/mL, were seeded in 12-well plates (Corning, New York, NY, USA) with complete medium (described in cell culture section). After EV-A71 viral adsorption, the cells were incubated in the absence or presence of different concentrations of 2b for 96 h, until the cytopatic effect CPE of the virus control reached 70-80%. Cells were then washed once with PBS 1 X and re-suspended in 100 µL of Annexin binding buffer plus 1 µL of Annexin V and 1 µL of PI. Then, the reaction was performed in the dark for 15 minutes at room temperature. Stained cells were then analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using a 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter, Pasadena, CA, USA). Cell apoptosis was analyzed using the software Summit Version 6.3.1.1, Beckman Coulter.

Virucidal Activity Assay
Compound 2b (20 µM) was incubated with 1 × 10 5 PFU/mL of EV-A71 at either 4 or 37 • C for 1 h. The mixture without a test sample was used as the control. At the end of the incubation period, samples were serially diluted in media, and titers were determined on Vero-76 cells at high dilutions, at which the compound was not active. Virus titers were determined by a plaque assay in Vero-76 cells.

Cell Pretreatment Assay
Vero-76 cell monolayers in 24-well plates were incubated with 20 µM concentration of the 2b or 50 µM of NM 107 for 2 h at 4 • C. After the removal of the compounds and two gentle washes, cells were infected with EV-A71. After virus adsorption to cells, the inoculum was removed and the cells were then overlaid with medium, incubated for 4 days at 37 • C, and then virus titers were determined by a plaque assay.

Adsorption Assays
Vero-76 cells grown in 24-well plates were infected with EV-A71, with an m.o.i. of 1, in the presence or absence of compound 2b. Multiwells were incubated for 120 min at 4 • C. The medium containing the unabsorbed virus was then removed, and cells were washed twice with PBS and overlayed with medium. Plaques were counted after 96 h of incubation at 37 • C.

Time-of-Addition Assay
The confluent monolayers of Vero-76 cells in 24-well tissue culture plates were infected for 1 h at room temperature with EV-A71 dilutions to give a final m.o.i. of 1. After adsorption, the monolayers were washed twice with maintenance medium and incubated with the same medium at 5% CO 2 and 37 • C (time zero). Vero-76 cells were treated with compound 2b (20 µM, approximately 10 times higher than the EC 50 ) or the reference for 1 h during the infection period (at −1 to 0) and at a specific time point, 0 to 2, 2 to 4, 4 to 6, 6 to 8, and 8 to 10 h post-infection. After each incubation period, the monolayers were washed two times with maintenance medium and incubated with fresh medium until 12 h post-infection. Then, the monolayers were frozen at -80 • C and the viral titers were determined by a plaque assay.

Penetration Assay
A 24-well tissue culture plate was seeded with Vero-76 cells (4 × 10 5 cells/well), which were then incubated overnight at 37 • C and 5% CO 2 . The cells were chilled on ice for 1 h, and the medium was removed. The cells were infected with 100 PFU (200 µL) of EV-A71 on ice for 120 min. The medium containing the unbound virus was then removed; various concentrations of compound 2b (100-0.8 µM) in medium were added, and the cells were incubated at 37 • C for 60 min to trigger endocytosis of the virus. The infected cells were then treated with alkaline phosphate-buffered saline (PBS; pH 11) for 1 min to inactivate any viruses that had not penetrated the cells, and then acidic PBS (pH 3) was immediately added to neutralize the mix. The neutralized medium was removed, and cells were overlayed with 0.75% methylcellulose in media and then incubated at 37 • C. After incubation at 37 • C for 96 h, cells were stained, and plaques were determined by counting. This penetration assay was performed according to the method [25,26].

Evaluation of Cell Monolayer Permeabilization (TEER)
The activity of compound 2b on intestinal cells monolayer permeability was assessed by measuring the transepithelial electrical resistance (TEER) value. Caco-2 cells (0.5 × 10 5 cells/well) were grown in 12 mm i.d. Transwell inserts (polycarbonate membrane, 0.4 µm pore size) and culture medium was dispensed in the apical (0.5 mL) and basolateral (1.5 mL) compartments of each well. Resistance was measured using a Millicell-ERS ohmmeter (Millicell-ERS system, Millipore, Bedford, MA, USA) as previously reported by Serreli et al., [27]. Once the cell monolayers were formed, only inserts with TEER values >300 Ω/cm 2 were chosen for the experiment. Then, compound 2b (final concentration 20 µM) and, to induce an increase in permeability, an oxysterol mixture (final concentration 60 µM) prepared as described by Serra et al. [28] were added to the culture medium. TEER values were subsequently measured at intervals of 0.25, 0.5, 0.75, 1, 2, 3, 6, 18, 24, and 48 h and are reported as a percentage of the corresponding TEER value at time zero (T = 0).

Statistical Analysis
Cell-based experiments were independently repeated at least three times. The data are reported as mean ± standard deviation (SD). The statistical significance values are defined as * p < 0.05, ** p < 0.01, *** p < 0.001. The statistical significance was calculated with the Mann-Whitney test performed in GraphPad Prism (San Diego, CA, USA).

Linear Regression Analysis
The extent of cell growth/viability and viral multiplication, at each drug concentration tested, were expressed as percentage of untreated controls. Concentrations resulting in 50% inhibition (CC 50 or EC 50 ) were determined by linear regression analysis.

In Silico Screening
The PreADMET (http://preadmet.bmdrc.kr/) tool was used to calculate druglikeness for the library of new compounds (1a-5a, 1b-5b). An ADME (absorption, distribution, metabolism and excretion) profile for all the new derivatives was predicted using the PreADMET and SwissADME (http://www.swissadme.ch/) tools. Two-dimensional structural models for each compound were drawn in ChemDraw Ultra version 12.0 (Cambridge Software) and SMILES (Simplified Molecular Input Line Entry System) strings of each compound were used as input for the predictions. Degree of plasma protein binding (PPB) was calculated with PreADMET and is classified as strongly bound if PPB% > 90% and weakly bound if PPB% < 90%. Blood-brain barrier (BBB) penetration was predicted by both the tools. BBB penetration is presented as a ratio of compound concentration in brain (C brain ) and compound concentration in blood (C blood ). Compounds are labeled as high, middle, or highly absorbing into the CNS when C brain /C blood values are >2, 2-0.1, and <0.1, respectively. SwissADMET evaluates whether a compound would be absorbed into the CNS or not. Lipophilicity was calculated by SwissADME and reported as Consensus Log P o/w (CLog P o/w ) as an average of different predictions from five different algorithms (iLOGP, XLOGP3, WLOGP, MLOGP, SILICOS-IT). Water solubility was evaluated by the SwissADME tool. It also evaluated compounds as targets of P-glycoprotein (P-gp) efflux. Gastrointestinal (GI) absorption was predicted by SwissADME, while PreADMET used Caco-2 cells as a GI absorption model and permeation was measured as nm/s. Permeation was classified as low, middle, or high for values <4 nm/s, 4-70 nm/s, and >70 nm/s, respectively. PreADMET predicts the toxicity of compounds on models of the Ames test against strains of Salmonella typhimurium TA100 and TA1535. Toxicity was calculated with or without consideration of liver metabolism as metabolic activation by a rat liver homogenate (S9 fraction). Results were produced as positive or negative mutagenicity.

Chemistry
Benzimidazole derivatives were designed and synthesized as reported in Scheme 1. o-Phenylenediamine (1-5) and the corresponding aldehyde (a and b) were mixed and reacted in a single-pot, simple condensation reaction at room temperature, obtaining derivatives 1a,b-5a,b.

Chemistry
Benzimidazole derivatives were designed and synthesized as reported in Scheme 1. o-Phenylenediamine (1-5) and the corresponding aldehyde (a and b) were mixed and reacted in a single-pot, simple condensation reaction at room temperature, obtaining derivatives 1a,b-5a,b. 1a,b-5a,b.

Antiviral Assays
All newly synthesized 2-phenyl-benzimidazole derivatives were evaluated for their broad-spectrum anti-enterovirus activity in cell-based assays. Several compounds exhibited significant inhibitory activity, with EC 50 values in the low micromolar range (i.e., <10 µM, Table 1). Concomitantly, moderate to low cytotoxicity was detected for almost all compounds, with CC 50 values mostly in the high micromolar range (>100 µM) in Vero-76 cells . Only 1a,b, and 3a,b showed cytotoxicity in the low micromolar range against Vero-76, HeLa, and LLC-MK2 cell monolayers.
Concerning the spectrum of antiviral activity, the most relevant results referred to the interesting activity of derivatives 2b and 2a against the broad panel of enteroviruses (Table 1). Compound 2a showed remarkable antiviral activity against Entero A, B, and C viruses with an EC 50 lower or equal to 10 µM. No activity was detected against EV-D68 (>100 µM) or CV-B4. The comparable interesting activity was determined for compound 2b against all the Entero strains tested. The same lack of activity was described for EV-D68 (>100 µM). Particularly, 2b resulted in being effective in reducing cytopathy induced by EV-A71 with an EC 50 of 3 µM and a selectivity index > 30. Further, derivatives 5a and 5b resulted in being active against Poliovirus 1 (PV-1) and EV-A71 with an EC 50 range of 12-31 and 10-6 µM, respectively.
From a structure-activity relationship (SAR) perspective, we can state the relevance of the substitution of the benzimidazole (BIZ) scaffold since derivatives 1a and 1b, with no substituents, are more endowed with cytotoxicity than with antiviral activity. At the same time, we detected high toxicity also when BIZ was substituted with two bigger chlorine atoms in positions 5 and 6 (series 3). In terms of cytotoxicity, the substitution with a moderate steric hindrance such as one bigger chlorine atom (series 2) and one or two smaller fluorine atoms (series 4 and 5) turned out as the best option. Non-toxic series 2 and 5 are also complemented with the best antiviral activity detected. Interestingly, the presence of one single chlorine atom on the BIZ scaffold provided series 2 derivatives a wider antiviral activity, as can be seen from Table 1, showing remarkable EC 50 values when tested against EV-A71, CV-B3, CV-B5, PV-1, and also Echo 9, which was successfully inhibited only by the derivatives 2a and 2b.
Regarding series 5, two fluorine atoms were placed in positions 5 and 6 on BIZ, whichendowed derivatives 5a and 5b withmore selective antiviral activity. Series 5 showed relevant EC 50 values when tested against PV-1, EV-A71, and EV-D68 (5b). To evaluate the weight of the sterical hindrance on position 4 of the phenyl substituent, we selected isopropyl (a) and tert-butyl moieties (b) for the purpose. It is clearly shown that the most hindered tert-butyl moiety lent an increased activity profile if compared with the parental derivative which brings the isopropyl substituent on the same 4 position. Focusing on EV-A71, compound 2b presented an EC 50 value of 3.5 µM, while the parental 2a value was recorded at 7 µM. It is even more striking when series 5 is considered, where 5b showed an EC 50 value of 6 µM, while 5a showed only 31 µM. Derivative 5b is also quite potent when inhibiting EV-D68 replication at 4 µM concentration, while the parental 5a is not active when tested against this Enterovirus. It can be concluded that the tert-butyl moiety provides the best antiviral activity to benzimidazoles that present one big or two small halogen atoms on the BIZ scaffold.
Based on the described results, 2b showed the most compelling profile, resulting in a promising antiviral activity against EV-A71 and extendedspectrum against CV-B3, CV-B4, CV-B5, Echo 9, and PV-1.Concomitantly, the general low cytotoxic profile of 2b was proved in the cell lines, sustaining the viral replication of several strains ( Table 2). Starting from these considerations and taking into account the recent EV-A71 epidemics that occurred in Europe, the concern about the potential emergence of EV-A71 as a worldwide health threat, and the lack of an established treatment, we then decided to select compound 2b and EV-A71 for additional investigations, as described in detail below.

Protective Effect of 2b on EV-A71 Infected Cells
To verify whether derivative 2b could interfere in EV-A71-induced apoptosis and preserve the vitality of monolayers, Vero-76 cells, growth in 12-well plates, were untreated or infected with an m.o.i. of 0.1 of EV-A71. After viral adsorption, the cells were incubated in the absence or presence of 20 and 80 µM of 2b. As shown in Figure 1, the intact cell monolayer suggests that both concentrations displayed no cytotoxic effect against cells ( Figure 1A-C), whereas, after infection, EV-A71 showed a cytopathic effect with morphological variations of monolayer and round-shaped cells representing the dead cells ( Figure 1D). On the contrary, treatment with 20 µM and 80 µM of the compound seems to protect cells from EV-A71 infection and no evident cytotoxic effects were detected up to a concentration of 80 µM. (Figure 1E,F). To validate this morphological result, the cells were stained with Annexin-V-fluorescein and propidium iodide and subsequently subjected to flow cytometry analysis.

Protective Effect of 2b on EV-A71 Infected Cells
To verify whether derivative 2b could interfere in EV-A71-induced apoptosis and preserve the vitality of monolayers, Vero-76 cells, growth in 12-well plates, were untreated or infected with an m.o.i. of 0.1 of EV-A71. After viral adsorption, the cells were incubated in the absence or presence of 20 and 80 µ M of 2b. As shown in Figure 1, the intact cell monolayer suggests that both concentrations displayed no cytotoxic effect against cells ( Figure 1A-C), whereas, after infection, EV-A71 showed a cytopathic effect with morphological variations of monolayer and round-shaped cells representing the dead cells ( Figure 1D). On the contrary, treatment with 20 µ M and 80 µ M of the compound seems to protect cells from EV-A71 infection and no evident cytotoxic effects were detected up to a concentration of 80 µ M. (Figure 1E,F). To validate this morphological result, the cells were stained with Annexin-V-fluorescein and propidium iodide and subsequently subjected to flow cytometry analysis. The dot plots in Figure 2 show the Vero-76 cell distribution within four different cell populations: live, apoptotic, late apoptotic, and necrotic cells, and represent one of three sets of independent experiments conducted. In the untreated cells, there was a minimal number of necrotic, early, and late apoptotic cells and this trend was very similar to the 20 µ M-treated cells (Figure 2A  The dot plots in Figure 2 show the Vero-76 cell distribution within four different cell populations: live, apoptotic, late apoptotic, and necrotic cells, and represent one of three sets of independent experiments conducted. In the untreated cells, there was a minimal number of necrotic, early, and late apoptotic cells and this trend was very similar to the 20 µM-treated cells (Figure 2A,B), whereas necrotic cells slightly increased in treated cells with 80 µM concentration if compared to untreated cells ( Figure 2C), confirming the minimal cytotoxic effect of our derivative. The A71 virus is able to induce necrosis in about 70% of untreated cells, while compound 2b effectively reduces the death of Vero-76 cells. When treated with 20 and 80 µM, EV-A71-infected cells are significantly protected from necrosis and apoptosis, displaying 90% and 80% of viability and only 10% and 20% of necrotic cells, respectively ( Figure 2E-G).

TEER Experiment
To ascertain any toxic effect of derivative 2b on human cells, we tested it on differentiated intestinal Caco-2 monolayers, commonly used to simulate the gut epithelium and to evaluate changes in intestinal permeability. Cells were treated with an oxysterols mixture (Oxy, 60 µM) as a negative control, and it was observed that it caused cellular redox imbalance, reflected in a significant alteration of the monolayer integrity with time ( Figure 3

TEER Experiment
To ascertain any toxic effect of derivative 2b on human cells, we tested it on differentiated intestinal Caco-2 monolayers, commonly used to simulate the gut epithelium

Yield Reduction Assay and Study of the Mechanism of Action
The antiviral activity of compound 2b was investigated in a yield reduction assay, in

Yield Reduction Assay and Study of the Mechanism of Action
The antiviral activity of compound 2b was investigated in a yield reduction assay, in order to ascertain the reduction invirus titer in the presence of the active compound. Non-cytotoxic concentrations of 100, 20, 4, and 0.8 µM were used and a dose-dependent reduction inthe titer was observed. An important reduction in theEV-A71 titer was detected also in a low concentration ( Figure 4A). To assess the effect of compound 2b on viral infectivity, a virucidal assay was performed. No significant differences between the titers of EV-A71 treated at the two different temperatures were observed ( Figure 4B). Compound 2b failed to affect the EV-A71 infectivity, suggesting that the inhibition of EV-A71 replication observed in cell-based assays could be due to interference with a step along the EV-A71 replication cycle. Furthermore, no inhibition was observed when cells were pre-incubated (2 h) with 20 µM concentration of derivative 2b and then infected with untreated EV-A71 (data not shown).
The kinetics of virus adsorption in the presence of derivative 2b was also evaluated. Low-temperature treatment permits the binding of viruses to the cell surface receptors but avoids the internalization of viral particles into the host cells. Accordingly, Vero-76 cells were incubated with EV-A71 (m.o.i. = 1) and compound 2b for 2 h at 4 • C, using compound concentrations of 20 µM. The treatment with compound 2b resulted in a detectable reduction in the virus titer in comparison to the untreated infected control ( Figure 4C). Furthermore, we assessed the potential mode of antiviral activity with a time-ofaddition assay ( Figure 4C). The experiment was performed in EV-A71-infected Vero-76 cells (m.o.i. = 1) exposed to the compound (20 µM) at different times of infection. Interestingly, Figure 4C showed that the compound exerted the greatest effect when added along with the virus. Moderate titer reduction is detectable until 2 h post-infection. However, a reduction in virus yield was detected if compared to the untreated control, even when the compound was added at the late stages of the growth cycle.
We performed a penetration assay to scan the mechanism of inhibition of the entry of EV-A71 by compound 2b into Vero-76 cells. The percentage of reduction in viral penetration was determined in relation to the untreated control.We found that compound 2b could effectively inhibit the viral penetration in a dose-dependent manner with an EC 50 of 6.2 µM ± 2. (Figure 4D).

Druglikeness, Pharmacokinetic and Toxicity Predictions
Theoretical druglikeness, pharmacokinetic, and toxicity properties prediction is a key step between in vitro and in vivo experiments. The PreADMET (http://preadmet. bmdrc.kr/) and SwissADME (http://www.swissadme.ch/) tools were employed to assess the newly synthesized library of compounds (1a-5a, 1b-5b) and results are reported in Table 3. On the base of Lipinski's"rule of five", all the tested compounds are suggested to be drug-like molecules. According to pharmacokinetic predictions, compounds are all strong binders to the plasma protein with %plasma protein binding (PPB) values higher than 90%. They are all suggested to be absorbed into the CNS with high BBB (blood-brain barrier) permeability scores. All molecules are supposed to be moderately water-soluble. Lipophilicity is predicted to be good in general terms, with CLog Po/w values ranging between 3.88 and 5.19. As a matter of fact, gastrointestinal absorbance of these compounds is expected to be from middle to high. With the sole exception of compounds 1a, 4a, and 5a, they are generally considered not to be a substrate for P-glycoprotein efflux. In order to assess whether these new molecules could be used for in vivo assays, a toxicity profile prediction was performed using the PreADMET tool. Most of our compounds were mutagenic in the Ames test either with or without metabolic activation, except for our lead compound 2b and derivative 3b. The human ether a go-go-related (hERG) gene encodes cardiac potassium channels, inhibition of which would prolong the QTc interval along with the risk of cardiac arrhythmias [29]. Toxicity predictions resulted in medium risk of hERG inhibition for our compounds. Before starting with eventualin vivoexperiments, it will be necessary to assess the concentration at which the hERG inhibition rate is troubling and compare it with the EC 50 values fromthe in vitroanalysis. All considered, the newly designed and synthesized compounds showed good properties of predicted pharmacokinetics and druglikeness, our lead compound 2b was proved to be a drug-like molecule, with the lowest toxicity profile coupled with discrete water solubility and a good lipophilicity property. Absorption and distribution are predicted to be valuable. Hence, compound 2b could be a good candidate for further in vivo assays. We performed a penetration assay to scan the mechanism of inhibition of the entry of EV-A71 by compound 2b into Vero-76 cells. The percentage of reduction in viral penetration was determined in relation to the untreated control.We found that compound 2b could effectively inhibit the viral penetration in a dose-dependent manner with an EC50 of 6.2 µ M ± 2. (Figure 4D).

Druglikeness, Pharmacokinetic and Toxicity Predictions
Theoretical druglikeness, pharmacokinetic, and toxicity properties prediction is a key step between in vitro and in vivo experiments. The PreADMET (http://preadmet.bmdrc.kr/) and SwissADME (http://www.swissadme.ch/) tools were employed to assess the newly synthesized library of compounds (1a-5a, 1b-5b) and results are reported in Table 3. On the base of Lipinski's"rule of five", all the tested compounds are suggested to be drug-like molecules. According to pharmacokinetic predictions, compounds are all strong binders to the plasma protein with %plasma protein binding (PPB) values higher than 90%. They are all suggested to be absorbed into the CNS with high BBB (blood-brain barrier) permeability scores. All molecules are supposed to be moderately water-soluble. Lipophilicity is predicted to be good in general terms, with CLog Po/w values ranging between 3.88 and 5.19. As a matter of fact, gastrointestinal absorbance of these compounds is expected to be from middle to high. With the sole exception of compounds 1a, 4a, and 5a, they are generally considered not to be a  (20 µM) was added at the indicated times. Viral yields were determined by a plaque assay. Dark columns, the viral yield for control cells; gray columns; viral yield for cells treated with 2b derivative. (D) Penetration assay. Dose-dependency of derivative 2b (dark columns) in reduction in EV-A71 penetration compared to non-treated sample control (gray columns). The results presented were obtained from three independent experiments. Data are mean ± SD. Table 3. Absorption, distribution, metabolism, excretion(ADME)and toxicity prediction of compounds (1a-5a, 1b-5b).

ADME Profile
Toxicity a

Discussion
In this study, we describe the promising anti-enteroviral activity of a selected group of new benzimidazoles. Among them, derivative 2b turned out to be endowed with a considerable broad-spectrum anti-enteroviral activity with a cytotoxic profile in the high micromolar range (Table 1). We documented the remarkable antiviral activity against the strain BrCr of EVA71, genogroup A.
This strainwas firstly isolated from a stool specimen of a patient with acute meningitis and from the brain of a patient who died of encephalitis [30]. This virus is a causal agent of severe CNS diseases, and for this reason, it was employed in our assays as a prototype and reference strain of EVA71.
To validate the suitability of compound 2b and to rule out its toxicity against human epithelial cells, its impact on Caco-2 cell monolayers permeability was evaluated. Caco-2 cells, coming from the human colonic epithelium, fully differentiate to enterocytes in vitro and are commonly used to evaluate the effect of nutrients as well as contaminants and drugs [31].
We tested compound 2b at 20 µM (active and non-cytotoxic concentration), and it was observed that it did not induce any significant change in permeability when compared to untreated cells, up to 48 h (the longest time of incubation). Therefore, it did not exert any toxic effects on epithelial cells, resulting as safe at the tested concentration. Derivative 2b, inter alia, was proved to be not only able to protect cell monolayers from EV-A71-induced cytopathogenicity, with an EC 50 value of 3 µM, but also Vero-76 cells when treated with 20 and 80 µM of 2b, also resulting as being significantly protected from cell death ( Figure 2). Generally, it has been demonstrated that EV-A71 can induce apoptosis [32,33]. In our hands, EV-A71 infection seems not to induce apoptosis but rather 70% of Vero-76 cells died from other forms of cell death such as necrosis or pyroptosis. Pyroptosis is a novel form of programmed cell death and an increasing number of studies have demonstrated that different viral infections [34][35][36], including EV-A71 infections, may activate this death pathway [37][38][39][40]. We can hypothesize that long-time (96 h, needed to reach optimal CPE) virus exposure leads to a switch from apoptosis to necrosis or that the pyroptosis process is involved. In any case, accurate investigations are needed. However, our goal was to understand whether the cells treated with 2b were protected from infection and, in parallel, to validate results obtained from the antiviral assay. Thus, we demonstrated that compound 2b, at both tested concentrations, can preserve the cells from death. In this study, we also showed that 2b was able to reduce the viral titer in a dose-dependent manner. Cells pretreated with 2b did not show inhibition of A71 replication, indicating that 2b does not act on cell receptors, as well as the treatment of viral particles not being able to determine the direct inactivation of the virion. Furthermore, we demonstrated that 2b reduces viral adsorption and penetration to Vero-76 cells by the time-of-addition assay and penetration assay when compared to untreated samples. Our derivative had its utmost effect when added during the infection period. Based on all these results, we can hypothesize that our compound can inhibit viral endocytosis by reducing viral attachment to and penetration into Vero monolayers. Compound 2b is endowed with low cytotoxicity against cell monolayers supporting the replication of EV-A71 but also against a large group of cell cultures, as reported in Table 2.
These results need to be interpreted in light of the strengths and limitations of our study. Some of the major strengths of our study are the characterization of a new class of compounds with a broad-spectrum anti-enterovirus activity and the identification of derivative 2b, which is endowed with remarkable anti-EV A71 activity. Preliminary mode of action studies showed that 2b exerts its activity during an early step of the viral cycle, also reducing the penetration phase. Moreover, the ADME and toxicity prediction of our derivatives suggested that 2b is endowed withlow toxicity, good lipophilicity, and discrete water solubility. Pharmacokinetic predictions showed that our lead compound can be easily absorbed into the CNS with high BBB permeability scores. This is an important prerequisite for the development of molecules that could take effect in the CNS. Since, at present, there is no effective treatment against EV-A71, maybe because many tested compounds failed due to a lack of the ability to penetrate the BBB, and monovalent vaccines are currently limited to the Chinese regions, this derivative could be considered as a good starting point for the development of effective candidates for treatment against EVA71. Considerable efforts have been made, by numerous scientists, to reproduce the human pathology induced by EV-A71 in animals such as cynomolgus and rhesus monkeys, as well as in laboratory mice and other mammals [41][42][43][44][45][46][47][48].
However, existing animal models of Enterovirus A71 infection do not reproduce, in animals, the complete spectrum of neurological hallmarks detected in humans [49] and the use of nonhuman primates as animal models is very tricky because of the ethical and cost problem.Another limit is that we documented antiviral activity against the strain BrCr of EVA71, genogroup A, and it would be important to validate our results against genotypes B and C, which are characterized by a wider diffusion [8].
That considered, further studies should be carried out to understand which animal model could be the most suitable to reproduce human EV-A71 neurological features. Meanwhile, it is necessary to clarify the mechanism of antiviral action of this promising compound.