Inhibition of enterovirus a71 by a novel 2-phenyl-benzimidazole derivative

Article abstract of DOI:10.3390/v13010058

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.

Full text of DOI:10.3390/v13010058

viruses
Article
Inhibition of Enterovirus A71 by a Novel 2-Phenyl-
Benzimidazole Derivative
Roberta Ibba 1,† , Antonio Carta 1,*, Silvia Madeddu 2 , Paola Caria 2, Gabriele Serreli 2 , Sandra Piras 1,
Simona Sestito 1 , Roberta Loddo 2 and Giuseppina Sanna 2, ,†
*
1
2
Department of Chemistry and Pharmacy, University of Sassari, Via Muroni, 23A, 07100 Sassari, Italy;
ribba@uniss.it (R.I.); piras@uniss.it (S.P.); ssestito@uniss.it (S.S.)
Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria Monserrato (Cagliari),
09042 Monserrato, Italy; silvia.madeddu@unica.it (S.M.); paola.caria@unica.it (P.C.);
gabriele.serreli@unica.it (G.S.); rloddo@unica.it (R.L.)
*
Correspondence: acarta@uniss.it (A.C.); g.sanna@unica.it (G.S.)
†
Equal contribution.
Abstract: 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
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
of 3
µ
M. Moreover, Vero-76 cells resulted in being significantly protected from necrosis and apoptosis
Citation: Ibba, R.; Carta, A.;
Madeddu, S.; Caria, P.; Serreli, G.;
Piras, S.; Sestito, S.; Loddo, R.;
Sanna, G. Inhibition of Enterovirus
A71 by a Novel 2-Phenyl-
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.
Benzimidazole Derivative. Viruses
2021, 13, 58. https://doi.org/
10.3390/v13010058
Academic Editor: Hung-Yao Ho
Received: 19 November 2020
Accepted: 29 December 2020
Published: 4 January 2021
Keywords: EV-A71; neurological complications; antivirals; benzimidazole derivatives; time course;
penetration assay; apoptosis assay; TEER
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
1. 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, Tai-
wan, 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 infec-
tions occurred also in European countries, such as the Netherlands, France, and Spain
(https://www.eurosurveillance.org). HFMD occurrences constitute a perpetual menace
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
in China, resulting in 7.2 million cases between 2008 and 2012 [3]. EV-A71 emerged as
Viruses 2021, 13, 58. https://doi.org/10.3390/v13010058
https://www.mdpi.com/journal/viruses

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responsible for about 80% of occurred severe cases and 93% of the deaths during this
time [
as the most clinically important enterovirus following the global eradication of poliomyeli-
tis [ ]. HFMD is a benign and common childhood disease, easily transmitted via the
4]. As a major neurotropic causative agent of HFMD, EV-A71 has replaced poliovirus
5,6
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 en-
cephalitis that may lead to cardiovascular collapse and pulmonary edema [
7]. Seven EV
A71 genogroups are known (A–G) [ ]. Genogroup A contains the prototype strain BrCr,
8
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 re-
ported 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], manage-
ment of severe EV-A71 infections is mainly aimed at alleviating disease symptoms using
supportive treatments such as mechanical cardiopulmonary support systems and the ad-
ministration 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
them turned out as active against representative Enteroviruses [18]. Previously reported
benzotriazole derivatives which showed antiviral activity against Coxsackievirus B5 [18
–15] and anti-infective screening [16,17]. Some of
]
and respiratory syncytial virus (RSV) [19] were used as prototypes for a scaffold simplifi-
cation 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 skele-
ton. 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 sub-
stituted 2-phenyl-benzimidazole derivatives (1a,b-5a,b) was designed, synthesized, and
tested against a panel of relevant enteroviruses.
2. Materials and Methods
2.1. Chemistry
2.1.1. Synthesis
All starting materials were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt,
Germany). Benzimidazole ring closure, to obtain derivatives 1a
as described in the literature [20] by mixing o-phenylenediamines (
aldehydes ( and ) in a ratio of 1:1,dissolvingthe mixture in a few mL of acetonitrile
,
b-
5a,
b, was carried out
1-5) and commercial
a
b
(CH3CN), and adding H2O2 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 CH3CN 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.

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2.1.2. Chemical Characterization
Compounds’ melting points (m.p.) were taken in open capillaries in a Köfler hot stage
and are uncorrected. Retention factors (Rf) 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
1
Avance III 400 NanoBay (400 MHz) instrument. 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). 13C-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.
2.2. Biology
2.2.1. Cells and Viruses
Cell lines were purchased from the American Type Culture Collection (ATCC). The
absence of mycoplasma contamination was checked periodically by the Hoechst staining
method. Cell lines supporting the multiplication of Enteroviruses were the following:
Monkey kidney (Vero-76) [ATCC CRL 1587 Cercopithecus Aethiops], Human cervix adeno-
carcinoma (HeLa cells) [ATCC CCL-2], and Macaca mulatta, monkey, rhesus (LLC-MK2)
[ATCC CCL-7]. Caco-2 cells (ECACC Salisbury, Wiltshire, UK) were cultured in Dul-
becco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated
bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, 100 U/mL penicillin,
and 100 mg/mL streptomycin, in monolayers at 37 ◦C in a humidified atmosphere of 5%
CO2, [21] replacing the medium twice a week. For experimental studies, Caco-2 cells, at
passage 21–40, were plated and used 18–21 dayspost-seeding, as differentiated entero-
cytes. Viruses were purchased from the American Type Culture Collection (ATCC). Viruses
representative of positive-sense, single-stranded RNAs (ssRNA+) were: Picornaviridae:
human enterovirus B [coxsackie type B4 (CV-B4), strain J.V.B. (ATCC VR-184), coxsackie
type B5 (CV-B5), strain Faulkner (ATCC VR-185), coxsackie type B3 (CV-B3), strain Nancy
(ATCC VR-30)], human enterovirus B [echovirus 9], human enterovirus A71 strain BrCr
(ATCC VR-1775),enterovirus C [poliovirus type-1 (PV-1), Sabin strain (Sb-1) Chat (ATCC
VR-1562)],and human enterovirus D 68 strain Fermon (ATCC VR-1826)].
2.2.2. Cytotoxicity Assays
HeLa and LLC-MK2 cells were seeded in 96-well plates at an initial density of
5 × 105 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 × 105 cells/mL, in

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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% CO2 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].
2.2.3. Antiviral Assays
Compounds’ activity against EV-A71 and EV-D68 was based on inhibition of virus-
induced 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
×
104 and 3
×
104 cells/well, respectively, and were allowed to form confluent
◦
monolayers by incubating overnight in growth medium at 37 C in a humidified CO2 (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
0
monolayers, as previously reported [24]. Plc (Pleconaril), NM107 (2 -C-methylcytidine),and
Rup (Rupintrivir) were used as references and internal controls.
2.2.4. 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% CO2, the inoculum was removed and replaced with fresh medium
containing the same concentration of compounds. After 96 h at 37 ◦C and 5% CO2, 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 (20-C-Methyl-Cytidine) was used as a reference compound.
2.2.5. 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 × 105 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 L of PI. Then, the reaction was performed in the dark for 15 minutes
µ
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.
2.2.6. Virucidal Activity Assay
Compound 2b (20
µM) was incubated with 1
×
105 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.

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2.2.7. Cell Pretreatment Assay
Vero-76 cell monolayers in 24-well plates were incubated with 20
µ
M concentration
of the 2b or 50 µ
M 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.
2.2.8. 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.
2.2.9. 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% CO2 and 37 ◦C (time zero). Vero-76 cells were treated with
compound 2b (20
µM, approximately 10 times higher than the EC50) 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.
2.2.10. Penetration Assay
A 24-well tissue culture plate was seeded with Vero-76 cells (4
which were then incubated overnight at 37 ◦C and 5% CO2. The cells were chilled on ice
for 1 h, and the medium was removed. The cells were infected with 100 PFU (200 L) 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 M) in medium were added, and the
×
105 cells/well),
µ
µ
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].
2.2.11. Evaluation of Cell Monolayer Permeabilization (TEER)
The activity of compound 2b on intestinal cells monolayer permeability was as-
sessed by measuring the transepithelial electrical resistance (TEER) value. Caco-2 cells
(
0.5 × 105 cells/well) were grown in 12 mm i.d. Transwell inserts (polycarbonate mem-
brane, 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
20 µM) and, to induce an increase in permeability, an oxysterol mixture (final concentration
60 M) prepared as described by Serra et al. [28] were added to the culture medium. TEER
Ω
/cm2 were chosen for the experiment. Then, compound 2b (final concentration
µ
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).

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2.2.12. 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).
2.2.13. 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 (CC50 or EC50) were determined by linear regression analysis.
2.3. 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 struc-
tural 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
(Cbrain) and compound concentration in blood (Cblood). Compounds are labeled as high,
middle, or highly absorbing into the CNS when Cbrain/Cblood 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 Po/w (CLog Po/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.
3. Experimental Section
3.1. 2-(4-Isopropylphenyl)-1H-Benzo[d]imidazole (1a)
Compound 1a (C16H16N2, MW 236.312) was obtained in a total yield of 88%; m.p.
1
206–207 ◦C; TLC (PE/EA 7/3): Rf 0.62. H-NMR (DMSO-d6):
δ 8.11 (2H, d,J = 8.6 Hz, H-
0
0
0
0
2 ,6 ), 7.66 (1H, d, J = 7 Hz, H-4), 7.61 (2H, d, J = 8.6 Hz, H-3 ,5 ), 7.54 (1H, d, J = 7 Hz, H-7),
7.21 (2H, m, H-5,6), 1.34 (9H, s, 3CH3). 13C-NMR (jmod, DMSO-d6):
δ
152.49 (C), 151.18 (C)
,
142.98 (C), 136.94 (C), 128.42 (C), 127.19 (2CH), 125.79 (2CH), 123.55 (CH), 122.50 (CH),
117.68 (CH), 111.16 (CH), 33.46 (CH), 22.99 (2CH3). ESI-MS (m/z):calcd. for C16H16N2
237.1386, found 237.1388 [M+H]+.
3.2. 5-Chloro-2-(4-Isopropylphenyl)-1H-Benzo[d]imidazole (2a)
Compound 2a (C16H15ClN2, MW 270.76) was obtained in a total yield of 75%; m.p.
◦
1
228-230 C; TLC (PE/EA 7/3): Rf 0.75. H-NMR (DMSO-d6):
δ 8.12 (2H, d, J = 8.4 Hz,
H-20,60), 7.66 (1H, d, J = 1.6 Hz, H-4), 7.61 (1H, d, J = 8.8 Hz, H-7), 7.40 (2H, d, J = 8.4 Hz,
H-30,50), 7.19 (1H, dd, Jortho = 8.6 Hz, Jmeta = 1.6 Hz, H-6), 2.95 (1H, q, aliphatic CH), 1.24
(6H, d, J = 6.8 Hz, 2CH3). 13C-NMR (jmod, DMSO-d6):
δ 152.90 (C), 150.61 (C), 127.46 (2C),

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126.86 (2CH), 126.67 (2CH), 126.06 (2C), 121.97 (CH), 116.01 (CH), 114.85 (CH), 33.32 (CH),
15.18 (2CH3). ESI-MS (m/z): calcd. for C16H15ClN2 271.0997, found 271.1000 [M+H]+.
3.3. 5,6-Dichloro-2-(4-Isopropylphenyl)-1H-Benzo[d]imidazole (3a)
Compound 3a (C16H14Cl2N2, MW 305.202) was obtained in a total yield of 87%; m.p.
◦
257-259 C; TLC (PE/EA 7/3): Rf 0.75. 1H-NMR (DMSO-d6):
δ
8.19 (2H, d, J = 8 Hz,
H-20,60), 7.96 (2H, s, H-4,7), 7.53 (2H, d, J = 8 Hz, H-30,50), 3.01 (1H, q, J = 6.8 Hz, CH), 1.26
(6H, d, J = 6.8 Hz, 2CH3). 13C-NMR (jmod, DMSO-d6):
153.19 (C), 152.38 (C), 135.09 (2C),
δ
127.70 (2CH), 127.60 (2CH), 126.43 (2C), 123.42 (C), 115.73 (2CH), 33.47 (CH), 23.46 (2CH3).
ESI-MS (m/z): calcd. for C16H14Cl2N2 305.0607, found 305.0608 [M+H]+.
3.4. 5-Fluoro-2-(4-Isopropylphenyl)-1H-Benzo[d]imidazole (4a)
Compound 4a (C16H15FN2, MW 254.302) was obtained in a total yield of 76%; m.p.
255 ◦C; TLC (PE/EA 7/3): Rf 0.56. 1H-NMR (acetone-d6):
δ
8.14 (2H, d, J = 8 Hz, H-20,60),
7.58 (1H, m, H-4), 7.44 (2H, d, J = 8 Hz, H-30,50), 7.32 (1H, d, Jortho = 8 Hz), 7.02 (1H, ddd,
Jpara = 1.8 Hz, Jortho = 8Hz, 3JH-F = 11.4 Hz, H-6), 3.01 (1H, q, J = 7 Hz, aliphatic-CH), 1.30
(6H, d, J = 7.2 Hz, 2CH3). 13C-NMR (APT, DMSO-d6):
δ 159.78 (C), 157.44 (C), 152.72 (C),
150.80 (C), 137.47 (1C, d, 1JC-F = 402 Hz, C-F), 127.12 (C), 126.96 (2CH), 126.50 (2CH), 115.45
(CH-7), 110.17 (1C, d, 2JC-F = 26 Hz, CH-6), 100.99 (1C, d, 2JC-F = 25 Hz, CH-4), 33.29 (CH),
23.56 (2CH3). ESI-MS (m/z): calcd. for C16H15FN2 255.1292, found 255.1289 [M+H]+.
3.5. 5,6-Difluoro-2-(4-Isopropylphenyl)-1H-Benzo[d]imidazole (5a)
Compound 5a (C16H14F2N2, MW 272.293) was obtained in a total yield of 67%; m.p.
1
235–237 ◦C; TLC (PE/EA 7/3): Rf 0.75. H-NMR (DMSO-d6):
δ
0
8.17 (2H, d, J = 8.4 Hz,
0
0
0
H-2 ,6 ), 7.82 (2H, t, J = 8.8 Hz, H-4,7), 7.54 (2H, d, J = 8 Hz, H-3 ,5 ), 3.01 (1H, m, J = 6.8 Hz,
CH), 1.26 (6H, d, J = 6.8 Hz, 2CH3). 13C-NMR (jmod, DMSO-d6):
δ 153.24 (C), 151.67 (C),
147.94 (2C, dd, 1JC-F = 242 Hz, 2JC-F = 16 Hz, 2CF),130.26 (2C), 127.57 (2CH), 127.41 (2CH),
122.94 (C), 102.52 (2CH, m, CH-4,7), 33.48 (CH), 23.45 (2CH3). ESI-MS (m/z): calcd. for
C16H14F2N2 273.1198, found 273.1195 [M+H]+.
3.6. 2-(4-(Tert-Butyl)phenyl)-1H-Benzo[d]imidazole (1b)
Compound 1b (C17H18N2, MW 250.338) was obtained in a total yield of 65%; m.p.
◦
1
239-241 C; TLC (PE/EA 7/3): Rf 0.50. H-NMR (DMSO-d6)0:
δ
0 8.11 (2H, d, J = 8.4 Hz,
0
0
H-2 ,6 ), 7.65 (1H, d, J = 7.2 Hz, H-4), 7.57 (2H, d, J = 8.4 Hz, H-3 ,5 ), 7.52 (1H, d, J = 6.8 Hz
,
H-7), 7.19 (2H, m, H-5,6), 1.34 (9H, s, 3CH3). 13C-NMR (jmod, DMSO-d6):
δ 152.53 (C),
151.24 (C), 143.82 (C), 134.92 (C), 127.41 (C), 126.19 (2CH), 125.70 (2CH), 122.29 (CH), 121.50
(CH), 118.69 (CH), 111.16 (CH), 34.56 (C), 30.96 (3CH3). ESI-MS (m/z): calcd. for C17H18N2
251.1543, found 251.1544 [M+H]+.
3.7. 2-(4-(Tert-Butyl)phenyl)-5-Chloro-1H-Benzo[d]imidazole (2b)
Compound 2b (C17H17ClN2, MW 284.783) was obtained in a total yield of 83%; m.p.
1
247–249 ◦C; TLC (PE/EA 7/3): Rf 0.76. H-NMR (acetone-d6):
δ 8.17 (2H, d, J = 8.4 Hz,
H-20,60), 7.62–7.59 (4H, m, H-4,7,30,50), 7.23 (1H, dd, 1J = 8.6 Hz, 2J = 1.6 Hz, 2 Hz, H-6),
1.38 (9H, s, 3CH3). 13C-NMR (jmod, acetone-d6):
δ
154.37 (C), 153.81 (C), 141.61 (C),
139.11 (C), 128.10 (C), 128.07 (C), 127.40 (2CH), 126.75 (2CH), 126.35 (CH), 116.83 (CH),
115.80 (CH), 35.45 (C), 31.45 (3CH3). ESI-MS (m/z): calcd. for C17H17ClN2 285.1153,
found 285.1157 [M+H]+.
3.8. 2-(4-(Tert-Butyl)phenyl)-5,6-Dichloro-1H-Benzo[d]imidazole (3b)
Compound 3b (C17H16Cl2N2, MW 319.228) was obtained in a total yield of 63%;
◦
m.p. 259–261 C; TLC (PE/EA 7/3): Rf 0.84. 1H-NMR (DMSO-d6):
δ
8.20 (2H, d,
H-20,60), 7.96 (2H, s, H-4,7), 7.68 (2H, d, J = 8.4 Hz, H-30,50), 1.35 (9H, s, 3CH3).
13C-NMR (jmod, DMSO-d6):
154.82 (C), 152.88 (C), 136.28 (2C), 127.12 (2CH), 126.15
J = 8.4 Hz
,
δ

Viruses 2021, 13, 58
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(2CH), 125.82 (2C), 124.09 (C), 115.89 (2CH), 34.81 (C), 30.80 (3CH3). ESI-MS (m/z): calcd.
for C17H16Cl2N2 319.0763, found 319.0766 [M+H]+.
3.9. 2-(4-(Tert-Butyl)phenyl)-5-Fluoro-1H-Benzo[d]imidazole (4b)
Compound 4b (C17H17FN2, MW 268.329) was obtained in a total yield of 84%;
m.p.>0300 ◦C; TLC (PE/EA 7/3): Rf 0.72. 1H-NMR (DMSO-d6):
δ 8.09 (2H, d, J = 8 Hz,
H-2 ,6 ), 7.57 (3H, d, J = 8.4 Hz, H-4,3 ,5 ), 7.38 (1H, d, J = 8.4 Hz, H-7), 7.05 (1H, m, H-6), 1.33
0
0
0
(9H, s, 3xCH3). 13C-NMR (jmod, DMSO-d6):
δ 159.83 (C), 157.50 (C), 153.22 (C), 152.60 (C),
137.22 (1C, d, 1JC-F = 401 Hz, C-F),126.44 (C), 126.28 (2CH), 125.87 (2CH), 115.50 (1C, d,
3JC-F = 10 Hz, CH-7), 110.38 (1C, 2JC-F = 25 Hz, CH-6), 100.95 (1C, 2JC-F = 26 Hz, CH-4), 34.55
(C), 30.84 (3CH3). ESI-MS (m/z): calcd. for C17H17FN2 269.1448, found 269.1448 [M+H]+.
3.10. 2-(4-(Tert-Butyl)phenyl)-5,6-Difluoro-1H-Benzo[d]imidazole (5b)
Compound 5b (C17H16F2N2, MW 286.319) was obtained in a total yield of 78%; m.p.
◦
1
257-259 C; TLC (PE/EA 7/3): Rf 0.78. H-NMR (DMSO-d6):
δ 8.14 (2H, d, J = 8.4 Hz,
H-2 ,6 ), 7.78 (2H, t, J = 8.8 Hz, H-4,7), 7.66 (2H, d, J = 8.4 Hz, H-3 ,5 ), 1.35 (9H, s, 3CH3). 13C-
0
0
0
0
0
δ =
154.65 (C-2), 152.25 (C-4 ), 147.56 (2C, dd, 1JC-F = 241 Hz, 2JC-F
NMR (jmod, DMSO-d6):
16 Hz, 2CF), 131.75 (C-3a,7a), 126.86 (C-20,60), 126.15 (C-30,50), 123.97 (C-10), 102.66 (2CH,
dd, 2JC-F = 15 Hz, CH-4,7), 34.80 (C), 30.81 (3CH3). ESI-MS (m/z): calcd. for C17H16F2N2
287.1354, found 287.1359 [M+H]+.
4. Results
4.1. Chemistry
Benzimidazole derivatives were designed and synthesized as reported in Scheme 1
.
o-Phenylenediamine 1-5) and the corresponding aldehyde ( and ) were mixed and
(
a
b
reacted in a single-pot, simple condensation reaction at room temperature, obtaining
derivatives 1a,b-5a,b.
Scheme 1. 1a,b-5a,b.
4.2. Antiviral Assays
All newly synthesized 2-phenyl-benzimidazole derivatives were evaluated for their
broad-spectrum anti-enterovirus activity in cell-based assays. Several compounds exhib-
ited significant inhibitory activity, with EC50 values in the low micromolar range (i.e.,
<10 µM, Table 1). Concomitantly, moderate to low cytotoxicity was detected for almost all
compounds, with CC50 values mostly in the high micromolar range (>100
cells. Only 1a , 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 in-
teresting activity of derivatives 2b and 2a against the broad panel of enteroviruses (Table 1)
µM) in Vero-76
,
b
.

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Table 1. Cytotoxicity and antiviral activity of benzimidazole (BIZ) derivatives and references against representatives of
Enterovirus (EV-A71, CV-B3, CV-B4, CV-B5, PV-1, EV-D68, and Echo9).
Cmp
Vero-76
EV-A71
CV-B3
CV-B4
CV-B5
PV-1
EC50
11.5
9
HeLa
EV-D68
LLC-MK2
Echo9
a
c
c
c
c
c
e
c
b
d
CC50
EC50
EC50
EC50
EC50
CC50
EC50
EC50
CC50
9
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
60/24 *
46/40 *
>100
>24
>40
7
>60
>46
17
>60
>46
>60
7
>9
>9
6
>6
>6.5
8
9
6.5
≥100
25.5
10
9
100
100
8
>100
>100
>8
>100
12
>100
3.5
>8
>9
>100
70
33
10
6
4
25.5/8 *
9
>25.5
>9
>25.5
>9
>25.5
2
>25.5
-
7
>7
6.4
>6.4
>100
>100
>100
4
12
>12
>29.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
≥100
20
>100
>100
>100
>100
29.5
>100
100
>100
>100
>100
31
12
>100
6
10
0.005 ±
2 ±
Plc
>100
2 ± 0.5
0.3
>100
0.1
0.001
0.005
NM 107
>100
>100
29
6
Rup
0.07
a–e
Compd conc (
µ
M) required to reduce by 50% the viability of mock-infected Vero-76, HeLa, and LLC-MK2, as determined by the MTT
b
method after 3 * days, 4 days, and 5 days, respectively. Compd conc (
from EV-A71- and EV-D68-induced cytopathogenicity, as determined by the MTT method at days 3 and 4-5 p.i., respectively. Compd
µ
M) required to achieve 50% protection of Vero-76 or HeLa cells
c
conc (µM) required to reduce by 50% the plaque number of CV-B3, CV-B4, CV-B5, PV-1,and Echo9 in Vero-76 and LLC-MK2 cells. Plc
(Pleconaril); NM107 (20-C-methylcytidine); Rup (Rupintrivir). Data represent mean values + SD for three independent determinations. For
values where SD is not shown, variation among samples was less than 15%.
Compound 2a showed remarkable antiviral activity against Entero A, B, and C viruses
with an EC50 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 EC50 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 EC50 range of 12–31 and
10–6 µM, respectively.
Compound 5b also showed an interesting EC50 against EV-D68 (4
resulted in being active against CV-B5 and PV-1 with an EC50 range of 7–9
while derivative 1a was endowed withmoderate activity against PV-1 (EC50 = 11.5
µ
M). Compound 1b
M, respectively,
M).
M and
µ
µ
Compounds 3a and 3b resulted in being cytotoxic and weakly active (3b CC50 = 9
EC50 = 2 µM) against CV-B5.
µ
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
a moderate steric hindrance such as one bigger chlorine atom (series
smaller fluorine atoms (series and ) turned out as the best option. Non-toxic series
and are also complemented with the best antiviral activity detected. Interestingly, the
presence of one single chlorine atom on the BIZ scaffold provided series derivatives
5
and
6
(series
3). In terms of cytotoxicity, the substitution with
2
) and one or two
4
5
2
5
2
a wider antiviral activity, as can be seen from Table 1, showing remarkable EC50 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.

Viruses 2021, 13, 58
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Regarding series
5
, two fluorine atoms were placed in positions
5
and
6
5
on BIZ,
showed
whichendowed derivatives 5a and 5b withmore selective antiviral activity. Series
relevant EC50 values when tested against PV-1, EV-A71, and EV-D68 (5b). To evaluate
the weight of the sterical hindrance on position 40 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 40 position. Focusing on
EV-A71, compound 2b presented an EC50 value of 3.5
recorded at 7 M. It is even more striking when series 5 is considered, where 5b showed
an EC50 value of 6 M, while 5a showed only 31 M. Derivative 5b is also quite potent
when inhibiting EV-D68 replication at 4 M concentration, while the parental 5a is not
µM, while the parental 2a value was
µ
µ
µ
µ
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).
◦
Table 2. Cytotoxicity of benzimidazole (BIZ) derivatives against selected cell lines .
Compounds
BHK-21
MDBK
Vero-76
HeLa
LLC-MK2
a
c
e
b
d
CC50
CC50
CC50
CC50
CC50
9
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
36
20
65
9.0
60/24 *
46/40 *
>100
6
9
6.5
>100
>100
8.0
80
100
100
8
>100
12
>100
2.5
>100
25.5/8 *
9
7
9
7
6.4
12
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
29.5
>100
100
>100
>100
>100
>100
p◦ Data represent mean values + SD for three independent determinations. For values where SD is not shown,
a–e
variation among samples was less than 15%.
Compound conc (µM) required to reduce by 50% the viability of
mock-infected BHK-21, MDBK, Vero-76, HeLa, and LLC-MK2, as determined by the MTT method after 3 * days,
4 days, and 5 days, respectively.
Starting from these considerations and taking into account the recent EV-A71 epi-
demics 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.
4.3. 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

Viruses 2021, 13, 58
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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.
Figure 1. Effect of 2b inhibitor (20 and 80
treated cells with 20 M ( ), treated cells with 80
(20 M) ( ), and infected treated cells (80 M) ( ). Pictures of cell morphology were taken at 72 h
post-infection using ZOE Fluorescent cell imager (Bio-Rad) (bar size = 100 m, magnification, 20 ).
µ
M) on the Vero-76-infected monolayers. Control (
A),
µ
B
µ
M ( ), infected cells ( ), infected treated cells
C
D
µ
E
µ
F
µ
×
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).
4.4. TEER Experiment
To ascertain any toxic effect of derivative 2b on human cells, we tested it on differ-
entiated 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), starting from 18 h of incubation, when the TEER value was about 65% of the
level of the untreated cells. TEER values measured in monolayers treated with compound
2b (20
µM) were instead not significantly different from control values throughout all the
time points, showing no enhancement of cell permeability.

Viruses 2021, 13, 58
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Cellstreated(80µM)
Control untreated cells
Cellstreated (20µM)
C
A
B
10 5
10 4
10 3
10 2
10 1
10 0
10 5
10 4
10 3
10 2
10 1
10 0
10 5
10 4
10 3
10 2
10 1
10 0
Late Apoptotic
Late Apoptotic
Late Apoptotic
Necrotic
Necrotic
Necrotic
Early Apoptotic
Early Apoptotic
Early Apoptotic
Live
Live
Live
100
101
102
103
104
105
100
101
102
103
104
105
100
101
102
103
104
105
Annexin V-FITC-Height Log_Comp
Annexin V-FITC-Height Log_Comp
Annexin V-FITC-Height Log_Comp
Virus-infected Cells
Cellstreated (80µM)+ Virus
E Cellstreated (20µM)+ Virus
F
D
10 5
10 5
10 4
10 3
10 2
10 1
10 0
10 5
10 4
10 3
10 2
10 1
10 0
Late Apoptotic
Early Apoptotic
Late Apoptotic
Early Apoptotic
Late Apoptotic
Early Apoptotic
Necrotic
Necrotic
Necrotic
10 4
10 3
10 2
10 1
10 0
Live
Live
Live
100
101
102
103
104
105
100
101
102
103
104
105
100
101
102
103
104
105
Annexin V-FITC-Height Log_Comp
Annexin V-FITC-Height Log_Comp
Annexin V-FITC-Height Log_Comp
G
100
#
***
***
100
80
60
40
20
0
live cells (%)
80
60
40
20
early apoptotic cells (%)
late apoptotic cells (%)
necrotic cells (%)
*
##
***
***
*
0
s
s
e
s
u
l
l
u
)
)
M
r
r
i
v
e
s
l
i
c
l
M
v
e


0
d
c
+
e
+
)
0
e
t
c
)
d
2
8
(
(
e
M
M
t
d
d
e
e
f


a
e
e
t
t
a
n
0
0
8
(
r
i
a
t
2
e
e
r
(
n
s
r
u
t
t
s
u
d
d
e
t
a
l
r
i
v
s
e
o
l
l
l
e
t
l
r
t
e
a
n
e
C
C
r
r
t
o
t
C
s
s
l
l
l
l
e
C
C
Figure 2. The inhibitory effect of compound 2b on EV-A71-induced apoptosis. The percentage of live, apoptotic, and
necrotic cells were measured by flow cytometry using the PI-annexin V assay. Dot plots show cell death in Vero-76 cells:
control (
A
), treated cells with 20
µ
M ( ), infected treated cells (20
B
), treated cells with 80
µ
M (
C
), infected cells (
D
µM) (E),
and infected treated cells (80
µM) (F). Percentage of live, apoptotic, and necrotic cells (G). Statistically significant differences
are expressed as follows: * = p < 0.05 vs. untreated cells; *** = p < 0.001 vs. untreated cells; # = p < 0.05 vs. cells treated
(20 µM) + virus; ## = p < 0.01 vs. cells treated (20 µM) + virus.
Figure 3. Measurement of cell monolayer permeabilization (transepithelial electrical resistance
(TEER)) assay. Caco-2 cell monolayers were incubated with OXY at 60
µM (black squares) as negative
control, derivative 2b at 20 M (blue triangles), and Control (red circles) as internal positive control.
µ
Statistically significant differences are expressed as follows: *** = p < 0.001 vs. Control. Each value
represents the mean ± SD of independent experiments (n = 3).

Viruses 2021, 13, 58
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4.5. 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
(data not shown).
The kinetics of virus adsorption in the presence of derivative 2b was also evaluated.
µM concentration of derivative 2b and then infected with untreated EV-A71
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-of-
addition 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 EC50 of
6.2 µM ± 2. (Figure 4D).
4.6. 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 pre-
diction 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 potas-
sium 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

Viruses 2021, 13, 58
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it with the EC50 values fromthe in vitroanalysis. All considered, the newly designed and
synthesized compounds showed good properties of predicted pharmacokinetics and drug-
likeness, 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.
Figure 4.
(
A
) Yield of infectious EV-A71 viruses produced in infected Vero-76 cells treated with 2b and NM 107. Vero-76
cells were infected with EV-A71 (m.o.i. 0.1). The infected cultures were treated with 2b, at indicated doses. Viral yields in
the culture supernatant were determined by a plaque assay at 96 h post-infection. * Statistically significant differences are
expressed (p < 0.05). (
B) Virucidal effect (expressed as plaque-forming units (PFU/mL) of derivative 2b (20 µM) against
◦
◦
EV-A71 virions at either 4 C or 37 C for 1 h. Dark columns, viral titer for viral and derivative 2b solution; white columns for
the viral titer of the untreated solution. (
for 1 h at 4 C with viruses at an m.o.i. = 1 in the presence of the compound (5
inoculated with EV-A71 (m.o.i. = 1) and then compound 2b (20
C
) Adsorption assay and time course experiment. Vero-76 cells were pre-adsorbed
EC50 concentration). Vero-76 cells were
M) 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. ( ) Penetration assay. Dose-dependency of derivative 2b (dark columns) in reduction in EV-A71 penetration
◦
×
µ
D
compared to non-treated sample control (gray columns). The results presented were obtained from three independent
experiments. Data are mean ± SD.

Viruses 2021, 13, 58
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Table 3. Absorption, distribution, metabolism, excretion(ADME)and toxicity prediction of compounds (1a-5a, 1b-5b).
ADME Profile
Toxicity a
Samples PPB (%) a
BBB Permeability
Caco-2 cells a
(nm/sec)
hERG Inhibition
Ames Test
Lipophilicity b
Consensus
Log Po/w
Water sol b
Solubility
Class
GI abs b
P-gp sub b
a
a
b
(Cbrain/Cblood
)
Pr.
Pr.
TA100
No S9 act
TA1535
No S9 act
Pr.
S9 act
S9 act
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
92
95
90
93
92
94
92
95
93
95
11.95
12.61
13.29
13.74
14.56
14.87
12.24
12.74
12.79
13.24
High
High
High
High
High
High
High
High
High
High
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
3.88
4.14
4.41
4.68
4.93
5.19
4.19
4.45
4.49
4.75
Soluble (m)
Soluble (m)
Soluble (m)
Soluble (m)
Soluble (m)
Soluble (m)
Soluble (m)
Soluble (m)
Soluble (m)
Soluble (m)
High
High
High
High
High
High
High
High
High
High
36.9
42.3
53.2
54.8
49.2
52.9
49.9
53.4
43.6
50.1
Yes
No
No
No
No
No
Yes
No
Yes
No
Medium
Medium
Medium
Medium
Medium
Medium
Medium
Medium
Medium
Medium
M
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
M
Neg
Pos
M
Pos
NM
M
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Pos
NM
M
Neg
Neg
Neg
Pos
M
M
Pos
M
Pos
Neg
a
Data predicted from the PreADMET tool; b Data predicted from the SwissADME tool. Pr. = prediction; (m) = moderate; sol = solubility; GI abs = gastrointestinal absorption; sub = substrate. Ames Test:
M = mutagen; NM = non-mutagen; S9 activation or non-activation = metabolic activation.

Viruses 2021, 13, 58
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5. 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 EC50 value of 3
and 80 M 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
µM, but also Vero-76 cells when treated with 20
µ
,
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
–36], including EV-A71 infections, may activate this death
pathway [37 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

Viruses 2021, 13, 58
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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 [41–48].
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. Mean-
while, it is necessary to clarify the mechanism of antiviral action of this promising compound.
Author Contributions: Conceptualization, G.S. (Giuseppina Sanna), R.I., and A.C.; formal analysis,
G.S. (Giuseppina Sanna), S.M., R.I., and A.C.; investigation, R.I., G.S. (Giuseppina Sanna), S.M., P.C.,
and G.S. (Gabriele Serreli); resources, A.C.; data curation, G.S. (Giuseppina Sanna), R.I., S.M., P.C.,
G.S. (Gabriele Serreli), and A.C.; writing—original draft preparation, G.S. (Giuseppina Sanna), R.I.;
writing—review and editing, G.S. (Giuseppina Sanna), R.I., S.M., G.S. (Gabriele Serreli), P.C., S.S., S.P.,
R.L., and A.C.; visualization, G.S. (Giuseppina Sanna), R.I., and A.C.; supervision, G.S. (Giuseppina
Sanna), A.C., funding acquisition, A.C. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by “Assessorato della Programmazione, Bilancio, Credito e Assetto
del territorio” of “Regione Autonoma della Sardegna” (Sardinia, Italy), grant number RASSR01499,
and by Ministry for University and Research (MIUR), grant number PRIN 2017Prot. 2017M8R7N9.
Data Availability Statement: Not applicable.
Acknowledgments: We acknowledge “Assessorato della Programmazione, Bilancio, Credito e As-
setto del territorio” of “Regione Autonoma della Sardegna” (Sardinia, Italy) for the funding support
with the grant named “Legge Regionale 7 agosto 2007: CRP1_574 (G.S.), and 22/41 del 2017”, grant
number RASSR01499. R.I. gratefully acknowledges the Sardinian Regional Government for the
financial support of the Ph.D. scholarship (P.O.R. Sardegna F.S.E.—Operational Programme of the
Autonomous Region of Sardinia, European Social Fund 2014–2020—Axis III Education and training,
Thematic goal 10, Investment Priority 10ii), Specific goal 10.5. G.S. gratefully acknowledges the Italian
Ministry for University and Research (MIUR) Grant PRIN 2017 Prot. 2017M8R7N9. We acknowledge
the CeSAR (Centro Servizi Ricerca d’Ateneo) core facility of the University of Cagliari and Rita Pillai
for assistance with the generation of the flow cytometry data.
Conflicts of Interest: The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest.
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