Design, synthesis, and evaluation of different scaffold derivatives against NS2B-NS3 protease of dengue virus

Article abstract of DOI:10.1007/s00044-020-02660-y

The number of deaths or critical health issues is a threat in the infection caused by Dengue virus, which complicates the situation, as only symptomatic treatment is the current solution. In this regard we have targeted the dengue protease NS2B-NS3 that is responsible for the replication. The series was designed with the help of molecular modeling approach using docking protocols. The series comprised of different scaffolds viz. cinnamic acid analogs (CA1–CA11), chalcone (C1–C10) and their molecular hybrids (Lik1–Lik10), analogs of benzimidazole (BZ1-BZ5), mercaptobenzimidazole (BS1-BS4), and phenylsulfanylmethylbenzimidazole (PS1-PS4). Virtual screening of various natural phytoconstituents was employed to determine the interactions of designed analogs with the residues of catalytic triad in the active site of NS2B-NS3. We have further synthesized the selected leads. The synthesized analogs were evaluated for the cytotoxicity and NS2B-NS3 protease inhibition activity and compared with known anti-dengue natural phytoconstituent quercetin as the standard. CA2, BZ1, and BS2 were found to be more potent and efficacious than the standard quercetin as evident from the protease inhibition assay.

Full text of DOI:10.1007/s00044-020-02660-y

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02660-y
ORIGINAL RESEARCH
Design, synthesis, and evaluation of different scaffold derivatives
against NS2B-NS3 protease of dengue virus
1
Lata R. Ganji1 Lekha Gandhi2 Venkataramana Musturi2 Meena A. Kanyalkar
●
●
●
Received: 17 June 2020 / Accepted: 27 October 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The number of deaths or critical health issues is a threat in the infection caused by Dengue virus, which complicates the
situation, as only symptomatic treatment is the current solution. In this regard we have targeted the dengue protease NS2B-
NS3 that is responsible for the replication. The series was designed with the help of molecular modeling approach using
docking protocols. The series comprised of different scaffolds viz. cinnamic acid analogs (CA1–CA11), chalcone (C1–C10)
and their molecular hybrids (Lik1–Lik10), analogs of benzimidazole (BZ1-BZ5), mercaptobenzimidazole (BS1-BS4), and
phenylsulfanylmethylbenzimidazole (PS1-PS4). Virtual screening of various natural phytoconstituents was employed to
determine the interactions of designed analogs with the residues of catalytic triad in the active site of NS2B-NS3. We have
further synthesized the selected leads. The synthesized analogs were evaluated for the cytotoxicity and NS2B-NS3 protease
inhibition activity and compared with known anti-dengue natural phytoconstituent quercetin as the standard. CA2, BZ1, and
BS2 were found to be more potent and efficacious than the standard quercetin as evident from the protease inhibition assay.
●
●
●
Keywords Dengue virus NS2B-NS3 protease Molecular modeling Protease inhibition
Introduction
Several scientists targeted some of the above vital
DENV proteins generating important synthetic leads with
variable degree of antiviral activity. Similarly, some nat-
ural phytoconstituents are reported as potential anti-DENV
agents. Among all the targets available, NS2B-NS3 is
considered as an important target of DENV. NS3 protease
is associated with co-factor NS2B via a Gly-Ser linker,
together believed to be involved in the DENV replication
activity. NS3 is a trypsin-like serine protease and com-
posed of two domains, N-terminal and C-terminal, con-
sisting together of 618 amino acids. The N-terminal known
as protease domain, consists of 1–180 residues whereas
C-terminal is known as helicase domain with 180–618
residues. Although, NS3 with NS2B is believed to be
responsible for the activity but the catalytic triad (His51,
Asp75, and Ser135) is located in the NS3 protease domain
[2–4]. It is reported that, only a part of NS2B is known to
be important for NS2B-NS3 protease activity. Molecules
that directly or indirectly inhibit this protease activity can
hinder DENV infection. In India, during recent outbreak,
Carica papaya leaves extract emerged as one of the ther-
apeutic options and is believed to increase the platelet
count in dengue-infected patients [5, 6]. The chief con-
stituents of papaya leaves extract include quercetin (i),
caffeic acid (ii), p-coumeric acid (iii), kaempferol (iv),
Dengue virus (DENV) is categorized as a pandemic, affect-
ing most of the population in India and the globe. DENV
belongs to Flaviviridae and the infection is transmitted by
mosquitoes’ specifically A. aegypti. DENV exists in five
different serotypes (DENV 1–5) [1] and protection against all
these types is a real challenge. DENV consists of positive
single-stranded RNA virus that encodes a polyprotein with
ten viral proteins, out of which three are structural (cap,
envelope and membrane) and seven are nonstructural (NS1,
NS2A, NS2B, NS3, NS4A, NS4B, and NS5).
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02660-y) contains supplementary
material, which is available to authorized users.
* Meena A. Kanyalkar
meenatul@gmail.com
1
Department of Pharmaceutical Chemistry, Prin. K. M. Kundnani
College of Pharmacy, Plot 23, Jote Joy Building Rambhau
Salgaonkar Marg, Cuffe Parade, Mumbai 400005, India
2
Department of Biotechnology and Bioinformatics, School of Life
Sciences, University of Hyderabad, Hyderabad, Telangana, India

Medicinal Chemistry Research
protocatechic acid (v), chlorogenic acid (vi), and 5, 7-
dimethoxy coumarin (vii). Other phytoconstituents con-
sidered are baicalein (viii), fistein (ix), hyperoside (x),
glabranine (xi), 7-O-methyl glabranine (xii), panduratin A
(xiii), and 4-hydroxy panduratin A (xiv) indicated in
Fig. 1a. We have also designed new leads from other
synthetic scaffolds that are reported to have inhibitory
activity against DENV viz. benzthiazole, benzimidazole,
and thioguanine derivatives (H [7, 8]) depicted in Fig. 1b.
Consequently, considering the structural features of these
scaffolds (Fig. 1c) used in designing the new series based
on natural and synthetic compounds, can further help to
generate synthetic lead that can inhibit DENV.
We have used molecular docking approach for virtual
screening of potential candidate molecules in NS2B-NS3
protease (PDB code: 2FOM) to get an insight toward its
binding interactions with the important residues of the
enzyme. In NS2B-NS3 protease complex, NS3 consists of
the catalytic triad of Ser135-His51-Asp75 [9]. Apart from
this triad, Gly151, Gly153, and Tyr161 are also reported as
important residues. If the inhibitor interacts with any or all
of these residues, they are believed to have the potential
activity toward dengue. In this view, we have designed few
scaffolds based on natural phytoconstituents that are ana-
logs of caffeic acid and chalcone along with molecular
hybrids of both. Likewise, we have designed analogs of
benzimidazole and mercaptobenzimidazole as well. For
docking protocol, as the selected crystal structure of protein
was devoid of any co-crystallized ligand, we have con-
structed the structure of NS2B-NS3 protease complexed
with an inhibitor using homology modeling approach. The
crystal structure of NS2B-NS3 protease from West Nile
virus (PDB code: 2FP7) [10] was found to be suitable for
this purpose. It belongs to the same family as that of DENV
and shares the structural similarity of catalytic triad and is
available in complex with the inhibitor tetrapeptide Bz-Nle-
Lys-Arg-Arg-H. The virtual screening of docked analogs
for favorable binding interactions along with synthetic
feasibility directed us to a few molecules, which were then
synthesized. Furthermore, the cytotoxic studies of these
analogs followed by in vitro antiviral evaluation against
NS2B-NS3 protease inhibition were performed.
Sigma Aldrich. The DENV2 serotype virus was obtained
from patient serum samples. Madin–Darby canine kidney
(MDCK) cells were obtained from National Centre for
Disease Control, New Delhi, India.
Methods
Computational studies
Computational studies were carried out with the modeling
package Discovery Studio v 3.1 (DS 3.1), Accelrys Inc.,
USA running on a Windows 7 platform. Docking studies
were carried out with Genetic Optimization for Ligand
Docking GOLD v 5.1.0 (CCDC, UK) running on a separate
Red Hat Enterprise Linux WS Workstation.
Preparation of enzyme and ligand for docking
The structure of available DENV2, which is a complex of
NS2B-NS3 protease (PDB code: 2FOM), is devoid of any
ligand. Thus, to get an insight into the binding cavity we
used coordinates of co-crystallized ligand from West Nile
virus (PDB code: 2FP7) belonging to the same family as
that of DENV and that shares the structural similarity of the
catalytic triad. It is available in complex with the inhibitor
tetrapeptide Bz-Nle-Lys-Arg-Arg-H. The structure of
DENV2 complexed with the inhibitor tetrapeptide Bz-Nle-
Lys-Arg-Arg-H was constructed by homology modeling
method described in Supplementary material.
Preparation of enzyme and ligand for docking The protein
complexed with ligand obtained from homology modeling
was used for molecular docking studies. The enzyme was
available in dimeric form; the monomeric unit of it was
prepared and used for docking studies. Using CHARMm
forcefield, the atom types and partial charges were defined
and the hydrogen atoms were added at pH 8.5 to the
monomeric unit and the formal charges for acidic and
basic amino acids were defined accordingly. The prepared
monomeric unit was then refined using CHARMm for-
cefield to a gradient of 0.1 kcal/mol/Å. Using the “Smart
Minimizer” module of DS 3.1 with gradient of 0.01 kcal/
mol/Å, the complexed ligand as well as the designed
analogs were energy minimized. The validation of the
docking protocol was done by redocking the complexed
ligand, followed by superimposition of the crystal struc-
ture of ligand with the docked pose that showed
RMSD 0.055.
Materials and methods
Materials
Substituted benzaldehyde, malonic acid, 4-aminoacetophe-
none, substituted o-phenylenediamine, acetic acid, pheny-
lacetic acid, and thiol and chloroacetic acid were purchased
from SD Fine Chem. Ltd., India. All other solvents used for
synthesis were of LR grade. Quercetin was purchased from
Docking protocol GOLD software program was used to
perform all calculations related to docking studies. This
program uses a genetic algorithm (GA) for detecting the
best-fit pose of ligand with respect to the protein, employing

Medicinal Chemistry Research
(a) Reported Natural Phytoconstituents
OH
OH
OH
O
B
O
HO
O
O
B
HO
HO
HO
O
O
OH
A
OH
A
OH
HO
OH
OH
OH
(iii)
(iv)
(i)
(ii)
HO
O
OH
COOH
OH
OCH3
O
HO
HO
O
O
O
H3CO
O
O
OH
HO
OH
OH
OH
OH
(vi)
(viii)
(vii)
OH
(v)
OH
OH
HO
O
HO
O
OH
OH
HO
O
H3CO
O
O
O
O
HO
OH
OH
O
OH
OH
O
(ix)
OH
O
OH
(x)
(xi)
(xii)
HO
OH
O
HO
OH
O
OH
OCH3
(xiii)
(xiv)
(b) Reported Synthetic Molecules
H
H
N
N
N
O
O2N
CN
N
R1
N
O
N
N
N
S
S
H
O
COOH
N
N
R2
Thioguanine derivative
Benzthiazole
Benzimidazole derivative
HOOC
(c) Designed target molecules scaffold
O
O
N
S
N
OH
R
N
R1
R
S
H2N
N
H
N
H
R
R
N
R
H
Cinnamic acid
Chalcone
Phenyl sulfanyl
benzimidzole
Benzimidazole
Mercaptobenzimidazole
Fig. 1 Reported natural phytoconstituents (a) and reported synthetic molecules (b) along with structural features of the designed target
molecules (c)

Medicinal Chemistry Research
20 GA run iterations. While running the 20 GA runs for
single structure, it uses a least-square routine as an attempt
to form as many of the interactive hydrogen bonds as
possible with the protein and the best fitted pose is assigned
the fitness score. All the 20 GA runs generate 20 different
poses of protein-ligand complex with the fitness score
according to the quality of the solution and the best-fit
analog is ranked with highest gold score. Similarly, all the
designed analogs were studied using GOLD v 5.1.0, to
explore the binding sites of these analogs within the tar-
geted protein.
3-(2-chlorophenyl) prop-2-enoic acid (CA2) White solid,
yield (83%), m.p. 216–217 °C. IR (KBr) νmax in cm−1:
2518, 1681, 1417, 1304 (COOH), 1616, 1588 (C=C alkene),
1
1469 (Ar–C=C), 1222 (Ar–Cl), 1207 (Ar–C–H); H-NMR
(DMSO-d6, 500 MHz) δ ppm 12.65 (s, 1H, COOH-1),
7.93–7.92 (d, 1H, H-3′), 7.90–7.86 (d, 1H, H-β), 7.55–7.54
(d, 1H, H-6′), 7.46–7.43 (t, 1H, H-4′), 7.41–7.38 (t, 1H, H-
5′), 6.63–6.59 (d, 1H, H-α). HPLC % purity = 99.82%.
3-(3-chlorophenyl) prop-2-enoic acid (CA3) White crystals,
yield (89%), m.p. 148–150 °C. IR (KBr) νmax in cm−1:2543,
1681, 1415,1300 (COOH), 1636, 1595 (C=C alkene), 1573
(Ar–C = C),1213 (Ar–Cl), 1138 (Ar–C–H); 1H-NMR
(DMSO-d6,500 MHz) δ ppm 13.09 (s, 1H, COOH-1),
7.90–7.89 (d, 1H, H-4′), 7.80 (s, 1H, H-2′), 7.70–7.65 (t, 1H,
H-5′), 7.59–7.55 (d, 1H, H-β), 7.44–7.41 (d, 1H, H-6′),
6.63–6.60 (d, 1H, H-α). HPLC % purity = 99.83%.
Synthesis The synthesis of designed analogs (Table 1) is
described in the synthetic protocol. The purity of starting
materials was assessed by determining their physical
constant (viz. melting point) and by thin-layer chromato-
graphy (TLC) on Merck silica gel F254 plates. The progress
of reactions was monitored by TLC. Physical constants
were determined using the Analab melting point apparatus
µThermoCal10 and the purity was analyzed by HPLC. The
structures of the synthesized analogs were characterized by
1H NMR and infrared spectroscopy (IR). NMR experi-
ments were recorded on a 600-MHz Varian NMR spec-
trometer in dimethylsulfoxide (DMSO)-d6 solvent, and
data were processed by using Bruker Topspin 2.1 and
Varian software. In proton NMR, 64 scans were recorded,
chemical shifts have been reported in parts per million
(ppm) using tetramethylsilane as an internal standard. IR
experiments were recorded on Bruker Alpha-T spectro-
meter with 44 scans, and the data were processed using
OPUS software.
3-(4-chlorophenyl) prop-2-enoic acid (CA4) White crystal-
line solid, yield (95%), m.p. 255–256 °C. IR (KBr) νmax in
cm−1: 2354, 1675, 1403, 1304 (COOH), 1623, 1589 (C=C
alkene), 1568 (Ar–C=C), 1204 (Ar–Cl), 1175 (Ar–C–H);
1H-NMR (DMSO-d6,500 MHz) δ ppm 12.46 (s, 1H,
COOH-1), 7.73–7.71 (d, 2H, H-3′, H-5′), 7.60–7.57 (d, 1H,
H-β), 7.47–7.42 (d, 2H, H-2′, H-6′), 6.57–6.54 (d, 1H, H-α).
HPLC % purity = 99.80%.
3-(2-methoxy-phenyl) prop-2-enoic acid (CA5) White
solid, yield (91%), m.p. 192 °C. IR (KBr) νmax in cm−1
:
1679, 1425, 1329 (COOH), 1618, 1489 (C=C alkene),
1462 (Ar–C=C), 1157, 1024 (C–O–C), 1102 (Ar–C–H);
1H-NMR (DMSO-d6, 500 MHz) δ ppm 12.29 (s, 1H,
COOH-1), 7.84–7.82 (d,1H, H-β), 7.67–7.66 (d, 1H, H-6′),
7.41–7.39 (t, 1H, H-5′), 7.09–7.08 (d, 1H, H-3′), 6.99–6.97
(t, 1H, H-4′), 6.52–6.49 (d, 1H, H-α), 3.86 (s, 3H, OCH3-
2′). HPLC % purity = 99.79%.
Synthesis of cinnamic acid analogs (CA1–CA11)
The scheme (Scheme 1) is based on a Knoevenagel con-
densation reaction using substituted benzaldehyde (9 mmol)
and dry malonic acid (24 mmol) in dry pyridine (20 ml)
along with few drops of piperidine. The solution was heated
at 80 °C on a water bath for 1 h. The reaction mixture was
cooled and then acidified with HCl solution (~1.5–2 ml) to
precipitate the product. Solid product was obtained, filtered,
and recrystallized from ethanol.
3-(3-methoxy-phenyl) prop-2-enoic acid (CA6) White
solid, yield (90%), m.p. 116–119 °C. IR (KBr) νmax in
cm−1: 2966, 1666, 1421, 1315 (COOH), 1620, 1412 (C=C
alkene), 1590 (Ar–C=C), 1167.9, 1109 (Ar–C–H), 1019.95
(C–O–C); 1H-NMR (DMSO-d6, 500 MHz) δ ppm 12.35 (s,
1H, COOH-1), 7.54–7.52 (d, 1H, H-6′), 7.31–7.28 (d, s,
2H, H-β, H-2′), 7.23–7.21 (t, 1H, H-5′), 6.96–6.95 (d, 1H,
H-α), 6.54–6.51 (d, 1H, H-4′), 3.76 (s, 3H, OCH3-3′).
HPLC % purity = 99.85%.
Chemistry
3-phenyl prop-2-enoic acid (CA1) White crystals, yield
(82%), m.p. 142 °C. IR (KBr) νmax in cm−1: 2825, 1671,
1448, 1311 (COOH), 1626, 1576 (C=C alkene), 1494
(Ar–C=C), 1175 (Ar–C–H); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 12.4 (s, 1H, COOH-1), 7.8 (d, 2H, H-2′,
H-6′), 7.69–7.68 (t, 2H, H-3′, H-5′), 7.61–7.58 (d, 1H, H-β),
7.42–7.41 (t, 1H, H-4′), 6.55–6.52 (d, 1H, H-α). HPLC %
purity = 99.81%.
3-(4-methoxy-phenyl) prop-2-enoic acid (CA7) White
solid, yield (95%), m.p. 179–180 °C. IR (KBr) νmax in
cm−1: 2476, 1670, 1427,1309 (COOH), 1621, 1510 (C=C
alkene), 1596.95 (Ar–C=C), 1170 (Ar–C–H), 1025
(C–O–C); 1H-NMR (DMSO-d6, 500 MHz) δ ppm 12.21 (s,
1H, COOH-1), 7.64–7.63 (d, 2H, H-2′, H-6′), 7.56–7.53 (d,

Medicinal Chemistry Research
Table 1 Structure of all designed analogs with their codes
Scaffolds
Code
R1
R2
R3
CA1
CA2
CA3
CA4
CA5
CA6
CA7
CA8
CA9
CA10
CA11
C1
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
OCH3
H
H
H
CA1– CA11
OCH3
H
H
H
OCH3
H
H
NO2
H
H
NO2
H
H
OH
H
H
OH
H
H
H
C2
Cl
H
H
H
C3
H
Cl
C4
OCH3
H
H
H
C1–C10
C5
OCH3
H
H
C6
H
OCH3
H
C7
H
NO2
H
C8
H
NO2
H
C9
H
OH
H
C10
LIK1
LIK2
LIK3
LIK4
LIK5
LIK6
LIK7
LIK8
LIK9
LIK10
H
OH
H
H
H
H
H
Cl
OCH3
H
H
H
H
OCH3
H
H
NO2
H
H
NO2
H
H
OH
H
H
OH
H
OCH3
H
H
OH
H
BZ1
BZ2
BZ3
BZ4
BZ5
CH3
H
H
H
H
H
H
CH3
Cl
CH3
NO2
CH3
H
CH3
BZ1–BZ5
CH2C6H5

Medicinal Chemistry Research
Table 1 (continued)
Scaffolds
Code
R1
R2
R3
BS1
BS2
BS3
BS4
H
H
H
H
H
H
H
H
H
Cl
NO2
CH3
PS1
PS2
PS3
PS4
H
H
H
H
H
H
H
H
H
Cl
NO2
CH3
O
O
O
O
O
O
O
Pyridine/
NaOH
Ethanol
OH
OH
Piperidine
H
OH
H2C
H
R
R
R
80 0
C
R
H2
N
H2N
4-Amino acetophenone
Substituted 4-Amino Chalcone
Substituted Benzaldehyde
Substituted Cinnamic Acid
Substituted Benzaldehyde
Malonic Acid
Scheme 2 Synthetic scheme for substituted chalcone analogs
Scheme 1 Synthetic scheme for substituted cinnamic acid analogs
1H, H-β), 6.98–6.96 (d, 2H, H-3′, H-5′), 6.39–6.36 (d, 1H,
H-α), 3.799 (s, 3H, OCH3- 4′). HPLC % purity = 98.88%.
5′), 7.10–7.09 (d, 1H, H-6′), 7.008 (s, 1H, H-2′), 6.82–6.81
(d, 1H, H-4′), 6.41–6.39 (d, 1H, H-α). HPLC % purity =
99.82%.
3-(3-nitrophenyl) prop-2-enoic acid (CA8) Yellow crystals,
yield (85%), m.p. 206–207 °C. IR (KBr) νmax in cm−1
:
3-(4-hydroxy-phenyl) prop-2-enoic acid (CA11) White
2523, 1684, 1420,1301 (COOH), 1633, 1519 (C=C
alkene), 1441 (Ar–C=C), 1357, 873 (NO2), 1093
solid, yield (50%), m.p. 208 °C. IR (KBr) νmax in cm−1
:
3490 (Ar-OH), 2576, 1669, 1448, 1311 (COOH), 1626,
1589 (C=C alkene), 1511, 1421 (Ar–C=C), 1104
(Ar–C–H); 1H-NMR (DMSO-d6,500 MHz) δ ppm 12.10 (s,
1H, COOH-1), 9.94 (s, 1H, OH-3′), 7.51–7.48 (d, 3H, H-β,
H-2′, H-6′), 6.80–6.78 (d, 1H, H-α), 6.29–6.27 (d, 2H, H-3′,
H-5′). HPLC % purity = 99.94%.
1
(Ar–C–H); H-NMR (DMSO-d6, 500 MHz) δ ppm 12.61
(s, 1H, COOH-1), 8.52 (s, 1H, H-2′), 8.25–8.23 (d, 1H, H-
4′), 8.19–8.18 (d, 1H, H-6′), 7.75–7.69 (d, t, 2H, H-β, H-5′),
6.77–6.73 (d, 1H, H-α). HPLC % purity = 98.59%.
3-(4-nitrophenyl) prop-2-enoic acid (CA9) Yellow solid,
yield (92%), m.p. 287 °C. IR (KBr) νmax in cm−1: 2513,
1683, 1425, 1225 (COOH), 1629, 1550 (C=C alkene),
Synthesis of chalcone analogs (C1–C10)
1
1305, 868 (NO2), 1225 (Ar–C=C), 1108 (Ar–C–H); H-
The scheme (Scheme 2) is based on the Claisen–Schmidt
condensation reaction to synthesize substituted chalcone.
Equimolar quantities (7.1 mmol) of 4-aminoacetophenone
and substituted benzaldehyde were dissolved in 30-ml
absolute ethanol. Sodium hydroxide solution (20%, 5 ml)
was added, and the resulting mixture was stirred at 10 °C for
2–4 h. The precipitate obtained was neutralized with HCl
and then filtered and recrystallized from ethanol.
NMR (DMSO-d6, 500 MHz) δ ppm 12.68 (s, 1H, COOH-
1), 8.24 (d, 2H, H-3′, H-5′), 7.98–7.97 (d, 2H, H-2′, H-6′),
7.70–7.68 (d, 1H, H-β), 6.75–6.73 (d, 1H, H-α). HPLC %
purity = 99.86%.
3-(3-hydroxy-phenyl) prop-2-enoic acid (CA10) Brown
solid, yield (71.5%), m.p. 203 °C. IR (KBr) νmax in cm−1
:
3377 (Ar-OH), 1668, 1450, 1332 (COOH), 1616, 1596
(C=C alkene), 1427 (Ar–C=C), 1178 (Ar–C–H); 1H-NMR
(DMSO-d6, 500 MHz) δ ppm 12.35 (s, 1H, COOH-1), 9.58
(s, 1H, OH-3′), 7.50–7.48 (d, 1H, H-β), 7.23–7.20 (t, 1H, H-
1-(4ʹ-Amino-phenyl)-3-phenyl-prop-2en-1-one (C1) Yel-
low crystals, yield (56%), m.p. 144 °C. IR (KBr) νmax in
cm−1: 3350 (NH2), 1650 (C=O), 1620, 1591 (C=C alkene),

Medicinal Chemistry Research
1555, 1451 (Ar–C=C), 1131 (Ar–C–H); 1H-NMR (DMSO-
d6, 500 MHz) δ ppm 7.94–7.93 (d, 2H, H-2′, H-6′),
7.86–7.84 (d, t, 3H, H-2, H-6, H-4), 7.44 (d, t, d, 4H, H-α,
H-3, H-5, H-β), 6.64–6.63 (d, 2H, H-3′, H-5′), 6.17 (s, 2H,
NH2). HPLC % purity = 99.18%.
1-(4ʹ-Amino-phenyl)-3-(3-nitro-phenyl)-prop-2en-1-one
(C7) Orange solid, yield (68%), m.p. 165–168 °C. IR
(KBr) νmax in cm−1: 3425, 3335 (NH2), 1632 (C=O), 1609
(C=C), 1530, 1344 (NO2); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 8.69 (s, 1H, H-2), 8.30–8.29 (d, 1H, H-4),
8.25–8.23 (d, 1H, H-6), 8.07–8.04 (d, 1H, H-β, J = 16 Hz),
7.98–7.97 (d, 2H, H-2′, H-6′), 7.76–7.71 (m, 2H, H-5, H-α,
J = 16 Hz), 6.68–6.66 (d, 2H, H-3′, H-5′), 6.17 (s, 2H,
NH2). HPLC % purity = 98.14%.
1-(4ʹ-Amino-phenyl)-3-(2-chloro-phenyl)-prop-2en-1-one
(C2) Yellow solid, yield (78%), m.p. 106–109 °C. IR
(KBr) νmax in cm−1: 3306, 3231 (NH2), 1643 (C=O), 1608
1
(C=C), 1178 (Ar–Cl); H-NMR (DMSO-d6, 500 MHz) δ
ppm 8.17–8.15 (d, 1H, H-6), 7.97–7.88 (m, 4H, H-β, J =
15.5 Hz, H-2′, H-6′, H-α, J = 15.5 Hz), 7.57–7.56 (d, 1H,
H-3), 7.48–7.45 (m, 2H, H-4, H-5), 6.68–6.67 (d, 2H, H-3′,
H-5′), 6.17 (s, 2H, NH2). HPLC % purity = 96.65%.
1-(4ʹ-Amino-phenyl)-3-(4-nitro-phenyl)-prop-2en-1-one
(C8) Orange solid, yield (60%), m.p. 182–184 °C. IR
(KBr) νmax in cm−1: 3459, 3338 (NH2), 1646 (C=O), 1615
(C=C), 1545, 1344 (NO2); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 8.28–8.27 (d, 2H, H-3, H-5), 8.13–8.11
(d, 2H, H-2, H-6), 8.06–8.03 (d, 1H, H-β, J = 16 Hz),
7.97–7.95 (d, 2H, H-2′, H-6′), 7.71–7.68 (d, 1H, H-α, J =
16 Hz), 6.67–6.66 (d, 2H, H-3′, H-5′), 6.16 (s, 2H, NH2).
HPLC % purity = 98.99%.
1-(4ʹ-Amino-phenyl)-3-(4-chloro-phenyl)-prop-2en-1-one
(C3) Yellow solid, yield (80%), m.p. 157–159 °C. IR
(KBr) νmax in cm−1: 3459, 3341 (NH2), 1630 (C=O), 1602
1
(C=C), 1175 (Ar–Cl); H-NMR (DMSO-d6, 500 MHz) δ
ppm 7.94–7.92 (d, 2H, H-2, H-6), 7.88–7.85 (m, 3H, H-2′,
H-6′, H-β, J = 16 Hz), 7.62–7.59 (d, 1H, H-α, J = 16 Hz),
7.52–7.50 (d, 2H, H-3, H-5), 6.66–6.65 (d, 2H, H-3′, H-5′),
6.08 (s, 2H, NH2). HPLC % purity = 98.31%.
1-(4ʹ-Amino-phenyl)-3-(3-hydroxy-phenyl)-prop-2en-1-one
(C9) Yellow powder, yield (62%), m.p. 203–208 °C. IR
(KBr) νmax in cm−1: 3343 (NH2), 3231 (OH), 1644 (C=O),
1582, 1557 (C=C alkene), 1439 (Ar–C=C), 1132
1
1-(4ʹ-Amino-phenyl)-3-(2-methoxy-phenyl)-prop-2en-1-one
(C4) Yellow powder, yield (83%), m.p.128 °C. IR (KBr)
νmax in cm−1: 3335, 3217 (NH2), 1243, 1022 (OCH3),
1644 (C=O, α, β-unsaturated), 1593 (C=C alkene), 1521
(Ar–C=C), 1172 (Ar–C–H); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 3.9 (s, OCH3-2), 6.15 (s, NH2-4′),
6.62–6.64 (d, H-3′, H-5′), 7.01-7.04 (d, H-α, t, H-5),
7.10–7.12 (d, H-3), 7.41–7.44 (t, H-4), 7.80–7.83 (d, H-β),
7.90–7.94 (d, H-6, H-2′, H-6′). HPLC % purity = 96.67%.
(Ar–C–H); H-NMR (DMSO-d6, 500 MHz) δ ppm 9.58 (s,
1H, OH-3), 7.91–7.89 (d, 2H, H-2′, H-6′), 7.76–7.73 (d,
1H, H-α), 7.53–7.50 (d, 1H, H-β), 7.26–7.21 (t, d, 2H, H-5,
H-6), 7.17 (s, 1H, H-2), 6.84–6.83 (d, 1H, H-4), 6.63–6.61
(d, 2H, H-3′, H-5′), 6.15 (s, 2H, NH2). HPLC % purity =
92.28%.
1-(4ʹ-Amino-phenyl)-3-(4-hydroxy-phenyl)-prop-2en-1-one
(C10) Yellow powder, yield (58%), m.p. 173 °C. IR (KBr)
νmax in cm−1: 3335 (NH2), 3214 (OH), 1635 (C=O), 1625,
1588 (C=C alkene), 1507 (Ar–C=C), 1163 (Ar–C–H); 1H-
NMR (DMSO-d6, 500 MHz) δ ppm 9.80 (s, 1H, OH-4),
8.20–8.19 (d, 2H, H-3, H-5), 7.91–7.90 (d, 1H, H-α),
7.57–7.54 (d, 2H, H-2′, H-6′), 6.9–6.8 (d, 2H, H-2, H-6),
6.63–6.61 (d, 1H, H-β), 6.58–6.56 (d, 2H, H-3′, H-5′), 6.2
(s, 2H, NH2). HPLC % purity = 98.98%.
1-(4ʹ-Amino-phenyl)-3-(3-methoxy-phenyl)-prop-2en-1-one
(C5) Brown powder, yield (75%), m.p. 145–146 °C. IR
(KBr) νmax in cm−1: 3341, 3218 (NH2), 1222, 1024
(OCH3), 1649 (C=O, α, β-unsaturated), 1582 (C=C
alkene), 1520 (Ar–C=C), 1171 (Ar–C-H); 1H-NMR
(DMSO-d6, 500 MHz) δ ppm 3.85 (s, OCH3-3), 6.06 (s,
NH2-4′), 6.66 (d, H-3′, H-5′), 7.01 (d, H-4), 7.35-7.39 (t, H-
5, d, H-6), 7.42 (s, H-2), 7.58–7.61 (d, H-α), 7.82–7.85 (d,
H-β), 7.94 (d, H-2′, H-6′).). HPLC % purity = 99.36%.
Synthesis of linkages of cinnamic acid (Lik1–Lik10)
Linkages of cinnamic acid with 4-aminoacetophenone and
substituted 4-amino chalcone were synthesized as shown in
Scheme 3. Cinnamic acid (10 mmol) was dissolved in 20 ml
of dimethylformamide (DMF), followed by addition of
triethanolamine (10 mmol). The solution was cooled in an
ice bath, and 10 mmol of amine (4-aminoacetophenone or
substituted 4-amino chalcone) was added. Then, a solution
comprising of 10 mmol of N, N′-dicyclohexylcarbodiimide
in 20 ml of dichloromethane was added in the reaction
mixture. The mixture was stirred at 0 °C for 30 min and then
1-(4ʹ-Amino-phenyl)-3-(4-methoxy-phenyl)-prop-2en-1-one
(C6) Yellow solid, yield (88%), m.p. 108–111 °C. IR
(KBr) νmax in cm−1: 3457, 3331 (NH2), 1630 (C=O), 1599
(C=C), 1259, 1025 (C–O–C); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 7.91–7.90 (d, 2H, H-2′, H-6′), 7.79–7.78
(d, 2H, H-2, H-6), 7.72–7.69 (d, 1H, H-β, J = 15.5 Hz),
7.61–7.58 (d, 1H, H-α, J = 15.5 Hz), 7.02–7.01(d, 2H, H-3,
H-5), 6.65–6.64 (d, 2H, H-3′, H-5′), 6.01 (s, 2H, NH2), 3.86
(s, 3H, OCH3). HPLC % purity = 97.55%.

Medicinal Chemistry Research
O
Scheme 3 Synthetic scheme for
linkage of cinnamic acid with 4-
aminoacetophenone
(Lik1–Lik8) and 4-amino
chalcone derivatives
O
O
O
TEA, DCC
N
H
OH
R
R
Stirring for 30 min at 00,
H2
N
2 hr at 370C
Substituted Cinnamic Acid
(Lik9–Lik10)
Linkage of Cinnamic acid with 4-Amino acetophenone
TEA, DCC
4-Amino acetophenone
O
O
Stirring for 30 min at 00,
R
OH
R
2 hr at 370C
H2N
O
Substituted Cinnamic Acid
Substituted 4-Amino Chalcone
O
R
N
H
Linkage of Cinnamic acid with Substituted Chalcone
at 27 °C for 2 h. DMF was evaporated under reduced
pressure, and the solution was diluted with 150 ml of water.
The product was extracted with ethyl acetate; the extract
was dried over sodium sulfate bed and evaporated. The
residues were further recrystallized from methanol.
H-IV, d, H-2′, H-6′), 7.95-7.97 (d, H-VI), 8.40–8.42 (d, H-
3′, H-5′). HPLC % purity = 99.86%.
1-(4ʹ-Acetyl-phenyl)-3-(4-methoxy-phenyl)-prop-2-en-amide
(Lik4) Yellowish white crystals, yield (72%), m.p.
190 °C. IR (KBr) νmax in cm−1: 1382.87, 1170.71,
1027.99 (OCH3), 3317.34, 779.19 (CO–NH), 2931.60,
2850.59, 1170.71 (CH3), 1704.96 (C=O), 1645.17,
1510.16 (C=C alkene), 1602.74, 1544.88 (Ar–C=C),
3271.05, 1089.71 (Ar–C–H); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 2.5 (s, H-1), 3.80 (s, OCH3-IV), 5.58 (s,
NH-4), 6.47 (d, H-III, H-V), 6.98–6.99 (d, H-5),
7.34–7.37 (d, H-II, H-VI), 7.49–7.51 (d, H-6, d, H-2′, H-
6′), 7.88–7.90 (d, H-3′, H-5′).
1-(4ʹ-Acetyl-phenyl)-3-(phenyl)-prop-2-en-amide
(Lik1)
White powder, yield (55%), m.p. 174–176 °C. IR (KBr)
νmax in cm−1: 3251.76, 763.76 (CO–NH), 2935.46,
2852.54, 1164.92, 1379.01 (CH3), 1706.88 (C=O),
1645.17, 1541.02 (C=C alkene), 1596.95 (Ar–C=C),
3060.82 (Ar–C–H); 1H -NMR (DMSO-d6, 500 MHz) δ
ppm 3.05 (s, H-1), 6.32–6.35 (d, H-6), 7.00–7.03 (d, H-7),
7.24–7.25 (d, III-H, VI-H), 7.28–7.32 (t, IV-H, d, H-2′, H-
6′), 7.33 (d, II-H, VI-H), 7.43–7.45 (d, H-3′, H-5′), 8.32 (s,
NH-4). HPLC % purity = 99.96%.
1-(4ʹ-Acetyl-phenyl)-3-(3-nitro-phenyl)-prop-2-en-amide
(Lik5) Yellowish white powder, yield (66%), m.p. 170 °C.
IR (KBr) νmax in cm−1: 1375.15, 736.76 (NO2), 3294.19,
865.98 (CO–NH), 2933.53, 2856.38, 1352.01 (CH3),
1701.10 (C=O), 1650.95, 1533.30 (C=C alkene), 1608.52
(Ar–C=C), 3082.04, 3041.53 (Ar–C–H); 1H-NMR
(DMSO-d6, 500 MHz) δ ppm 2.52 (s, H-1), 4.15 (s, NH-
4), 6.86–6.89 (d, H-5), 7.66–7.69 (d, H-6), 7.71–7.74 (t, H-
V), 8.06–8.08 (d, H-2′, H-6′, H-VI), 8.22–8.24 (d, H-3′, H-
5′), 8.42 (s, H-II), 8.46–8.48 (d, H-IV). HPLC % purity =
98.58%.
1-(4ʹ-Acetyl-phenyl)-3-(4-chloro-phenyl)-prop-2-en-amide
(Lik2) Buff powder, yield (78%), m.p. 220–221 °C. IR
(KBr) νmax in cm−1: 821.62 (Ar–Cl), 3325.05 (CO–NH),
2943.17, 2850.59, 1375 (CH3), 1706.88 (C=O), 1623.95,
1537.16 (C=C alkene), 1569.95 (Ar–C=C), 1164.92
1
(Ar–C–H); H-NMR (DMSO-d6, 500 MHz) δ ppm 2.2 (s,
H-1), 5.58 (s, NH-4), 6.68–6.71 (d, H-5), 7.49–7.51 (d, H-
III, H-V), 7.52–7.55 (d, H-6, H-II, H-VI), 7.61–7.63 (d, H-
2′, H-6′), 8.40–8.43 (d, H-3′, H-5′). HPLC % purity =
94.54%.
1-(4ʹ-Acetyl-phenyl)-3-(3-hydroxy-phenyl)-prop-2-en-amide
(Lik7) White flakes, yield (65%), m.p. 227–229 °C. IR
(KBr) νmax in cm−1: 3328.91 (OH), 3267, 827.41
(CO–NH), 2929.67, 2850.59, 1166.85, 1309.58 (CH3),
1782.10 (C=O), 1623.95 (C=C alkene), 1571.88
(Ar–C=C), 1087.78 (Ar–C–H); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 2.50 (s, H-1), 5.581 (s, NH-4), 6.84–6.86
(d, H-6, d, H-2′, H-6′), 7.29–7.32 (t, H-V, d, H-IV),
7.67–7.69 (d, H-5, s, H-II), 8.43–8.44 (d, H-VI, H-3′, H-5′),
10.16 (s, OH-III). HPLC % purity = 98.68%.
1-(4ʹ-Acetyl-phenyl)-3-(2-methoxy-phenyl)-prop-2-en-amide
(Lik3) Yellowish powder, yield (67%), m.p. 172 °C. IR
(KBr) νmax in cm−1: 1164.92, 1080.06 (Ar–OCH3),
3253.69 (CO–NH), 2937.38, 2852.52, 891.05 (CH3),
1704.96 (C=O), 1647.10, 1539.09 (C=C alkene), 1596.95
(Ar–C=C), 3060.80, 1228.57 (Ar–C–H); 1H-NMR
(DMSO-d6, 500 MHz) δ ppm 2.52 (s, H-1), 3.85 (s,
OCH3-II) 5.6 (s, NH-4), 6.64–6.67 (d, H-5), 6.77–6.80 (d,
H-III), 6.99–7.00 (d, H-6), 7.07–7.11 (t, H-V), 7.48–7.51 (t,

Medicinal Chemistry Research
1-(4ʹ-Acetyl-phenyl)-3-(4-hydroxy-phenyl)-prop-2-en-amide
(Lik8) Yellowish white powder, yield (72%), m.p.
188–189 °C. IR (KBr) νmax in cm−1: 3324 (OH), 2955, 892
(CO–NH), 2927, 2850, 1159 (CH3), 1672 (C=O), 1626
(C=C alkene), 1574 (Ar–C=C), 1088 (Ar–C–H); 1H -NMR
(DMSO-d6, 500 MHz) δ ppm 2.48 (s, H-a), 6.54–6.57 (d,
Hα), 6.76–6.78 (d, H-3, H-5), 6.94–6.95 (d, H-2, H-6),
7.17–7.20 (d, H-β), 7.40–7.42 (d, H-2′, H-6′), 8.390–8.403
(d, H-3′, H-5′), 9.577 (s, OH-4), 5.552 (s, NH). HPLC %
purity = 97.16%.
was neutralized with ammonia hydroxide solution to
precipitate the product, filtered and recrystallized from
methanol.
2-Methyl-1H-benzimidazole (BZ1) Dark brown powder,
yield (82%), m.p. 175 °C. IR (KBr) νmax in cm−1: 3300
(NH), 1626 (C=N), 1420 (C–N), 2900 (Ar–C–H), 1504
(Ar–C=C), 3000 (CH3); 1H-NMR (DMSO-d6, 500 MHz) δ
ppm 12.39 (s, NH-1), 3.34 (s, CH3-2), 6.51–6.55 (d, H-4),
7.42 (t, H-5), 7.68–7.69 (t, H-6), 7.58–7.61 (d, H-7). HPLC
% purity = 98.47%.
1-{4ʹ-[3-(2ʹʹ-methoxy-phenyl)-acryloyl]-phenyl}-3-phenyl-
prop-2en-amide (Lik9) White powder, yield (56%), m.p.
174–176 °C. IR (KBr) νmax in cm−1:3251.76, 763.76
(CO–NH), 2935.46, 2852.54, 1164.92, 1379.01 (CH3),
1706.88 (C=O), 1645.17, 1541.02 (C=C alkene), 1596.95
(Ar–C=C), 3060.82 (Ar–C–H); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 8.32 (s, 1H, NH), 7.45–7.43 (d, 2H, H-2′,
H-6′), 7.33 (d, 2H, 2-H, 6-H), 7.32–7.28 (t, d, 3H, 4-H,
H-3′, H-5′), 7.25–7.24 (t, 2H, 3-H, 5-H), 7.03–7.00 (d, 1H,
H-β), 6.35–6.32 (d, 1H, H-α), 3.05 (s, 1H, H-a). HPLC %
purity = 96.24%.
4-Chloro-2-methyl-1H-benzimidazole (BZ2) Buff powder,
yield (78%), m.p. 211–212 °C. IR (KBr) νmax in cm
−1:3014 (NH), 1618 (C=N), 1456 (C–N), 2896 (Ar–C–H),
1545 (Ar–C=C), 803 (Ar–C–Cl); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 12.35 (s, NH-1), 2.45 (s, CH3-2),
7.09–7.10 (d, H-4), 7.41–7.42 (d, H-5), 7.47 (s, H-7). HPLC
% purity = 98.07%.
4-Nitro-2-methyl-1H-benzimidazole (BZ3) Orange red
powder, yield (72%), m.p. 227–229 °C. IR (KBr) νmax in
1
cm−1: 3436 (NH), 1624 (C=N), 1334, 1512 (NO2) H-
1-{4ʹ-[3-(3ʹʹ-hydroxy-phenyl)-acryloyl]-phenyl}-3-phenyl-
prop-2en-amide (Lik10) Yellow crystals, yield (52%), m.p.
185–186 °C. IR (KBr) νmax in cm−1: 2937.38, 2852.52,
1080.06 (Ar–OCH3) 3253.69 (CO–NH), 1704.96 (C=O),
1647.10, 1539.09 (C=C alkene), 1596.95 (Ar–C=C),
3060.82 (Ar–C–H); 1H-NMR (DMSO-d6, 500 MHz) δ ppm
8.42–8.40 (d, 2H, H-2, H-6), 7.97–7.95 (t, 3H, H-3, H-4, H-
5), 7.51–7.48 (m, 4H, H-2′, H-3′, H-5′, H-6′), 7.11–6.99 (m,
4H, H-3″, H-4″, H-5″, H-6″), 6.80–6.77 (d, 2H, H-β, H-β′),
6.67–6.64 (d, 2H, H-α, H-α′), 5.60 (s, 1H, NH), 3.81 (s, 3H,
OCH3). HPLC % purity = 88.76%.
NMR (DMSO-d6, 500 MHz) δ ppm 12.85 (s, NH-1), 2.54
(s, CH3-2), 7.59–7.61 (d, H-4), 8.02-8.04 (d, H-5), 8.33 (s,
H-7). HPLC % purity = 99.75%.
4-Methyl-2-methyl-1H-benzimidazole
(BZ4) Blackish
brown powder, yield (85%), m.p. 183–185 °C. IR (KBr)
νmax in cm−1: 3433.47 (NH), 1620.21 (C=N), 1440.04
(C–N), 3355.86 (Ar–C–H), 1497.72 (Ar–C=C), 2919.86,
1
2856 (Ar–C–CH3); H-NMR (DMSO-d6, 500 MHz) δ ppm
12.78 (s, NH-1), 2.62 (s, CH3-2), 2.43 (s, CH3-6), 7.05 (d,
H-5), 7.38 (d, H-4), 8.17 (s, H-7). HPLC % purity =
97.97%.
Synthesis of substituted benzimidazole analogs (BZ1–BZ5)
2-Benzyl-1H-benzimidazole (BZ5) Dark brown shine pow-
Substituted o-phenylenediamine (20 mmol) and acetic acid
or phenylacetic acid (30 mmol) were refluxed in presence of
20 cc 4-N HCl for 30–40 min (Scheme 4). The completion
of the reaction is monitored by TLC, the reaction mixture
der, yield (63%), m.p. 191 °C. IR (KBr) νmax in cm−1
:
3048 (NH), 1587 (C=N), 1456 (C–N), 2732 (Ar–C–H),
1
1535 (Ar–C=C), 2835, 2922 (CH3); H-NMR (DMSO-d6,
500 MHz) δ ppm 12.37 (s, NH-1), 4.18 (s, CH2), 7.47–7.48
(d, 4, 7-H), 7.29–7.30 (t, 5,6-H), 7.125–7.138 (m, 2′, 6′-H,
3′, 5′-H), 7.240 (t, 4′-H). HPLC % purity = 99.28%.
O
N
+
Con. HCl,
CH3
H3
C
OH
Synthesis of benzyl sulfanyl benzimidazole analogs
(BS1–BS4)
N
H
R
Acetic Acid
Methyl benzimidazole derivatives
NH2
NH2
Basification with Conc. ammonium hydroxide solution
Substituted o-phenylenediamine (19 mmol), carbon dis-
ulfide (22 mmol), and potassium hydroxide (22 mmol) in
presence of ethanol and water were refluxed for 1 h
(Scheme 5). After completion of the reaction, 20 ml of
warm water and glacial acetic acid were added to precipitate
the intermediate, recrystallized from methanol. To the
N
R
4-Substituted Ortho Phenylene Diamine
N
H
R
O
HO
Benzyl benzimidazole derivatives
Phenyl Acetic Acid
Scheme 4 Synthetic scheme for substituted benzimidazole analogs

Medicinal Chemistry Research
Scheme 5 Synthetic scheme for
substituted benzyl
sulfanylbenzimidazole analogs
NH2
NH2
N
N
CS2, KOH
Cl
SH
S
Reflux at 800C
N
H
R
Dry DMF,
Sodium in
N
H
R
R
4-Substituted Ortho Phenylene Diamine
Intermediate
Substituted benzyl sulfanyl
benzimidazole derivatives
Dry Methanol,
stir at 370C
NH2
Scheme 6 Synthetic scheme for
substituted phenyl-sulfanyl
Potassium Phosphate,
Ethanol, stir at 370C
S
N
Chloroacetic acid,
Conc. HCl
N
methyl benzimidazole analogs
N
H
R
Heat to Reflux
N
H
Cl
R
NH2
R
Substi
benzi
tuted
ph
enyl su
lfan
yl m
ethy
l
4-Substituted
Ortho Phenylene Diamine
mida
zole
deriv
ative
s
SH
Intermediate
intermediate (15 mmol) in 25 ml of dry DMF, a solution of
sodium (15 mmol) in dry methanol (7.5 ml) was added and
stirred for 20 min at room temperature. To this, benzyl
chloride (15 mmol) was added drop-wise, stirring was
continued till the reaction completed. The final product was
precipitated by pouring the reaction mixture into crushed ice
water, filtered, and air-dried.
NMR (DMSO-d6, 500 MHz) δ ppm 2.6 (s, CH3-6), 2.7 (s,
CH3-4′), 4.3 (s, CH2), 7–7.6 (m, H-4′, H-2′, H-6′, H-4, H-5,
H-7). HPLC % purity = 98.32%.
Synthesis of phenyl-sulfanyl methyl benzimidazole analogs
(PS1–PS4)
Substituted o-phenylenediamine (26 mmol), chloroacetic
acid (20.8 mmol), and hydrochloric acid 5 ml were refluxed
until the reaction completed (Scheme 6). The reaction
mixture was poured in water and basified using an ammo-
nium hydroxide solution to precipitate the intermediate. An
equimolar quantity of intermediate, thiol in presence of
potassium phosphate, and ethanol were stirred at room
temperature until reaction completed that was observed by
TLC. The reaction mixture was diluted with water and
extracted with ethyl acetate. The organic layer was dried on
anhydrous sodium sulfate and removed in a vacuum.
2-(Benzyl-sulfanyl)-1H-benzimidazole (BS1) Dark brown
shine crystals, yield (58%), m.p. 192 °C. IR (KBr) νmax in
cm−1: 3300 (NH), 1626 (C=N), 1415 (C–N), 3016
(Ar–C–H), 1524 (Ar–C=C), 2956, 2814 (CH2), 706 (C–S);
1H-NMR (DMSO-d6, 500 MHz) δ ppm 12.549 (s, NH-1),
4.543 (s, CH2), 7.413–7.425 (d, 4, 7, 2′, 6′-H), 7.093–7.107
(t, 5, 6-H), 7.271–7.296 (t, 3′, 5′-H), 7.211–7.235 (t, 4′-H).
HPLC % purity = 94.99%.
2-(Benzyl-sulfanyl-6-chloro)-1H-benzimidazole
(BS2)
Brown shine crystals, yield (67%), m.p. 139–140 °C. IR
(KBr) νmax in cm−1: 3321 (NH), 1622 (C=N), 1423
(C–N), 3045 (Ar–C–H), 1528 (Ar–C=C), 2961, 2853
(CH2), 825 (C–Cl); 1H-NMR (DMSO-d6, 500 MHz) δ ppm
12.732 (s, NH-1), 4.542 (s, CH2), 7.115–7.129 (d, 4-H),
7.410–7.422 (5, 2′, 6′-H), 7.489 (s, 7-H), 7.273–7.298 (t, 3′,
5′-H), 7.227–7.239 (t, 4′-H). HPLC % purity = 99.17%.
2-(Phenyl-sulfanyl-methyl)-1H-benzimidazole (PS1) Dark
brown powder, yield (51%), m.p. 282 °C. IR (KBr) νmax in
cm−1: 3382 (NH), 1625 (C=N), 1455 (C–N), 3056
(Ar–C–H), 1533 (Ar–C=C), 2964, 2853 (CH2), 736 (C–S);
1H-NMR (DMSO-d6, 500 MHz) δ ppm 12.513 (s, NH-1),
4.428 (s, CH2), 7.169 (d, 4, 7-H), 7.120–7.129 (t, 5, 6-H),
7.383–7.396 (d, 1′, 6′-H) 7.465–7.473 (t, 3′, 5′-H),
7.264–7.289 (t, 4′-H). HPLC % purity = 99.50%.
2-(Benzyl-sulfanyl-6-nitro)-1H-benzimidazole (BS3) Brown
powder, yield (77%), m.p. 223–225 °C. IR (KBr) νmax in
cm−1: 3584, 3326 (NH), 1628 (C=N), 1438 (C–N), 3103
(Ar–C–H), 1594 (Ar–C=C), 2924, 2818 (CH2), 698 (C–S),
1517, 1316 (N–O); 1H-NMR (DMSO-d6, 500 MHz) δ ppm
2.534 (s, CH2), 7.257–7.271 (d, H-3′, H-5′), 7.613-7.599 (t,
d, H-4′, H-2′, H-6′), 7.846 (s, H-4), 8.038–8.026 (d, H-5),
8.332 (s, H-7), 13.023 (s, NH). HPLC % purity = 95.92%.
2-(Phenyl-sulfanyl-methyl-6-chloro)-1H-benzimidazole
(PS2) Buff powder, yield (58%), m.p. 155 °C. IR (KBr)
νmax in cm−1: 3388 (NH), 1621 (C=N), 1468 (C–N), 3098
(Ar–C–H), 1582 (Ar–C=C), 2928, 2854 (CH2), 702 (C–S),
1
807 (C–Cl); H-NMR (DMSO-d6, 500 MHz) δ ppm 12.23
(s, NH-1), 4.25 (s, CH2), 7.51–7.52 (d, 4-H), 7.21–7.24 (5,
2′, 6′-H), 7.32 (s, 7-H), 7.15–7.16 (t, 3′, 5′-H), 7.28–7.32 (t,
4′-H). HPLC % purity = 99.62%.
2-(Benzyl-sulfanyl-6-methyl)-1H-benzimidazole
(BS4)
Dark brown powder, _ield (75%), m.p. 232–234 °C. IR
(KBr) νmax in cm−1: 3033.20 (NH), 1625.61 (C=N),
1425.63 (C–N), 1520.94 (Ar–C=C), 2715.74 (CH2),
2970.17, 2762.89 (CH3), 693.35 (C–S), 2604.37 (C–H); 1H-
2-(Phenyl-sulfanyl-methyl-6-nitro)-1H-benzimidazole
(PS3) Light brown powder, yield (54%), m.p. 196 °C. IR
(KBr) νmax in cm−1: 3567 (NH), 1629 (C=N), 1451

Medicinal Chemistry Research
(C–N), 3110 (Ar–C–H), 1593 (Ar–C=C), 2923, 2852
(CH2), 733 (C–S), 1515, 1341 (N–O); H-NMR (DMSO-
d6, 500 MHz) δ ppm 12.883 (s, NH-1), 4.499 (s, CH2),
7.493–7.506 (d, 4-H), 7.603 (d, 5-H), 8.336 (s, 7-H),
8.026–8.040 (d, 2′, 6′-H), 7.352–7.377 (t, 3′, 5′-H),
7.260–7.285 (t, 4′-H).
separated. The purification was performed using Ni-NTA
column chromatography. The Ni-NTA agarose resin was
pre-equilibrated with binding buffer and the supernatant
was incubated with resin for 2–3 h at 4 °C. The supernatant
was passed through column and washed with lysis buffer
containing 20–50-mM imidazole. The protein was eluted in
lysis buffer containing 250-mM imidazole. The protein
fractions were pooled and dialyzed at 4 °C using 10-kDa
cutoff membrane against dialysis buffer (100-mM Tris, pH
8.0, 150-mM NaCl, and 5% glycerol). Protein concentration
was determined by the Bradford assay using BSA as a
standard, and the aliquots were stored at −80 °C in 25%
glycerol [1].
1
2-(Phenyl-sulfanyl-methyl-6-methyl)-1H-benzimidazole
(PS4) Brown powder, yield (62%), m.p. 252–254 °C. IR
(KBr) νmax in cm−1: 3035.37 (NH), 1622.85 (C=N),
1445.57 (C–N), 1586.58 (Ar–C=C), 2858.71 (CH2),
2918.35 (CH3), 739.45 (C–S); 1H-NMR (DMSO-d6,
500 MHz) δ ppm 2.4 (s, CH3), 9.6 (s, NH-1), 4.6 (s, CH2),
7–7.4 (m, 3′, 5′-H, 4′-H, 7-H), 7.6 (d, 5-H), 7.8 (d, 2′, 6′-H).
HPLC % purity = 93.24%.
DENV2 NS2B-NS3 protease inhibition assay
Cytotoxicity studies
The enzymatic inhibition was assayed using a fluorogenic
substrate, Bz-Nle-Lys-Arg-Arg-AMC (Bachem) in a 96-
well plate. Briefly, 50 µl of reaction containing purified
NS2B-NS3 protease in assay buffer (100-mM Tris–HCl pH
9, 1-mM CHAPS and 20% glycerol) with different dilutions
of the analogs (10–500 µM) was pre-incubated for 15 min at
37 °C. Finally, 20 µM of substrate was added to the reaction
mixture and incubated at 37 °C for 1 h. The substrate
hydrolysis was monitored as relative fluorescence units
(RFUs) at an excitation and emission wavelengths of 380
and 460 nm, respectively, in a multimode microplate reader
(Bio-Rad). The assay was performed twice in duplicates,
and IC50 values were calculated using nonlinear regression
models in Graph Pad Prism 5.01 software. Prior to the
inhibitory assays, protease assay without analogs was per-
formed to monitor the activity of NS2B-NS3. Protease
activity was measured as the increase in RFUs. Quercetin
was used as a reference compound.
MTT-Formazan assay (3-(4, 5-dimethylthiozol-2-yl)-3, 5-
diphenyl tetrazolium bromide) was used to determine the
cytotoxicity of all synthesized analogs [11]. MDCK cells
were grown in 96-well plates for 24 h till 85–90% con-
fluency. After 24 h, the media of serially diluted compounds
(tenfold dilution) was added to replace the plates. After 16 h
of incubation, the medium was removed and 100-µL MTT
solution was added to each well and incubated at 37 °C for
4 h. The supernatant layer was removed followed by addi-
tion of 100 µL of DMSO to dissolve formazan crystals.
Absorbance was measured at 540 nm in a microplate reader.
The equation used to normalize the data is:
Cell viabilityð%Þ ¼ ðSample value À blank controlÞ
=ðCell control À blank controlÞ Â 100:
A dose–response curve was plotted via nonlinear
regression (curve fit). The cytotoxic concentration i.e., CC50
was defined as the concentration required to reduce cell
viability by 50%.
Result and discussion
Molecular docking
Expression and purification of DENV2 NS2B-NS3 protease
In NS2B-NS3 protease complex of DENV, the NS3 protein
consists of the catalytic triad of Ser135-His51-Asp75. These
catalytic residues take a form of an oxyanion stabilized by
hydrogen bond formed between the hydroxyl group of
serine with imidazole nitrogen of histidine, which in turn is
hydrogen-bonded to the aspartic carboxyl group [9]. The
catalytic mechanism known to be involved is nucleophilic
attack of the oxygen atom in serine hydroxyl group, which
in turn results into the cleavage of electrophilic carbon in
the peptide bond. Consequently, a tetrahedral intermediate
is formed, and this is stabilized by hydrogen bonds invol-
ving Gly151, Gly153, and Tyr161. Hence, it is believed that
any molecule, which can interact with these residues, can
DENV2 NS2B-NS3 plasmid construct was cloned in
pRSET Vector A. The cloned plasmid was transformed in
to Escherichia coli Bl21(DE3) cells and grown in Luria
Bertani media containing ampicillin (100 µg/ml) at 37 °C
with continuous shaking until the OD600 reached to 0.6.
Expression was induced by 0.6-mM IPTG, and cells were
further grown for 18 h at 18 °C. The cells were pelleted at
8000 rpm for 10 min and resuspended in lysis buffer (100-
mM Tris, pH 8.0, 150-mM NaCl, 10-mM imidazole, and
5% glycerol). The cells were lysed on ice by sonication
using ultrasonic processor. The lysate was subjected to
centrifugation at 10000 rpm for 45 min and supernatant was

Medicinal Chemistry Research
inhibit the function of DENV protease. We have docked
the reported natural phytoconstituents showing anti-
dengue activity, along with our designed analogs in the
reconstructed protein 2FOM considering quercetin as
standard. It was observed that quercetin showed interac-
tions with all the catalytic triad residues via hydrogen
bonding, which is in the agreement with its reported
activity against DENV. The hydroxyl group of the B-ring
of quercetin interacted with Asp75 whereas hydroxyl group
at A-ring interacted with Phe130. The hydroxyl group of
pyrone ring interacted with His51 and Ser135, while car-
bonyl group of the pyrone ring interacted with Ser135. In
case of other phytoconstituents caffeic acid, protocatechic
acid, and coumarin, they showed interactions with two of
the catalytic triad residues along with other residues such as
Tyr150, Gly151, and Gly153. Whereas p-coumeric acid,
showed no interaction with catalytic triad but interacted
with Gly151, that is known as the important residue in
stabilizing the tetrahedral intermediate. Baicalein showed
hydrophobic interaction with His51 and remaining phyto-
constituents did not interact with triad residues but
showed interactions with other important residues Tyr150,
Gly151, and Gly153. The docking interactions of natural
phytoconstituents are shown in Supplementary material
(Supplementary Table S1).
Depending on the molecular interaction results of
phytoconstituents with the catalytic triad, different scaf-
folds were designed. Cinnamic acid analogs (CA1–CA11)
were designed based on caffeic acid and p-coumaric acid
scaffolds. Similarly, chalcone series (C1–C10) was
designed based on quercetin scaffold, which is a flavone,
a cyclized form of hydroxychalcone. The molecular
hybrid approach was used to link caffeic acid and chal-
cone (Lik1–Lik10), both of the scaffolds being antivirals.
Many synthetic molecules based on benzimidazole
scaffold are also reported as well as patented for their
antiviral activity [12]. Based on these observations, dif-
ferent benzimidazole analogs were designed (BZ1–BZ5,
BS1–BS4, and PS1–PS4). We have docked our designed
analogs in NS2B-NS3 protease to get insight of their
binding interactions with the above important residues
(Supplementary Table S2). It was observed that most of
the designed analogs showed similar pattern of interac-
tions comparable to the phytoconstituents discussed
above. In cinnamic acid analogs, the acidic carbonyl
group interacted with carboxylic hydrogen of Ser135
while the substituents (such as chloro, methoxy, nitro,
and hydroxyl) showed interaction with Asp75. The aro-
matic ring had the hydrophobic interaction with His51. In
chalcone analogs, the free amino group interacted with
Ser135. Apart from triad residue they were observed to
interact with other residues such as Gly151, Gly153,
Tyr161, Tyr150, Phe150, Pro132, and Ser131. In case of
Fig. 2 Docked poses of quercetin and caffeic acid (a) and all the
natural phytoconstituents occupying and interacting with the catalytic
triad (His51-Asp75-Ser135) of NS2B-NS3 protease of DENV (b), and
all the designed analogs occupying the catalytic triad of NS2B-NS3
protease of DENV (c)

Medicinal Chemistry Research
Fig. 3 Detailed view of catalytic
triad (a) and molecular
interactions of quercetin (b),
caffeic acid (c), and designed
analog CA2 (d)
linkage of cinnamic acid, the amide bond interacted with
Ser135, Tyr150, and the aromatic ring had the hydro-
phobic interaction with His51. In benzimidazole, benzyl
sulfanyl benzimidazole, and phenyl sulfanyl benzimida-
zole analogs, the heteroatom in the ring showed interac-
tions with Tyr150 and Phe130 while the aromatic ring
showed hydrophobic interaction with His51. The sub-
stituted functional groups on the aromatic ring of all the
above-mentioned rings interacted mainly with Ser135.
The above observations indicated that the designed ana-
logs of different scaffolds showed interactions with the
catalytic triad as well as other important residues. This
might lead to the inhibition of NS2B-NS3 protease of
DENV. The docking interactions of phytoconstituents
and designed analogs are shown in Figs. 2 and 3 and their
2D diagrams are shown in Fig. 4.
Synthesis
The computational studies of 14 natural phytoconstituents
showed interactions with DENV protease residues. Based
on molecular docking results, we have synthesized 44
analogs of different scaffolds viz. eleven cinnamic acid
analogs (CA1–CA11), ten chalcone analogs (C1–C10), ten
molecular hybrids of cinnamic acid and chalcone
(Lik1–Lik10), and different other scaffolds based on ben-
zimidazole (BZ1–BZ5, BS1–BS4, and PS1–PS4). The
structures of all the synthesized molecules were confirmed
after structural characterization by spectral technique such
as IR and 1H NMR spectroscopy. The IR spectra had shown
the respective absorption bands for different functional
groups, along with differentiating bands for aliphatic
alkenes and aromatic double bonds. 1H NMR spectra,

Medicinal Chemistry Research
Fig. 4 2D representation of
molecular interactions: quercetin
(a), caffeic acid (b), and CA2 (c)
further confirmed the structure, based on the predictable
delta values by protons of aromatic and aliphatic carbons of
analogs.
NS2B-NS3 protease Inhibition
The in vitro protease inhibition assay was carried out as a
quantitative evaluation to identify the inhibitory effect of
our synthesized analogs. It is based on the measurement of
fluorescence reflected by the cleavage of substrate due to
the enzymatic activity of protease. Throughout the assay,
protease enzyme concentration and the substrate con-
centration were maintained at 50-nM and 20 µM, respec-
tively. The inhibitory activity was measured by recording
the fluorescence as RFU and every concentration was
recorded in duplicate. It was compared with quercetin as
standard drug by observing its effect at different con-
centration levels i.e., 10, 30, 50, 100, and 500 µM. The
synthesized analogs at different concentrations between 10
and 500 µM were also measured in similar way. Experi-
mentally used concentrations and measured RFU values
Anti-dengue activity
Cytotoxicity studies
All the synthesized analogs were tested for cytotoxicity
using the MTT-Formazan assay [11]. The concentration
of analogs causing 50% reduction in cell viability (CC50)
was measured. The outcome of this study indicated that
the synthesized analogs had no serious effect on MDCK
cells (CC50 values are shown in Supplementary Table
S3). Further, the anti-dengue activity of these nontoxic
analogs was evaluated using the NS2B-NS3 protease
inhibition assay.

Medicinal Chemistry Research
Table 2 Result of molecular testing against NS2B-NS3 protease
inhibition assay
group was observed to form the hydrogen bond with the
catalytic as well as the important amino-acid residues.
Moreover, the aromatic ring of all designed analogs
showed the hydrophobic interaction with triad residue as
expected and, it was observed that incorporating more
than three hydrophobic rings did not show additive
effect. Analogs with various substituents such as elec-
tron-withdrawing, electron-donating were considered.
Substitution of chloro, methyl, and methoxy functional
groups at ortho and meta position was observed to have
additive effect for protease inhibition.
Two analogs from benzimidazole scaffold (BS2 and
BZ1) and one from cinnamic acid scaffold (CA2) showed
more potent protease inhibition compared to standard
quercetin, while CA1 showed comparable potency,
which was in agreement with the docking results. The
comprehensive examination revealed that two chloro
substituted analogs (BS2 and CA2) and two unsub-
stituted analogs (BZ1 and CA1) showed more potency
than the standard quercetin. The docking results indi-
cated that CA2 interacted with all the amino acids in the
catalytic triad by forming direct hydrogen bond. The free
carboxyl group showed interaction with Ser135, chloro
group showed hydrogen bond with Asp75 and Gly153,
whereas the aromatic ring showed hydrophobic interac-
tion with His51 (π–π). Similar pattern of interactions was
observed with the standard. In case of unsubstituted
cinnamic acid (CA1), the free carboxyl group interacted
with Ser135. Similar pattern of interactions was observed
in case of benzimidazole scaffold too. The other analogs,
which showed comparable inhibitory activity to that of
standard, had different substituents like methyl, methoxy,
hydroxyl, and nitro groups (PS2, BZ4, C9, CA7, C10,
and C8) but all these interacted similarly with the cata-
lytic triad by forming the hydrogen bond with the
Ser135. Along with Ser135, some also interacted with
Tyr161 and Gly151.
Other analogs, which interacted with the catalytic triad
residues and having IC50 value comparable to standard, are
CA3, CA6, CA9, CA10, C7, BS1, BS4, and PS4 but these
were not considered for discussion due to their poor
experimental r2 values. The remaining analogs, which were
active at very high concentrations in the protease inhibition
assay, did not show interaction with the catalytic triad,
which might be the reason for their inactivity.
It was also noted that when the aromatic ring was
attached/fused with additional heterocyclic ring it reflected a
higher activity, as observed with benzimidazole analogs
showing better IC50 values. Whereas, increasing the chain
length for synergistic activity as in case of linkage analogs,
did not show any additional benefit. It was pragmatic that,
the analogs, which interacted with all the triad residues,
showed better IC50 values.
Sr. no.
Compound code
IC50 (µM)
Std quercetin
CA1
CA2
CA7
CA11
C4*
28.73
35.07
27.33
57.68
217.8
113.3
97.52
47.91
73.36
25.84
43.45
22.19
136.2
42.77
1
2
3
4
5
6
C8
7
C9
8
C10
9
BZ1
10
11
12
13
BZ3
BS2
BS3
PS2
*p = 0.3457
were used to determine the inhibitory concentration (IC50)
of the individual drug. The standard quercetin IC50 value
was 28.73 µM, in case of cinnamic acid analogs CA2 was
showing the lower IC50 value than the standard 27.33 µM
whereas CA1 and CA7 were showing the comparable IC50
value to that of the standard 35.07 and 57.68 µM, respec-
tively. In chalcone analogs, C9 was showing the compar-
able IC50 value with 47.91 µM, C10 and C9 were shown
IC50 values of 73.36 and 97.52 µM. In case of benzimida-
zole analogs, BZ1 and BS2 were showing the lower IC50
value than the standard 25.84 and 22.19 µM, respectively,
and BZ4 and PS2 showing the IC50 values of 43.45 and
42.77 µM, respectively. The evaluated IC50 values are
shown in Table 2, and Fig. 5 shows the nonlinear regression
curve fit representation. The analogs whose IC50 values
were lower and comparable with standard with the r2 above
0.9 were considered for further discussion. The recorded
fluorescence reading in duplicate as RFU and biological
replicates (n ≥ 2) was considered for analysis. For assays,
p < 0.05 (Mann–Whitney U test and Student’s t test) was
considered statistically significant and the analogs showing
p > 0.3 shown in the Table 2 by indicating with * mark.
The designed analogs were developed based on the
important structural features of reported natural phyto-
constituents, and the activity results were in agreement
with the observations. Although, the scaffolds tend to
exhibit varied degree of NS2B-NS3 protease inhibition
but some similar pattern of interactions was observed
with respect to the scaffolds. For e.g., in case of cinnamic
acid analogs, the free carboxyl group was interacting
with the catalytic triad along with the other crucial
amino-acid residues. In chalcone analogs, the free amino

Medicinal Chemistry Research
Fig. 5 Plot of % DENV2 NS2B/
NS3pro inhibition vs. log
concentration of standard
quercetin (a), CA2 (b), BZ1 (c),
BS2 (d), and overlay of
quercetin and CA2 (e)
Conclusion
cinnamic acid analogs showed better IC50 value and more
potency in protease inhibition compared to that of stan-
dard quercetin [13–17].
The severity of dengue infection is increasing day by day
and the unavailability of specific medication is making
the scenario more unmanageable. Many scientists across
the globe are continuously contributing their knowledge
to give a breakthrough in the treatment of DENV. Our
studies in the lead optimization focused on the natural
phytoconstituents, designing the analogs by considering
the important structural features, and develop a promis-
ing NS2B-NS3 protease inhibitor. In computational stu-
dies, we found most of the designed analogs showed
interaction with the triad residues of the enzyme, in a
similar manner as that of reported phytoconstituents.
Among all the designed analogs, benzimidazole and
Acknowledgements MAK acknowledges Indian Council of Medical
Research (ICMR), New Delhi (58/27/2007-BMS) for computational
facility and Department of Science and Technology (DST-FIST), New
Delhi (SR/FST/College-264/2015(C)) for spectroscopic facility at Prin
K M Kundnani College of Pharmacy, Mumbai. LRG acknowledges
National NMR Facility provided by Tata Institute of Fundamental
Research (TIFR), Colaba, Mumbai.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.

Medicinal Chemistry Research
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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