Design, synthesis and evaluation of chalcones as H1N1 Neuraminidase inhibitors

Article abstract of DOI:10.1007/s00044-017-2124-2

A series of chalcone derivatives (1a–2i) were designed based on isoliquiritigenin (the most active natural chalcone non-competitive neuraminidase (NA) inhibitor). Molecular modeling studies revealed that isoliquiritigenin and its designed analogs occupied

Full text of DOI:10.1007/s00044-017-2124-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2124-2
ORIGINAL RESEARCH
Design, synthesis and evaluation of chalcones as H1N1
Neuraminidase inhibitors
Anand S. Chintakrindi1 Devanshi J. Gohil2 Sweta T. Kothari2
●
●
●
Abhay S. Chowdhary2 Meena A. Kanyalkar
1
●
Received: 30 August 2017 / Accepted: 2 December 2017
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract A series of chalcone derivatives (1a–2i) were
designed based on isoliquiritigenin (the most active natural
chalcone non-competitive neuraminidase (NA) inhibitor).
Molecular modeling studies revealed that isoliquiritigenin
and its designed analogs occupied 430-loop cavity of NA
and interacted favorably with catalytic site residues. The
favorable derivatives were synthesized and evaluated for
cytotoxicity and for inhibition of cytopathic effect by H1N1
virus. The inhibitory effect was further quantified by hea-
magglutinition (HA) assay, H1N1-NA inhibition, and
kinetics of inhibition. The HA assay showed compound 1e
has the lowest EC50 of 1.71 nM. In contrast, the H1N1-NA
inhibition assay showed compound 1f has the best activity
with the IC50 of 3.58 μM. Enzyme kinetic study suggested
that the inhibition mechanism of compounds 1f and 2f was
non-competitive.
Introduction
Influenza is a viral infection caused by influenza virus,
which has resulted in many human fatalities since antiquity.
The influenza viruses are RNA viruses of Orthomyxoviridae
family (Chintakrindi et al. 2012) and are of three types—A,
B, and C. Type A influenza viruses are further classified
into subtypes according to the combinations of various virus
surface antigenic glycoproteins, haemagglutinin (HA), and
neuraminidase (Sialidase, NA) (WHO 2014). According to
World Health Organization (WHO) among many subtypes
of influenza A viruses, currently influenza A (H1N1), A
(H3N2), A (H5N1), and A (H9N2) subtypes are circulating
among humans and pose an increasing pandemic threat
(WHO 2015a, b). HA binds to host cell sialic acid an
endogenous ligand (Gong et al. 2007) leading to the
attachment and subsequent penetration of virus into the
target cell (Colman et al. 1989). NA is responsible for
cleaving the terminal sialic acid from the receptor in order
to release the newly formed virions from the infected cell
(Oxford et al. 2002). The highly conserved active site of NA
and its role in influenza virus replication makes it an
interesting target for the development of newer inhibitors.
The current commercially available drugs, NA inhibitors
such as oseltamivir (OMV) and zanamivir (ZMV) have
gained remarkable success in the treatment of influenza.
Although ZMV is highly effective, its use is limited due to
respiratory distress associated with its inhalational delivery
(Williamson and Pegram 2000). Being orally active, OMV
overcomes this limitation, but the production cost is high
owing to its tedious synthesis from the expensive starting
material, shikimic acid (Magano 2009). Furthermore,
influenza virus has high potential to develop resistance
against both these drugs. Thus, it becomes necessary to
search for novel inhibitors with an alternate mechanism of
●
●
●
Keywords Chalcones H1N1 Neuraminidase Mutually
●
●
non-competitive inhibitor Docking Enzyme kinetics
* Meena A. Kanyalkar
meenatul@gmail.com
1
Department of Pharmaceutical Chemistry, Prin. K. M. Kundnani
College of Pharmacy, Cuffe Parade, Mumbai 400005, India
2
Department of Virology, Haffkine Institute for Training, Research
and Testing, Parel, Mumbai 400012, India

Med Chem Res
inhibition such as the design of inhibitors that bind to an
alternate site of NA enzyme. Two such sites are 150-loop
and 430-loop that play an important role in the function of
NA (Russell et al. 2006).
Noncompetitive inhibitors bind to allosteric site of
enzyme and alter the conformation of enzyme thereby
inhibiting the binding of natural substrates or endogenous
ligands resulting in loss of enzyme’s catalytic activity. This
alternate mechanism of inhibition circumvents the problem
of resistance to the inhibitors binding to enzyme’s catalytic
site. The competitive NA inhibitors like OMV and ZMV are
transition state analogs of sialic acid (SA) (Kim et al. 1999).
The reports indicate that development of NA inhibitors so
far has revolved around theory of transition state analogs.
Interestingly, several chalcones from plant sources have
been reported as non-competitive inhibitors of H1N1-NA
(Dao et al. 2011; Dao et al. 2010; Nguyen et al. 2011; Ryu
et al. 2010). However, these chalcones display weak anti-
influenza activity. Hence, analogs of these natural inhibitors
could be designed using rational drug design approach to
identify the potential neuraminidase inhibitors which may
have better anti-influenza activity.
Chalcones which consist of two aromatic rings connected
by an α,β-unsaturated carbonyl group are important pre-
cursors for synthetic manipulations. Chalcones can bind
effectively to many kinds of receptor, and exhibit diverse
biological activities, such as anti-cancer, anti-infective, anti-
inflammatory, anti-oxidant, and anti-angiogenic effects
(Singh et al. 2014). Thus based on reported chalcones, we
designed a series of analogs of chalcones [nine 4′-ami-
nochalcones (1a–1i) and nine 3′-hydroxy chalcones
(2a–2i)] and studied their binding mode using molecular
docking studies. Furthermore, we synthesized fifteen ana-
logs and evaluated ten of these analogs in vitro for cyto-
toxicity, inhibition of cytopathic effect (CPE) by H1N1
virus, H1N1-NA inhibition. Moreover, we explored the
binding mode of the compounds to NA by kinetic
experiments.
Fig. 1 Catalytic site of H1N1-NA comprising of 430-loop, 150-loop,
and SA cavity
Glu277, Arg292, Asn294, Asn347, Arg371, and Tyr406).
The SA cavity comprises of group of residues involved in
catalytic activity of NA. ZMV docked in SA cavity of
catalytic site. The initial validation studies with the docking
protocol could satisfactorily reproduce (RMSD 0.5236) the
binding conformation of the ligand ZMV in the H1N1-NA
crystal structure (PDB ID: 3B7E) (Xu et al. 2008). During
catalysis of NA cleavage of α-ketosidic linkage between
terminal host cell sialic acid and HA occurs. Thus, we
docked sialic acid in SA cavity of NA to investigate resi-
dues critical to its binding and the catalytic activity. The
observation focuses on the carboxylate group of sialic acid,
which shows characteristic bi-dentate interaction with
Arg371 in addition to the H-bond interactions with Arg118.
The terminal hydroxyl groups of glycerol side-chain of
sialic acid form H-bond with the side-chains of Glu276 and
Arg292. The hydroxyl group forms H-bond with Asp151
whereas carbonyl of acetamido side-chain forms H-bond
with Arg152.
We have used blind docking technique for docking of
isoliquiritigenin (Fig. 2), the most active natural chalcone
(Ryu et al. 2010) in NA to gain insights into the non-
competitive inhibition pattern.
Results and discussion
Computational studies
Blind docking is a technique in which the ligands are
docked to entire protein surface and useful when prior
knowledge of binding region is missing, often the case with
noncompetitive inhibitors. Since this chalcone is a non-
competitive inhibitor, it was anticipated that it would dock
into a cavity other than SA cavity. As expected, this com-
pound did not bind to SA cavity. However, the binding was
confined to specific cavity of catalytic site namely, the 430-
loop cavity (Fig. 3a). Although this binding mode may not
be a classical allosteric binding observed in noncompetitive
The catalytic site of NA is divided into three parts as illu-
strated in Fig. 1.
The left region comprise of 150-loop cavity (Gly147,
Thr148, Val149, Lys150, Asp151, and Arg152), top region
encompass of 430-loop cavity (Asn325, Gly348, Ser369,
Ser370, Trp403, Ile427, Gly429, Gln430, Pro431, Lys432,
Glu433, Asn435, Thr436, Ile437, Trp438, and Thr439) and
central region consist of SA cavity (Arg118, Glu119,
Asn146, Asp151, Arg152, Tyr178, Ile222, Glu227, Glu276,

Med Chem Res
inhibition, but can be termed as mutually noncompetitive as
it is not occupying the true catalytic site domain. To inspect
this mutually noncompetitive binding, isoliquiritigenin was
docked in NA along with SA. In this case, isoliquiritigenin
does not overlap with SA and occupies the same region i.e.,
430-loop cavity in catalytic site in the same manner when
docked in absence of SA (Fig. 3b).
This observation implies that isoliquiritigenin might have
an alternate binding mode that is distinct from known drugs
and sialic acid. The ring A of isoliquiritigenin interacts with
hydrophobic side chain of Trp403, Ile427, Pro431, and
Lys432. The hydroxy group on ring A forms H-bond with
backbone carbonyl oxy (–C=O) of Gly429. The carbonyl
group forms H-bond with the backbone (–NH) of Arg371.
The ring B has has hydrophobic and electrostatic interaction
with side-chain of Arg371. The hydroxy groups on ring B
form H-bonds with side-chain of Asn325 and backbone
carbonyl oxy (–C=O) of Gly348. The non-bonded inter-
action energies of isoliquiritigenin within the catalytic site
were calculated to identify the energetically favorable cat-
alytic site residues, Fig. 4.
Figure 4 highlight the catalytic site residues which have
favorable non-bonded interaction with isoliquiritigenin,
noticeably with Asp151, Glu277, Ser370, and Arg371. These
residues are critical for NA’s catalytic activity since site-
directed mutagenesis studies of NA have emphasized the
importance of Asn146, Arg152, Trp178, Asp198, Glu276,
Glu277, Ser370, Arg371, and Tyr 406 in NA’s catalytic
activity (Kobasa et al. 1997; Lentz et al. 1987). Furthermore,
docking studies with SA (vide supra) suggests that Asp151
helps in binding of SA in the catalytic cavity of active site.
These results suggest that by the virtue of above H-bonding
and non-bonded interactions, isoliquiritigenin presumably
could hinder with NA’s catalytic activity and thus inhibit NA
albeit with an alternate binding mode in NA active site.
Various analogs (Table 1) of backbone of iso-
liquiritigenin i.e., chalcone were designed by varying sub-
stituent on both phenyl rings and docked in H1N1-NA
active site in order to investigate the effect of various
functional group substitution on the binding pattern. These
substituents impart varied electronic and lipophilic character
Fig. 2 Isoliquiritigenin
Fig. 3 Docked pose of isoliquiritigenin (stick model) in a absence and b presence of sialic acid (SA) (ball and stick model). The inlet shows
binding interactions of isoliquiritigenin (ball and stick model) with catalytic site residues (stick model) of H1N1-NA
Fig. 4 Histogram of non-
bonded interaction energies (van
der Waal (vdW), electrostatic
and total) of isoliquiritigenin
with the catalytic site residues

Med Chem Res
Table 1 Designed analogs of chalcone (1-(substitutedphenyl)-3-
(substitutedphenyl)-prop-2-en-1-one)
B-ring with Lys432 is responsible for their reverse orien-
tation with respect to isoliquiritigenin. The hydrophobic
interaction of B-ring of compounds 2a–2e, and 2g–2i with
Trp403 and Pro430 keeps them in the hydrophobic hole.
This dual orientation of the designed analogs could be
attributable to higher flexibility of enone chain of chalcones.
In addition, all docked analogs, either have hydrophobic/
electrostatic interaction with Arg371 side-chain or form H-
bond backbone of Arg371. The comprehensive analysis of
these non-bonded interactions of designed analogs with
active site residues is presented in Table 2.
Sr. no.
Compound
R1
R2
R3
R4
R5
1
1a
1b
1c
1d
1e
1f
Cl
H
H
H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
2
H
Cl
H
H
These observations imply that the designed molecules
display additional interactions, which may help in tighter
binding and could have better activity.
3
H
H
Cl
H
4
OCH3
H
H
H
H
5
OCH3
H
H
H
6
H
OCH3
H
H
Chemistry
7
1g
1h
1i
NO2
H
H
H
8
NO2
H
H
H
The target compounds 1a–2i were synthesized via Clasein-
Schmidt reaction (Zerong 2010) of equimolar quantities of
substituted benzaldehydes and substituted acetophenones
under the catalysis of NaOH/ethanol. Briefly, NaOH solu-
tion was added drop wise to mixture of substituted ben-
zaldehyde and substituted acetophenones in ethanol. The
temperature of the reaction was maintained below 10 °C
throughout the addition of NaOH solution. The temperature
and rate of addition of NaOH solution affects the yield and
appearance of chalcone. In fact for compounds 1i and 2i,
which were prepared from 4-nitrobenzaldehyde, the tem-
perature was maintained below 5 °C, as increase in tem-
perature resulted in decreased yield and charring of the
target chalcone. The structures of the synthesized com-
9
H
NO2
H
H
10
11
12
13
14
15
16
17
18
2a
2b
2c
2d
2e
2f
Cl
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
Cl
H
H
H
H
Cl
H
OCH3
H
H
H
H
OCH3
H
H
H
H
OCH3
H
H
2g
2h
2i
NO2
H
H
H
NO2
H
H
H
H
NO2
H
to the designed analogs that may improve their pharmaco-
dynamic and pharmacokinetic profile.
pounds were characterized by nuclear magnetic resonance
13
spectroscopy (1H NMR and
C NMR), infrared spectro-
1
Detailed analysis of binding interactions of above docked
analogs with the catalytic site residues demonstrates that all
compounds occupy 430-loop cavity of the catalytic site
similar to the parent chalcone, isoliquiritigenin. The docked
pose of compounds 1f, 1i, and 2f is equivalent to iso-
liquiritigenin in which A-ring projects into the hydrophobic
hole of 430-loop cavity. Compounds 1f and 2f have p-
methoxy substituent on A-ring, which interacts, with
hydrophobic side-chains of Trp403, Ile427, Pro431, and
Lys432. The amino group of 1f and hydroxyl group of 2f on
B-ring form H-bond with Ser369 and Asn325 respectively.
For compound 1i, the p-nitro group on A-ring has electro-
static interaction with Trp403 and amino group on B-ring
form H-bond with Ser369. However, although all other
compounds (1a–1e, 1g, 1h, 2a–2e, and 2g–2i) lie on axis
similar to isoliquiritigenin, they are oriented in opposite
direction, so that A-ring of these compounds occupies the
region on the right side of hydrophobic hole in the 430-loop
cavity. For compounds 1a–1e, 1g, 1h, either the H-bond of
amino group with Glu433 or hydrophobic interaction of
scopy (IR) (figure not shown). In the H NMR spectra of
chalcones olefinic protons H-α and H-β appeared as doub-
lets in the range of δ 7.58–7.87 and δ 7.69–8.13, respec-
tively. Trans-stereochemistry of enone moiety of chalcones
was confirmed by coupling constant of vinyl hydrogen
(15.5–17 Hz). The IR spectra of chalcones showed carbonyl
absorption in the range of 1627–1661 cm-1 and an olefinic
C=C in the range 1570–1609 cm-1.
In vitro evaluation
The synthesized compounds were tested for cytotoxicity
using the MTT-Formazan assay (Mosmann 1983). The
cytotoxicity effect of these compounds was determined by
measuring the concentration that caused 50% reduction in
cell viability (CC50). Compounds 1c, 2a, 2b, and 2h with
CC50 values of 51.58, 27.85, 38.15, and 6.53 µM respec-
tively, showed cytotoxicity. Thus, these compounds were
not selected for further evaluation. However, Compound 1c
was elected for H1N1-NA inhibition assay to check effect

Med Chem Res
Table 2 Detailed analysis of binding interactions of docked analogs with catalytic site residues
Sr. no.
Compound
Pro326
Ser369
Arg371b
Trp403b
Ile427b
Arg428
Gly429b
Pro431b
Lys432b
Glu433
1
1a
1b
1c
1d
1e
1fa
1g
1h
1ia
2a
2b
2c
2d
2e
2fa
2g
2h
2i
–
–
–
–
–
–
H
–
–
H
–
–
–
–
–
–
–
–
–
H, π–a
π–π
π–σ
–
–
–
H
H
H
H
H
–
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–σ
π–σ
π–σ
π–σ
π–a
π–a
–
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
π–a
H
H
H
H
H
–
2
–
π–a
π–π
π–σ
3
–
π–+, π–a
π–a
π–π
π–σ
4
–
π–π
π–σ
5
–
π–a
π–π
π–σ
–
6
–
π–+, π–a
C–A, π–a
π–+, π–a
π–+, π–a
H, π–+, π–a
H, π–+, π–a
H, π–+, π–a
π–a
–
π–a
–
7
–
π–π
π–σ
–
H
H
–
H
H
–
8
π–a
–
π–π
H, π–a
π–a
–
9
π–+, π–a
π–π, π–σ
π–π, π–σ
π–π, π–σ
–
C–A
–
10
11
12
13
14
15
16
17
18
–
π–σ
H
H
H
–
H
H
H
–
–
π–a
–
–
π–σ
–
π–a
π–a
π–a
π–a
–
π–σ
–
H, π–a
π–π, π–σ
–
π–a
–
H
–
H
–
π–+, π–a
C–A, π–a
π–+, π–a
π–+, π–a
π–a
–
–
H, π–σ
H, π–σ
H, π–σ
–
–
H
H
H
–
–
–
–
–
π–π, π–σ
–
–
π–a
H hydrogen bond, C–A cation–anion interaction (electrostatic), π–+ pi–cation interaction (electrostatic), π–π pi–pi interaction (hydrophobic), π–a
pi–alkyl interaction (hydrophobic), π–σ pi–sigma interaction (hydrophobic)
a
Compounds having docked orientation similar to isoliquiritgenin
b
Catalytic site residues interacting with isoliquiritigenin
of cytotoxic compound on NA activity. The anti-influenza
activity of non-cytotoxic compounds was further evaluated
for reduction of cytopathic effect (CPE) by H1N1 virus in
Madin-Darby canine kidney (MDCK) cells. OMV was used
as a standard for competitive inhibition while quercetin
(QR) the most potent reported natural non-competitive
inhibitor (Jeong et al. 2009) was used as a standard for non-
competitive inhibition.
Figure 5 shows the effect of compound 1e and OMV on
CPE by H1N1 virus in MDCK cells. There were plenty of
gaps with distorted morphology in virus-infected (Fig. 5b)
but not in mock-infected cells (Fig. 5a). Treatment of cells
with compound 1e or OMV, however, reduced the number
of gaps and cell morphology was restored (Fig. 5c, d). The
reduction of CPE was quantified by comparing the HA titer
of virus in cells treated with compounds vs. the HA titer in
untreated cells. The antiviral effect of these compounds was
ascertained by measuring the concentration that caused 50%
reduction in log2HA titer (EC50) determined using regres-
sion analysis, Table 3. Hemagglutinition assay illustrated
that our compounds effectively suppress the viral titer.
Compound 1e exhibited highest activity with EC50 value of
1.71 nM, better than the commercially used drug, OMV
(EC50 = 7.1 nM). The ratio of CC50/EC50 (selectivity index)
was used to analyze the compound’s selectivity. The high
selectivity index values of these compounds indicated that
they are effective to inhibit the virus growth without
affecting the living host cells.
We also assessed H1N1-NA inhibitory of the com-
pounds. The IC50 values of the evaluated analogs ranged
from 3.58 to 12.36 µM against H1N1-NA (Table 3). The
NA inhibitory activity of these compounds was ascertained
by measuring the concentration required to inhibit 50% of
the enzyme activity (IC50) (Fig. 6).
The antiviral evaluation and NA inhibition studies indi-
cate that the tested compounds are effective in inhibiting
CPE of H1N1 virus and overall viral replication than inhi-
biting the neuraminidase enzyme per se. These findings
suggest that our compounds may have an additional unex-
plored mode of action in controlling virus replication. A
comprehensive examination of NA inhibition data suggests
that among the tested aminochalcones, para-substitution is
favored on A-ring over meta-substitution as compounds 1f
and 1i display higher NA inhibition than their correspond-
ing meta-isomers. With this in view, we evaluated only
para-substituted 3′-hydroxychalcones (compounds 2c, 2f,
and 2i) for CPE inhibition and NA inhibition. Moreover,
methoxy substituent on A-ring is preferential over chloro
and nitro substituent since compounds 1e, 1f, and 2f exhibit
better NA inhibitory activity than rest of the compounds.

Med Chem Res
Fig. 5 The antiviral effects of compound 1e and OMV in cytopathic
effect reduction assay. a Mock-inoculated MDCK cells show normal
morphology and no gaps in the cells. b Virus-infected MDCK cells
without compound 1e. There are many gaps and abnormal
morphology. c Virus-infected MDCK cells with 1e. There are
remarkably reduced gaps and morphology restored. d Virus-infected
MDCK cells with oseltamivir
Compounds 1e, 1f, and 2f have high SI value also indi-
cating selectivity.
Table 3 In vitro evaluation of tested compounds
Sr. no. Compound
CC50 (µM) EC50 (nM) SI
IC50 (µM)
We have performed enzyme kinetics study on few
selected synthesized analogs (compounds 1f and 2f, the
most active based on NA inhibition among hydroxy and
aminochalcone respectively) to confirm the non-competitive
inhibition of designed analogs as demonstrated by compu-
tational studies while OMV and QR were used as standard
for competitive and non-competitive inhibition. As expec-
ted, OMV, a transition state analog of SA shows competi-
tive inhibition.
The Lineweaver-Burk plot (Fig. 7) of 1/V vs. 1/[S] for
OMV (Ki = 2.8 × 10−4 µM) has same y-axis intercept along
with higher value of x-axis intercept indicating its compe-
titive inhibition. However, in case of QR (Ki = 8.69 µM),
compound 1f (Ki = 5.58 µM) and 2f (Ki = 8.69 µM), the y-
axis intercept value increases while x-axis intercepts does
not change signifying their noncompetitive inhibition.
1
1a
170.6
150.4
51.6
2.36
2.36
CT
72288
63729
–
6.64
5.56
10.25
2
1b
3
1c
4
1e
180.4
199.9
127.9
112.6
116.0
292.4
169.8
253.8
713.4
1.71
2.76
5.26
4.62
15.64
8.62
12.65
84.46
7.1
105497 4.10
5
1f
72428
24316
24372
7417
3.58
8.26
7.86
12.36
5.25
8.65
7.75
6
1h
7
1i
8
2c
9
2f
33921
13423
3007
10
11
12
2i
Quercetin
Oseltamivir
100478 6.48 × 10−4
All compounds were examined in a set of duplicated experiment; EC50
values represent the concentration that resulted in 50 % log2HA viral
titer reduction; SI selectivity index was generated by the ratio of CC50
and EC50; IC50 values of compounds represent the concentration that
caused 50% enzyme activity loss, CT cytotoxic

Med Chem Res
Fig. 6 Effects of oseltamivir
(OMV), compounds 1a–1c,
1e–1f, 1h–1i, 2c, 2f, 2i and
quercetin (QR) on H1N1-NA for
the hydrolysis of substrate
Fig. 7 Lineweaver–Burk plots for the inhibition of Oseltamivir (OMV), Quercetin (QR), compounds 1f and 2f on H1N1-NA for the hydrolysis of
substrate in the presence of increasing concentrations of inhibitors for lines from bottom to top
These findings indicate that synthesized compounds are
non-competitive inhibitors of H1N1-NA and show better
activity than quercetin, the natural cyclic analog. A sum-
mary of the inhibition constant (Ki) values for compounds
showed that these results agree with those of IC50.
Conclusion
The emergence of drug resistance for the current competi-
tive inhibitors of NA such as OMV necessitates the search
for an alternative approach in the treatment of influenza. In

Med Chem Res
this view, we selected isoliquiritgenin the most active nat-
ural chalcone NA non-competitive inhibitor and docked it
in H1N1-NA catalytic site using blind docking approach.
The docking studies revealed that docked isoliquiritigenin
430-loop cavity, which was distinct from binding of sialic
acid in H1N1-NA suggesting non-classical non-competitive
(mutually non-competitive) inhibition wherein the active
site is large enough to accommodate inhibitor and substrate
simultaneously. Interaction energy analysis of docked iso-
liquiritigenin in H1N1-NA catalytic site showed that it had
favorable interactions with residues involved in catalytic
activity of NA and by virtue of these interactions; this
natural chalcone could hinder with NA’s catalytic activity
and inhibit NA albeit with an alternate binding mode in NA
active site. Eighteen analogs of isoliquiritigenin i.e. chal-
cone were designed by varying substituent on phenyl rings
and docked in H1N1-NA. The docking studies demon-
strated that all compounds occupied 430-loop cavity of the
catalytic site similar to their parent natural chalcone but
displayed interactions with additional catalytic site residues,
which may help in tighter binding. Fifteen of these designed
analogs were then synthesized and characterized by IR, 1H-
MDCK cells, obtained from National Center for Disease
Control were maintained in Minimal Essential Medium
(MEM, Gibco, by Life Technologies) supplemented with
10% fetal bovine serum (Gibco, by Life Technologies),
100 U/mL Penicillin and 0.5 mg/mL Streptomycin (Hi-
Media Laboratories, India). The Influenza A/Pune /2009
(H1N1) virus was obtained from National Institute of Vir-
ology and propagated in MDCK cells in the presence of 10
µg/mL trypsin.
Computational studies
Computational studies were carried out with the modeling
package Discovery Studio v 3.1 (DS 3.1) (Accelrys Inc. San
Diego) running on a Red Hat Enterprise Linux WS work-
station. Docking studies were carried out with “Libdock”
(Diller and Merz 2001) module of DS 3.1. Interaction
energy for docked ligand was calculated with CHARMm
forcefield using “Simulation” module of DS 3.1.
Preparation of receptor/ ligand system for docking and
validation of the docking protocol
1
NMR and 13C-NMR spectroscopy. The IR, H-NMR, and
13C-NMR spectra confirmed the structures of designed
analogs. Cytotoxicity studies showed that no tested com-
pounds show any significant cytotoxicity except for 1c. The
CPE inhibition studies portrayed that the compounds
effectively suppressed the CPE of H1N1 virus in a manner
similar to standard drug OMV. Heamagglutinition assay
illustrated that our compounds efficiently reduced the viral
titer. Compound 1e exhibited highest activity better than the
commercially used drug, OMV. The H1N1-NA inhibition
studies demonstrated that compound 1f exhibited highest
activity but lower than OMV. The antiviral and NA inhi-
bition studies indicated that the tested compounds are
effective in inhibiting CPE of H1N1 virus and overall viral
replication than inhibiting the neuraminidase enzyme
per se. suggesting our compounds may have an additional
unexplored mode of action in controlling virus replication.
The enzyme kinetics study on representative compounds 1f
and 2f, the most active among 4′-aminochalcones and 3′-
hydroxychalcones respectively confirmed the non-
competitive inhibition indicated by docking studies.
The crystal structure of the enzyme H1N1-NA in complex
with ZMV was taken from the protein data bank (3B7E)
(Xu et al. 2008). The enzyme is a dimer; however for
docking studies only the monomer unit was used. All water
molecules were deleted and hydrogens were added. The
system was refined using the CHARMm forcefield
(Momany and Rone 1992) to a gradient of 0.1 kcal/mol/Å.
The docking protocol was validated by reproduction of
binding pose of ZMV in the H1N1-NA.
Different scaffolds used as ligands were also energy
minimized using the “Smart Minimzer” method in energy
minimization with the CHARMm forcefield to a gradient of
0.01 kcal/mol/Å.
Docking studies using Libdock
The docking parameters were optimized to reproduce the X-
ray pose of ZMV in the PDB structure. The final optimized
docking parameters of Libdock mainly consist of the fol-
lowing: (1) The “number of hotspots” set to 10,000; (2) the
docking tolerance set to 0.25; (3) the “Apolar SASA Cutoff”
to 20, (4) the “Polar SASA Cutoff” to 20 and (5) the
“conformation method” set to “BEST”; other parameters
were kept on default settings. The binding site was defined
as entire protein surface.
Experimental section
Materials
All chemicals, reagents used for synthesis were purchased
from SD Fine Chemicals Pvt. Ltd, India. OMV and Quer-
cetin (QR) were obtained from Clearsynth Labs Pvt. Ltd,
India and Sigma-Aldrich Co. LLC, USA respectively.
Synthesis
The series of analogs of chalcone were synthesized by
Clasein-Schmidt condensation using Scheme 1. Equimolar

Med Chem Res
Scheme 1 Synthesis of substituted chalcones (1-(substitutedphenyl)-3-(substitutedphenyl)prop-2-en-1-ones)
quantities (7.1 mmol) of substituted acetophenone and
substituted benzaldehyde were dissolved in absolute etha-
nol. Sodium hydroxide solution (20%, 5 mL) was added to
this solution at 10 °C and the resulting mixture was stirred
at room temperature (temperature increased gradually). The
precipitate obtained was neutralized with conc. HCl and
then filtered and recrystallized using ethanol. All the com-
pounds were characterized by spectroscopic methods (Syam
et al. 2012).
(C, C-1″), 133.83 (C, C-3″), 131.37 (CH, C-2′, C-6′),
130.61 (CH, C-5″), 129.53 (CH, C-4″), 127.64 (CH, C-6″),
127.54 (CH, C-2″), 125.27 (C, C-1′), 124.13 (CH, C-2),
112.81 (CH, C-3′, C-5′).
(E)-1-(4-aminophenyl)-3-(4-chlorophenyl)prop-2-en-1-one
(1c)
Yellow solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 4-chlorobenzaldehye and 4-
aminoacetophenone) yield (80%), m.p. 157–159 °C. IR
(KBr) νmax 3459, 3341 (NH2), 1630 (C=O), 1602 (C=C),
(E)-1-(4-aminophenyl)-3-(2-chlorophenyl)prop-2-en-1-one
(1a)
1
1175 (Ar–Cl); H-NMR (DMSO-d6, 500 MHz) δ ppm 7.93
Yellow solid (ethanol) (the title compound was prepared
according to Scheme 1 using 2-chlorobenzaldehye and 4-
aminoacetophenone), yield (78%), m.p. 106–109 °C. IR
(KBr) νmax 3306, 3231 (NH2), 1643 (C=O), 1608 (C=C),
1178 (Ar–Cl); 1H-NMR (dimethylsulfoxide (DMSO)-d6,
500 MHz) δ ppm 8.16 (d, 1H, H-6″), 7.97–7.88 (m, 4H, H-
3, J = 15.5 Hz, H-2′, H-6′, H-2, J = 15.5 Hz), 7.57 (d, 1H,
H-3″), 7.48–7.45 (m, 2H, H-4″, H-5″), 6.68 (d, 2H, H-3′, H-
5′), 6.17 (s, 2H, NH2); 13C-NMR (DMSO-d6, 500 MHz) δ
ppm 185.61 (C=O, C-1), 154.29 (C, C-4′), 136.17 (CH, C-
3), 134.02 (C, C-2″), 132.82 (C, C-1″), 131.43 (CH, C-3″),
131.36 (CH, C-2′, C-6′), 129.98 (CH, C-4″), 128.43 (CH,
C-6″), 127.70 (CH, C-5″), 125.33 (C, C-1′), 124.98 (CH, C-
2), 112.87 (CH, C-3′, C-5′).
(d, 2H, H-2″, H-6″), 7.88–7.85 (m, 3H, H-2′, H-6′, H-3, J =
16 Hz), 7.60 (d, 1H, H-2, J = 16 Hz), 7.51 (d, 2H, H-3″, H-
5″), 6.66 (d, 2H, H-3′, H-5′), 6.08 (s, 2H, NH2); 13C-NMR
(DMSO-d6, 500 MHz) δ ppm 185.77 (C=O, C-1), 154.00
(C, C-4′), 139.96 (CH, C-3), 134.43 (C, C-4″), 134.21 (C,
C-1″), 131.24 (CH, C-2′, C-6′), 130.23 (CH, C-3″, C-5″),
128.90 (CH, C-2″, C-6″), 125.30 (C, C-1′), 123.30 (CH, C-
2), 112.81 (CH, C-3′, C-5′).
(E)-1-(4-aminophenyl)-3-(3-methoxyphenyl)prop-2-en-1-
one (1e)
Yellow solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 3-methoxybenzaldehye and 4-
aminoacetophenone) yield (84%), m.p. 145–146 °C. IR
(KBr) νmax 3450, 3354 (NH2), 1644 (C=O), 1606 (C=C),
(E)-1-(4-aminophenyl)-3-(3-chlorophenyl)prop-2-en-1-one
(1b)
1
1259, 1043 (C–O–C); H-NMR (DMSO-d6, 500 MHz) δ
Yellow solid (ethanol) (the title compound was prepared
according to Scheme 1 using 3-chlorobenzaldehye and 4-
aminoacetophenone), yield (72%), m.p. 121–123 °C. IR
(KBr) νmax 3420, 3331 (NH2), 1627 (C=O), 1580 (C=C),
ppm 7.94 (d, 2H, H-2′, H-6′), 7.84 (d, 1H, H-3, J =
15.5 Hz), 7.60 (d, 1H, H-2, J = 15.5 Hz), 7.42 (s, 1H, H-2″),
7.39–7.35 (m, 2H, H-5″, H-6″), 7.01 (d, 1H, H-4″), 6.66 (d,
2H, H-3′, H-5′), 6.06 (s, 2H, NH2), 3.85 (s, 3H, OCH3);
13C-NMR (DMSO-d6, 500 MHz) δ ppm 189.02 (C=O, C-
1), 162.75 (C, C-3″), 157.03 (C, C-4′), 144.51 (CH, C-3),
139.71 (C, C-1″), 134.30 (CH, C-2′, C-6′), 132.94 (CH, C-
5″), 128.45 (CH, C-6″), 125.83 (C, C-1′), 124.37 (CH, C-2),
119.16 (CH, C-4″), 116.15 (CH, C-2″), 115.87 (CH, C-3′,
C-5′), 58.37 (OCH3).
1
1176 (Ar–Cl); H-NMR (DMSO-d6, 500 MHz) δ ppm 8.00
(s, 1H, H-2″), 7.96–7.91 (m, 3H, H-2′, H-6′, H-3, J =
15.5 Hz), 7.77 (t, 1H, H-5″), 7.60 (d, 1H, H-2, J = 15.5 Hz),
7.48 (d, 2H, H-4″, H-6″), 6.66 (d, 2H, H-3′, H-5′), 6.07 (s,
2H, NH2); 13C-NMR (DMSO-d6, 500 MHz) δ ppm 185.74
(C=O, C-1), 154.09 (C, C-4′), 139.74 (CH, C-3), 137.53

Med Chem Res
(E)-1-(4-aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-
(E)-1-(3-hydroxyphenyl)-3-(2-chlorophenyl)prop-2-en-1-
one (1f)
one (2a)
Yellow solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 4-methoxybenzaldehye and 4-
aminoacetophenone) yield (88%), m.p. 108–111 °C. IR
(KBr) νmax 3457, 3331 (NH2), 1630 (C=O), 1599 (C=C),
Yellow solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 2-chlorobenzaldehye and 3-
hydroxyacetophenone) yield (88%), m.p. 156–158 °C. IR
(KBr) νmax 3341 (OH), 1652 (C=O), 1590 (C=C), 1187
1
1
1259, 1025 (C–O–C); H-NMR (DMSO-d6, 500 MHz) δ
(Ar–Cl); H-NMR (DMSO-d6, 500 MHz) δ ppm 9.85 (s,
ppm 7.91 (d, 2H, H-2′, H-6′), 7.79 (d, 2H, H-2″, H-6″), 7.71
(d, 1H, H-3, J = 15.5 Hz), 7.60 (d, 1H, H-2, J = 15.5 Hz),
7.02 (d, 2H, H-3″, H-5″), 6.66 (d, 2H, H-3′, H-5′), 6.01 (s,
2H, NH2), 3.86 (s, 3H, OCH3); 13C-NMR (DMSO-d6,
500 MHz) δ ppm 185.96 (C=O, C-1), 160.89 (C, C-4″),
153.72 (C, C-4′), 141.39 (CH, C-3), 131.87 (CH, C-2′, C-
6′), 131.02 (CH, C-2″, C-6″), 127.86 (C, C-1′), 125.64 (C,
C-1″), 119.98 (CH, C-2), 114.39 (CH, C-3′, C-5′), 112.78
(CH, C-3″, C-5″), 55.36 (OCH3).
1H, OH), 8.23 (d, 1H, H-6″), 8.04 (1H, H-3, J = 15.5 Hz),
7.94 (d, 1H, H-2, J = 15.5 Hz), 7.68 (d, 1H, H-6′), 7.60 (d,
1H, H-3″), 7.52–7.46 (m, 3H, H-4″, H-2′, H-5″), 7.41 (t, 1H,
H-5′), 7.10 (d, 1H, H-4′); 13C-NMR (DMSO-d6, 500 MHz)
δ ppm 188.99 (C=O, C-1), 157.85 (C, C-3′), 138.76 (CH,
C-3), 138.44 (CH, C-5′), 134.40 (C, C-2″), 132.34 (C,
C-1″), 131.94 (CH, C-6′), 130.04 (CH, C-3″), 129.94 (CH,
C-4″), 128.62 (CH, C-6″), 127.69 (CH, C-5″), 124.98
(C, C-1′), 120.65 (CH, C-4′), 119.79 (CH, C-2), 114.76
(CH, C-2′).
(E)-1-(4-aminophenyl)-3-(3-nitrophenyl)prop-2-en-1-one
(1h)
(E)-1-(3-hydroxyphenyl)-3-(3-chlorophenyl)prop-2-en-1-
one (2b)
Orange solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 3-nitrobenzaldehye and 4-
aminoacetophenone) yield (68%), m.p. 165–168 °C. IR
(KBr) νmax 3425, 3335 (NH2), 1632 (C=O), 1609 (C=C),
Yellow solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 3-chlorobenzaldehye and 3-
hydroxyacetophenone) yield (84%), m.p. 108–110 °C. IR
(KBr) νmax 3405 (OH), 1651 (C=O), 1583 (C=C), 1185
1
1530, 1344 (NO2); H-NMR (DMSO-d6, 500 MHz) δ ppm
1
8.69 (s, 1H, H-2″), 8.30 (d, 1H, H-4″), 8.24 (d, 1H, H-6″),
8.06 (d, 1H, H-3, J = 16 Hz), 7.98 (d, 2H, H-2′, H-6′),
7.76–7.71 (m, 2H, H-5″, H-1, J = 16 Hz), 6.67 (d, 2H, H-3′,
H-5′), 6.17 (s, 2H, NH2); 13C-NMR (DMSO-d6, 500 MHz)
δ ppm 185.64 (C=O, C-1), 154.29 (C, C-4′), 148.47 (C, C-
3″), 138.92 (CH, C-3), 137.15 (C, C-1″), 134.78 (CH, C-
6″), 131.43 (CH, C-2′, C-6′), 130.35 (CH, C-5″), 125.42 (C,
C-1′), 125.04 (CH, C-2), 124.12 (CH, C-4″), 122.67 (CH,
C-2″), 112.81 (CH, C-3′, C-5′).
(Ar–Cl); H-NMR (DMSO-d6, 500 MHz) δ ppm 9.82 (s,
1H, OH), 8.10 (s, 1H, H-2″), 7.98 (1H, H-3, J = 15.5 Hz),
7.84 (d, 1H, H-6″), 7.72–7.68 (m, 2H, H-2, J = 15.5 Hz, H-
6′), 7.54–7.48 (m, 3H, H-4″, H-2′, H-5″), 7.40 (t, 1H, H-5′),
7.10 (d, 1H, H-4′); 13C-NMR (DMSO-d6, 500 MHz) δ ppm
189.06 (C=O, C-1), 157.83 (C, C-3′), 142.08 (CH, C-3),
138.85 (CH, C-5′), 136.99 (C, C-1″), 133.89 (C, C-3″),
130.67 (CH, C-6′), 130.10 (CH, C-5″), 129.88 (CH, C-4″),
128.02 (CH, C-6″), 127.79 (CH, C-2″), 123.81 (C, C-1′),
120.52 (CH, C-4′), 119.79 (CH, C-2), 114.83 (CH, C-2′).
(E)-1-(4-aminophenyl)-3-(4-nitrophenyl)prop-2-en-1-one
(1i)
(E)-1-(3-hydroxyphenyl)-3-(4-chlorophenyl)prop-2-en-1-
one (2c)
Orange solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 4-nitrobenzaldehye and 4-
aminoacetophenone) yield (60%), m.p. 182–184 °C. IR
(KBr) νmax 3459, 3338 (NH2), 1646 (C=O), 1615 (C=C),
Yellow solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 4-chlorobenzaldehye and 3-
hydroxyacetophenone) yield (78%), m.p. 148–152 °C. IR
(KBr) νmax 3322 (OH), 1645 (C=O), 1589 (C=C), 1171
1
1545, 1344 (NO2); H-NMR (DMSO-d6, 500 MHz) δ ppm
1
8.28 (d, 2H, H-3″, H-5″), 8.12 (d, 2H, H-2″, H-6″), 8.04
(1H, H-3, J = 16 Hz), 7.98 (d, 2H, H-2′, H-6′), 7.70 (d, 1H,
H-1, J = 16 Hz), 6.67 (d, 2H, H-3′, H-5′), 6.16 (s, 2H,
NH2); 13C-NMR (DMSO-d6, 500 MHz) δ ppm 190.07
(C=O, C-1), 152.35 (C, C-4′), 147.80 (C, C-4″), 141.70
(CH, C-3), 139.11 (C, C-1″), 134.71 (CH, C-2′, C-6′),
131.65 (C, C-1′), 129.63 (CH, C-2″, C-6″), 127.69 (CH, C-
2), 123.93 (CH, C-3′, C-5′), 114.55 (CH, C-3″, C-5″).
(Ar–Cl); H-NMR (DMSO-d6, 500 MHz) δ ppm 9.82 (s,
1H, OH), 7.96–7.89 (m, 3H, H-2″, H-6″, H-3, J = 16 Hz),
7.73 (d, 1H, H-2, J = 16 Hz), 7.66 (d, 1H, H-6′), 7.55 (d,
2H, H-3″, H-5″), 7.48 (t, 1H, H-2′), 7.40 (t, 1H, H-5′), 7.09
(d, 1H, H-4′); 13C-NMR (DMSO-d6, 500 MHz) δ ppm
189.00 (C=O, C-1), 157.82 (C, C-3′), 142.37 (CH, C-3),
138.99 (CH, C-5′), 135.13 (C, C-4″), 133.70 (C, C-1″),
130.54 (CH, C-2″, C-6″), 129.88 (CH, C-6′), 128.96 (CH,

Med Chem Res
C-3″, C-5″), 123.02 (C, C-1′), 120.45 (CH, C-4′), 119.70
(CH, C-2), 114.76 (CH, C-2′).
H-5″), 3.85 (s, 3H, OCH3); 13C-NMR (DMSO-d6,
500 MHz) δ ppm 189.05 (C=O, C-1), 161.39 (C, C-4″),
157.76 (C, C-3′), 143.88 (CH, C-3), 139.39 (CH, C-5′),
130.79 (CH, C-2″, C-6″), 129.82 (CH, C-6′), 127.38 (C, C-
1″), 120.11 (C, C-1′), 119.79 (CH, C-4′), 119.48 (CH, C-2),
114.64 (CH, C-2′), 114.48 (CH, C-3″, C-5″), 55.40 (OCH3).
(E)-1-(3-hydroxyphenyl)-3-(2-methoxyphenyl)prop-2-en-1-
one (2d)
Yellow solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 2-methoxybenzaldehye and 3-
hydroxyacetophenone) yield (73%), m.p. 122–124 °C. IR
(KBr) νmax 3398 (OH), 1658 (C=O), 1579 (C=C), 1254,
1047 (C–O–C); 1H-NMR (DMSO-d6, 500 MHz) δ ppm
9.81 (s, 1H, OH), 8.02 (d, 1H, H-3, J = 16 Hz), 7.97 (d, 1H,
H-6″), 7.82 (d, 1H, H-2, J = 16 Hz), 7.60 (d, 1H, H-6′),
7.49–7.45 (m, 2H, H-5″, H-2′), 7.39 (t, 1H, H-5′), 7.14 (d,
1H, H-4″), 7.08–7.04 (m, 2H, H-3″, H-4′), 3.92 (s, 3H,
OCH3); 13C-NMR (DMSO-d6, 500 MHz) δ ppm 189.34
(C=O, C-1), 158.33 (C, C-2″), 157.76 (C, C-3′), 139.23
(CH, C-3), 138.51 (CH, C-5′), 132.31 (CH, C-6″), 129.91
(CH, C-6′), 128.71 (CH, C-4″), 122.99 (C, C-1″), 122.17
(C, C-1′), 120.78 (CH, C-4′), 120.23 (CH, C-2), 119.48
(CH, C-5″), 114.61 (CH, C-2′), 111.86 (CH, C-3″), 55.78
(OCH3).
(E)-1-(3-hydroxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-on
(2h)
Orange solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 3-nitrobenzaldehye and 3-
hydroxyacetophenone) yield (72%), m.p. 160–166 °C. IR
(KBr) νmax 3406 (OH), 1658 (C=O), 1580 (C=C), 1526,
1
1350 (NO2); H-NMR (DMSO-d6, 500 MHz) δ ppm 9.85
(s, 1H, OH), 8.79 (s, 1H, H-2″), 8.37 (d, 1H, H-4″), 8.30 (d,
1H, H-6″), 8.12 (d, 1H, H-3, J = 16 Hz), 7.86 (d, 1H, H-2, J
= 16 Hz), 7.77 (t, 1H, H-5″), 7.72 (d, 1H, H-6′), 7.53 (s, 1H,
H-2′), 7.42 (t, 1H, H-5′), 7.11 (d, 1H, H-4′); 13C-NMR
(DMSO-d6, 500 MHz) δ ppm 189.03 (C=O, C-1), 157.82
(C, C-3′), 148.46 (C, C-3″), 141.26 (CH, C-3), 138.70 (C,
C-1″), 136.65 (CH, C-5′), 135.06 (CH, C-6″), 130.35 (CH,
C-6′), 129.91 (CH, C-5″), 125.01 (CH, C-4″), 124.66 (CH,
C-2″), 123.08 (C, C-1′), 120.65 (CH, C-4′), 119.89 (CH, C-
2), 114.83 (CH, C-2′).
(E)-1-(3-hydroxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-
one (2e)
Yellow solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 3-methoxybenzaldehye and 3-
hydroxyacetophenone) yield (81%), m.p. 157–160 °C. IR
(KBr) νmax 3383 (OH), 1656 (C=O), 1570 (C=C), 1258,
1051 (C–O–C); 1H-NMR (DMSO-d6, 500 MHz) δ ppm
9.82 (s, 1H, OH), 7.88 (d, 1H, H-3, J = 15.5 Hz), 7.71 (d,
1H, H-2, J = 15.5 Hz), 7.66 (d, 1H, H-6′), 7.49 (m, 2H, H-
2″, H-2′), 7.45 (d, 1H, H-6″), 7.41–7.37 (m, 2H, H-5″, H-5′),
7.09–7.04 (m, 2H, H-4″, H-4′), 3.85 (s, 3H, OCH3); 13C-
NMR (DMSO-d6, 500 MHz) δ ppm 189.24 (C=O, C-1),
159.72 (C, C-3″), 157.79 (C, C-3′), 143.88 (CH, C-3),
139.05 (CH, C-5′), 136.14 (C, C-1″), 130.01 (CH, C-6′),
129.91 (CH, C-5″), 122.58 (C, C-1′), 121.69 (CH, C-4′),
120.37 (CH, C-2), 119.70 (CH, C-6″), 116.79 (CH, C-2′),
114.71 (CH, C-4″), 113.78 (CH, C-2″), 55.36 (OCH3).
(E)-1-(3-hydroxyphenyl)-3-(4-nitrophenyl)prop-2-en-1-one
(2i)
Orange solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 4-nitrobenzaldehye and 3-
hydroxyacetophenone) yield (68%), m.p. 177–180 °C. IR
(KBr) νmax 3363 (OH), 1661 (C=O), 1587 (C=C), 1516,
1
1340 (NO2); H-NMR (DMSO-d6, 500 MHz) δ ppm 9.86
(s, 1H, OH), 8.31 (d, 1H, H-3″, H-5″), 8.20 (d, 1H, H-2″, H-
6″), 8.09 (d, 1H, H-3, J = 15.5 Hz), 7.82 (d, 1H, H-2, J =
15.5 Hz), 7.70 (d, 1H, H-6′), 7.51 (s, 1H, H-2′), 7.42 (t, 1H,
H-5′), 7.12 (d, 1H, H-4′); 13C-NMR (DMSO-d6, 500 MHz)
δ ppm 188.99 (C=O, C-1), 157.85 (C, C-3′), 148.08 (C, C-
4″), 141.22 (CH, C-3), 140.23 (C, C-1″), 138.64 (CH, C-5′),
129.97 (CH, C-6′), 129.88 (CH, C-2″, C-6″), 126.28 (C, C-
1′), 123.97 (CH, C-3″, C-5″), 120.74 (CH, C-4′), 119.86
(CH, C-2), 114.80 (CH, C-2′).
(E)-1-(3-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-
one (2f)
In vitro evaluation
Yellow solid, (ethanol) (the title compound was prepared
according to Scheme 1 using 4-methoxybenzaldehye and 3-
hydroxyacetophenone) yield (90%), m.p. 142–144 °C. IR
(KBr) νmax 3366 (OH), 1650 (C=O), 1600 (C=C), 1257,
1058 (C–O–C); 1H-NMR (DMSO-d6, 500 MHz) δ ppm
9.78 (s, 1H, OH), 7.86 (d, 2H, H-2″, H-6″), 7.75–7.68 (m,
2H, H-3, H-2, J = 17 Hz), 7.63 (d, 1H, H-6′), 7.46 (s, 1H,
H-2′), 7.38 (t, 1H, H-5′), 7.07–7.03 (m, 3H, H-4′, H-3″,
Cytotoxicity
MDCK cells were grown in 96-well plates for 24 h. The
plates were replaced with media containing serially diluted
compounds. After 16 h of incubation, the medium was
removed and 100 µL MTT (3-(4,5-dimethylthiozol-2-yl)-
3,5-dipheryl tetrazolium bromide), solution was added to

Med Chem Res
each well and incubated at 37 °C for 4 h. After removal of
supernatant, 100 µL of DMSO was added to dissolve for-
mazan crystals. Absorbance was measured at 540 nm in a
microplate reader.(Jeong et al. 2009)
plate. H1N1 virus was diluted five-fold with the assay
buffer. To the plate, 25 μL of the diluted virus was mixed
with the compounds and incubated at 37 °C for 20 min. The
substrate was diluted at 1:1000 in assay buffer immediately
before use. Then 10 μL of the diluted substrate were added
to each well. The reaction mixtures were incubated at room
temperature for 15 min and then activated by adding 60 μL
of accelerator. The chemiluminescent signal was quantified
immediately by microplate reader (An et al. 2009). A 50%
inhibitory concentration (IC50), relative to the activity in
positive control i.e., the reaction mixture well containing
virus but no test compound, was determined to measure
inhibitory activity of test compounds.
CPE reduction assay
The standard H1N1 virus (100 µL) was inoculated onto near
confluent MDCK cell monolayers for 1 h in 24-well plates
at 37 °C under 5% CO2 atmosphere. The solution was
removed and the cells replaced with MEM containing 10
µg/mL trypsin and candidate compounds at different con-
centration. The cultures were incubated for 3–4 days at 37 °
C under 5% CO2 atmosphere until the cells in the infected,
untreated control well showed complete viral CPE. All
compounds were assayed for virus inhibition in duplicate
(Jeong et al. 2009). After 3–4 days, the supernatant from
each well was removed and tested for reduction in hea-
magglutinition (HA) titer of virus as compared to HA titer
of virus in untreated control well (virus control). The HA
titer was determined by means of HA assay.
Kinetics assay
To 96-well microtiter plate, 25 μL of OMV or QR or
compounds 1f and 2f (two conc. bracketing IC50) was pre-
incubated with 25 μL diluted H1N1 virus at 37 °C for
20 min. This was followed by addition of substrate (6.25,
12.5, 25, 50, 100 μM). Kinetic characterization for the
hydrolysis of substrate catalyzed by H1N1-NA was carried
out by measuring by chemiluminescent signal of hydrolysis
product. The parallel control experiment was performed
without compounds in the mixture.
Heamagglutinition assay
Serial twofold dilutions of supernatant of infected cells
(100 μL) were prepared using PBS in a 96-well plate. A 50
μL of 0.75% guinea pig red blood cells was added to each
well. After 30 min incubation at 4 °C followed by 30 min
incubation at room temperature, Heamagglutinition (uni-
form reddish color across the well) and precipitation of red
blood cells (dot in the center of well) was observed. The
virus’s HA titer is a simple number of the highest dilution
factor that produced heamagglutinition (Hosseini et al.
2012). The %log2HA titer (Serkedjieva and Velcheva 2003)
reduction for each compound was calculated using Eq. 1
Acknowledgements M.A.K. thanks Indian Council of Medical
Research (ICMR), New Delhi for funding computational facilities at
Prin. K. M. Kundnani College of Pharmacy through Adhoc research
scheme (58/27/2007-BMS). A.S.C. also thanks ICMR, New Delhi for
Senior Research Fellowship (58/27/2007-BMS). D.J.G., S.T.K., and
A.S.C. acknowledge National Center for Disease Control (NCDC),
New Delhi, for providing Madin-Darby canine kidney (MDCK) cell
line and thank National Institute of Virology for providing seasonal
Influenza A (H1N1) Pune Isolate for neuraminidase inhibition assay.
Compliance with ethical standards
%log2HA titre reduction
ꢂ
ꢀ
ꢁꢃ
ð1Þ
log2HA titre of virus in sample
Conflict of interest The authors declare that they have no competing
interest.
¼
1À
Â100
log2HA titre of virus in virus control
The antiviral effect of the compounds was ascertained by
measuring concentration required for 50% log2HA titer
reduction (EC50).
References
An J, Lee DC, Law AH, Yang CL, Poon LL, Lau AS, Jones SJ (2009)
A novel small-molecule inhibitor of the avian influenza H5N1
virus determined through computational screening against the
neuraminidase. J Med Chem 52:2667–2672
Neuraminidase inhibition assay
Chintakrindi A, D’Souza C, Kanyalkar M (2012) Rational develop-
ment of neuraminidase inhibitor as novel anti-flu drug. Mini Rev
Med Chem 12:1273–1281
Colman PM, Tulip WR, Varghese JN, Tulloch PA, Baker AT, Laver
WG, Air GM, Webster RG (1989) Three-dimensional structures
of influenza virus neuraminidase-antibody complexes. Philos
Trans R Soc Lond B Biol Sci 323:511–518
Dao TT, Nguyen PH, Lee HS, Kim E, Park J, Lim SI, Oh WK (2011)
Chalcones as novel influenza A (H1N1) neuraminidase inhibitors
from Glycyrrhiza inflata. Bioorg Med Chem Lett 21:294–298
Neuraminidase inhibitory activity was determined by the
NA-Star influenza neuraminidase inhibitor resistance
detection kit (Applied Biosystems). The assay was per-
formed as per the NA-Star assay kit protocol. Briefly, 25 μL
of synthesized compounds (conc. ranging from 1000 to 1
µM) or QR (conc. ranging from 1000 to 1 µM) or OMV
(conc. ranging from 1000 to 1 nM) at two times the desired
concentration was added in duplicate to a 96-well microtiter

Med Chem Res
Dao TT, Tung BT, Nguyen PH, Thuong PT, Yoo SS, Kim EH, Kim
SK, Oh WK (2010) C-methylated flavonoids from Cleistocalyx
operculatus and their inhibitory effects on novel influenza A
(H1N1) neuraminidase. J Nat Prod 73:1636–1642
Diller DJ, Merz Jr. KM (2001) High throughput docking for library
design and library prioritization. Proteins 43:113–124
Gong J, Xu W, Zhang J (2007) Structure and functions of influenza
virus neuraminidase. Curr Med Chem 14:113–122
Hosseini S, Amini E, Kheiri M, Mehrbod P, Shahidi M, Zabihi E
(2012) Anti-influenza activity of a novel polyoxometalate deri-
vative (POM-4960). Int J Mol Cell Med 1:21–29
Oxford JS, Novelli P, Sefton A, Lambkin R (2002) New millennium
antivirals against pandemic and epidemic influenza: the neur-
aminidase inhibitors. Antivir Chem Chemother 13:205–217
Russell RJ, Haire LF, Stevens DJ, Collins PJ, Lin YP, Blackburn GM,
Hay AJ, Gamblin SJ, Skehel JJ (2006) The structure of H5N1
avian influenza neuraminidase suggests new opportunities for
drug design. Nature 443:45–49
Ryu YB, Kim JH, Park SJ, Chang JS, Rho MC, Bae KH, Park KH,
Lee WS (2010) Inhibition of neuraminidase activity by poly-
phenol compounds isolated from the roots of Glycyrrhiza ura-
lensis. Bioorg Med Chem Lett 20:971–974
Jeong HJ, Ryu YB, Park SJ, Kim JH, Kwon HJ, Park KH, Rho MC,
Lee WS (2009) Neuraminidase inhibitory activities of flavonols
isolated from Rhodiola rosea roots and their in vitro anti-
influenza viral activities. Bioorg Med Chem 17:6816–6823
Kim CU, Chen X, Mendel DB (1999) Neuraminidase inhibitors as
anti-influenza virus agents. Antivir Chem Chemother 10:141–154
Kobasa D, Rodgers ME, Wells K, Kawaoka Y (1997) Neuraminidase
hemadsorption activity, conserved in avian influenza A viruses,
Serkedjieva J, Velcheva M (2003) In vitro anti-influenza virus activity
of the pavine alkaloid (–)-thalimonine isolated from Thalictrum
simplex L. Antivir Chem Chemother 14:75–80
Singh P, Anand A, Kumar V (2014) Recent developments in biolo-
gical activities of chalcones: a mini review. Eur J Med Chem
85:758–777
Syam S, Abdelwahab SI, Al-Mamary MA, Mohan S (2012) Synthesis
of chalcones with anticancer activities. Molecules 17:6179–6195
World Health Organisation (2015a) Avian influenza in humans. http://
www.who.int/influenza/human_animal_interface/avian_
influenza/en/. Accessed 31 Oct 2015
does not influence viral replication in ducks.
71:6706–6713
J Virol
Lentz MR, Webster RG, Air GM (1987) Site-directed mutation of the
active site of influenza neuraminidase and implications for the
catalytic mechanism. Biochemistry 26:5351–5358
World Health Organisation (2014) Influenza (Seasonal)—Fact sheet
N°211.
http://www.who.int/mediacentre/factsheets/fs211/en/.
Magano J (2009) Synthetic approaches to the neuraminidase inhibitors
zanamivir (Relenza) and oseltamivir phosphate (Tamiflu) for the
treatment of influenza. Chem Rev 109:4398–4438
Accessed 31 Oct 2015
World Health Organisation (2015b) Swine influenza in humans. http://
www.who.int/influenza/human_animal_interface/swine_
influenza/en/. Accessed 31 Oct 2015
Williamson JC, Pegram PS (2000) Respiratory distress associated with
Zanamivir. N Engl J Med 342:661–662
Momany FA, Rone R (1992) Validation of the general purpose
QUANTA®3.2/CHARMm® force field.
13:888–900
J
Comput Chem
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63
Xu X, Zhu X, Dwek RA, Stevens J, Wilson IA (2008) Structural
characterization of the 1918 influenza virus H1N1 neuraminidase.
J Virol 82:10493–10501
Nguyen TNA, Dao TT, Tung BT, Choi H, Kim E, Park J, Lim S-IL,
Oh WK (2011) Influenza A (H1N1) neuraminidase inhibitors
from Vitis amurensis. Food Chem 124:437–443
Zerong W (2010) Claisen-Schmidt condensation. In: Zerong W (ed)
Comprehensive organic name reactions and reagents. Wiley,
Hoboken, NJ, pp 660–664