Benzimidazole Derivatives as Novel Zika Virus Inhibitors

Article abstract of DOI:10.1002/cmdc.202000124

We have synthesized 50 benzimidazole (BMZ) derivatives with 1,2-phenylenediamines and aromatic aldehydes under mild oxidation conditions by using inexpensive, nontoxic inorganic salt sodium metabisulfite in a one-pot condensation reaction and screened their ability to interfere with Zika virus (ZIKV) infection utilizing a cell-based phenotypic assay. Seven BMZs inhibited an African ZIKV strain with a selectivity index (SI=CC₅₀/EC₅₀) of 9–37. Structure-activity relationship analysis demonstrated that substitution at the C-2, N-1, and C-5 positions of the BMZ ring were important for anti-ZIKV activity. The hybrid structure of BMZ and naphthalene rings was a structural feature responsible for the high anti-ZIKV activity. Importantly, BMZs inhibited ZIKV in human neural stem cells, a physiologically relevant system considering the severe congenital anomalies, like microcephaly, caused by ZIKV infection. Compound 39 displayed the highest antiviral efficacy against the African ZIKV strain in Huh-7 (SI>37) and neural stem cells (SI=12). Compound 35 possessed the highest activity in Vero cells (SI=115). Together, our data indicate that BMZs derivatives have to be considered for the development of ZIKV therapeutic interventions.

Full text of DOI:10.1002/cmdc.202000124

Accepted Article
Title: Benzimidazole Derivatives as Novel Zika Virus Inhibitors
Authors: Bui Thi Buu Hue, Phuong Hong Nguyen, Tran Quang De, Mai
Van Hieu, Eunji Jo, Nguyen Van Tuan, Than Thi Thoa, Le
Duc Anh, Nguyen Hoang Son, Danh La Duc Thanh, Myrielle
Dupont-Rouzeyrol, Regis Grailhe, and Marc P Windisch
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000124
Link to VoR: https://doi.org/10.1002/cmdc.202000124
01/2020

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
Benzimidazole Derivatives as Novel Zika Virus Inhibitors
Bui Thi Buu Hue,*[a]+ Phuong Hong Nguyen,[b,c]+ Tran Quang De,[a] Mai Van Hieu,[a] Eunji Jo,[b] Nguyen
Van Tuan,[a] Than Thi Thoa,[b] Le Duc Anh,[a] Nguyen Hoang Son,[a] Danh La Duc Thanh,[a] Myrielle
Dupont-Rouzeyrol,[d] Regis Grailhe,[e] Marc P. Windisch*[b,c]
[a]
Dr. Bui Thi Buu Hue* (0000-0002-4635-933X); Dr. Tran Quang De, Mai Van Hieu (0000-0002-5077-2899); Nguyen Van Tuan, Le Duc Anh, Nguyen Hoang
Son, Danh La Duc Thanh
Department of Chemistry, College of Natural Sciences
Can Tho University
Campus II, 3/2 street, Ninh Kieu District, Can Tho City, Vietnam
E-mail: btbhue@ctu.edu.vn
[b]
[c]
Phuong Hong Nguyen; Than Thi Thoa; Eunji Jo; Dr. Marc P. Windisch*
Discovery Biology Division, Applied Molecular Virology Laboratory
Institut Pasteur Korea
696, Seongnam 13488, Republic of Korea
Phuong Hong Nguyen; Dr. Marc P. Windisch* (0000-0003-1874-8503)
Division of Bio-Medical Science and Technology
University of Science and Technology
Daejeon 34141, Republic of Korea
Email: marc.windisch@ip-korea.org
[d]
[e]
Dr. Myrielle Dupont-Rouzeyrol
URE Dengue and Arboviruses
Institut Pasteur de Nouvelle-Calédonie
98800 Noumea, New Caledonia
Dr. Regis Grailhe
Technology Development Platform
Institut Pasteur Korea
696, Seongnam 13488, Republic of Korea
+These authors contributed equally to this work.
Supporting Information for this article is given via a link at the end of the document.
Abstract: We synthesized 50 benzimidazole (BMZ) derivatives with
1,2-phenylenediamines and aromatic aldehydes under mild oxidation
conditions utilizing inexpensive, non-toxic inorganic salt sodium
metabisulfite by one-pot condensation reaction and screened their
ability to interfere with Zika virus (ZIKV) infection utilizing a cell-based
phenotypic assay. Seven BMZs inhibited an African ZIKV strain with
increased number of microcephaly cases and other congenital
abnormalities in fetuses and newborns.[1] Additionally, ZIKV
poses a health risk due to its association with neural-inflammatory
diseases such as Guillain-Barré syndrome and ophthalmological
complications in adults.[2] Furthermore, recent data demonstrate
that the virus can be sexually transmitted by hiding in testes for
long periods.[3] There are neither vaccines nor virus-specific
therapeutics available to treat ZIKV-infected patients. To address
this medical need, novel ZIKV interventions suitable for
prevention and therapeutic purposes are urgently needed.
Benzimidazoles (BMZs) are recognized as a useful chemical
scaffold for the development of pharmaceutical molecules of
biological interest. BMZs have applications in diverse therapeutic
areas, including anti-bacterial,[4] anti-fungal,[5] anti-histaminic,[6]
anti-inflammatory,[7] and anti-HIV.[8] Additionally, BZMs were
recently reported to be potent inhibitors of hepatitis C virus
(HCV).[9] Yet, there are no reports of the activity of this type of
compound against ZIKV, which belongs to the same viral family
as HCV. Therefore, we designed and synthesized a series of
BMZs and screened for their ability to inhibit ZIKV infection. One
of the best documented methods for synthesizing BMZs is the
condensation of o-phenylenediamine with aldehydes. Various
reagents, including NaHSO3,[10] Na2S2O3,[11] Na2S2O4,[12] and
oxone,[13] have been utilized to promote this type of condensation.
The use of Na2S2O5 to construct disubstituted BMZs with two
different functional groups at positions 1 and 2 of the BMZ ring
has also been described, but this protocol requires the use of a
toxic and high boiling point solvent such as DMF at a high
a
selectivity index (SI=CC50/EC50) of 9-37. Structure-activity
relationship analysis demonstrated substitution at the C-2, N-1, and
C-5 positions of the BMZ ring were important for anti-ZIKV activity.
The hybrid structure of BMZ and naphthalene rings was a structural
feature responsible for high anti-ZIKV activity. Importantly, BMZs
inhibited ZIKV in human neural stem cells, a physiologically relevant
system considering the severe congenital anomalies, like
microcephaly, caused by ZIKV infection. Compound 39 displayed the
highest antiviral efficacy against the African ZIKV strain in Huh-7 (SI
>37) and neural stem cells (SI=12). Compound 35 possessed the
highest activity in Vero cells (SI=115). Together, our data indicate that
BMZs derivatives have to be considered for the development of ZIKV
therapeutic interventions.
Introduction
Zika virus (ZIKV) was declared a global health concern in 2016 by
the World Health Organization. Over 2 billion people worldwide
are at risk of contracting ZIKV, a member of the Flaviviridae family
that was recently found to be responsible for a dramatically
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
temperature for a prolonged reaction time.[14] In a previous study,
we reported the synthesis of BZMs by a one-pot condensation
reaction between 1,2-phenylenediamines and aromatic
aldehydes under a mild oxidation condition utilizing inexpensive
and non-toxic inorganic salt sodium metabisulfite.[15]
Based on this protocol, we report the design and synthesis of
50 1,2-disubstituted BZMs derivatives, including 29 new
derivatives, and screened for their inhibition of ZIKV infection
using a cell-based phenotypic high-content platform. This assay
utilizes a multi-parametric readout to directly monitor viral
replication in host cells, simultaneously evaluating cell viability to
rapidly score compounds for activity. We also conducted a
structure-activity relationship (SAR) study and further validated
the antiviral activity of selected BMZs in human neural stem cells
using an Asian ZIKV strain and by conducting mechanism of
action studies.
positions.[9c, 15-16] Considering these previous reports and the fact
that ZIKV belongs to the same viral family as HCV, we devised
strategies to synthesize a library of 1,2-disubstituted BMZs and
screened for their anti-ZIKV activity. We changed the electron
density of the pharmacophoric group by introducing bioisosteric
groups, including the CF3 group in the C-5 position and different
aryl groups in the C-2 position, and substituting benzyl or alkyl
groups at the N-1 position of the BMZ ring. Fifty structurally
diverse 1,2-disubstituted BMZs (1-50), of which 29 were novel
BMZ structures, were successfully synthesized with good to
excellent yields (64-94%) based on a one-pot condensation
reaction between N-substituted o-phenylenediamines 60 with
substituted aromatic aldehydes 51-59 (Scheme 1).[15] The
appropriate diamine 60 was readily prepared following reported
procedures,[16] aldehydes 51-56 were obtained commercially, and
aldehydes 57-59 were prepared via a tandem Stobbe reaction
and cyclization starting from the corresponding substituted
benzaldehyde.[17] In all cases, the reactions proceeded
consistently and were completed within 2 h to generate the
desired products with excellent isolated yields (78-94%). Only in
the case of N-morpholinylpropyl o-phenylenediamines were
longer reaction times (3-4 h) and lower yields (64-75%) observed
(9, 10, 16-19, 44) (Table S1).
Results and Discussion
Structural design and synthesis
Of the seven positions of the BMZ heterocycle, most anti-HCV
BMZ-based compounds bear functional groups at the 1, 2, and 5
Scheme 1. Synthesis protocol of compounds 1-50.
Evaluation of anti-ZIKV activity
(DRC) analysis of the selected BMZs is shown in Fig. 1C. As
BMZs are reported to have inhibitory effects on flaviviruses,[18] we
evaluated the activity of the selected compounds against
infectious HCV to determine compound specificity (Table S2). Of
these, three derivatives (23, 24, and 35) showed poor activity
(SI<5), and one derivative (39) showed no anti-HCV activity.
Human hepatoma Huh-7 cell monolayers were treated with BMZ
derivatives, mycophenolate acid (MPA), an immunosuppressant,
used as a positive control, or vehicle (0.5% DMSO) for 1 h before
infection with the African ZIKV strain MR766 with a multiplicity of
infection (MOI) of 0.8 (Fig. 1A). Screening results are summarized
in Table 1 (full data set of 50 BMZs presented in Table S1).
Seventeen BMZs showed activity against ZIKV with a selectivity
index (SI) >3. Of these, seven compounds (23, 24, 30, 32, 34, 35,
and 39) exhibited high anti-viral activity with a SI>9. As shown in
Fig. 1B, 76% of cells in DMSO-treated wells were infected
compared with <1% of those treated with MPA, whereas the
seven selected representative BMZs inhibited ZIKV to different
extents at non-cytotoxic concentrations. Dose-response curve
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
Table 1. Anti-ZIKV activity of BMZ derivatives.
SI[b]
R1
R2
R3
EC50 (μM)[a]
CC50 (μM)[a]
Compound
Category A
3[e]
4[e]
H
CF3
CF3
H
b
b
d
a
53
53
53
54
4.0±1.4
2.5±1.1
12.7±1.2
9.6±1.1
3.2
3.8
1.2
2.6
8[e]
33.0±1.1
28.1±2.7
40.0±1.3
74.0±1.1
11
Category B
20
H
CF3
H
a
a
b
b
c
c
51
51
51
51
51
51
53.3±5.7
61.4±1.1
6.1±1.2
97.9±51.1
82.6±1.3
27.0±1.1
>418
1.8
1.3
21[e]
22[e]
23
4.4
CF3
H
24.7±2.0
13.3±1.1
>16.9
9.2
24[e]
25[e]
123.1±1.2
ND
NA[d]
1.9
ND[c]
CF3
28
CF3
e
51
302.0±1.1
564.0±10.6
Category C
30[e]
CF3
H
a
c
c
d
d
e
52
52
52
52
52
52
7.5±1.1
18.5±1.1
8.5±1.3
48.3±1.3
6.1±1.2
21.0±1.3
146.0±1.3
263.0±1.2
18.0±1.6
684.5±1.2
55.5±1.2
75.0±1.0
19.5
14.2
2.1
32
33[e]
CF3
H
34
14.2
9.1
35[e]
CF3
CF3
36
3.5
Category D
37
39
H
H
a
b
b
a
b
d
/
57
57
57
58
58
58
/
~30.0
1.9±1.0
16.9±1.4
2.5±0.7
10.0±1.3
3.6±2.5
0.8±0.1
57.1±1.5
70.9±1.4
87.7±1.1
2.6±0.8
19.3±1.1
4.0±1.6
>150.0
~1.9
37.3
4.6
40
CF3
CF3
H
45
1.0
46
1.9
48
CF3
/
1.1
MPA
>35.0
[a]EC50 and CC50 values were calculated by 10-point DRC analysis in triplicate. Experiments were repeated at least two times independently. [b]SI = CC50/EC50 [c]ND,
.
not determined. [d]NA, not applicable (compounds were tested but CC50 and EC50 values could not be calculated). [e]Compounds were previously synthesized and
published. See the experimental section. A full data set for the 50 BMZs can be found in the Supporting Information.
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
Fig. 1. Screening of BMZs for anti-ZIKV activity. (A) Screening schedule. Huh-7 cells were plated in 384-well format for 18 h prior to pre-treatment with serially
diluted BMZs with a 3-fold serial dilution for 1 h (t = -1 h) before inoculation (t = 0 h). Cells were infected with ZIKV for 24 h before fixation, immunofluorescence
staining, and 10-point DRC analysis. (B) Representative images showing ZIKV envelope antigen (green) and cell nuclei (red). Rates of infection are shown for
DMSO, MPA, and selected BMZs as indicated (140 μM (23), 88 μM (24), 28 μM (30), 87 μM (32), 97 μM (34), 57 μM (35), and 18 μM (39)). Scale bar, 0.1 μm. (C)
Representative DRCs of selected compounds and reference inhibitor MPA. Percentage ZIKV inhibition and cell viability are shown in black and red, respectively.
Structure-activity relationship (SAR) analysis
of the vanillin moiety at the C-2 position by the 4-pyridinyl
substituent (compound 23) led to a 4.4-fold increase in SI value.
The importance of a heterocyclic moiety at the C-2 and the
CF3 group at the C-5 positions are again found in category C with
the C-2 substituted pyrrole. The presence of these substituents
led to good activity and good selectivity (SI up to 19.5) (30, 32,
34, and 35). Without the CF3 group, no activity was observed (29
and 31). It seems that the CF3 group at the C-5 leads to a
decrease in activity only when combined with an electron
releasing groups at N-1 such as 4-OMe-benzyl or propyl (25 and
28 in category B or 33 and 36 in category C).
For derivatives in category D bearing the naphthalenyl
moieties at the C-2 position of the BMZ ring (37-50), all
compounds with the CF3 substituents at the C-5 positions (38, 40,
44, 45, 48, 49, 50) did not show any activity. The presence of the
substituents such as simple benzyl (37, 38 and 49) or 4-OCH3-
benzyl (41), or morpholylpropyl (44) at the N-1 position, also did
not lead to any difference either. The introduction of the 2-
Based on different substituents at the C-2 position of the BMZ
ring, BMZs 1-50 were classified into four categories A-D (Table
1). Structurally diverse substituents were introduced to the N-1
position of the BMZ ring, including simple benzyl, substituted
benzyl (4-OMe-benzyl, 2-chloro-benzyl, and 3-pyridinylmethyl), or
alkyl (3-morpholylpropyl and propyl), as well as the CF3 group at
the C-5 position. As a result, variations in the activity of
compounds in each category may be attributed to different groups
at these N-1 and C-5 positions.
The ZIKV screening results presented in Table 1 show that
compounds in category A (1-19) with C-2-substituted phenyl
exhibited good activity, with up to EC50 = 2.5 µM for the 4-hydroxy-
substituted compound 4, but had poor selectivity (SI<5).
Category B contains derivatives with the 4-pyridine moiety at
the C-2 position, which showed good selectivity up to 16.9
(compounds 23, 24). Compared to compound 4, the substitution
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
chlorobenzyl group at the N-1 position (39), however, could
remarkably produce significant improvement with the EC50 up to
1.9 µM and SI up to 37.3. The presence of the CF3 group at the
C-5 position, however, seemed to be worse in this case (40). It is
also noted that the change of substituents within the naphthalene
moiety with the free OH group being protected as the methoxy
group (46) led to the loss of the activity.
In summary, seven BMZs (23, 24, 30, 32, 34, 35, and 39) were
identified as potent inhibitors of an African ZIKV strain (Fig. 2). It
seems that the aromatic heterocyclic ring, such as pyridine or
pyrrole at the C-2 position and 2-Cl-benzyl, 4-OCH3-benzyl, or 3-
pyridinylmethyl at the N-1 position, in combination with a CF3
group at the C-5 position of the BMZ ring play essential roles in
anti-ZIKV activity. Notably, the hybrid structure of BMZ and
naphthalene rings, with a free OH group at the C-4 position, three
methoxy groups at the C-5, C-6, and C-7 positions of the
naphthalene moiety, and 2-Cl-benzyl at the N-1 position of the
BMZ ring (39), is the novel structural feature that produces anti-
ZIKV activity comparable to the positive control MPA. Thus,
bioisosteric groups such as CF3 at C-5 and Cl at N-1 substituents
are necessary for enhancing anti-ZIKV activity.
Table 2. Validation of anti-ZIKV activity of selected BMZs in human neural stem
cells.
Compound
EC50 (μM)[a]
CC50 (μM)[a]
SI[b]
23
24
3.0±1.4
3.4±1.5
18.2±1.3
17.5±1.2
10.4±1.1
32.8±1.1
114.5±1.1
28.1±1.7
7.3±2.2
6.1
5.1
30
2.2±1.3
4.7
32
4.2±1.3
7.8
34
21.0±1.3
4.7±2.0
5.4
35
5.9
39
0.6±1.5
12.1
25.0
MPA
0.02±0.003
0.5±0.07
[a]EC50 and CC50 values were calculated by 10-point DRC analysis in triplicate.
Experiments were repeated at least two times independently. [b]SI = CC50/EC50
.
1 5 0
1 0 0
5 0
Fig. 2 Potent anti-ZIKV structures of evaluated BMZs.
0
Evaluation of anti-ZIKV activity in human neural stem cells
k
M
M
M
_
M
1
M
M
M
M
M
M
c










o
7
0
7
6
7
6
6
2
_
5
2
_
M
ZIKV infection can dramatically affect fetal neural development,
and strong evidence indicates that neural stem cells or
progenitors and neurons support ZIKV entry and RNA
replication.[19] Therefore, the antiviral activity of the selected BMZs
(23, 24, 30, 32, 34, 35, and 39) was tested in primary human
neural stem cells, a model physiologically resembling the situation
in patients. Except for 34, all tested compounds exhibited good
antiviral activity with EC50 at low micromolar levels. The most
potent compound (39) showed a submicromolar EC50 and SI of
12.1 (Table 2).
1
5
_
1
_
_
_
_
_
0
_
5
9
9
3
A
A
3
3
0
3
5
3
3
P
P
2
2
3
3
M
M
Fig. 3 BMZs protect against cytopathic effects induced by ZIKV infection. Cell
protection was evaluated by assessing cell viability in ZIKV-infected Vero cells
in the presence of BMZs (23, 30, 35, and 39) and MPA at the indicated
concentrations. CPE inhibition is shown as a percentage, and the positive
control MPA was set to 100% inhibition of CPE.
Evaluation of antiviral activity using an Asian ZIKV strain
To assess differences in the susceptibility of viral strains to BMZs,
we evaluated selected compounds using an Asian ZIKV strain. All
selected BMZs showed potent antiviral activity with an average SI
of ~68 and maximum SI of 115.3 (35) (Table 3). Notably, whereas
all tested compounds exhibited remarkably improved EC50
against the Asian ZIKV strain as compared with the African ZIKV
strain (Table 1), the EC50 of compound 39 remained almost the
same. This could be attributed to significant phenotypic
differences in the replication characteristics between African and
Asian lineage ZIKV strains, leading to the observed differences in
the activity of the tested compounds, especially 39.[21]
Evaluation of cell-protective effects of BMZs in ZIKV-infected
cells
As ZIKV infection induces cytopathic effects (CPEs),[20] we tested
whether treatment with BMZs could prevent CPEs induced by
ZIKV replication. Based on the results of our primary screen and
human neural stem cell experiments, we selected four
compounds for testing (23, 30, 35, and 39). Compound 24 was in
the same category as 23. Upon pre-treatment of Vero cells with
MPA, 100% of CPEs were inhibited (Fig. 3). Selected BMZs (23,
30, 35, and 39) reduced CPEs in a dose-dependent manner.
Compared with the positive control MPA, 35 at 17 µM, which is
below the CC50 of 50 µM, showed the highest CPE inhibition.
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
ZIKV MR-766, as this strain shows higher infection rates, is more
virulent, and causes more severe brain damage than the current
Asian lineage.[22] Cells were treated at the indicated time points
with compound 39 (Fig. 4A). As controls, the nucleoside
replication inhibitor MPA and NH4Cl, an early virus entry inhibitor
that blocks acidification of the late endosome and prevents fusion
of the flaviviruses envelope to the endosomal membrane, were
tested side-by-side. We found that MPA strongly inhibited ZIKV at
all time points, whereas NH4Cl was active predominantly at early
time points (-1, 0, and 1 h; Fig. 4B, left). The inhibition pattern of
compound 39 was similar to that of MPA, and passaging the viral
supernatants to naive target cells confirmed these results (Fig. 4B,
right). Importantly, these experiments were conducted at non-
cytotoxic compound concentrations (Fig. 4C). Next, we
investigated whether BMZs blocked the pre- or post-entry step in
the viral life cycle (Fig. 4D). Therefore, we delivered compounds
during the entire infection period (30 hours post-infection (hpi)) or
removed them at 3 hpi (t = 3 h), after viral entry had occurred.
NH4Cl inhibited infection when removed at 3 hpi, whereas the
inhibitory effects of compound 39 and MPA were significantly
reduced when they were removed at 3 hpi (Fig. 4D, left). After
transferring viral supernatants to naïve target cells, we observed
similar trends in ZIKV inhibition (Fig. 4D, right). Together, these
results indicate that BMZs inhibit ZIKV RNA replication.
Table 3. Validation of antiviral activity of selected BMZs using an Asian ZIKV
strain.
Compound
EC50 (μM)[a]
CC50 (μM)[a]
SI[b]
23
24
0.2±0.2
0.5±0.3
0.5±0.1
0.3±0.2
1.3±0.4
0.3±0.09
1.5±0.1
0.06±0.05
24.4±1.3
29.1±1.6
20.7±2.5
20.1±3.2
97.4±1.6
34.6±2.0
>9.4
112.0
58.0
30
41.4
32
67.0
34
74.9
35
115.3
>6.3
39
MPA
>5.9
>98.3
[a]EC50 and CC50 values were calculated by 10-point DRC analysis in triplicate.
Experiments were repeated at least two times independently. [b]SI = CC50/EC50
.
Mechanism of action studies
Lastly, to determine which stage of the viral life cycle was inhibited
by BMZs, we conducted a time-of-addition study with the African
Figure 4. Time-of-addition study with ZIKV. (A) Experimental scheme. (B) Graph showing ZIKV inhibition in passage 1 (p1, left panel) and p2 (right panel) cells. As
controls, NH4Cl (2 mM, black), MPA (25 μM, white), and DMSO (0.5%) were compared with BMZ compound 39 (25 μM, gray). Compounds were added at different
time points pre- or post-infection as indicated. (C) Cell viability in the presence of inhibitors was evaluated using a commercial kit. (D) ZIKV inhibition in p1 (left
panel) and p2 (right panel) cells with or without drug removal. Cells were treated with the indicated drugs for 1 h before infection and incubated for 30 h (black) or
removed at t = 3 h (grey). The percentage inhibition is shown for a representative experiment (n = 3). ***pꢀ<0.001, **pꢀ<0.01, or *p <0.05.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
4-(1-Benzyl-1H-benzo[d]imidazol-2-yl)-2-methoxyphenol (1): Yield
91% as light brown solid; mp 140-142°C; IR νmax (KBr): 2927, 1599, 1495,
1410, 1283, 1238, 1118, 735; ESI-MS found m/z 331.0 [M+H]+ (calcd.
331.14, C21H19N2O2); 1H-NMR (500 MHz, DMSO-d6): 9.51 (s, 1H), 7.69 (d,
J = 7.0 Hz, 1H), 7.42 (d, J = 7.0 Hz, 1H), 7.31 (t, J = 7.0 Hz, 2H), 7.26-7.19
(m, 4H), 7.14 (dd, J = 2.0 Hz, J = 8.25 Hz, 1H), 7.04 (d, J = 7.5 Hz, 2H),
6.86 (d, J = 8.0 Hz, 1H), 5.57 (s, 2H), 3.66 (s, 3H); 13C-NMR (125 MHz,
DMSO-d6): 153.6, 148.2, 147.5, 142.6, 137.2, 136.1, 128.8, 127.4, 125.9,
122.3, 122.0, 121.9, 120.8, 118.9, 115.6, 112.9, 110.7, 55.4, 47.5.
Conclusions
For the first time, we synthesized a series of 50 structurally
diverse BMZ derivatives through an efficient and environmentally
friendly one-pot route and screened compounds for their anti-
ZIKV activity. The synthetic protocol is general, simple, fast, using
inexpensive and easily available Na2S2O5 which could be an
effective method for large-scale synthesis. Seven compounds
efficiently inhibited ZIKV RNA replication in different human and
non-human cell lines. The aromatic heterocyclic ring at the C-2
position and 2-Cl-benzyl, 4-OCH3-benzyl, or 3-pyridinylmethyl at
the N-1 position, in combination with a CF3 group at the C-5
position of the BMZ ring (23, 24, 30, 32, 34, 35) play essential
roles in anti-ZIKV activity. Notably, the hybrid structure of BMZ
and naphthalene ring with bioisosteric groups such as CF3 at C-
5 and Cl at N-1 substituents (39) is the novel structural feature
that produces anti-ZIKV activity comparable to the positive control
MPA. Importantly, BMZs inhibited ZIKV in human neural stem
cells, which are physiologically the most relevant system, as ZIKV
infection is associated with severe congenital anomalies like
microcephaly. Compound 39 displayed the highest antiviral
efficacy against the African ZIKV strain in terms of SI values in
Huh-7 (SI>37) and neural stem cells (SI=12), whereas compound
35 possessed the highest activity against the Asian ZIKV strain
(SI=115) in Vero cells. Furthermore, a time-of-addition study
indicated that BMZs inhibited ZIKV RNA replication. Although
further intensive compound optimization and biological studies
are required, BMZs are a new chemical class for anti-ZIKV drug
development, and both 35 and 39 are favorable lead compounds
for further optimization as well as suitable chemical tools to study
the viral life cycle.
2-Methoxy-4-(1-(pyridin-3-ylmethyl)-1H-benzo[d]imidazol-2-yl)phenol
(7): Yield 80% as pale yellow solid; mp 149-151C; IR (KBr) νmax: 2922,
1595, 1488, 1458, 1428, 1280, 1223, 1026, 810, 743; ESI-MS m/z 331.9
[M+H]+, 329.9 [M+H]; 1H-NMR (300 MHz, CDCl3): 8.57 (d, J = 6.9 Hz, 1H),
8.51 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.20-7.37 (m, 6H), 7.04 (dd, J1 = 8.4
Hz, J2 = 2.4 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 6.69 (brs, 1H, -OH), 5.50 (s,
2H), 3.82 (s, 3H); 13C-NMR (125 MHz, DMSO-d6): 153.6, 148.7, 148.3,
147.8, 147.6, 142.6, 135.8, 133.9, 132.8, 123.7, 122.4, 122.1, 122.0, 120.8,
119.0, 116.0, 113.0, 110.7, 55.5, 45.3.
2-Methoxy-4-(1-(3-morpholinopropyl)-1H-benzo[d]imidazol-2-
yl)phenol (9): Yield 64% as white solid; mp 240–242C; IR νmax (KBr):
2951, 2914, 2850, 1596, 1498, 1465, 1312, 1271, 1176, 1119, 1032, 761;
ESI-MS found m/z 368.1 [M+H]+ (calcd. 368.19, C21H26N3O3); 1H-NMR
(500 MHz, CDCl3) 7.80 (q, J = 3.0 Hz, 1H), 7.42 (q, J = 3.0 Hz, 1H), 7.32
(d, J = 1.5 Hz, 1H), 7.27–7.32 (m, 2H), 7.17 (dd, J = 1.5; 8.0 Hz, 1H), 7.01
(d, J = 8.0 Hz, 1H), 4.36 (t, J = 7.5 Hz, 2H), 3.95 (s, 3H), 3.63 (t, J = 4.3
Hz, 4H), 2.29 (s, 4H), 2.25 (t, J = 6.8 Hz, 2H), 1.89-1.95 (m, 2H); 13C-NMR
(125 MHz, CDCl3): 153.7, 147.3, 147.0, 143.0, 135.8, 122.6, 122.5, 122.4,
122.2, 119.8, 114.5, 112.4, 110.0, 66.9, 56.2, 55.4, 53.6, 42.7, 26.6.
2-Methoxy-4-(1-(3-morpholinopropyl)-5-(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)phenol (10): Yield 68% as white solid; mp 186-
188C; IR (KBr) νmax cm-1: 2952, 2826, 1621, 1599, 1534, 1485, 1429,
1328, 1277, 1227, 1152, 1112, 1046, 853, 817. ESI-MS found m/z 436.1
[M+H]+ (calcd. 436.18, C22H25F3N3O3); 1H-NMR (500 MHz, CDCl3): 8.07 (s,
1H, 7.51-7.55 (m, 2H), 7.31 (d, J = 2.0 Hz, 1H), 7.18 (dd, J = 2.0; 8.5 Hz,
1H), 7.02 (d, J = 8.0 Hz, 1H), 4.42 (t, J = 7.3 Hz, 2H), 3.95 (s, 3H), 3.65 (s,
4H), 2.29 (q, J = 9.5 Hz, 6H), 1.93 (t, J = 7.0 Hz, 2H); 13C-NMR (125 MHz,
CDCl3): 154.6, 146.8, 146.2, 141.3, 136.7, 124.9, 123.8, 122.7, 121.1,
120.6, 118.4, 116.4, 116.3, 113.7, 111.3, 109.3, 65.6, 55.1, 54.2, 52.4,
41.8, 29.9.
Experimental section
General information
Reactions were monitored by thin-layer chromatography on 0.2-mm pre-
1
coated silica gel 60 F254 plates (Merck). H NMR and 13C NMR spectra
1-(2-Chlorobenzyl)-2-(3-fluorophenyl)-1H-benzo[d]imidazole
(14):
were measured with Bruker Avance 300 MHz, Bruker Avance 500 MHz,
and Bruker Avance 600 MHz spectrometers. Mass spectrometry data were
recorded on an 1100 series LC-MSD-Trap-LS Agilent spectrometer, and
HRESI-MS observation was performed on a Bruker MicrOTOF-Q mass
spectrometer. FT-IR was conducted using the KBr pellet method with a
Thermo Nicolet 6700. Chemical shifts are given in parts per million relative
to tetramethylsilane (Me4Si, δ = 0); J values are given in Hz.
Yield 85% as white solid; mp 163-165°C; IR(KBr) νmax: 3059, 2923, 1614,
1585, 1517, 1476, 1441, 1390, 1269, 1201, 1154, 1043, 903, 793, 744,
691; HR-ESI-MS found m/z 337.0910 [M+H]+ (calcd. 337.0908,
C20H15N2ClF)); 1H-NMR (300 MHz, CDCl3): 7.91 (d, J = 8.1 Hz, 1H), 7.50
(dd, J1 = 8.1 Hz, J2 = 1.2 Hz, 1H), 7.37-7.46 (m, 4H), 7.35 (dd, J1 = 8.1 Hz,
J2 = 1.2 Hz, 1H), 7.31 (d, J = 1.2 Hz, 1H), 7.14-7.22 (m, 3H), 6.76 (dd, J1
= 7.5 Hz, J2 = 0.9 Hz, 1H), 5.52 (s, 2H); 13C-NMR (125 MHz, DMSO-d6):
162.9, 160.9, 151.92, 151.90, 142.4, 135.9, 133.8, 132.2, 132.1, 131.3,
130.93, 130.87, 129.6, 129.4, 127.7,127.3, 124.89, 124.87, 124.9, 123.2,
122.5, 119.5, 116.9, 116.7 115.8, 115.6, 110.9, 45.7.
Chemistry
General procedure for the synthesis of BMZ derivatives (1-50): A
mixture of aldehyde (0.2 mmol), diamine (0.2 mmol), and Na2S2O5 (0.6
mmol) in ethanol (1.0 mL) was stirred at 80°C for 0.5-2 h. The reaction was
quenched with 5 mL saturated NaHCO3 and the reaction mixture was
extracted with ethyl acetate (3 × 10 mL). The combined organic layers
were washed with H2O (30 mL) and brine (30 mL) and dried over Na2SO4,
and the solvent was evaporated under reduced pressure. Purification by
column chromatography on silica gel was performed using a mixture of
hexane and ethyl acetate as solvent to afford the desired BMZ products.
2-(3-Fluorophenyl)-1-(3-morpholinopropyl)-1H-benzo[d]imidazole
(16): Yield 75% as white solid; mp 60-62C; IR (KBr) νmax: 3375, 2952,
2849, 2814, 1616, 1587, 1451, 1325, 1279, 1140, 1114, 917, 791, 747;
ESI-MS found m/z 340.0 [M+H]+ (calcd. 340.18, C20H23FN3O); 1H-NMR
(500 MHz, CDCl3)  7.81-7.84 (m, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.44 –
7.51 (m, 3H), 7.29-7.33 (m, 2H), 7.19-7.23 (m, 1H), 4.38 (t, J = 7.3 Hz, 2H),
3.61 (t, J = 4.8 Hz, 4H), 2.28 (s, 4H), 2.23 (t, J = 6.8 Hz, 2H), 1.89-1.95 (m,
2H); 13C-NMR (125 MHz, CDCl3) δ 163.7 (161.7), 152.2, 143.0, 135.7,
132.8 (132.7), 130.5 (130.4), 125.1 (125.0), 123.1, 122.6, 120.1, 116.8
(116.7), 116.5 (116.4), 110.2, 66.8, 55.1, 53.5, 42.6, 26.4.
1
All compounds were ≥95% pure according to high-resolution H-NMR of
500 MHz (see spectra in Supporting Information).
Spectral data of compounds 2-6, 8, 15, 21, 22, 24, 25, 30, 31, 33, 35,[15]
11-13,[23] and 20[24] are described in previous literature.
5-(Trifluoromethyl)-2-(3-fluorophenyl)-1-(3-morpholinopropyl)-1H-
benzo[d]imidazole (17): Yield 70% as white solid; mp 120-122C; IR
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
(KBr) νmax: 3066, 2957, 2859, 2807, 1615, 1589, 1436, 1331, 1264, 1224,
1151, 1113, 1049, 883, 860, 822, 786. ESI-MS found m/z 408.0 [M+H]+
(calcd. 408.17, C21H22F4N3O); 1H-NMR (500 MHz, CDCl3) 8.09 (s, 1H),
7.52 – 7.58 (m, 4H), 7.49 (q, J = 4.7 Hz, 1H), 7.23-7.27 (m, 1H), 4.43 (t, J
= 7.3 Hz, 2H), 3.61 (t, J = 4.0 Hz, 4H), 2.28 (s, 4H), 2.44 (t, J = 4.5 Hz, 2H),
1.89-1.95 (m, 2H); 13C-NMR (125 MHz, CDCl3) 163.7 (161.8), 154.1
(154.0), 142.5, 137.7, 132.1 (132.0), 130.7 (130.6), 125.1 (125.0), 125.9
(125.3, 124.8), 123.7, 119.89 (119.86, 119.83), 117.89 (117.86, 117.83),
116.6 (116.4), 110.7, 66.7, 55.0, 53.5, 42.8, 26.3.
1604; 1472; 1335; 1148; 1120; 864; 836; 804. ESI-MS found m/z 306.0
(calcd. 306.12, C16H15F3N3); 1H-NMR (500 MHz, DMSO-d6): 8.82 (dd, J =
4.5 Hz, 2H, 8.11 (s, 1H, 7.98 (d, J = 8.5 Hz, 1H, 7.82 (d, J = 4.5, 2H, 7.66
(d, J = 8.5, 1H, 4.39 (t, J = 7.5, 2H, 1.70 (d, J = 15, 2H, 0.74 (t, J = 7.0,
3H); 13C-NMR (125 MHz, DMSO-d6): 152.7; 150.3; 141.8; 138.1; 137.3;
123.8; 123.4; 123.2; 119.6; 117.0; 112.4; 45.9; 22.6; 10.7.
1-Benzyl-2-(1H-pyrrol-2-yl)-1H-benzo[d]imidazole (29): Yield 92% as
white solid; mp 149-151C; IR (KBr) νmax: 3414, 3057, 2923, 2856, 1601,
1577, 1497, 1453, 1392, 1356, 1135, 927, 731; HR-ESI-MS found m/z
274.1341 [M+H]+ (calcd. 274.1344, C18H16N3); 1H-NMR (500 MHz, DMSO-
d6): 7.64 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.32 (t, J = 7.5 Hz,
2H), 7.16-7.26 (m, 3H), 7.08 (d, J = 7.5 Hz, 2H), 6.97 (s, 1H), 6.49 (s, 1H),
6.15 (d, J = 3Hz, 1H), 5.71 (s, 2H); 13C-NMR (125 MHz, DMSO-d6): 147.1,
142.5, 137.0, 136.1, 128.7, 127.3, 126.0, 122.0, 121.9, 121.6, 120.7, 118.1,
110.1, 109.8, 109.3, 47.0.
2-(2,4-dimethoxyphenyl)-1-(3-morpholinopropyl)-1H-
benzo[d]imidazole (18): Yield 72% as yellow solid; mp 92-94C; IR (KBr)
νmax: 3054, 2953, 2850, 2811, 1614, 1580, 1534, 1457, 1388, 1279, 1209,
1160, 1116, 1030, 833, 747; ESI-MS found m/z 382.1 [M+H]+ (calcd.
382.21, C22H28N3O3); 1H-NMR (500 MHz, DMSO) 7.61 (d, J = 8.0 Hz, 1H),
7.59 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H),
7.19 (t, J = 7.5 Hz, 1H), 6.75 (d, J = 2.0 Hz, 1H), 6.68 (dd, J = 3.0; 8.5 Hz,
1H), 4.07 (t, J = 7.3 Hz, 2H), 3.85 (s, 3H), 3.78 (s, 3H), 3.41 (t, J = 4.0 Hz,
4H), 2.09 (m, 6H), 1.64-1.69 (m, 2H); 13C-NMR (125 MHz, DMSO) 162.1,
158.3, 151.2, 142.8, 135.0, 132.6, 121.9, 121.3, 118.9, 112.2, 110.4, 105.4,
98.5, 66.1, 55.6, 55.5, 54.7, 52.9, 41.5, 25.7.
1-(4-Methoxybenzyl)-2-(1H-pyrrol-2-yl)-1H-benzo[d]imidazole
(32):
Yield 87% as white solid; mp 123-125°C; IR (KBr) νmax: 3278, 2923, 2854,
1655, 1447, 1405, 1312, 1137, 1088, 1040, 759; HR-ESI-MS found m/z
304.1444 [M+H]+ (calcd. 304.1450, C19H18N3O); 1H-NMR (300 MHz,
CDCl3): 11.02 (s, 1H), 7.75 (dd, J1 = 6.3 Hz, J2 = 0.9 Hz, 1H), 7.18-7.30
(m, 3H), 7.11 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 1.2 Hz, 1H), 6.87 (d, J = 8.4
Hz, 2H), 6.54 (m, 1H), 6.28 (m, 1H), 5.58 (s, 2H); 13C-NMR (125 MHz,
DMSO-d6): 158.5, 147.0, 142.6, 136.0, 128.7, 127.3, 121.9, 121.8, 121.5,
120.8, 118.1, 114.2, 110.2, 109.8, 109.3, 55.0, 46.5.
5-(Trifluoromethyl)-2-(2,4-dimethoxyphenyl)-1-(3-morpholinopropyl)-
1H- benzo[d]imidazole (19): Yield 64% as yellow solid; mp 112-114C;
IR (KBr) νmax: 2947, 2815, 1616, 1578, 1536, 1466, 1436, 1330, 1211,
1158, 1117, 1029, 880, 844, 803. ESI-MS found m/z 450.1 [M+H]+ (calcd.
450.20, C23H27F3N3O3); 1H-NMR (500 MHz, CDCl3) 8.07 (br, 1H), 7.52 (brs,
2H), 7.41 (d, J = 8.5 Hz, 1H), 7.26 (s, 1H), 6.62 (dd, J = 2.0; 8.5 Hz, 1H),
6.58 (d, J = 8.0 Hz, 1H), 4.18 (t, J = 7.8 Hz, 2H), 3.88 (s, 3H), 3.79 (s, 3H),
3.60 (t, J = 4.5 Hz, 4H), 2.23 (s, 4H), 2.18 (q, J = 5.2 Hz, 2H), 1.80 (t, J =
7.0, 2H); 13C-NMR (125 MHz, CDCl3) 162.8, 158.6, 153.8, 142.7, 137.3,
132.8, 126.0, 124.5 (124.2, 123.9), 119.2 (119.1), 117.5 (117.4), 112.0,
110.2, 105.2, 98.8, 66.8, 55.7, 55.6, 55.2, 53.4, 42.4, 26.1.
1-(Pyridine-3-ylmethyl)-2-pyrrolyl-1H-benzo[d]imidazole (34): Yield
87% as white solid; mp 189-191C; IR (KBr) νmax: 3433, 3172, 2924, 2854,
1736, 1617, 1577, 1500, 1456, 1401, 1131, 1028, 829, 746, 708; HR-ESI-
MS found m/z 275.1293 [M+H]+ (calcd. 275.1297, C17H15N4); 1H-NMR (500
MHz, DMSO-d6): 11.87 (s, 1H, N-H), 8.46 (dd, J1 = 4.5 Hz, J2 = 1.5 Hz,
1H), 8.40 (d, J = 1.5 Hz, 1H), 7.66 (dd, J1 = 7.0 Hz, J2 = 1.0 Hz, 1H), 7.55
(dd, J1 = 7.0 Hz, J2 = 1.5 Hz, 1H), 7.37 (td, J1 = 8.0 Hz, J2 = 2.0 Hz,
1H),7.31-7.33 (m, 1H), 7.18-7.25 (2H), 6.97 (m, 1H), 6.55-6.57 (m, 1H),
6.17-6.19 (m, 1H), 5.78 (s, 2H); 13C-NMR (125 MHz, DMSO-d6): 148.6,
147.7, 146.9, 142.5, 135.8, 133.8, 132.6, 123.8, 122.2, 111.0, 121.7, 118.2,
110.1, 109.8, 109.4, 44.8.
1-(2-Chlorobenzyl)-2-(pyridin-4-yl)-5-(trifluoromethyl)-1H-
benzo[d]imidazole (23): Yield 80% as pale yellow solid; mp 150-152C;
IR (KBr) νmax:3057, 2962, 2928, 1600, 1473, 1443, 1386, 1326, 1162, 1109,
1044, 923, 811, 756, 695, 659; HR-ESI-MS found m/z 388.0835 [M+H]+
(calcd. 388.0828, C20H14N3ClF3); 1H-NMR (300 MHz, CDCl3): 8.75 (d, J =
5.1 Hz, 2H), 8.20 (s, 1H), 7.58 (dd, J1 = 4.5 Hz, J2 = 1.8 Hz, 2H), 7.54 (dd,
J1 = 4.8 Hz, J2 = 1.2 Hz, 1H), 7.50 (d, J = 0.9 Hz, 1H), 7.31-7.35 (m, 2H),
7.19 (td, J1 = 7.8 Hz, J2 = 1.2 Hz, 1H), 6.68 (d, J = 7.8 Hz, 1H), 5.58 (s,
2H); 13C-NMR (125 MHz, DMSO-d6): 153.2, 150.2, 141.9, 138.3, 136.8,
133.2, 131.5, 129.7, 129.6, 127.8, 127.6, 125.9, 123.8, 123.5, 123.0, 120.1,
117.24, 117.21, 112.4, 54.8.
1-Propyl-2-(1H-pyrrol-2-yl)-5-(trifluoromethyl)-1H-benzimidazole (36):
Yield 78% as white solid; mp 109-111C; IR (KBr) νmax: 3132; 2974; 2874;
1624; 1589; 1500; 1330; 1234; 1149; 1111; 924; 810; 729; ESI-MS m/z
294.0 [M+H]+; 1H-NMR (500 MHz, DMSO-d6): 11.90 (s, 1H), 7.88 (s, 1H),
7.82 (d, J = 8.5, 1H), 7.52 (dd, J = 8.5, 1H), 7.04 (t, J = 1.75, 1H), 6.79 (d,
J = 4.5, 1H), 6.30 (d, J = 6, 1H), 4.44 (t, J = 7.5, 2H), 1.81 (d, J = 14.5, 2H),
0.93 (t, J = 7.5, 3H); 13C-NMR (125 MHz, DMSO-d6, δ ppm): 148.8, 141.9,
138.3, 122.8, 122.5, 122.2, 120.3, 118.2, 114.9, 111.1, 110.5, 109.8, 45.4,
22.4, 10.9.
1-(Pyridin-3-ylmethyl)-2-(pyridin-4-yl)-1H-benzo[d]imidazole
(26):
Yield 83% as brownish yellow wax; IR (KBr) νmax: 2923, 2852, 1600, 1458,
1416, 1392, 1331, 1317, 821, 771, 758, 670; HR-ESI-MS found m/z
287.1289 [M+H]+ (calcd. 287.1297, C18H15N4); 1H-NMR (300 MHz, CDCl3):
8.75 (d, J = 5.4 Hz, 2H), 8.59 (d, J = 4.2 Hz, 1H), 8.50 (s, 1H), 7.91 (d, J =
7.2 Hz, 1H), 7.59 (dd, J1 = 4.5 Hz, J2 = 1.5 Hz, 2H), 7.25-7.40 (m, 5H), 5.53
(s, 2H); 13C-NMR (125 MHz, DMSO-d6): 150.5, 150.2, 142.5, 137.5, 136.6,
136.2, 128.8, 127.5, 126.0, 123.4, 123.1, 122.6, 119.7, 111.3, 47.5.
3-(1-Benzyl-1H-benzo[d]imidazol-2-yl)-6,7,8-trimethoxynaphthalen-1-
ol (37): Yield 81% as pale yellow solid; mp 94-96C; IR (KBr) νmax: 3346;
2931; 1722, 1612; 1453; 1352; 1253; 1106; 1024; 731; ESI-MS found m/z
441.0 [M+H]+ (calcd. 441.18, C27H25N2O4); 1H-NMR (500 MHz, DMSO-d6,
δ ppm): 9.64 (1H, s, -OH, 7.72 (1H, d, J = 7 Hz, 6.51 (1H, s, 7.47 (1H, d, J
= 7.5 Hz, 7.22-7.30 (5H, m, 7.18 (1H, s, 7.03-7.04 (3H, m), 5.68 (2H, s, -
CH2-, 4.00 (3H, s, -OMe, 3.91 (3H, s, -OMe, 3.87 (3H, s, -OMe); 13C-NMR
(125 MHz, DMSO-d6, δ ppm): 153.5; 153.13; 153.12; 148.1; 142.7; 141.0;
137.0; 135.9; 132.2; 128.7; 127.8; 127.4; 126.2; 122.6; 122.2; 119.2;
118.7; 113.7; 111.1; 108.3; 104.2; 62.2; 60.8; 55.7; 47.6.
1-(Pyridin-3-ylmethyl)-2-(pyridin-4-yl)-5-(trifluoromethyl)-1H-
benzo[d]imidazole (27): Yield 88% as white solid; mp 128-130C; IR
(KBr) νmax: 3372, 3048, 2922, 1602, 1436, 1412, 1325, 1233, 1167, 1117,
1048, 839, 814, 706; ESI-MS m/z 355.0 [M+H]+ (calcd. 355.12,
C19H14F3N4); 1H-NMR (500 MHz, DMSO-d6): 8.77 (d, J = 5.0 Hz, 2H, 8.45
(d, J = 4.0 Hz, 1H, 8.32 (s, 1H, 8.18 (s, 1H, 7.87 (d, J = 8.5 Hz, 1H, 7.77
(d, J = 5.5 Hz, 2H, 7.66 (d, J = 8.5 Hz, 1H, 7.32 (d, J = 8.0 Hz, 1H, 7.28 (t,
J = 8.0 Hz, 1H, 5.80 (s, 2H); 13C-NMR (125 MHz, DMSO-d6): 152.9; 150.3;
149.0; 147.8; 141.9; 138.2; 136.7; 134.0; 131.8; 123.8; 123.6; 123.3;
120.1; 117.2; 112.5; 45.6.
3-(1-Benzyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-6,7,8-
trimethoxynaphthalen-1-ol (38): Yield 80% as pale yellow solid; mp 130-
132C; IR (KBr) νmax : 3302, 2948, 1616, 1491, 1470, 1331, 1261, 1157,
1104, 1026, 888, 806, 728; ESI-MS found m/z 509.0 [M+H]+ (calcd. 509.17,
1
C28H24F3N2O4); H-NMR (500 MHz, DMSO-d6, δ ppm): 8,10 (1H, s, 7,72
(1H, d, J = 8,5 Hz, 7,68 (1H, d, J = 1,5 Hz, 7,58 (1H, dd, J1 = 8,5 Hz, J2 =
1,5 Hz), 7,28-7,31 (2H, m, 7,23-7,25 (1H, m, 7,20 (1H, s, 7,02-7,04 (3H,
m, 5,74 (2H, s, 3,98 (3H, s, 3,91 (3H, s) ; 3,86 (3H, s); 13C-NMR (125 MHz,
1-Propyl-2-(pyridin-4-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazole
(28): Yield 87% as white solid; mp 110-112°C; IR (KBr) νmax: 2960; 2880;
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
DMSO-d6, δ ppm): 155,5; 153,6; 153,2; 148,2; 142,1; 141,3; 138,3; 136,5;
132,2; 128,8; 127,6; 127,1; 126,2; 123,9; 123,4; 119,3; 119,0; 116,6;
114,0; 112,3; 108,1; 104,3; 62,3; 60,8; 55,8; 47,9.
1578, 1472, 1331, 1256, 1155, 1111, 1024, 919, 883, 868, 808. ESI-MS
found m/z 546.1 [M+H]+ (calcd. 546.22, C28H31F3N3O5); 1H-NMR (500 MHz,
CDCl3)  9.60 (s, 1H, -OH), 8.11 (brs, 1H), 7.61 (d, J = 1.5 Hz, 1H), 7.55
(brs, 2H) 7.03 (d, J = 1.5 Hz, 1H), 7.00 (s, 1H), 4.48 (t, J = 7.3 Hz, 2H),
4.20 (s, 3H), 3.98 (d, J = 3.0 Hz, 6H), 3.54 (t, J = 4.5 Hz, 4H), 2.23 (t, J =
7.0 Hz, 6H), 1.88-194 (m, 2H); 13C-NMR (125 MHz, CDCl3) δ 150.8, 149.5,
148.9, 143.4, 137.8, 136.1, 133.1, 127.6, 123.6, 120.0, 114.8, 112.9, 108.6,
105.7, 103.5, 99.2, 62.0, 57.7, 56.6, 51.2, 50.3, 48.7, 38.1, 21.6.
3-(1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)-6,7,8-
trimethoxynaphthalen-1-ol (39): Yield 85% as pale yellow solid; mp 140-
142C; IR (KBr) νmax: 3307, 2929, 1615, 1575, 1457, 1263, 749; HR-ESI-
MS found m/z 475.1425 [M+H]+ (calcd. 475.1425 C27H24ClN2O); 1H-NMR
(500 MHz, DMSO-d6, δ ppm): 9.67 (s, 1H), 7.76 (d, J = 7.5 Hz, 1H), 7.57
(d, J = 1.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 8.0, 1H), 7.32-
7.21 (m, 4H), 7.14 (s, 1H), 6.95 (d, J = 1.5 Hz, 1H), 6.63 (d, J = 7.5 Hz,
1H), 5.70 (s, 2H), 3.98 (s, 3H), 3.90 (s, 3H), 3.85 (s, 3H); 13C-NMR (125
MHz, DMSO-d6, δ ppm): 153.5, 153.3, 153.2, 148.1, 142.7, 141.1, 135.9,
134.1, 132.1, 131.4, 129.6, 129.3, 127.7, 127.6, 127.3, 122.9, 122.4, 119.3,
118.6, 113.7, 110.8, 108.0, 104.1, 62.2, 60.8, 55.7, 45.8.
1-Benzyl-5-(trifluoromethyl)-2-(4,6,7-trimethoxynaphthalen-2-yl)-1H-
benzo[d]imidazole (45): Yield 71% as white wax; IR (KBr) νmax: 2936,
2834, 1610, 1503, 1436, 1329, 1261, 1221, 1160, 1115, 1049, 1013, 929,
809, 732. ESI-MS found m/z: 493.1 [M+H]+ (calcd. 493.17, C28H24F3N2O3);
1H-NMR (500 MHz, DMSO-d6, δ ppm): 8.11 (s, 1H), 7.81 (s, 1H), 7.76 (d,
J = 8.5 Hz, 1H), 7.59 (t, J = 9.5 Hz, 1H), 7.44 (s, 1H, =CH-), 7.36 (s, 1H),
7.31 (t, J = 14.5 Hz, 2H), 7.25 (t, J = 14.5 Hz, 1H), 7.06 (d, J = 7.5 Hz, 3H),
5.76 (s, 2H), 3.90 (s, 3H), 3.89 (s, 3H), 3.83 (s, 3H); 13C-NMR (125 MHz,
DMSO-d6, δ ppm): 156.2, 154.0, 150.2, 150.1, 142.1, 138.5, 136.8, 129.4,
128.9, 127.6, 126.2, 126.1, 125.0, 123.9, 123.3, 123.1, 120.6, 120.3, 119.3,
116.5, 112.1, 107.3, 103.2, 100.0, 55.5, 55.5, 55.430. 48.0.
3-(1-(2-Chlorobenzyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-
6,7,8- trimethoxynaphthalen-1-ol (40): Yield 80% as pale yellow solid;
mp 185-187C; IR (KBr) νmax: 3301, 2939, 1624, 1577, 1480, 1446, 1334,
1110, 748; ESI-MS found m/z 543.0 [M+H]+ (calcd. 543.13,
C28H23ClF3N2O4); 1H-NMR (500 MHz, DMSO-d6): 9.71 (s, 1H), 8.13 (s,
1H), 7.66 (d, J = 8.5 Hz, 1H), 7.60-7.57 (m, 2H), 7.52 (d, J = 8.0 Hz, 1H),
7.33 (t, J = 7.8 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.16 (s, 1H), 6.96 (d, J =
1.0 Hz, 1H), 6.68 (d, J = 7.5 Hz, 1H), 5.77 (s, 2H), 3.98 (s, 3H), 3.90 (s,
3H), 3.85 (s, 3H); 13C-NMR (125 MHz, DMSO-d6): 155.6, 153.6, 153.2,
148.2, 142.1, 141.3, 138.3, 133.6, 132.1, 131.4, 129.7, 129.5, 127.8, 127.4,
126.9, 119.5, 118.9, 116.7, 113.9, 112.0, 107.9, 104.2, 62.2, 60.8, 55.8,
47.8.
1-(2-Chlorobenzyl)-2-(4,6,7-trimethoxynaphthalen-2-yl)-1H-
benzo[d]imidazole (46): Yield 71% as pale yellow wax; IR (KBr) νmax
:
3006, 2929, 2829, 1609, 1504, 1480, 1450, 1260, 1218, 1162, 1009, 850,
746, ESI-MS found m/z: 459.0 [M+H]+) (calcd. 459.15, C27H24ClN2O3); 1H-
NMR (500 MHz, DMSO-d6): 7.78 (t, J = 8.5 Hz), 7.71 (s, 1H), 7.53 (t, J =
9 Hz, 1H), 7.47 (t, J = 8 Hz, 1H), 7.42 (s, 1H), 7.29 (m, 5H), 6.97 (d, J =
1.5 Hz, 1H), 6.71 (d, J = 6.5 Hz, 1H), 5.70 (s, 2H), 3.89 (s, 3H), 3.88 (s,
3H), 3.78 (s, 3H)); 13C-NMR (125 MHz, DMSO-d6): 153.9, 153.8, 150.2,
149.9, 142.7, 136.2, 134.4, 131.3, 129.7, 129.4, 129.4, 127.8, 127.3, 125.5,
122.9, 122.4, 120.3, 120.0, 119.3, 110.7, 107.2, 102.9, 100.4, 55.5, 55.5,
55.2, 45.97.
6,7,8-Trimethoxy-3-(1-(4-methoxybenzyl)-1H-benzo[d]imidazol-2-
yl)naphthalen-1-ol (41): Yield 82% as pale yellow solid; mp 93-95C; IR
(KBr) νmax: 3295, 2933, 1631, 1613, 1513, 1460, 1349, 1253, 1177, 1112,
1027, 907, 821, 741; ESI-MS found m/z 471.0 [M+H]+ (calcd. 471.19,
C28H27N2O5); 1H-NMR (500 MHz, DMSO-d6): 9.69 (s, 1H, -OH), 7.70-7.72
(m, 1H), 7.67 (d, J = 1.5 Hz, 1H), 7.48-7.50 (m, 1H), 7.21-7.25 (m, 3H),
7.05 (d, J = 1.5 Hz, 1H), 6.97 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.5 Hz, 2H),
5.59 (s, 2H), 4.00 (s, 3H), 3.92 (s, 3H), 3.856 (s, 3H), 3.67 (s, 3H); 13C-
NMR (125 MHz, DMSO-d6): 158.5, 153.5, 153.1, 153.0, 148.1, 142.7,
141.0, 135.8, 132.2, 128.8, 127.9, 127.6, 122.5, 122.1, 119.1, 118.7, 114.1,
113.7, 111.2, 108.3, 104.2, 62.2, 60.8, 55.7, 55.0, 47.0.
1-(Pyridin-3-ylmethyl)-2-(4,6,7-trimethoxynaphthalen-2-yl)-1H-
benzo[d]imidazole (47): Yield 82% as organe solid; mp 194-196C; IR
(KBr) νmax: 2926, 2854, 1612, 1580, 1512, 1455, 1369, 1257, 1212, 1160,
1009, 739, ESI-MS found m/z 426.0 [M+H]+) (calcd. 426.18, C26H24N3O3);
1H-NMR (500 MHz, DMSO-d6): 8.43 (t, J = 6 Hz, 1H), 8.33 (d, J = 1.5 Hz,
1H), 7.79 (s, 1H), 7.75 (m, 1H), 7.59 (m, 1H), 7.45 (s, 1H), 7.37 (t, J = 12
Hz, 2H), 7.28 (m, 3H), 7.09 (d, J = 1 Hz, 1H), 5.76 (s, 2H), 3.91 (s, 3H),
3.90 (s, 3H), 3.89 (s, 3H,); 13C-NMR (125 MHz, DMSO-d6): 153.9, 153.7,
150.1, 149.9, 148.7, 147.9, 142.7, 135.9, 134.1, 132.8, 129.4, 125.7, 123.7,
122.71, 122.3, 120.4, 119.9, 119.2, 110.9, 107.3, 103.4, 100.4, 55.5, 55.5,
55.4, 45.4.
6,7,8-Trimethoxy-3-(1-(pyridin-3-ylmethyl)-1H-benzo[d]imidazol-2-
yl)naphthalen-1-ol (42): Yield 85% as pale yellow solid; mp 129-131C;
IR (KBr) νmax: 3352, 2999, 2932, 1604, 1572, 1386, 1317, 1253, 1119,
1022, 869; 746; ESI-MS found m/z 442.0 [M+H]+ (calcd. 442.18,
C26H24N3O4); 1H-NMR (500 MHz, DMSO-d6): 9.69 (1H, s, -OH), 8.43 (1H,
d, J = 7 Hz), 8.30 (1H, s), 7.73 (1H, m), 7.47 (1H, s), 7.55 (1H, m), 7.35
(1H, d, J = 8 Hz), 7.26-7.30 (3H, m), 7.23 (1H, s), 7.03 (1H, s), 5.73 (2H,
s), 4.00 (3H, s), 3.92 (3H, s), 3.87 (3H, s, -OMe); 13C-NMR (125 MHz,
DMSO-d6): 153.6, 153.14, 153.10, 148.8, 148.2, 147.8, 142.7, 141.1,
135.7, 134.1, 132.6, 132.2, 127.7, 123.7, 122.8, 122.3, 119.3, 118.7, 113.8,
111.0, 108.2, 104.2, 62.2, 60.8, 55.7, 45.3.
1-(Pyridin-3-ylmethyl)-5-(trifluoromethyl)-2-(4,6,7-
trimethoxynaphthalen-2-yl)-1H-benzo[d]imidazole (48): Yield 73% as
white wax; IR (KBr) νmax: 494.0 2926, 2954, 1739, 1615, 1513, 1436, 1327,
1261, 1215, 1161, 1108, 1011, 925, 813, ESI-MS found m/z [M+H]+ (calcd.
494.17, C27H23F3N3O3); 1H-NMR (500 MHz, DMSO-d6): 8.44 (t, J = 6 Hz,
1H), 8.34 (d, J = 2 Hz, 1H, 8.12 (s, 1H), 7.84 (t, J = 14 Hz, 2H), 7.62 (t, J
= 9.5 Hz, 1H), 7.46 (s, 1H), 7.40 (s, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.29 (t, J
= 13 Hz, 1H), 7.10 (d, J = 1 Hz, 1H), 5.83 (s, 2H), 3.91 (s, 3H), 3.90 (s,
6H); 13C-NMR (125 MHz, DMSO-d6): 156.08, 154.00, 150.17, 150.06,
148.85, 147.87, 142.13, 138.22, 134.09, 132.32, 129.35, 124.92, 123.79,
120.60, 120.24, 119.37, 116.55, 112.02, 107.35, 103.17, 100.38, 55.52,
55.48, 55.45. 45.69.
3-(1-Benzyl-5-methoxy-1H-benzo[d]imidazol-2-yl)-6,7,8-
trimethoxynaphthalen-1-ol (43): Yield 81% as pale yellow solid; mp 110-
112C; IR (KBr) νmax: 3354; 2923; 2852; 1618; 1486; 1382; 1266; 11123;
1027; 817. ESI-MS found m/z 471.1 [M+H]+ (calcd. 471.19, C28H27N2O5);
1H-NMR (500 MHz, DMSO-d6): 9.71 (s, 1H, -OH), 7.62 (s, 1H), 7.33 (d, J
= 9.0 Hz, 1H), 7.22-7.28 (m, 4H), 7.12 (s, 1H), 6.99-7.02 (m, 3H), 6.86 (dd,
J1 = 8.5 Hz, J2 = 1.2 Hz, 1H), 5.6 (s, 2H), 3.97 (s, 3H), 3.89 (s, 3H), 3.84
(s, 3H), 3.79 (s, 3H); 13C-NMR (125 MHz, DMSO-d6): 156.1, 153.6, 153.5,
153.3, 148.3, 143.6, 141.5, 137.2, 132.3, 130.6, 128.9, 127.7, 126.3, 118.8,
114.5, 113.8, 112.5, 111.7, 108.4, 104.4, 101.9, 62.4, 61.0, 55.9, 55.7,
47.8.
3-(1-Benzyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-5,8-
dimethoxynaphthalen-1-ol (49): Yield 79% as white solid; mp 173-
175C; IR (KBr) νmax: 3425, 2938, 1619, 1506, 1455, 1397, 1329, 1255,
1154, 1103, 1047, 932, 803, 725, HR-ESI-MS found m/z 479.1585 [M+H]+
(calcd. 479.1583, C27H22N2O3F3)); 1H-NMR (300 MHz, CDCl3): 9.57 (s, 1H,
OH), 8.19 (s, 1H), 8.05 (d, J = 1.5 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.30-
7.35 (m, 5H), 7.12 (dd, J1 = 8.1 Hz, J2 = 2.1 Hz, 2H), 6.77 (d, J = 8.4 Hz,
1H), 6.67 (d, J = 8.7 Hz, 1H), 5.59 (s, 2H), 4.03 (s, 3H), 3.82 (s, 3H)); 13C-
NMR (125 MHz, DMSO-d6): 155.2, 154.4, 149.4, 142.0, 138.6, 136.5,
3-(5-(Trifluoromethyl)-1-(3-morpholinopropyl)-1H-benzo[d]imidazol-
2-yl)-6,7,8-trimethoxynaphthalen-1-ol (44): Yield 68% as yellowish
brown solid; mp 78-80C; IR (KBr) νmax: 3353, 2940, 2853, 2816, 1616,
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
128.8, 127.5, 127.1, 125.9, 119.5, 116.6, 115.6, 113.8,112.1, 110.5, 106.0,
105.3, 56.5, 55.7, 48.0.
incubated until 24 hpi at 37°C in an incubator followed by 4%
paraformaldehyde fixation and IFA.
3-(1-(2-Chlorobenzyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-
5,8-dimethoxynaphthalen-1-ol (50): Yield 87% as white solid; mp 183-
185C; IR (KBr) νmax:3352, 2927, 2852, 1745, 1619, 1501, 1442, 1395,
1330, 1256, 1157, 1105, 1053, 934, 805, HR-ESI-MS found m/z 513.1192
[M+H]+ (calcd. 513.1193, C27H21N2O3ClF3)); 1H-NMR (500 MHz, DMSO-
d6): 9.66 (s, 1H, OH), 7.76 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 2.0 Hz, 1H),
7.62 (dd, J1 = 4.0 Hz, J2 = 1.5 Hz, 1H), 7.60 (dd, J1 = 3.5 Hz, J2 = 1.0 Hz,
1H), 7.38 (td, J1 = 8.5 Hz, J2 = 1.5 Hz, 1H), 7.27 (td, J1 = 7.5 Hz, J2 = 1.0
Hz, 1H), 7.21 (d, J = 1.5 Hz, 1H), 6.96 (d, J = 8.5 Hz, 1H), 6.88 (d, J = 8.5
Hz, 1H), 6.68 (dd, J1 = 7.5 Hz, J2 = 1.0 Hz, 1H), 5.74 (s, 2H), 3.98 (s, 3H),
3.77 (s, 3H)); 13C-NMR (125 MHz, DMSO-d6): 155.1, 154.4, 149.41,
149.38, 142.0, 138.7, 133.8, 131.8, 129.7,129.3, 127.9, 127.2, 127.0,
126.8, 119.6, 116.8, 115.6, 113.3, 111.9, 110.4, 106.1, 105.3, 56.5, 55.6,
46.5.
Evaluation of anti-ZIKV activity in human neural stem cells: Cells were
plated in 384-well format for 18 h before infection. For neural stem cells,
plates were coated with poly-L-ornithine (Sigma, P4957) and laminin
(Sigma, L2020) 1 day before plating cells. Cells were pre-treated with
compounds in a 3-fold serial dilution with 10 concentrations prior to
infection of ZIKV MR766 with a MOI of 10. Cells were fixed at 72 hpi, and
ZIKV infection was evaluated by IFA. EC50 (μM), CC50 (μM), and SI values
were calculated by DRC analysis in triplicate.
Plaque assay: Vero cells were cultured in growth medium as described
above. Cells were incubated with ZIKV for 1 h, washed, and overlaid with
a mixture of 1% (w/v) agar (Sigma) and 1% MEM. The assay was
terminated by fixing cells with 37% formaldehyde for 4 h. After fixation, the
overlay was removed and stained with 1× crystal violet to count ZIKV
plaques. Infectious virus titer (PFU/mL) was determined using the
following formula: number of plaques × dilution factor × (1 / inoculation
volume).
Biological methods
Cell culture and reagents: Vero (ATCC CCL-81) and Huh-7 cells (JCRB
Cell Bank, JCRB-0403) were maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin
and streptomycin (P/S) (GIBCO #11995-065), and 2 mM L-glutamine
(GIBCO #11500626). MPA was purchased from Sigma (#sig-sml0284-10).
H9 human embryonic stem cell-derived cells were purchased from
Invitrogen (#N7800-100). Cell cultures and human neural stem cells were
maintained and expanded according to the manufacturer's protocol.[25]
Time-of-addition study: Time-of-addition experiments were conducted
as previously reported.[31] Briefly, Huh-7 cells at passage 1 (p1) were
plated in 384-well format for 18 h (t = 18 h). Cells were infected at a MOI
of 10 at 4°C and then washed at 1.5 hpi. Compounds were treated at
different time points as indicated. At 30 hpi, the supernatant of p1 cells was
transferred to naïve Huh-7 cells (p2). p1 cells were fixed and evaluated by
IFA. p2 cells infected at 24 hpi were fixed and stained.
Virus preparation: The African ZIKV strain (MR766) was purchased from
ATCC (USA, VR-1838). The Asian ZKIV strain was isolated from a patient
in New Caledonia (NC-2014-843).[26] Viral stocks were amplified in Vero
cells (ATCC, Manassas, USA) and quantified according to the standard
plaque assay methodology.[27] Viral stock titers were determined by plaque
assay on Vero cells. The quantity of the viral stock was also confirmed by
RT-PCR using primers for NS1 regions (5’-TGACTCCCCTCGTAGACTG-
3’ and 3’-CTCTCCTTCCACTGATTTCCAC-5’).
Cell viability: Cell viability due to CPEs and compound cytotoxicity was
assessed using a CellTiter-Glo cell viability kit (Promega, G7570). To
examine cytopathic effects, Vero cells were plated in a 384-well white plate
for 18 h. Cells were treated with compounds prior to ZIKV infection at a
MOI 0.2. Cells were analyzed using the kit at 3 dpi. Percent inhibition was
calculated using values of maximum infectivity and background derived
from infected cultures treated with 0.5% DMSO (0% inhibition) and the
EC100 of MPA (100% inhibition) as references using the following formula:
(DMSO - sample) / (DMSO - EC100) × 100. To test compound cytotoxicity,
Huh-7 cells were plated in a 384-well plate for 18 h. Cells were treated with
different concentrations (12.5-200 μM) for 30 h and assayed using
CellTiter-Glo.
Immunofluorescence analysis: ZIKV infection was evaluated using
immunofluorescence analysis (IFA). Cells were fixed with 4%
paraformaldehyde, permeabilized using phosphate-buffered saline with
0.1% TritonX-100, and blocked using 2.5% bovine serum albumin (BSA).
Mouse monoclonal α-E (4G2)23, purchased from ATCC (# HB-112),[28]
was diluted in 2.5% BSA and incubated with cells for 2 h. Secondary
antibody goat anti-mouse Alexa488 (Life Technologies, #A28175) and
nuclear stain were used at a dilution of 1:1500 in 2.5% BSA. For 384-well
plates, six fields per well were imaged using the 10× objective on a
PerkinElmer Opera High Content Imaging System (Perkin-Elmer, Waltham,
MA, USA), and images were analyzed using in-house imaging software.[29]
Percent inhibition was calculated using values of maximum infectivity and
background derived from infected cultures treated with 1% DMSO (0%
inhibition) and the EC100 of positive controls (100% inhibition) as
Graphs and statistics: Prism v5.0 (GraphPad Software Inc., La Jolla, CA)
software was used to prepare graphs, calculate EC50 and CC50 values, and
determine statistical significance.
Acknowledgements
This research was funded by the Vietnam National Foundation for Science
and Technology Development (104.01-2018.51) and a National Research
Foundation of Korea (NRF) grant funded by the Korean government (MSIT,
NRF-2017M3A9G6068246).
references using the following formula: (DMSO - sample) / (DMSO - EC100
)
× 100%.[30] The antiviral activity of tested compounds was evaluated by
DRC analysis, and EC50 and CC50 values were calculated using Prism v5.0
software (GraphPad Software, Inc., La Jolla, CA, USA). Experiments were
conducted in triplicate with at least three independent repetitions. Cell
viability was assessed by determining the total number of Hoechst 33342-
stained cells.
Conflict of interest
The authors declare no conflict of interest.
Compound screening: Huh-7 cells were seeded in 384-well black wall
optical bottom plates (Sigma, CLS3828) in DMEM supplemented with 10%
fetal bovine serum and 1% P/S overnight. Cells were pre-treated with
tested compounds dissolved in DMSO and further diluted in DMEM in a
1:3 serial dilution at 10 concentrations in triplicate for 1 h at 37°C.
Approximately 1 h after compound pre-treatment, 15 μL ZIKV inoculum
(MOI 0.8) was added to each well (final volume 50 μL). Infected cells were
Keywords: Zika virus • phenotypic screening • benzimidazoles,
sodium metabisulfite
• o-phenylenediamine • aldehydes •
antivirals • replication
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
S. Park, E. Jo, H. Y. Kim, Y. S. Bae, M. P. Windisch, Antiviral Res. 2016,
132, 287-295.
References:
[1] a) S. A. Rasmussen, D. J. Jamieson, M. A. Honein, L. R. Petersen, N. Engl.
J. Med. 2016, 374, 1981-1987; b) J. Lei, G. Hansen, C. Nitsche, C. D. Klein,
L. Zhang, R. Hilgenfeld, Science 2016, 353, 503-505.
[2] S. K. Desai, S. D. Hartman, S. Jayarajan, S. Liu, G. I. Gallicano, Am. J.
Stem Cells 2017, 6, 13-22.
[3] K. Rausch, B. A. Hackett, N. L. Weinbren, S. M. Reeder, Y. Sadovsky, C. A.
Hunter, D. C. Schultz, C. B. Coyne, S. Cherry, Cell Rep. 2017, 18, 804-815.
[4] Y. K. Yoon, M. A. Ali, A. C. Wei, T. S. Choon, R. Ismail, Eur. J. Med. Chem.
2015, 93, 614-624.
[5] N. T. Chandrika, S. K. Shrestha, H. X. Ngo, S. Garneau-Tsodikova, Bioorg.
Med. Chem. 2016, 24, 3680-3686.
[6] R. Iemura, T. Kawashima, T. Fukuda, K. Ito, G. Tsukamoto, J. Med. Chem.
1986, 29, 1178-1183.
[7] M. Mader, A. de Dios, C. Shih, R. Bonjouklian, T. Li, W. White, B. L. de
Uralde, C. Sánchez-Martinez, M. del Prado, C. Jaramillo, Bioorg. Med.
Chem. Lett. 2008, 18, 179-183.
[8] A. Rao, A. Chimirri, E. De Clercq, A. M. Monforte, P. Monforte, C.
Pannecouque, M. Zappalà, IL Farmaco 2002, 57, 819-823.
[9] a) L. Delang, M. Froeyen, P. Herdewijn, J. Neyts, Antiviral Res. 2012, 93,
30-38; b) H. I. El Diwani, H. T. Abdel-Mohsen, I. Salama, F. A. F. Ragab, M.
M. Ramla, S. A. Galal, M. M. Abdalla, A. Abdel-Wahab, M. A. El Demellawy,
Chem. Pharm. Bull. 2014, 62, 856-866; c) K. Ding, A. Wang, M. A.
Boerneke, S. M. Dibrov, T. Hermann, Bioorg. Med. Chem. Lett. 2014, 24,
3113-3117.
[10] a) Y. K. Yoon, M. A. Ali, A. C. Wei, A. N. Shirazi, K. Parang, T. S. Choon,
Eur. J. Med. Chem. 2014, 83, 448-454; b) K. Y. Yeong, M. A. Ali, C. W. Ang,
S. C. Tan, H. Osman, Tetrahedron Lett. 2014, 55, 4697-4700.
[11] H. Siddiqui, R. Farooq, B. P. Marasini, R. Malik, N. Syed, S. T. Moin, M. I.
Choudhary, Bioorg. Med. Chem. 2016, 24, 3387-3395.
[12] P. Roy, A. Pramanik, Tetrahedron Lett. 2013, 54, 5243-5245.
[13] J. R. Gillard, P. L. Beaulieu, Organic Syntheses 2012, 89, 131-142.
[14] a) Y. K. Yoon, M. A. Ali, A. C. Wei, T. S. Choon, H. Osman, K. Parang, A. N.
Shirazi, Bioorg. Med. Chem. 2014, 22, 703-710; b) Y. K. Yoon, M. A. Ali, A.
C. Wei, T. S. Choon, K. Y. Khaw, V. Murugaiyah, H. Osman, V. H. Masand,
Bioorg. Chem. 2013, 49, 33-39; c) S. Ahn, Y. B. Cha, M. Kim, K. H. Ahn, Y.
C. Kim, Synth. Met. 2015, 199, 8-13; d) D. Karthik, K. J. Thomas, J. H. Jou,
Y. L. Chen, Dyes Pigm. 2016, 133, 132-142.
[15] B. T. B. Hue, H. M. Nguyen, M. Van Hieu, D. L. D. Thanh, N. H. Son, T. Q.
De, H. Morita, Heterocycles 2019, 98, 650-665.
[16] D. N. Kommi, P. S. Jadhavar, D. Kumar, A. K. Chakraborti, Green Chem.
2013, 15, 798-810.
[17] H. T. B. Bui, Q. T. K. Ha, W. K. Oh, D. D. Vo, Y. N. T. Chau, C. T. K. Tu, E.
C. Pham, P. T. Tran, L. T. Tran, H. V. Mai, Tetrahedron Lett. 2016, 57, 887-
891.
[18] a) M. Tonelli, G. Paglietti, V. Boido, F. Sparatore, F. Marongiu, E. Marongiu,
P. La Colla, R. Loddo, Chem. Biodivers. 2008, 5, 2386-2401; b) R. Raut, H.
Beesetti, P. Tyagi, I. Khanna, S. K. Jain, V. U. Jeankumar, P. Yogeeswari,
D. Sriram, S. Swaminathan, Virol. J. 2015, 12, 16.
[19] H. Tang, C. Hammack, S. C. Ogden, Z. Wen, X. Qian, Y. Li, B. Yao, J. Shin,
F. Zhang, E. M. Lee, K. M. Christian, R. A. Didier, P. Jin, H. Song, G. L.
Ming, Cell Stem Cell 2016, 18, 587-590.
[20] R. Delvecchio, L. M. Higa, P. Pezzuto, A. L. Valadao, P. P. Garcez, F. L.
Monteiro, E. C. Loiola, A. A. Dias, F. J. Silva, M. T. Aliota, E. A. Caine, J. E.
Osorio, M. Bellio, D. H. O'Connor, S. Rehen, R. S. de Aguiar, A. Savarino,
L. Campanati, A. Tanuri, Viruses 2016, 8, 322.
[21] a) P. Österlund, M. Jiang, V. Westenius, S. Kuivanen, R. Järvi, L. Kakkola,
R. Lundberg, K. Melén, M. Korva, T. Avšič–Županc, Sci. Rep. 2019, 9, 1-
15; b) K. Esser-Nobis, L. D. Aarreberg, J. A. Roby, M. R. Fairgrieve, R.
Green, M. Gale, J. Virol. 2019, 93, e00640-00619.
[22] a) D. Udenze, I. Trus, N. Berube, V. Gerdts, U. Karniychuk, infections,
Emerging Microbes Infect. 2019, 8, 1098-1107; b) Q. Shao, S. Herrlinger,
Y.-N. Zhu, M. Yang, F. Goodfellow, S. L. Stice, X.-P. Qi, M. A. Brindley, J.-F.
Chen, Development 2017, 144, 4114-4124.
[23] C. Xie, X. Han, J. Gong, D. Li, C. Ma, Org. Biomol. Chem. 2017, 15, 5811-
5819.
[24] J. E. Sadig, R. Foster, F. Wakenhut, M. C. Willis, J. Org. Chem. 2012, 77,
9473-9486.
[25] M. I. Onyshchenko, I. G. Panyutin, I. V. Panyutin, R. D. Neumann, Stem
Cells Int. 2012, 2012, 634914.
[26] E. Calvez, O. O'Connor, M. Pol, D. Rousset, O. Faye, V. Richard, A.
Tarantola, M. Dupont-Rouzeyrol, Emerging Microbes Infect. 2018, 7, 159.
[27] J. A. Bernatchez, Z. Yang, M. Coste, J. Li, S. Beck, Y. Liu, A. E. Clark, Z.
Zhu, L. A. Luna, C. D. Sohl, B. W. Purse, R. Li, J. L. Siqueira-Neto,
Antimicrob. Agents Chemother. 2018, 62, e00725-00718.
[28] D. Shum, J. L. Smith, A. J. Hirsch, B. Bhinder, C. Radu, D. A. Stein, J. A.
Nelson, K. Fruh, H. Djaballah, Assay Drug Dev. Technol. 2010, 8, 553-570.
[29] C. Ko, W. J. Park, S. Park, S. Kim, M. P. Windisch, W. S. Ryu, Antiviral Ther.
2015, 20, 835-842.
[30] M. Lee, J. Yang, E. Jo, J. Y. Lee, H. Y. Kim, R. Bartenschlager, E. C. Shin,
Y. S. Bae, M. P. Windisch, Sci. Rep. 2017, 7, 44676.
[31] a) J. Zmurko, R. E. Marques, D. Schols, E. Verbeken, S. J. Kaptein, J.
Neyts, PLoS Neglected Trop. Dis. 2016, 10, e0004695; b) M. Lee, J. Yang,
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000124
ChemMedChem
FULL PAPER
Entry for the Table of Contents
First report of 1,2-disubstituted benzimidazole (BMZ) derivatives as potent Zika virus (ZIKV) inhibitors. BMZs were synthesized through
an efficient and environmentally friendly one-pot condensation between 1,2-phenylenediamines and aromatic aldehydes utilizing
inexpensive and non-toxic inorganic salt sodium metabisulfite. In vitro screening of 50 1,2-disubstituted BMZ derivatives showed their
ability to interfere with ZIKV infection in human neural stem cells. Compound 39 displayed the highest antiviral efficacy against the
African ZIKV strain in term of selectivity index (SI) in Huh-7 (SI>37) and neural stem cells (SI=12), whereas compound 35 possessed
the highest activity against the Asian ZIKV strain (SI=115) in Vero cells. Furthermore, a time-of-addition study indicated that BMZs
inhibit ZIKV RNA replication.
12
This article is protected by copyright. All rights reserved.