In quest of small-molecules as potent non-competitive inhibitors against influenza

Article abstract of DOI:10.1016/j.bioorg.2021.105139

A series of scaffolds namely aurones, 3-indolinones, 4-quinolones and cinnamic acid-piperazine hybrids, was designed, synthesized and investigated in vitro against influenza A/H1N1pdm09 virus. Designed molecules adopted different binding mode i.e., in 430-cavity of neuraminidase, unlike sialic acid and oseltamivir in molecular docking studies. All molecules reduced the viral titer and exhibited non-cytotoxicity along with cryo-protective property towards MDCK cells. Molecules (Z)-2-(3′-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2f), (Z)-2-(4′-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2g) and 2-(2′-Methoxy-phenyl)-1H-quinolin-4-one (3a) were the most interesting molecules identified in this research, endowed with robust potencies showing low-nanomolar EC₅₀ values of 4.0 nM, 6.7 nM and 4.9 nM, respectively, compared to reference competitive and non-competitive inhibitors: oseltamivir (EC₅₀ = 12.7 nM) and quercetin (EC₅₀ = 0.56 μM), respectively. Besides, 2f, 2g and 3a exhibited good neuraminidase inhibitory activity in sub-micromolar range (IC₅₀ = 0.52 μM, 3.5 μM, 1.3 μM respectively). Moreover, these molecules were determined as non-competitive inhibitors similar to reference non-competitive inhibitor quercetin unlike reference competitive inhibitor oseltamivir in kinetics studies.

Full text of DOI:10.1016/j.bioorg.2021.105139

Bioorganic Chemistry 114 (2021) 105139
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
In quest of small-molecules as potent non-competitive inhibitors
against influenza
Khushboo Malbaria, Priyanka Sahab, Mamta Chawla-Sarkarb, Shanta Duttab, Swita Raia,
,
*
Mamata Joshic, Meena Kanyalkara
a Department of Pharmaceutical Chemistry, Prin K M Kundnani College of Pharmacy, Cuffe Parade, Mumbai 400005, India
b Division of Virology, ICMR-National Institute of Cholera and Enteric Diseases (ICMR-NICED), Beleghata, Kolkata 700010, India
c National Facility for High Field NMR, Tata Institute of Fundamental Research (TIFR), Colaba, Mumbai 400005, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of scaffolds namely aurones, 3-indolinones, 4-quinolones and cinnamic acid-piperazine hybrids, was
designed, synthesized and investigated in vitro against influenza A/H1N1pdm09 virus. Designed molecules
adopted different binding mode i.e., in 430-cavity of neuraminidase, unlike sialic acid and oseltamivir in mo-
lecular docking studies. All molecules reduced the viral titer and exhibited non-cytotoxicity along with cryo-
protective property towards MDCK cells. Molecules (Z)-2-(3′-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2f),
(Z)-2-(4′-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2g) and 2-(2′-Methoxy-phenyl)-1H-quinolin-4-one (3a)
were the most interesting molecules identified in this research, endowed with robust potencies showing low-
nanomolar EC50 values of 4.0 nM, 6.7 nM and 4.9 nM, respectively, compared to reference competitive and
non-competitive inhibitors: oseltamivir (EC50 = 12.7 nM) and quercetin (EC50 = 0.56 µM), respectively. Besides,
2f, 2g and 3a exhibited good neuraminidase inhibitory activity in sub-micromolar range (IC50 = 0.52 µM, 3.5
µM, 1.3 µM respectively). Moreover, these molecules were determined as non-competitive inhibitors similar to
reference non-competitive inhibitor quercetin unlike reference competitive inhibitor oseltamivir in kinetics
studies.
430-cavity
Neuraminidase
Non-competitive inhibition
Pandemic H1N1
Influenza
1. Introduction
and adjacent sugar residue [4,5]. The virulence and replication of a
pandemic H1N1 strain has been reported to be greater than that for a
Influenza A/H1N1pdm09, despite being a mild pandemic, remains a
critical challenge to public health in terms of mortality and morbidity. In
addition, one cannot rule out further inter genotypic reassortment
events with currently circulating high pathogenic avian influenza strains
such as H5N1 and H9N2 [1–3]. The influenza life cycle depends on an
accurate balance between the functionality of two viral surface glyco-
proteins viz. Hemagglutinin (HA) and Neuraminidase (NA). HA being a
fusion protein, facilitates the fusion of endocytosed virus particle with
the host cell endosomal membrane after binding to the surface receptors
containing sialic acid (SA). This brings about the viral internalization.
seasonal H1N1 strain, and this difference increases the severity of the
disease [6,7].
The anti-influenza drugs currently used as treatment, such as osel-
tamivir (OMV) and zanamivir (ZMV), have the structural similarity to
SA and consequently similar binding pattern to the NA enzyme. But,
mutations in NA gene limits their use as competitive inhibitors due to
resistance [8,9]. This reinforces the requirement of an alternate
approach in design and synthesis of new molecules to be developed as
potential anti-influenza agents. This could be achieved by discovery of
newer scaffolds that are structurally dissimilar to OMV/ZMV and have
different binding pattern in the enzyme cavity. The resistance problem
NA being an exosialidase, cleaves the α-ketosidic linkage between SA
Abbreviations: CAPi, Cinnamic acid-piperazine; CC, Cell control; CV, Crystal violet; CPE, Cytopathic effect; DCM, Dichloromethane; DMF, Dimethyl formamide;
DMSO, Dimethylsulfoxide; FBS, Foetal Bovine Serum; HA, Hemagglutinin; HA titer, Hemagglutination titer; HAI, Hemagglutination inhibition; LR, Laboratory
Reagent; MDCK, Madin-Darby Canine Kidney cells; MEM, Minimum Essential Medium; NA, Neuraminidase; OMVC, Oseltamivir Carboxylate; OMVP, Oseltamivir
Phosphate; PDB, Protein Data Bank; QR, Quercetin; SA, Sialic Acid; VC, VirusControl.
* Corresponding author at: Department of Pharmaceutical Chemistry, Prin. K. M. Kundnani College of Pharmacy, Plot 23, Jote Joy Building, Rambhau Salgaonkar
Marg, Cuffe Parade, Mumbai 400005, India.
E-mail address: ma.kanyalkar@kmkcp.edu.in (M. Kanyalkar).
https://doi.org/10.1016/j.bioorg.2021.105139
Received 18 November 2020; Received in revised form 14 May 2021; Accepted 28 June 2021
Available online 1 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
towards competitive inhibitors of NA i.e., OMV and ZMV, due to mu-
tations could be avoided by developing non-competitive inhibitors
which inhibit the enzyme by binding allosterically to the target enzyme.
There are two cavities present adjacent to SA/OMV binding site in NA
viz., 150-cavity and 430-cavity. These two cavities could be considered
as the potential alternate binding sites in NA.
crystal structure of NA from the pandemic 2009 influenza A/
H1N1pdm09 strain indicated that it lacks the 150-loop in its active site
(akin to closed conformation in contrast to the open conformation
observed in seasonal H1N1-NA influenza attributed to the presence of
150-loop) [29–33]. The initial docking validation studies could
acceptably replicate (RMSD 0.32 Å) the binding pose of the co-
crystallized ligand viz. oseltamivir carboxylate (OMVC) in the influ-
enza A/H1N1pdm09-NA X-ray crystal structure (PDB ID: 3TI6). The
There are reported molecules that bind to 150-cavity and 430-cavity
demonstrating potent inhibition of various NA subtypes [10,11]. In fact,
in recent years, discovery of various molecules, by our lab team, probing
150-cavity and 430-cavity led us to a series of chalcones showing non-
competitive inhibition mechanism towards H1N1-NA and H5N1-NA
along with certain other scaffolds, showing anti-influenza activities
against H1N1-NA [12–15]. Among several reported antiviral phyto-
constituents and our lab team’s findings, we have selected chalcones
cleavage of α-ketosidic linkages between SA and adjacent sugar residue
occurs during NA catalyses [8,34]. Thus, to understand the interaction
pattern and critical residues involved in SA binding, we docked sialic
acid in SA catalytic cavity of NA (Fig. 1a). It was observed that the
carbonyl of N-acetamido group formed H-bond with R152 while the
deoxy formed H-bond with E277. The terminal hydroxyl groups of
glycerol sidechain formed H-bond with R118, E119, and Y406. The
carboxylate group formed H-bond with the sidechain of R292 along with
a bi-dentate interaction with R371. The hydroxyl group formed H-bond
with R151.
that is characterized by α, β-unsaturated carbonyl functional groups.
Nonetheless, chalcones show weaker anti-influenza activity along with
cytotoxicity [16–19]. This scaffold can be modified to various cyclized
derivatives such as aurones, flavones, 3-indolinones and 4-quinolones,
for cyclized rigidification has been proven to improve the activity of a
lead molecule [20]. Chalcones, the chosen parent scaffold, can corre-
To ensure about the unbiased search of binding site of our designed
molecules, we explored all the putative binding pockets for docking in
NA enzyme. This strategy is often applied when prior knowledge of the
binding mode of the molecule is unknown [35,36]. Initially, we docked
quercetin, active natural non-competitive anti-influenza agent [37] in
NA to gain insight into its non-competitive inhibition behaviour. As
anticipated from its non-competitive nature, quercetin did not bind to
SA cavity, rather its binding was confined to 430-cavity within the active
site than other putative binding pockets. Moreover, to ensure whether
quercetin occupy distinctly different region than SA binding site or not,
we have docked it into the active site of NA already complexed with
OMVC/SA. It was observed that it still bound the same way as observed
in absence of OMVC/SA. This confirmed that quercetin occupied a very
distinct site and did not overlap with the SA binding site that further
signified existence of an alternate binding region for quercetin within
the catalytic site discrete from available drugs and SA (Fig. 1b). The two
hydroxyl groups of flavone ring of quercetin formed H-bond with N347
spondingly be compared with other α, β-unsaturated carbonyl functional
group containing phytoconstituent showing antiviral activity such as
cinnamic acid derivatives [21–23]. Further, piperazine moiety, having
wide variety of activity [24,25], is a commonly used scaffold in various
drugs, and thus had been selected and linked with cinnamic acid scaffold
by molecular hybridization approach [26]. Therefore, considering the
above facts and modifications, we report here the design, synthesis and
evaluation of a series of four scaffolds, namely aurones, 3-indolinones, 4-
quinolones and cinnamic acid-piperazine (CAPi) hybrids against influ-
enza A/H1N1pdm09 virus. The above-mentioned selected scaffolds are
structurally dissimilar to the transition state of sialo-glycoconjugate
being hydrolysed by viral NA, the substrate for NA. They were
designed with introducing substituents with varied functional groups
attributing electronic, steric and volume effect to the molecules.
Cytopathic effect (CPE) inhibition and hemagglutination inhibition
(HAI) assays were executed on the molecules that were selected based
on docking results and cytotoxicity, to screen them for further extensive
evaluation studies on the basis of % CPE inhibition and % HA titer
reduction of virus with candidate molecules. All the screened molecules
were subjected to cell-based assay to determine their EC50 values fol-
lowed by enzyme-based assay to determine their IC50 values. Addi-
tionally, mechanism of inhibition of the molecules was determined by
enzyme kinetics studies which showed non-competitive inhibition when
compared with competitive inhibitor viz. OMV. This strengthens our
idea of developing potential anti-influenza agents and propose better
therapeutic and prophylactic upshots than what current anti-influenza
drugs can offer us.
and K432 along with π-alkyl interaction with R371 and P431. The hy-
droxyl of phenyl ring formed H-bond with I427 and phenyl ring had
π
-alkyl interaction with R371, I427, P431 and K432.
Additionally, non-bonded interaction energies were calculated for
quercetin with the catalytic site residues. Fig. 2 demonstrates that
quercetin has favourable non-bonded interactions with the catalytic site
residues that included R118, I149, R152, N294, N347, S370, R371,
W403, Y406, R430, P431, L432, T436, I437 and W438. The previously
reported site-directed mutagenesis studies highlighted the importance of
N146, R152, W178, E276, E277, S370, R371, W403, Y406 in the cata-
lytic activity of NA [9,38], out of which quercetin showed non-bonded
interactions with R152, S370, R371, W403 and Y406. Moreover, it
was observed that R152 is involved in the binding of SA to active site of
NA (vide supra) in molecular docking studies. Therefore, it could be
presumed from the above outcomes that on account of H-bonding and
non-bonded interactions, quercetin cause hinderance in the catalytic
activity of NA thereby inhibiting it, nonetheless, with an alternate
binding mode in the NA enzyme.
Also, there has been studies conducted to suggest the corelation
between influenza and SARS-CoV-2 [27]. It was demonstrated that
influenza caused increase in the SARS-CoV-2 transmission along with
enabling the spread of COVID-19 in Europe during early 2020. More
generally, taking into account the resistance issue of influenza, it is
imperative to develop agents with alternate mechanism of action that
this current work is all about that can combat this very contributing
factor of influenza to the spread of COVID-19.
The designed molecules were then docked in influenza A/
H1N1pdm09-NA enzyme to have knowledge of how substituting
various functional groups at different positions and having the scaffold
changed affect their binding mode in NA enzyme. The introduction of
substituents may improve the pharmacokinetic and pharmacodynamic
properties of the molecule by virtue of imparting electronic and lipo-
philic characters [39].
2. Results and discussion
2.1. Computational studies demonstrating alternate binding mode in NA
enzyme
The comprehensive analysis of molecular docking studies high-
lighted that the binding affinity of our designed molecules was confined
to the 430–cavity similar to quercetin, which was considered as the
standard for non-competitive inhibition, unlike SA and OMVC as shown
in Fig. 3. In the series of aurones, the benzofuranone ring of all molecules
occupied the hydrophobic cavity (W403, I427, P431, K432). Molecules
The active site of N1-NA was classified into three cavities for suit-
ability: (i) the SA catalytic cavity (R118, E119, N146, D151, R152,
Y178, I222, E227, E276, E277, R292, N294, N347, R371, and Y406); (ii)
the 150-cavity (N146-R152) and (iii) the 430-cavity (N325, P326, G348,
S369, S370, W403, I427, G429, R430-T439) [13,28]. Interestingly, the
2

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
Fig.1. Docked poses of (a) sialic acid (orange colour) in SA cavity and (b) oseltamivir carboxylate (green colour) in SA cavity and quercetin (yellow colour) in 430-
cavity of H1N1-NA enzyme. The SA cavity is represented in green colour and 430-cavity is represented in yellow colour in the surface model. The figure was prepared
using Discovery Studio visualizer tool. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig.2. Histogram of interaction energies (van der Waal i.e., vdW and Electrostatic) of quercetin with the catalytic site residues.
1a-1c showed H-binding with K432. 1d showed H-bond interactions
with R371 and N347; while 1e had electrostatic interaction with W403
and salt bridge with D283. The docked conformations of 3-indolinone
molecules 2a-2e, 2g and 2h revealed that the 3-indolinone ring of
these molecules had hydrophobic interactions with I427, P431 and K432
along with hydrogen bond and hydrophobic interactions with R371;
while the phenyl ring interacted hydrophobically with P326. However,
molecule 2f had reverse orientation in which the phenyl ring showed
hydrophobic interactions with I427, P431 and K432 along with inter-
action of its m-chloro group with R371 and W403; while the 3-indoli-
none ring had hydrogen bond and hydrophobic interaction with R371,
which could be responsible for its higher activity. The molecules 3a, 3c-
3f, 3h and 3i were docked such that commonly the 4-quinolone ring of
these molecules had hydrophobic interactions with R371, I427, P431
and K432 along with hydrogen bond and hydrophobic interaction with
R371; while phenyl ring showed interaction with N347. 3b and 3g were
docked in a different orientation than others having their phenyl ring
interacting hydrophobically with I427, P431 and K432 and with R371 as
well showing hydrogen bond and hydrophobic interaction; while the 4-
quinolone ring showed hydrogen bond interactions with R118 and I149.
The molecules 4a-4e were docked with their phenyl ring interacting
hydrophobically with I427, P431 and K432 along with hydrogen bond
and hydrophobic interaction with R371. The piperazine ring of CAPi
hybrid molecules had hydrogen bond interactions with N347 and S369.
It is evident that additionally, all the designed molecules have either H-
bonding or hydrophobic interaction with backbone and sidechain of
R371. Detailed analysis of binding interactions of the molecules with NA
enzyme is depicted in Table 1.
The observations from computational studies recommended that the
molecules might bind tight to the NA enzyme attributed to their addi-
tional interactions, consequently leading to better anti-influenza
activity.
The pharmacological activity and the performance of a drug candi-
date can be predicted to some extent by determining their ADME
properties. Accurate prediction of in vivo pharmacological activity of a
potential drug molecule is the ultimate aim of in-silico ADME studies
[40]. Various descriptors were evaluated for ADME properties of
candidate molecules. None of the designed molecules violated Lipinski’s
rule of five indicating towards their drug-likeness. All the molecules’
molecular weight was in the range values of 130–725, donor HB
3

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
Fig.3. Docked poses of designed molecules in 430-cavity (top) in surface model of H1N1-NA enzyme. 3D docking model and corresponding 2D schematic diagram of
docking model of representative (a) aurone, (b) 3-indolinone, (c) 4-quinolone and (d) cinnamic acid-piperazine hybrid.
–
(hydrogen bond) and acceptor HB were in the range of 0–6 and 2–20
respectively. Solvent accessible surface area (SASA) is an indication of
the partition coefficient and aqueous solubility which was found to be in
the range of 300–1000 for all the deigned molecules [41]. The
bioavailability of a molecule is considerably determined by QPlogPo/w
which was found to be favourable in the range of ꢀ 2 to 6.5. The ioni-
zation potential parameter is indicative of distribution of molecule that
affects the molecule’s availability for further physical, chemical or
biological reactions. The solute ionization potential (eV) was found to be
in the range of 7.9–10.5 for all the molecules. It is indicated that the
designed molecules possess favourable pharmacokinetic properties
evident from the overall in-silico ADME results.
1640–1645 cmꢀ 1. The exocyclic C C in aurones and 3-indolinones was
–
characterized by a band near 1660–1667 cmꢀ 1, whereas the endocyclic
ꢀ 1
–
–
–
C
C in 4-quinolones was near 1600–1620 cm . The C C stretch in
–
CAPi hybrids was around 1600–1615 cmꢀ 1. 1H and 13C NMR spectra of
molecules represented predictable delta values for all aliphatic and ar-
omatic protons and carbons, respectively. In 1H NMR spectra of syn-
thesized molecules, the vinylic proton of aurones and 3-indolinones (H-
10) appeared in the range of δ 6.90–7.11 and δ 6.63–7.20, respectively
as singlet, while the pyridone ring proton (H-3) in 4-quinolones showed
chemical shift value in the range of δ 6.02–7.28 as singlet. The downfield
appearance of vinylic proton (H-10) of 3-indolinones is the character-
istics of Z isomer. We interpret this to be due to the H-10 proton being
deshielded by the neighbouring carbonyl at C-3 position [42–45]. The
NH of 3-indolinones and 4-quinolones appeared downfield around δ
10–12, while the piperazine NH appeared around δ 1.5–1.6. The
chemical shift value of vinylic carbon (C-10) of aurones were in the
range of δ 108.38–113.76 [46] represented in 13C NMR which is the
characteristics of thermodynamically more stable Z isomer. Mass spectra
of representative synthesized molecules gave the predicted m/z peak
corresponding to their estimated molecular weight. The HPLC purity of
molecules was > 95%. Therefore, it was confirmed that the anticipated
structures of the synthesized molecules are correct and are pure (Spectra
in Supplementary data).
2.2. Chemistry
Based on the outcomes of computational studies of newly designed
four scaffolds; with respect to their interactions with the enzyme, their
binding poses and affinity; twenty-seven molecules corresponding to
four scaffolds were synthesized viz. five aurones (1a-1e), eight 3-indoli-
nones (2a-2h), nine 4-quinolones (3a-3i), and five CAPi hybrids (4a-
4e). The target molecules were synthesized as shown in Scheme 1 and
Scheme 2. The IR spectra demonstrated expected absorption bands for
the functional groups of the synthesized molecules. All the aurone
molecules showed cyclic C O stretching near 1300 cmꢀ 1. The absorp-
–
tion band for NH stretching in 3-indolinone molecules was observed
near 3450–3100 cmꢀ 1, while that for 4-quinolones was observed near
3232–3080 cmꢀ 1 and that for CAPi hybrids was observed near 3130
2.3. Anti-influenza evaluation
Oseltamivir phosphate (OMVP) was considered as standard for
cellular assays, viz. MTT, CPE, HAI and CV assays, as the esterase
enzyme present in cells convert it to its active metabolite i.e., oseltamivir
carboxylate (OMVC) [47,48], while oseltamivir carboxylate was
cmꢀ 1. The IR band for carbonyl C O stretch in aurones and 3-indoli-
–
–
nones was around 1650 cmꢀ 1, while that in 4-quinolones was around
1715 cmꢀ 1. Carbonyl C O stretch for CAPi hybrids was observed near
–
–
4

Table 1
Detailed analysis of binding interactions of docked molecules with amino acid residues of H1N1-NA.
Residues
R118*
E119*
I149
D151*
R152*
R224*
R292*
P326
N347
S369
S370
R371*
W403
I427
R430
P431
K432
E433
Molecules
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
2f
H
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
H
–
–
–
–
–
–
–
–
H
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
π
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
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H,
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–
–
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H
–
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–
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–
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H
H
H
H
H
H
–
–
–
H
–
–
–
–
–
–
–
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–
–
–
–
–
–
–
–
H
–
–
–
–
H
–
–
–
–
H
–
–
–
–
–
–
H,
H
π
-a
–
–
–
H
–
H
–
–
–
H
π
–
π
–
–
–
–
–
π
–
–
–
–
–
–
–
π
π
–
–
–
–
–
-
π
H
–
π
-a (2)
–
–
H (2),
H
π
-a
π
–
π
π
π
π
π
π
π
π
–
π
π
–
π
π
π
π
π
π
π
-a
π
–
π
π
π
π
π
π
π
π
π
π
π
–
π
π
π
π
π
π
π
π
π
π
-a (2)
H,
H
π-a (2)
–
–
–
–
–
-a,
π-
π
–
H (2)
–
H (2),
π
-a
–
H
–
H,
H,
H,
π
π
π
-a (2)
-a
-a (2)
-a (2)
-a (2)
-a (3)
-a (2)
-a
π
π
π
π
π
-a (2)
–
π
π
–
π
–
π
π
–
–
–
–
–
–
–
–
π
–
–
–
–
–
-a
H
–
-a,
-a,
π
-
-
σ
σ
-a
-a (2)
–
–
-a
–
H
–
π
-a
-
π
,
π-a (2)
–
–
–
–
π
-a,
π
-
σ
-a
-a (2)
-a (2)
–
–
-a (2)
–
–
H
–
H,
H,
H,
H,
H,
H,
H,
H
π
π
π
π
π
π
π
-a,
π
-
σ
(2)
-a
–
–
–
–
-a (3), halogen
-a
-a (2)
-a
H,
H,
π
-a
–
2g
2h
3a
3b
3c
3d
3e
3f
–
-a (2)
-a
H
–
–
-a,
-a,
-a
π
-
-
σ
σ
-a (2)
-a (2)
-a (2)
-a (3)
-a (2)
π
-a (2)
–
–
–
H
–
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-a
π
-a (2)
-a
–
H
H
–
π
–
–
–
–
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–
–
–
–
–
–
–
-a
H
–
H
–
π
–
–
H
H
-a
-a (2)
-a
H (2),
π
-a
–
–
-a,
π
-
-
σ
σ
H,
–
π-a (2)
–
H
–
-a
π
-a
–
–
π
–
–
-
π
–
H
–
H,
π
-a,
π
(2)
-a
-a (3)
-a (2)
-a (3)
-a (2)
-a (2)
H,
π-a (2)
–
–
-a
-a
–
π
π
-a
-a
-a
-a (2)
-a
π
-a (2)
–
3g
3h
3i
H
–
–
–
-a,
π
-
σ
H,
H,
π
-a
–
H
H
–
–
H
–
H (2),
π
-a,
π-
σ
-a
π
-a (2)
–
–
-
σ
–
H,
H,
H,
π
π
π
-a,
π
-
σ
-a (2)
π
π
π
π
π
π
-a (2)
–
4a
4b
4c
4d
4e
–
–
H
–
-
σ
-
σ
-
σ
-
σ
–
–
H (2)
–
-a
-a
-a
-a
-a
-a
-a
–
–
H (2)
–
–
π
-
σ
H,
H,
π
-a
-a
-a
–
–
–
–
–
–
H,
π
-a,
π
-
σ
H
π
-a
–
–
H (2)
–
π
-a
π
-a
π
-a
-a (2)
Halogen
H: Hydrogen bond;
bonds.
π-π: pi- pi interaction (hydrophobic); π-a: pi-alkyl interaction (hydrophobic); π-σ: pi-sigma interaction (hydrophobic); *Catalytic site residues interacting with SA. Figures in brackets indicate number of

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
Scheme 1. Synthesis of aurone (1a-1e) from 2-hydroxychalcone; 3-indolinone (2a-2h) and 4-quinolone (3a-3i) from 2-aminochalcone. Reagents and conditions
used: (i) 20% NaOH, absolute ethanol, 10 ◦C, 4–5 h; (ii) Hg(OAC)2, pyridine, 110 ◦C, 1–2 h; (iii) acetic acid, 10% w/w Amberlyst-15, 80 ◦C, 2–5 h; (iv) DMSO, iodine
crystal, reflux, 30 min.
directly used as standard for enzymatic assays viz. NA inhibition assay
and enzyme kinetics studies. OMVP/OMVC and quercetin (QR, the re-
ported natural non-competitive inhibitor[37]) were used as standards
for representing the competitive and non-competitive inhibition,
respectively.
carried out as qualitative evaluation study to assess the degree of viral
inhibitory activity of the molecules after comparison with OMVP as
standard drug. Interestingly, CPEs in the form of loss of cell adhesion
that is the characteristics of influenza A/H1N1pdm09 virus infection
were found to be considerably reduced in our synthesized molecule-
treated infected cells, further asserting a robust cyto-protective and
anti-influenza property of these molecules. The degree of inhibition of
viral CPE by all the evaluated molecules is depicted in supplementary
table, Table S1. All the molecules showed at least 50% CPE inhibition
suggestive of the substantial ability of molecules to reduce the viral load.
Subsequently, they were carried forward for further assays.
2.3.1. Cytotoxicity studies of all the synthesized molecules
Cytotoxicity studies determined the concentration of synthesized
molecules responsible for 50% reduction in cell viability (CC50). The
results of MTT-Formazan assay for cytotoxicity study of twenty-seven
synthesized molecules indicated that they had no serious effect on
MDCK cells except for molecule 4d having CC50 of 40 µM (Table 2). The
anti-influenza activity of these non-cytotoxic molecules was further
evaluated for decrease in cytopathic effect (CPE) of influenza A/
H1N1pdm09 strain in MDCK cells in presence of the molecules.
2.3.3. Hemagglutination inhibition (HAI) to determine viral titer reduction
by molecules
The CPE inhibition was further assessed by comparing the HA titer of
virus in virus control well with the HA titer of virus in molecule-treated
wells. Relative viral load viz. the HA titer of molecule-treated infected
cells was represented as ‘% HA titer reduction’ considering the
2.3.2. Cytopathic effect (CPE) inhibition of all the molecules
CPE inhibition assay of all twenty-seven synthesized molecules was
6

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
Scheme 2. Synthesis of cinnamic acid-piperazine hybrid (4a-4e). Reagents and conditions: (v) pyridine/piperidine, 100 ◦C, 2 h; (vi) DCM, thionyl chloride, DMF,
reflux, 24 h; (vii) DCM, TEA, 0 ◦C till reaction completed monitored by TLC.
infectivity, in terms of HA titer, of the vehicle-treated infected control as
100% in each replicate of triplicate experiments. OMVP and QR were
used as standards, and %HA viral titer reduction data for all the mole-
cules are summarized in Table 3. As evident from data, most of the
molecules caused robust and persistent reduction in the viral titer at
least by 50%, as shown in Fig. 4. The results of HAI assay illustrated that
our molecules efficiently suppress the viral titer showing weak-to-
moderate-to-high potencies compared to standards (OMVP = 85 ± 4%
and QR = 79 ± 4%, at 100 µM) further directing towards the anti-
influenza activity of the molecules. The structure–activity relationship
was tough to draw, attributed to the vague outline between these mol-
ecules and reduction rates of %HA titer. Next, the molecules were car-
ried further for quantitative evaluation of their anti-influenza activity.
50% of the enzyme activity (IC50). Dose response curves depicting the
effect of our molecules on H1N1-NA enzyme are shown in Fig. 6.
The IC50 values of the evaluated molecules ranged from 0.52 ± 0.01
µM to 24.6 ± 1.3 µM. OMVC and QR, the standards chosen in enzyme-
based assay, has IC50 value of 1.9 ± 0.1 nM and 8.7 ± 0.1 µM, respec-
tively (Table 2). Though the potent molecules discussed in CV assay had
their IC50 values lower than OMVC, they certainly tend to exhibit
excellent activity with their IC50 values at low-micromolar level,
intriguingly, better than QR. The trend of efficacy of molecules seen in
CV assay is followed in NA inhibition assay as well, the potent molecules
2f (IC50 = 0.52 ± 0.01 µM), 2g (IC50 = 3.5 ± 0.1 µM) and 3a (IC50 = 1.3
± 0.2 µM) possessed lowest IC50 values than other molecules of the se-
ries. Interestingly, the combined results of CV assay and NA inhibition
studies suggested that the evaluated molecules suppress overall viral
replication than inhibiting the NA enzyme per se. These results pointed
towards the fact that there might be an accompanying mechanism by
which our evaluated molecules control virus replication indicated by
their potency in vitro.
2.3.4. Cell-based crystal violet (CV) assay to quantify the effective
concentration of molecules
CV assay determined quantitatively the effective concentration of
tested molecules that resulted in 50% cell survival (EC50). Consistent
with the marked attenuated viral HA titer, the tested molecules notably
showed significant degrees of cell survival in CV assay (Table 2). Mol-
ecules 2f (3-indolinone with m-chloro group on benzyl ring), 2g (3-
indolinone with p-chloro group on benzyl ring) and 3a (4-quinolone
with o-methoxy group on phenyl ring) having EC50 of 4.0 ± 0.1 nM, 6.7
± 0.1 nM and 4.9 ± 0.1 nM, respectively, had highest activity with their
EC50 values better than commercial drug i.e., OMVP (EC50 = 12.7 ± 0.3
nM) as well as QR (EC50 = 0.56 ± 0.01 µM). Molecules 1d (aurone with
p-chloro group on benzyl ring) and 2b (3-indolinone with m-methoxy
group on benzyl ring) with EC50 = 39 ± 2 nM and 25 ± 1 nM, respec-
tively, however were slightly less active than OMVP, but better than QR,
still could be believed to have remarkable anti-influenza property. The
effects of representative potent molecules along with standards on % cell
survival infected with pdmH1N1 virus are shown in Fig. 5. These results
suggested that these molecules could hamper overall replication of virus
in MDCK cells. The high selectivity index values of these compounds
indicated that they decreased the virus replication without adverse ef-
fect on the living host cells.
2.3.6. Enzyme kinetics studies to ascertain mechanism of inhibition of
molecules
Enzyme kinetics study was performed on eleven most active mole-
cules (based on CV and NA inhibition assay) to validate the non-
competitive inhibition of designed molecules as exhibited by docking
studies. In this study, OMVC and QR were used as standards for
competitive and non-competitive inhibition, respectively. Unsurpris-
ingly, OMVC being structurally similar and transition state analogue of
SA, showed competitive inhibition evident from the plots exhibited in
Fig. 7 which shows Lineweaver-Burk plot of 1/V versus 1/[S]. The plots
were obtained by considering absence of inhibitor molecule (i.e., at 0
nM concentration) and at two concentrations bracketing the IC50 value
of the inhibitor molecule. Increasing concentrations of OMVC (Ki = 2.8
× 10-4 µM) resulted in collection of lines with increased Km while the
V
max remained unaffected i.e., they have equivalent y-axis intercept but
with higher value of x-axis intercept. This indicated its competitive in-
hibition. However, increasing concentrations of QR (Ki = 5.1 µM)
resulted in collections of lines wherein the Km remained unaffected
while Vmax showed reduction i.e., they have equivalent x-axis intercepts
along with increase in the y-axis intercept values. This demonstrated its
anticipated non-competitive inhibition as reported in literature.
Interestingly, in case of our evaluated molecules, the Lineweaver-
2.3.5. Enzyme-based NA inhibition to determine inhibitory concentration of
molecules
H1N1-NA inhibition by twenty-five molecules, based on CV assay,
was determined by calculating the concentration required to inhibit
7

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
Table 2
Table 3
In-vitro evaluation of tested molecules.
Reductions of %HA titer of pdmH1N1 virus by aurone (1a-1e), 3-indolinone (2a-
2h), 4-quinolone (3a-3i), CAPi hybrids (4a-4e); and standard competitive and
non-competitive inhibitors oseltamivir phosphate and quercetin, respectively.
Sr.
Molecules
CC50
EC50 (µM)
SI
IC50 (µM)
Ki (µM)
No.
(µM)
Molecules
HA titer Reduction (%)a
Molecules
HA titer Reduction (%)a
1
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
2f
193 ±
>100
–
Not tested
9.3 ± 0.1
14.9 ± 1.2
1.8 ± 0.1
Not tested
20.9 ± 1.3
22.4 ± 1.3
10.5 ± 1.2
19.8 ± 1.3
19.4 ± 1.2
Not tested
10.5 ± 0.7
Not tested
11.2 ± 2.8
Not tested
Not tested
2.6 ± 0.1
Not tested
Not tested
Not tested
2.8 ± 0.2
1
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
2f
31 ± 2%
82 ± 5%
64 ± 3%
81 ± 4%
12 ± 2%
75 ± 4%
70 ± 3%
70 ± 3%
60 ± 2%
63 ± 4%
89 ± 4%
65 ± 3%
72 ± 4%
84 ± 4%
50 ± 2%
3c
17 ± 1%
20 ± 2%
19 ± 2%
30 ± 3%
82 ± 5%
20 ± 2%
54 ± 2%
52 ± 2%
58 ± 3%
41 ± 2%
54 ± 2%
42 ± 3%
85 ± 4%
79 ± 4%
2
171 ±
1.5 ± 0.9
1.0 ± 0.1
117
118
3d
3e
1
3
120 ±
3f
1
3g
4
113 ±
(39 ± 2) ×
103
2.9 ×
103
–
3h
3i
1
5
111 ±
>100
4a
1
4b
4c
6
263 ±
5.1 ± 0.9
52
1
4d
4e
7
292 ±
(25 ± 1) ×
103
1.2 ×
104
2g
2h
3a
3b
1
OMVP
QR
8
287 ±
11.9 ± 1.3
24
1
9
128 ±
0.17 ±
0.05
752
88
a
Reduction of %HA titer of pdmH1N1 virus (mean ± std dev of three ex-
periments) in presence of 100 µM of inhibitors, measured by hemagglutination
inhibition assay.
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
269 ±
3.1 ± 0.1
1
272 ±
(4.0 ±
6.8 ×
104
0.52 ±
0.01
1
0.1) × 103
(6.7 ±
2.4. Induced fit docking (IFD)
2g
2h
3a
3b
3c
3d
3e
3f
192 ±
2.8 ×
3.5 ± 0.1
2.0 ±
1
0.1) × 103
0.25 ±
104
0.003
115 ±
1
462
15.7 ± 1.2
1.3 ± 0.2
24.3 ± 1.3
3.8 ± 0.1
12.2 ± 1
33.7 ± 2.3
IFD is an accurate and robust docking technique that takes into ac-
count the ligand and protein flexibility. Validation of IFD process was
done by superimposing the docked oseltamivir structure over the osel-
tamivir obtained from crystal structure of NA enzyme (PDB 3TI6). RMSD
value for all the heavy atoms was obtained as 0.17 Å. Furthermore, the
receptor binding of oseltamivir was found similar to that in the crystal
structure. The interactions formed by both crystal structure and docked
oseltamivir are: amide functional group of oseltamivir forming H-bond
with R152; amino group forming salt bridges with E119, D151 and E277
and H-bond with E119 and E227; carboxylate moiety forming H-bonds
with R118, R292 and R371 as shown in Fig. 1b (vide supra). IFD was
executed on five most potent molecules to have an insight towards the
accurate binding of these molecules when the enzyme is in its optimal
conformation. IFD scores were found to be consistent with experimental
CV assay results as shown in Table 4.
0.01
286 ±
1
(4.9 ±
5.8 ×
104
–
2.1 ± 0.2
Not tested
53.6 ± 9
2.8 ± 1
0.1) × 103
>100
235 ±
1
277 ±
1
>100
>100
>100
>100
–
200 ±
1
–
181 ±
1
–
18.4 ± 1.2
16.9 ± 1.4
8.7 ± 0.1
6.4 ± 0.1
16.7 ± 1.2
15 ± 2
Not tested
Not tested
7.1 ± 0.2
8.4 ± 0.1
Not tested
Not tested
Not tested
Not tested
163 ±
1
–
3g
3h
3i
163 ±
1
0.22 ±
0.01
742
2
117 ±
1
78 ± 1
118 ±
1
>100
–
4a
4b
4c
155 ±
1
3.6 ± 0.2
5.1 ± 0.1
7.8 ± 0.1
43
21
18
2.5. Structure-activity relationship (SAR)
106 ±
1
24.6 ± 1.3
18.4 ± 1.2
All the deigned scaffolds fitted well in the 430-cavity which was
believed to be the alternate binding region within the catalytic active
site. However, the scaffolds exhibited varied degree of anti-influenza
activity. The prototype scaffold selected i.e., chalcone, having uncycl-
137 ±
1
26
27
4d
4e
40 ± 1
189 ±
1
>100
–
28
12 ± 2
Not tested
Not tested
6.7 ± 0.9
11.2 ± 1.7
ized α, β-unsaturated carbonyl system, showed weaker anti-influenza
28
29
QR
254 ±
1
0.56 ±
0.01
453
8.72 ± 0.1
5.1 ± 0.1
activity as evident from literature. Comprehensive examination of the
anti-influenza evaluation of our designed scaffolds suggested that
cyclization of chalcone not only reduced cytotoxicity but also caused the
improvement in activity. It was observed that 5-exo cyclocondensation
of 2-amino chalcone to yield 3-indolinone enhanced the activity than 6-
endo cyclocondensation to yield 4-quinolone. 3-indolinones were found
to be even more active than the oxidative cyclized product of 2-hydroxy
chalcone viz. aurone. Thus, it can be said that the isosteric replacement
of nitrogen in 3-indolinone to oxygen in aurone reduced the activity.
Incorporation/presence of phenyl rings provides hydrophobic sites for
interaction between inhibitor molecule and the enzyme. The higher
activity of 3-indolinones, 4-quinolones and aurones may be attributed to
the presence of another phenyl ring that imparted more hydrophobicity
to the molecules indicating towards their prominent interactions with
the hydrophobic 430-cavity in docking studies. At the same time, when
OMV
713 ±
1
(12.7 ±
5.6 ×
(1.9 ±
(2.8 ±
0.3) × 103
104
0.1) × 103
0.3) × 104
MTT assay and crystal violet experiments performed in triplicates to give CC50
and EC50 values respectively, NA-inhibition and enzyme kinetics experiments
performed in duplicates to give IC50 and Ki values respectively; CC50 (mean ±
std dev) values represent the concentration of molecules that showed 50%
cytotoxicity; EC50 (mean ± std dev) values represent the concentration that
resulted in 50% cell survival after infection in presence of inhibitor; SI =
Selectivity Index was generated by the ratio of CC50 and EC50; IC50 (mean ± std
dev) values of molecules represent the concentration that caused 50% enzyme
activity loss; Ki represents the enzyme inhibitor constant.
Burk plot of 1/V versus 1/[S] resulted in group of lines as observed in
QR, signifying their mechanism of enzyme inhibition to be non-
competitive. Fig. 7 displays the plot for the representative potent
molecules.
cinnamic acid (scaffold similar to chalcone having
α, β-unsaturated
carbonyl system) was linked to piperazine moiety, it improved the ac-
tivity than cinnamic acid itself. This may be attributed to decrease in the
8

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
Fig.4. Histogram of % HA titer reduction of virus treated with candidate molecules showing the effect of oseltamivir phosphate (OMVP, green colour), quercetin
(QR, yellow colour) and evaluated molecules on pdmH1N1 virus yield. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
acidity of the molecule. The scaffolds were designed by substitution of
various electron-withdrawing and electron-donating groups. Among
tested molecules, substituting the benzyl ring with chloro group
enhanced activity, out of which meta and para substituted chloro group
containing 3-indolinones, 2f and 2g respectively, showed potency better
than oseltamivir. While for 4-quinolones, electron-donating methoxy
group at ortho position (3a) showed better activity than oseltamivir.
Hence, it can be said that for 5-exo cyclocondensation of chalcones,
chloro group substitution at benzyl ring enhanced the activity while for
6-endo cyclocondensation, methoxy group substitution at phenyl ring
showed better activity than the standard drug. Overall SAR studies
pointed towards a better understanding of effect of presence of phenyl
ring at 1–2 carbon lengths, 5-exo cyclocondensation, chloro substitution
along with decreased acidity of the designed molecules towards their
anti-influenza activity against influenza A/H1N1pdm09 virus.
actively than inhibiting the NA enzyme alone. Among the tested mole-
cules, 2f, 2g and 3a were found to be most potent anti-influenza agents
having low-nanomolar EC50 values 4.02 nM, 6.72 nM and 4.96 nM,
respectively, that were better compared to reference competitive and
non-competitive inhibitors: oseltamivir (EC50 = 12.7 nM) and quercetin
(EC50 = 0.56 µM). 2f, 2g and 3a also exhibited good NA inhibitory ac-
tivity in sub-micromolar range (IC50 = 0.52 µM, 3.46 µM, 1.31 µM
respectively). In addition, enzyme kinetics studies suggested that the
mechanism of inhibition of our designed molecules is non-competitive
that was highlighted by in-silico studies as well wherein the molecules
bound to the alternate binding site. Owing to the fact that current
commercial drugs work like charm in wild-type influenza, the potent
molecules of present study can therefore be certainly used as an adjunct
therapy in combination with oseltamivir or zanamivir for improved
antiviral efficiency. This further strengthens our idea of developing
potential anti-influenza agents in future which can be used as an alter-
native in wake of oseltamivir resistance owing to their non-competitive
inhibition mechanism.
Interesting to note here is that the active site of NA comprises of 430-
cavity where our designed molecules bound. This binding region is
distinctly different from oseltamivir and sialic acid binding site. There-
fore, the molecules displayed non-competitive nature of inhibition.
Since, this binding region of the molecules is adjacent to catalytic site
and is present within the same active site, these molecules could
correspondingly be called “mutually non-competitive” agents.
Thus, it can be concluded that the current work has generated few
potential anti-influenza molecules especially for influenza A/
H1N1pdm09 strain. The approach used here is completely different than
the currently available marketed drugs. The potent molecules can be
subsequently developed to be used either alone or in combination with
current NA inhibitors for better management and eradication of influ-
enza virus.
3. Conclusion
In current backdrop of COVID pandemic, our research is focused on
another pandemic virus viz. swine flu – pandemic H1N1, which created
havoc in 2009. Although, the current floating strains are not virulent
and vaccine is available for its defense but the lesson learnt from COVID
crisis is not to underestimate the strength of the virus. The impulsiveness
of a potential influenza A pandemic outbreak and rapid unpredictable
emergence of influenza A virus resistance to current anti-influenza drugs
are urging the researchers and scientists to come up with newer strate-
gies to overcome resistance and develop better options in terms of
treatment than what current drugs can offer us. Based on the results
obtained in the present study, we can say that subset of the drug mol-
ecules showed substantial antiviral activity against influenza A/
H1N1pdm09 virus. Computational studies identified the molecules as
binders of 430-cavity, an alternate binding region within the active site,
similar to quercetin which was the standard for non-competitive inhi-
bition, but distinct from OMV in SA binding site which was the standard
for competitive inhibition. The non-cytotoxic molecules efficiently
reduced the viral titer and remarkably restricted viral replication more
4. Experimental section
4.1. Materials
The chemicals, reagents and catalyst Amberlyst-15 employed for
synthesis were purchased from SD Fine chem Ltd. and Sigma Aldrich
Chemicals Pvt Ltd. All the solvents used for synthesis were of LR grade.
Oseltamivir phosphate (OMVP) was obtained as a gift sample from Cipla
Ltd, India. Oseltamivir carboxylate (OMVC) and quercetin (QR) was
purchased from Clearsynth Labs, Ltd and Sigma Aldrich Chemicals Pvt
Ltd, respectively. The strain of A(H1N1)pdm09 virus Kolkata isolate [A/
human/India/Kol-7251/2018(H1N1); accession number: MN508979]
was obtained from ICMR-National Institute of Cholera and Enteric Dis-
eases (ICMR-NICED), Kolkata, India. Madin-Darby Canine Kidney
(MDCK) cells were obtained from National Institute of Virology (NIV),
Pune, India.
9

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
Fig.5. Effects of oseltamivir phosphate (OMVP), quercetin (QR) and representative potent molecules 2f, 2g and 3a on % cell survival infected with H1N1 virus.
4.2. Methods
to optimize the hydrogen-bonding network in the complex using Pro-
tAssign program in Maestro. Minimization was implemented using
OPLS3e forcefield.
4.2.1. Modelling and system preparation for modelling
The X-ray crystal structure of OMV-complexed pandemic H1N1-NA
enzyme was imported from RCSB protein data bank (PDB ID: 3TI6),
with a resolution of 1.69 Å [32]. Since NA is a homotetramer, monomer
unit of the enzyme (3TI6) was considered for docking calculations in
computational studies. Protein Preparation Wizard module of Maestro
SiteMap [36,52] tool was applied to explore the potential binding
sites and characterize them. Different molecules were designed with
introducing substituents with varied functional groups attributing
electronic, steric and volume effect to the scaffolds. All the designed
molecules were built using the 2D sketcher available with Maestro 11.5
¨
¨
11.5, Schrodinger LLC, New York, USA [49] was employed to optimize
(Schrodinger LLC NY 2016) and their geometries were optimised using
the geometry of the enzyme. The crystallographic waters were removed,
hydrogen atoms were added to the enzyme structure consistent with a
pH of 6.5, which is the optimum pH for NA enzymatic activity [50]. N-
and C-termini were capped. Ca2+ is reported to be crucial for enzyme
activity and stability, therefore, two Ca2+ ions were retained: one whose
binding site is close to the active site which helps in holding the active
site in appropriate conformation for substrate binding; while the other
whose binding site was found in 1918 N1 and 2009 swine-origin N1
structures [50,51]. The terminal rotamer states were set automatically
for Asn, Gln and His, as well as tautomeric and protonation states of His
LigPrep v3 module [53] with OPLS3e forcefield and docked in the pu-
tative binding sites using Glide XP [35,54]. Best docking scores were for
the molecules showing binding in the 430-cavity of influenza A/
H1N1pdm09-NA enzyme than other putative binding pockets. Addi-
tionally, induced fit docking (IFD) [55] of the best molecules was
executed in this binding pocket since the receptor may not be in an
optimal conformation to bind the inhibitor molecules. The validation of
docking protocol was done by replicating the binding pose and in-
teractions of co-crystallized ligand i.e., oseltamivir in the X-ray crystal
structure.
10

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
Fig.6. Effects of oseltamivir carboxylate (OMVC), quercetin (QR) and evaluated molecules on H1N1-NA for the hydrolysis of substrate.
Fig.7. Lineweaver–Burk plots for the inhibition of oseltamivir carboxylate (OMVC), quercetin (QR) and representative evaluated molecules on H1N1-NA for the
hydrolysis of substrate in the presence of increasing concentrations of tested molecules (two conc. bracketing IC50) for lines from bottom to top.
11

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
5), 116.29 (CH, C-4′), 113.76 (CH, C-10), 112.63 (CH, C-2′), 55.66
(OCH3). MS: calcd for C16H12O3 m/z = 252, found 253.1. HPLC peak
purity: 99.6%.
Table 4
IFD scores, EC50 and IC50 values of potent inhibitor molecules.
Molecules
EC50 (µM)
IC50 (µM)
IFD scores
(Z)-2-(4′-Methoxy-benzylidene)-benzofuran-3-one (1b). Yellow
solid, yield (52%), m.p. 137–139 ◦C. 1H NMR (DMSO‑d6, 600 MHz,
25 ◦C, TMS) δ ppm 7.96–7.94 (d, 2H, H-2′, H-6′), 7.78–7.75 (d, t, 2H, H-
4, H-5), 7.53–7.52 (d, 1H, H-7), 7.31–7.28 (t, 1H, H-6), 7.08–7.06 (d,
2f
(4.0 ± 0.1) × 103
(6.7 ± 0.1) × 103
(4.9 ± 0.1) × 103
0.56 ± 0.01
0.52 ± 0.01
3.5 ± 0.1
ꢀ 861.6
ꢀ 858.9
ꢀ 860.5
ꢀ 857.7
ꢀ 858.0
2g
3a
1.3 ± 0.2
QR
OMV
8.7 ± 0.1
(12.7 ± 0.3) × 103
(1.9 ± 0.1) × 103
2H, H-3′, H-5′), 6.91 (s, 1H, H-10), 3.82 (s, 3H, OCH3); 13C NMR
◦
–
(DMSO‑d , 200 MHz, 25 C, TMS) δ ppm 183.74 (C O, C-3), 165.61 (C,
–
6
C-4′), 161.34 (C, C-8), 145.63 (C, C-2), 137.79 (CH, C-6), 133.89 (CH, C-
4), 124.90 (CH, C-2′, C-6′), 124.64 (C, C-1′), 124.28 (CH, C-9), 121.62
(CH, C-5), 115.195 (CH, C-7), 113.65 (CH, C-10), 113.23 (CH, C-3′, C-
5′), 55.88 (OCH3). MS: calcd for C16H12O3 m/z = 252, found 253.1.
HPLC peak purity: 97.3%.
The physicochemical properties, viz. absorption, distribution, meta-
bolism and elimination (ADME), of the designed molecules were pre-
¨
dicted in-silico by QikProp 3.3 (Schrodinger LLC, New York, USA) [56].
The out file of LigPrep, employed to optimize the designed molecules,
was employed as the input for QikProp to predict the molecular de-
scriptors as well as properties of the molecules that are physically sig-
nificant and pharmaceutically relevant. Comparison between properties
of the designed molecules with those of 95% of overall known drugs was
done using the range values of each molecular descriptor provided by
QikProp.
(Z)-2-(3′-Chloro-benzylidene)-benzofuran-3-one (1c). Yellow
solid, yield (53%), m.p. 165–168 ◦C. 1H NMR (DMSO‑d6, 600 MHz,
25 ◦C, TMS) δ ppm 8.02 (s, 1H, H-2′), 7.95–7.94 (d, 1H, H-6′), 7.82–7.78
(d, t, 2H, H-4, H-5), 7.58–7.56 (d, 1H, H-7), 7.54–7.50 (d, t, 2H, H-4′, H-
5′), 7.34–7.31 (t, 1H, H-6), 6.93 (s, 1H, H-10). MS: calcd for C15H9O2Cl
m/z = 256.5, found 257.0. HPLC peak purity: 98.7%.
(Z)-2-(4′-Chloro-benzylidene)-benzofuran-3-one (1d). Yellow
solid, yield (62%), m.p. 154–160 ◦C. 1H NMR (DMSO‑d6, 600 MHz,
25 ◦C, TMS) δ ppm 8.00–7.99 (d, 2H, H-2′, H-6′), 7.81–7.79 (d, t, 2H, H-
4, H-5), 7.57–7.53 (d, d, 3H, H-3′, H-5′, H-7), 7.33–7.30 (t, 1H, H-6),
6.94 (s, 1H, H-10); 13C NMR (DMSO‑d6, 150 MHz, 25 ◦C, TMS) δ ppm
4.2.2. Chemistry
The starting materials and solvents utilized for synthesis were
assessed for their purity by determining their physical constants (viz.
melting and boiling point) and by thin-layer chromatography (TLC) on
Merck silica gel F254 plates. We report here the synthesis of four scaffolds
namely aurones, 3-indolinones, 4-quinolones and CAPi hybrids (Scheme
1 and Scheme 2). Synthesis was carried out in Carousel 6-reaction sta-
tion parallel synthesizer by Radleys. TLC was employed to monitor the
synthetic reaction progress. Physical constants (melting point) of the
reaction products were determined by Analab melting point apparatus
µThermoCal10. Infrared spectroscopy (IR), 1H NMR and 13C NMR were
employed for structural characterization of the synthesized molecules.
Mass spectra (MS) and HPLC purity of the synthesized molecules were
measured as well. IR experiments were recorded on an inhouse Bruker
Alpha-T spectrometer with 44 scans, and data were processed by OPUS
software. NMR experiments were recorded on 800 MHz Bruker Avance
spectrometer and 600 MHz Varian spectrometer using DMSO‑d6 and
CDCl3 solvent, and the data were processed using Bruker Topspin 2.1
and Varian software. 64 scans were recorded for 1D proton NMR (in 600
MHz NMR spectrometer), while for carbon NMR, 2064 scans (in 150
MHz NMR spectrometer) and 500 scans (in 200 MHz NMR spectrom-
eter) were recorded. LC-MS/MS were recorded on Water make Mass
Spectrometry LCMS: water alliance quadrupole mass. HPLC was per-
formed on Agilent 1200 series HPLC system.
–
–
183.59 (C O, C-3), 165.44 (C, C-8), 146.47 (C, C-2), 137.87 (CH, C-6),
134.65 (C, C-4′), 132.97 (C, C-1′), 130.86 (C, C-4), 129.21 (CH, C-3′, C-
5′), 124.44 (CH, C-2′, C-6′), 124.19 (C, C-9), 120.74 (CH, C-5), 113.31
(CH, C-7), 111.04 (CH, C-10). MS: calcd for C15H9O2Cl m/z = 256.5,
found 257.1. HPLC peak purity: 97.4%.
(Z)-2-(4′-Nitro-benzylidene)-benzofuran-3-one (1e). Yellow solid,
yield (48%), m.p. 211–215 ◦C. 1H NMR (DMSO‑d6, 600 MHz, 25 ◦C,
TMS) δ ppm 8.40–8.38 (d, 2H, H-3′, H-5′), 8.26–8.25 (d, 2H, H-2′, H-6′),
7.82–7.79 (d, t, 2H, H-4, H-5), 7.58–7.56 (d, 1H, H-7), 7.36–7.33 (t, 1H,
H-6), 7.11 (s, 1H, H-10). MS: calcd for C15H9NO4 m/z = 267, found
268.0.
4.2.2.2. General procedure for synthesis of 3-indolinones (2a- 2h). The
scheme is based on 5-exo cyclic condensation of 2-amino chalcone in
presence of Amberlyst-15 as a catalyst [57] as shown in Scheme 1. 0.075
mol of 2-amino chalcone derivatives were dissolved in 3–5 mL of acetic
acid. To this solution 10% w/w Amberlyst-15 was added. The mixture
was stirred at 80 ◦C for 2–5 h until starting material was not detected by
TLC (Hexane:Ethyl acetate; 3:2). The product was filtered and recrys-
tallized from absolute ethanol. The catalyst was obtained back by
washing with methanol, which could be reused.
4.2.2.1. General procedure for synthesis of aurones (1a–1e). 2-Hydrox-
ychalcones were primarily synthesized followed by their oxidative
cyclization to obtain aurones (Scheme 1). 2-Hydroxychalcones and
mercuric acetate [Hg(OAC)2] were dissolved in pyridine (15–20 mL) in
equimolar (0.002 mol) quantities at 27 ◦C. The reaction mixture was
kept on stirring at 110 ◦C for 1–2 h. Completion of reaction was moni-
tored by TLC (Hexane:Ethyl acetate; 3:2). The cooled reaction mixture
was poured into ice-cold water and acidified with dil. HCl (10% aqueous
solution). The precipitated solid was extracted with dichloromethane or
ethyl acetate, the extracts were dried over sodium sulphate bed and the
solvent was evaporated to give a solid which was recrystallized from
absolute ethanol.
(Z)-2-(2′-Methoxy-benzylidene)-1,2-dihydro-indol-3-one
(2a).
Pale yellow solid, yield (52%), m.p. 173–175 ◦C. 1H NMR (DMSO‑d6,
600 MHz, 25 ◦C, TMS) δ ppm 11.24 (s, 1H, NH), 7.93 (d, 1H, H-4), 7.67
(d, 1H, H-6′), 7.60 (d, 1H, H-7), 7.52 (t, 1H, H-6), 7.35 (t, 1H, H-4′),
7.00–6.90 (m, 4H, H-3′, H-5, H-5′, H-10), 3.69 (s, 3H, OCH3); 13C NMR
◦
–
(DMSO‑d , 200 MHz, 25 C, TMS) δ ppm 186.30 (C O, C-3), 157.49 (C,
–
6
C-2′), 144.97 (C, C-2), 135.61 (CH, C-6), 135.21 (C, C-8), 133.53 (CH, C-
4′), 128.25 (CH, C-6′), 124.01 (CH, C-4), 123.01 (CH, C-5′), 122.57 (C,
C-1′), 121.41 (C, C-9), 119.61 (CH, C-5), 112.81 (CH, C-7), 111.91 (CH,
C-3′), 108.38 (C-10), 54.53 (OCH3). MS: calcd for C16H13NO2 m/z =
251, found 250.96. HPLC peak purity: 99.7%.
(Z)-2-(3′-Methoxy-benzylidene)-1,2-dihydro-indol-3-one
(2b).
(Z)-2-(3′-Methoxy-benzylidene)-benzofuran-3-one (1a). Yellow
solid, yield (64%), m.p. 117–120 ◦C. 1H NMR (DMSO‑d6, 600 MHz,
25 ◦C, TMS) δ ppm 7.81–7.78 (d, t, 2H, H-4, H-5), 7.59–7.55 (d, d, s, 3H,
H-6′, H-7, H-2′), 7.43–7.40 (t, 1H, H-5′), 7.33–7.30 (t, 1H, H-6),
Pale yellow solid, yield (64%), m.p. 117–120 ◦C. 1H NMR (DMSO‑d6,
600 MHz, 25 ◦C, TMS) δ ppm 11.26 (s, 1H, NH), 7.94 (d, 1H, H-4), 7.62
(d, 1H, H-7), 7.53 (t, 1H, H-6′), 7.24–7.19 (m, 3H, H-2′, H-5′, 6′), 6.96 (t,
1H, H-5), 6.86 (d, 1H, H-4′), 6.65 (s, 1H, H-10), 3.65 (s, 3H, OCH3); 13C
7.04–7.03 (d, 1H, H-4′), 6.90 (s, 1H, H-10), 3.81 (s, 3H, OCH3); 13C NMR
◦
–
–
NMR (DMSO‑d6, 200 MHz, 25 C, TMS) δ ppm 188.23 (C O, C-3),
◦
–
–
(DMSO‑d , 200 MHz, 25 C, TMS) δ ppm 184.15 (C O, C-3), 165.96 (C,
6
159.72 (C, C-3′), 144.37 (C, C-2), 137.06 (C, C-8), 136.67 (C, C-1′),
C-3′), 159.92 (C, C-8), 146.86 (C, C-2), 138.21 (C, C-1′), 133.58 (CH, C-
6), 130.55 (CH, C-4), 124.80 (CH, C-5′), 121.28 (C, C-9), 117.12 (CH, C-
135.61 (CH, C-6), 126.61 (CH, C-5′), 125.01 (CH, C-6′), 124.01 (CH, C-
12

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
4), 121.24 (C, C-9), 119.61 (CH, C-5), 114.79 (CH, C-4′), 114.65 (CH, C-
2′), 112.81 (CH, C-7), 110.41 (CH, C-10), 55.25 (OCH3). MS: calcd for
C16H13NO2 m/z = 251, found 253.98. HPLC peak purity: 99.5%.
4′), 112.81 (CH, C-7), 110.41 (CH, C-10). MS: calcd for C15H11NO2 m/z
= 237, found 237.99.
(Z)-2-(4′-Methoxy-benzylidene)-1,2-dihydro-indol-3-one
(2c).
4.2.2.3. General procedure for synthesis of 4-quinolones (3a–3i). 4-
quinolones were obtained by 6-endo cyclocondensation of 2-amino
chalcone derivatives using iodine and dimethylsulfoxide (DMSO) as
shown in Scheme 1. 2-Amino chalcone derivatives were suspended in
DMSO (10 mL) and a crystal of iodine was added to it. The reaction
mixture was refluxed for 30 min and then diluted with water. The solid
obtained was filtered off, washed with 20% sodium thiosulphate and
recrystallized from absolute ethanol.
Pale yellow solid, yield (52%), m.p. 137–140 ◦C. 1H NMR (DMSO‑d6,
600 MHz, 25 ◦C, TMS) δ ppm 11.25 (s, 1H, NH), 7.94 (d, 1H, H-4), 7.62
(d, 1H, H-7), 7.55–7.49 (t, d, 3H, H-6, H-2′, H-6′), 6.97–6.95 (d, 3H, H-5,
H-3′, H-5′), 6.64 (s, 1H, H-10), 3.70 (s, 3H, OCH3); 13C NMR (DMSO‑d6,
◦
′
–
150 MHz, 25 C, TMS) δ ppm 188.23 (C O, C-3), 159.85 (C, C-4 ),
–
145.34 (C, C-2), 137.06 (C, C-8), 135.61 (CH, C-6), 130.22 (CH, C-2′, C-
6′), 129.72 (C, C-1′), 124.01 (CH, C-4), 121.24 (C, C-9), 119.61 (CH, C-
5), 113.98 (CH, C-3′, C-5′), 112.81 (CH, C-7), 110.56 (CH, C-10), 55.35
(OCH3). MS: calcd for C16H13NO2 m/z = 251, found 254.30. HPLC peak
purity: 99.2%.
2-(2′-Methoxy-phenyl)-1H-quinolin-4-one (3a). White solid, yield
(57%), m.p. 102–104 ◦C. 1H NMR (DMSO‑d6, 600 MHz, 25 ◦C, TMS) δ
ppm 11.50 (s, 1H, NH), 7.98 (d, 1H, H-6), 7.65 (d, 1H, H-9), 7.59–7.51
(t, t, 2H, H-7, H-8), 7.37 (d, 1H, H-6′), 7.28 (t, 1H, H-4′), 7.00 (d, 1H, H-
3′), 6.94–6.88 (t, 1H, H-5′), 6.02 (s, 1H, H-3), 3.74 (s, 3H, OCH3); 13C
(Z)-2-(3′, 4′, 5′-Trimethoxy-benzylidene)-1,2-dihydro-indol-3-one
(2d). Pale yellow solid (56%), m.p. 138–141 ◦C. 1H NMR (DMSO‑d6,
600 MHz, 25 ◦C, TMS) δ ppm 11.25 (s, 1H, NH), 7.93 (d, 1H, H-4), 7.62
(d, 1H, H-7), 7.52 (t, 1H, H-6), 6.95 (t, 1H, H-5), 6.84 (s, 2H, H-2′, H-6′),
6.64 (s, 1H, H-10), 3.73–3.64 (d, 9H, OCH3); 13C NMR (DMSO‑d6, 150
◦
–
NMR (DMSO‑d6, 200 MHz, 25 C, TMS) δ ppm 182.44 (C O, C-4),
–
155.01 (C, C-2′), 141.08 (C, C-10), 140.71 (C, C-2), 137.01 (CH, C-4′),
133.43 (CH, C-8), 130.32 (CH, C-6′), 125.01 (CH, C-6), 124.21 (C, C-1′),
123.83 (C, C-5), 123.31 (CH, C-7), 122.88 (CH, C-5′), 119.07 (CH, C-9),
110.53 (CH, C-3′), 106.19 (CH, C-3), 55.20 (OCH3). MS: calcd for
◦
′
′
–
–
MHz, 25 C, TMS) δ ppm 188.23 (C O, C-3), 154.24 (C, C-3 , C-5 ),
144.29 (C, C-2), 143.34 (C, C-4′), 137.06 (C, C-8), 135.61 (CH, C-6),
131.95 (C, C-1′), 124.01 (CH, C-4), 121.24 (C, C-9), 119.61 (CH, C-5),
112.81 (CH, C-7), 110.06 (CH, C-10), 107.52 (CH, C-2′, C-6′),
60.68–56.20 (OCH3). MS: calcd for C18H17NO4 m/z = 311, found
311.92. HPLC peak purity: 99.4%.
C
16H13NO2 m/z = 251, found 251.99. HPLC peak purity: 99.9%.
2-(3′-Methoxy-phenyl)-1H-quinolin-4-one (3b). White solid, yield
(64%), m.p. 130–134 ◦C. 1H NMR (DMSO‑d6, 600 MHz, 25 ◦C, TMS) δ
ppm 11.65 (s, 1H, NH), 8.09 (d, 1H, H-6), 7.76 (d, 1H, H-9), 7.71–7.63
(t, t, 2H, H-7, H-8), 7.45 (t, 1H, H-5′), 7.36 (d, 1H, H-6′), 7.27 (s, 1H, H-
2′), 7.09 (d, 1H, H-4′), 6.31 (s, 1H, H-3), 3.75 (s, 3H, OCH3); 13C NMR
(Z)-2-(2′-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2e). Pale
yellow solid, yield (58%), m.p. 136–138 ◦C. 1H NMR (DMSO‑d6, 600
MHz, 25 ◦C, TMS) δ ppm 10.91 (s, 1H, NH), 8.21 (d, 1H, H-4), 7.49 (d,
2H, H-3′, H-6′), 7.36–7.30 (m, 2H, H-6, H-4′), 7.24 (t, 1H, H-5),
◦
–
–
(DMSO‑d , 150 MHz, 25 C, TMS) δ ppm 182.44 (C O, C-4), 161.82 (C,
6
C-3′), 156.61 (C, C-2), 141.64 (C, C-10), 138.82 (C, C-1′), 133.43 (CH, C-
8), 125.01 (CH, C-6), 123.31 (CH, C-7), 122.56 (CH, C-4′), 122.41 (C, C-
5), 119.61 (CH, C-6′), 118.41 (CH, C-2′), 115.36 (CH, C-9), 108.21 (CH,
C-5′), 105.71 (CH, C-3), 55.50 (OCH3). MS: calcd for C16H13NO2 m/z =
251, found 251.97. HPLC peak purity: 97.6%.
7.20–7.16 (t, s, 2H, H-5′, H-10), 6.98 (d, 1H, H-7); 13C NMR (DMSO‑d6,
◦
–
–
150 MHz, 25 C, TMS) δ ppm 181.92 (C O, C-3), 148.98 (C, C-8),
138.01 (C, C-2′), 134.65 (C, C-1′), 133.47 (CH, C-6′), 129.59 (CH, C-4′),
129.47 (CH, C-6), 128.98 (CH, C-3′), 126.64 (C, C-2), 126.43 (CH, C-5′),
122.76 (CH, C-4), 121.10 (CH, C-5), 118.34 (C, C-9), 112.91 (CH, C-7),
104.28 (CH, C-10). MS: calcd for C15H10NOCl m/z = 255.5, found
255.92. HPLC peak purity: 94.5%.
2-(4′-Methoxy-phenyl)-1H-quinolin-4-one (3c). White solid, yield
(64%), m.p. 156–159 ◦C. 1H NMR (DMSO‑d6, 600 MHz, 25 ◦C, TMS) δ
ppm 11.66 (s, 1H, NH), 8.09 (d, 1H, H-6), 7.84–7.80 (d, 2H, H-3′, H-5′),
7.76 (d, 1H, H-9), 7.71–7.63 (t, t, 2H, H-7, H-8), 7.13–7.12 (d, 2H, H-2′,
H-6′), 6.32 (s, 1H, H-3), 3.75 (s, 3H, OCH3); 13C NMR (DMSO‑d6, 150
(Z)-2-(3′-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2f). Pale
yellow solid, yield (53%), m.p. 101–103 ◦C. 1H NMR (DMSO‑d6, 600
MHz, 25 ◦C, TMS) δ ppm 10.65 (s, 1H, NH), 8.11 (d, 1H, H-4), 7.79 (d,
1H, H-6′), 7.60 (s, 1H, H-2′), 7.33–7.30 (t, 2H, H-4′, H-5′), 7.18 (t, 1H, H-
◦
′
–
–
MHz, 25 C, TMS) δ ppm 182.44 (C O, C-4), 158.80 (C, C-4 ), 151.79
(C, C-2), 141.66 (C, C-10), 133.43 (CH, C-8), 128.79 (CH, C-2′, C-6′),
128.29 (C, C-1′), 125.01 (CH, C-6), 123.31 (CH, C-7), 122.41 (C, C-5),
115.36 (CH, C-9), 114.33 (CH, C-3′, C-5′), 105.65 (CH, C-3), 55.35
(OCH3). MS: calcd for C16H13NO2 m/z = 251, found 251.97. HPLC peak
purity: 96.7%.
5), 7.09 (t, 1H, H-6) 6.88–6.86 (d, 2H, H-7, H-10); 13C NMR (DMSO‑d6,
◦
–
–
200 MHz, 25 C, TMS) δ ppm 180.04 (C O, C-3), 148.95 (C, C-8),
135.11 (C, C-1′), 133.52 (C, C-3′), 133.21 (CH, C-2′), 131.13 (CH, C-6′),
130.65 (CH, C-4′), 129.47 (CH, C-6), 128.48 (CH, C-5′), 127.78 (C, C-2),
122.76 (CH, C-4), 121.10 (CH, C-5), 118.31 (C, C-9), 112.91 (CH, C-7),
107.98 (CH, C-10). MS: calcd for C15H10NOCl m/z = 255.5, found
255.92. HPLC peak purity: 99.9%.
2-(3′, 4′, 5′-Trimethoxy-phenyl)-1H-quinolin-4-one (3d). White
solid, yield (75%), m.p. 162–164 ◦C. 1H NMR (DMSO‑d6, 600 MHz,
25 ◦C, TMS) δ ppm 11.65 (s, 1H, NH), 8.09 (d, 1H, H-6), 7.77 (d, 1H, H-
9), 7.71–7.63 (t, t, 2H, H-7, H-8), 6.92 (s, 2H, H-2′, H-6′), 6.31 (s, 1H, H-
3), 3.84–3.75 (d, 9H, OCH3); 13C NMR (DMSO‑d6, 150 MHz, 25 ◦C, TMS)
(Z)-2-(4′-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2g). Pale
yellow solid, yield (62%), m.p. 156–160 ◦C. 1H NMR (DMSO‑d6, 600
MHz, 25 ◦C, TMS) δ ppm 10.67 (s, 1H, NH), 8.11 (d, 1H, H-4), 7.73 (d,
2H, H-2′, H-6′), 7.43 (d, 2H, H-3′, H-5′), 7.25 (t, 1H, H-6), 7.04 (t, 1H, H-
′
′
–
–
δ ppm 182.44 (C O, C-4), 153.72 (C, C-3 ), 152.86 (C, C-2, 5 ), 141.68
(C, C-10, C-4′), 141.64 (CH, C, C-8, C-1′), 133.43 (CH, C-6), 125.01 (CH,
C-7), 123.31 (C, C-5), 122.41 (CH, C-9), 115.36 (CH, C-3), 104.99 (CH,
C-2′), 104.08 (CH, C-6′), 60.68–56.26 (OCH3). MS: calcd for C18H17NO4
m/z = 311, found 311.91. HPLC peak purity: 99.8%.
5), 6.88–6.85 (d, 2H, H-7, H-10); 13C NMR (DMSO‑d6, 200 MHz, 25 ◦C,
′
–
–
TMS) δ ppm 180.02 (C O, C-3), 148.95 (C, C-8), 135.41 (C, C-1 ),
134.48 (CH, C-3′, C-5′), 132.16 (C, C-4′), 129.47 (CH, C-6), 129.22 (CH,
C-2′, C-6′), 127.07 (CH, C-4), 122.76 (CH, C-5), 121.10 (C, C-9), 118.31
(CH, C-7), 112.91 (C, C-2), 107.68 (CH, C-10). MS: calcd for C15H10NOCl
m/z = 255.5, found 255.92. HPLC peak purity: 99.3%.
2-(2′-Chloro-phenyl)-1H-quinolin-4-one (3e). Off-white solid, yield
(56%), m.p. 116–119 ◦C. 1H NMR (DMSO‑d6, 600 MHz, 25 ◦C, TMS) δ
ppm 11.62 (s, 1H, NH), 8.09 (d, 1H, H-6), 7.76 (d, 1H, H-9), 7.70 (t, 1H,
H-8), 7.64 (t, 1H, H-7), 7.59 (d, 1H, H-3′), 7.49 (t, 1H, H-4′), 7.42 (d, 1H,
H-6′), 7.24 (t, 1H, H-5′), 6.14 (s, 1H, H-3); 13C NMR (DMSO‑d6, 150
(Z)-2-(3′-Hydroxy-benzylidene)-1,2-dihydro-indol-3-one
(2h).
Pale yellow solid, yield (56%), m.p. 130–132 ◦C. 1H NMR (DMSO‑d6,
600 MHz, 25 ◦C, TMS) δ ppm 11.26 (s, 1H, NH), 9.87 (s, 1H, OH), 7.94
(d, 1H, H-4), 7.62 (d, 1H, H-7), 7.53 (t, 1H, H-6), 7.14 (t, 1H, H-5′), 7.07
◦
–
–
MHz, 25 C, TMS) δ ppm 182.44 (C O, C-4), 141.08 (C, C-10), 140.53
(C, C-2), 136.39 (C, C-1′), 133.43 (C, C-2′), 133.29 (CH, C-8), 130.79
(CH, CC-4′), 129.90 (CH, C-3′), 129.40 (CH, C-6′), 126.00 (CH, C-5′),
125.01 (CH, C-6), 123.83 (C, C-5), 123.31 (CH, C-7), 119.07 (CH, C-9),
113.95 (CH, C-3). MS: calcd for C15H10NOCl m/z = 255.5, found 258.90.
HPLC peak purity: 96.7%.
(d, 1H, H-6′), 6.96 (t, 1H, H-5), 6.65–6.63 (d, 3H, H-2′, H-4′, H-10); 13C
◦
–
–
NMR (DMSO‑d6, 200 MHz, 25 C, TMS) δ ppm 188.23 (C O, C-3),
156.32 (C, C-3′), 144.37 (C, C-2), 137.06 (CH, C-8), 136.74 (C, C-1′),
135.61 (CH, C-6), 130.04 (CH, C-5′), 124.85 (CH, C-6′), 124.01 (CH, C-
4), 121.24 (C, C-9), 119.69 (CH, C-2′), 119.61 (CH, C-5), 116.55 (CH, C-
13

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
2-(3′-Chloro-phenyl)-1H-quinolin-4-one (3f). Off-white solid, yield
(CH, C-2, C-6), 116.40 (CH, C-8), 45.9 (CH2, C-2′, C6′), 42.2 (CH2, C-3′,
C-5′). MS: calcd for C13H16N2O m/z = 216, found 216.99. HPLC peak
purity: 93.2%.
(53%), m.p. 109–110 ◦C. 1H NMR (DMSO‑d6, 600 MHz, 25 ◦C, TMS) δ
ppm 11.55 (s, 1H, NH), 7.99 (d, 1H, H-6), 7.66 (d, 1H, H-9), 7.61–7.53
(t, t, 2H, H-7, H-8), 7.46 (d, 1H, H-6′), 7.3–7.2 (m, 3H, H-2′, H-4′, H-5′),
6.21 (s, 1H, H-3); 13C NMR (DMSO‑d6, 150 MHz, 25 ◦C, TMS) δ ppm
3-(2-Methoxy-phenyl)-1-piperazin-1-yl-propenone (4b). White
solid, yield (57%), m.p. 224–225 ◦C. 1H NMR (CDCl3, 600 MHz, 25 ◦C,
TMS) δ ppm 7.94 (d, 1H, H-7), 7.5 (d, 1H, H-6), 7.33 (t, 1H, H-5), 6.96
(d, t, d, 3H, H-3, H-4, H-8), 3.8 (s, 1H, H-2), 3.7 (bd, 6H, H-2′, H-3′, H-5′,
H-6′), 1.6 (s, 1H, NH); 13C NMR (CDCl3 200 MHz, 25 ◦C, TMS) δ ppm
–
182.44 (C O, C-4), 156.61 (C, C-2), 141.64 (C, C-10), 138.36 (CH, C-
–
1′), 134.03 (C, C-3′), 133.43 (CH, C-8), 130.39 (CH, C-4′), 129.51 (CH,
C-5′), 128.68 (CH, C-2′), 125.20 (CH, C-6′), 125.01 (CH, C-6), 123.31
(CH, C-7), 122.41 (C, C-5), 115.36 (CH, C-9), 105.71 (CH, C-3). MS:
calcd for C15H10NOCl m/z = 255.5, found 255.28. HPLC peak purity:
85.5%.
–
166.4 (C O, C-9), 158.2 (C, C-2), 139.1 (CH, C-7), 130.9 (CH, C-4),
–
129.2 (CH, C-6), 124.0 (C, C-1), 120.6 (CH, C-5), 117.4 (CH, C-8), 111.1
(CH, C-3), 55.5 (OCH3), 45.6 (CH2, C-2′, C6′), 42.1 (CH2, C-3′, C-5′). MS:
calcd for C14H18N2O2 m/z = 246, found 246.16. HPLC peak purity:
99.8%.
2-(4′-Chloro-phenyl)-1H-quinolin-4-one (3 g). Off-white solid,
yield (59%), m.p. 170–179 ◦C. 1H NMR (DMSO‑d6, 600 MHz, 25 ◦C,
TMS) δ ppm 11.66 (s, 1H, NH), 8.09 (d, 1H, H-6), 7.84–7.83 (d, 2H, H-3′,
H-5′), 7.76 (d, 1H, H-9), 7.71–7.63 (t, t, 2H, H-7, H-8), 6.92–6.91 (d, 2H,
H-2′, H-6′), 6.32 (s, 1H, H-3); 13C NMR (DMSO‑d6, 200 MHz, 25 ◦C,
3-(4-Methoxy-phenyl)-1-piperazin-1-yl-propenone (4c). White
solid, yield (66%), m.p. 240–242 ◦C. 1H NMR (CDCl3, 600 MHz, 25 ◦C,
TMS) δ ppm 7.6 (d, 1H, H-7), 7.4 (d, 2H, H-2, H-6), 6.9 (d, 2H, H-3, H-5),
6.7 (d, 1H, H-8), 3.8 (s, 1H, H-4), 3.7 (bd, 6H, H-2′, H-3′, H-5′, H-6′),
1.57 (s, 1H, NH); 13C NMR (CDCl3 200 MHz, 25 ◦C, TMS) δ ppm 166.0
–
TMS) δ ppm 182.44 (C O, C-4), 151.79 (C, C-2), 141.66 (C, C-10),
–
135.80 (C, C-1′), 134.23 (C, C-4′), 133.43 (CH, C-8), 129.41 (CH, C-3′, C-
5′), 129.22 (CH, C-2′, C-6′), 125.01 (CH, C-6), 123.31 (CH, C-7), 122.41
(C, C-5), 115.36 (CH, C-9), 105.65 (CH, C-3). MS: calcd for C15H10NOCl
m/z = 255.5, found 255.91. HPLC peak purity: 99.3%.
–
(C O, C-9), 161.0 (C, C-4), 143.3 (CH, C-7), 129.4 (CH, C-2, C-6), 127.7
–
(C, C-1), 114.2 (CH, C-3, C-5), 113.8 (CH, C-8), 55.3 (OCH3), 45.5 (CH2,
C-2′, C6′), 42.1 (CH2, C-3′, C-5′). MS: calcd for C14H18N2O2 m/z = 246,
found 246.17. HPLC peak purity: 99.9%.
2-(3′-Nitro-phenyl)-1H-quinolin-4-one (3 h). Pale yellow solid,
yield (49%), m.p. 195–196 ◦C. 1H NMR (DMSO‑d6, 600 MHz, 25 ◦C,
TMS) 11.53 (s, 1H, NH), 8.02–7.9 (m, 3H, H-6, H-4′, H-6′), 7.73 (s, 1H,
H-2′), 7.65 (d, 1H, H-9), 7.60–7.52 (t, t, 2H, H-7, H-8), 7.45 (t, 1H, H-5′),
6.21 (s, 1H, H-3); 13C NMR (DMSO‑d6, 200 MHz, 25 ◦C, TMS) δ ppm
3-(3,4-Dimethoxy-phenyl)-1-piperazin-1-yl-propenone
(4d).
White solid, yield (70%), m.p. 241–242 ◦C. 1H NMR (CDCl3, 600 MHz,
25 ◦C, TMS) δ ppm 7.6 (d, 1H, H-7), 7.1 (d, 1H, H-6), 7.0 (s, 1H, H-2), 6.8
(d, 1H, H-5), 6.7 (d, 1H, H-8), 3.93 (s, 1H, H-4), 3.91 (s,1H, H-3), 3.8
182.44 (C O, C-4), 156.61 (C, C-2), 147.93 (C, C-3 ), 141.64 (C, C-10),
(bd, 6H, H-2′, H-3′, H-5′, H-6′), 1.58 (s, 1H, NH); 13C NMR (CDCl3 200
′
–
–
138.46 (C, C-1′), 133.82 (CH, C-5′), 133.43 (CH, C-8), 128.82 (CH, C-6′),
125.21 (CH, C-4′), 125.01 (CH, C-6), 123.47 (CH, C-2′), 123.31 (CH, C-
7), 122.41 (C, C-5), 115.36 (CH, C-9), 105.71 (CH, C-3). MS: calcd for
MHz, 25 C, TMS) δ ppm 165.9.0 (C O, C-9), 150.8 (C, C-3), 149.1 (C,
◦
–
–
C-4), 143.7 (CH, C-7), 127.9 (C, C-1), 122.0 (CH, C-8), 114.0 (CH, C-6),
111.1 (CH, C-5), 109.9 (CH, C-2), 55.9 (OCH3), 45.6 (CH2, C-2′, C6′),
42.1 (CH2, C-3′, C-5′). MS: calcd for C15H20N2O3 m/z = 276, found
276.28. HPLC peak purity: 99.9%.
C
15H10N2O3 m/z = 266, found 266.93. HPLC peak purity: 98.3%.
2-(4′-Nitro-phenyl)-1H-quinolin-4-one (3i). Pale yellow solid, yield
(45%), m.p. 195–196 ◦C. 1H NMR (DMSO‑d6, 600 MHz, 25 ◦C, TMS) δ
ppm 11.56 (s, 1H, NH), 8.22–8.21 (d, 2H, H-3′, H-5′), 8.00 (d, 1H, H-6),
7.79–7.77 (d, 2H, H-2′, H-6′), 7.67 (d, 1H, H-9), 7.62–7.54 (t, t, 2H, H-7,
3-(4-Chloro-phenyl)-1-piperazin-1-yl-propenone (4e). Off-white
solid, yield (76%), m.p. 275–276 ◦C. 1H NMR (CDCl3, 600 MHz,
25 ◦C, TMS) δ ppm 7.6 (d, 1H, H-7), 7.4 (d, 2H, H-3, H-5), 7.3 (d, 2H, H-
2, H-6), 6.8 (d, 1H, H-8), 3.76 (bd, 6H, H-2′, H-3′, H-5′, H-6′), 1.57 (s,
1H, NH).
H-8), 6.32 (s, 1H, H-3); 13C NMR (DMSO‑d6, 150 MHz, 25 ◦C, TMS) δ
′
–
–
ppm 182.44 (C O, C-4), 151.79 (C, C-2), 151.79 (C, C-4 ), 147.79 (C, C-
1′), 142.32 (C, C-10), 141.66 (CH, C-8), 133.43 (CH, C-2′), 128.76 (CH,
C-6′), 125.01 (CH, C-6), 124.31 (CH, C-3′), 124.21 (CH, C-5′), 123.31
(CH, C-7), 122.41 (C, C-5), 115.36 (CH, C-9), 105.65 (CH, C-3).
4.2.3. In vitro evaluation
4.2.3.1. Cells and virus
4.2.2.4. General procedure for synthesis of cinnamic acid-piperazine hy-
brids (4a – 4e). Synthetic scheme is shown in Scheme 2. Substituted
cinnamic acid (1 mol) was taken in organic solvent (dichloromethane,
DCM) and thionyl chloride (1.2 mol) was added followed by addition of
catalytic amount of dimethylformamide (DMF). The reaction mixture
was refluxed for 24 h. Completion of reaction was monitored by TLC
(Hexane:Ethyl acetate:methanol; 3:2:0.5). After completion of reaction,
solvent was evaporated to remove excess of thionyl chloride and to get
cinnamoyl chloride in the form of an amorphous powder. Unsubstituted
piperazine (3 mol) was dissolved in DCM and triethylamine (TEA) (3
equivalent) was added to it and stirred at 0 ◦C. Substituted cinnamoyl
chloride (1 mol) was dissolved in DCM and added dropwise to above
stirred solution at 0 ◦C. Completion of reaction was monitored by TLC
(Hexane : Ethyl acetate : methanol; 3:2:0.5). After completion, the
precipitate obtained was filtered and washed with water. The resulting
solid product was suspended in 5% sodium bicarbonate solution to
remove any acid impurity. The final product was recrystallized from
absolute alcohol.
4.2.3.1.1. Maintenance of MDCK cells. Madin-Darby canine kidney
(MDCK) cells were grown in minimum essential medium (MEM, Gibco,
by Life Technologies) complemented with 10% foetal bovine serum
(FBS, Gibco, by Life Technologies) and 1% Antibiotic (10,000 U/mL
penicillin and 0.5 mg/mL streptomycin) (Gibco, by Life Technologies).
4.2.3.1.2. Preparation of virus stock. The influenza A(H1N1)pdm09
virus was propagated in MDCK cells in the presence of 2 µg/mL tosyl
phenylalanyl chloromethyl ketone (TPCK)-trypsin. The stock of virus
was obtained by adding 500 µL of A(H1N1)pdm09 virus to 85–90%
confluent MDCK cells after removing the medium from flask and incu-
bated for 1 h at 37 ◦C with 5% CO2 to maximize the viral adsorption to
the cells. Subsequently, 4.5 mL of viral growth medium (2 µg/mL TPCK-
trypsin + MEM) was added and incubated at above mentioned condi-
tions for 5–7 days. Supernatant was then collected based on cytopathic
effect of the virus and stored at ꢀ 80 ◦C. These were repeated several
times for adequate virus stock.
4.2.3.2. Cytotoxicity studies. Cytotoxicity studies of the synthesized
molecules and standard drug viz. oseltamivir phosphate (OMVP) were
carried out by MTT-Formazan assay [58]. MDCK cells were inoculated
into 96-well plates and incubated at 37 ◦C with 5% CO2 for 24 h until
grown to 90% confluency. The media was then replaced with serum-free
DMEM (Dulbecco’s Modified Eagle Medium, without phenol red) con-
taining serially diluted molecules (1000, 500, 250, 200, 125, 100, 40,
3-Phenyl-1-piperazin-1-yl-propenone (4a). White solid, yield
(61.85%), m.p. 264–265 ◦C. 1H NMR (CDCl3, 600 MHz, 25 ◦C, TMS) δ
ppm 7.7 (d, 1H, H-7), 7.5 (d, 2H, H-2, H-6), 7.3 (m, 3H, H-3, H-4, H-5),
6.8 (d, 1H, H-8), 3.8 (bd, 6H, H-2′, H-3′, H-5′, H-6′), 1.6 (s, 1H, NH); 13C
◦
–
NMR (CDCl3 200 MHz, 25 C, TMS) δ ppm 165.74 (C O, C-9), 143.7
–
(CH, C-7), 135 (C, C-1), 129.9 (CH, C-4), 128.8 (CH, C-3, C-5), 127.8
14

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
10, 1, 0.1 µM corresponding to 10-folds, 5-folds and 2-folds dilutions).
density (OD) was measured at 570 nm. The percentage cell survival in
wells treated with molecules was calculated in reference to the unin-
fected untreated control i.e., CC and plotted against the molecule’s
concentration. EC50 was calculated using a linear regression analysis
tool. The experiment was performed in triplicate for each candidate
molecule.
After 16 h of incubation, the media was removed and 100 μL of a 0.5
mg/mL MTT (3-(4,5-Dimethylthiozol-2-yl)-3,5-dipheryl tetrazolium
bromide, Sigma-Aldrich) solution was added to each well and incubated
at 37 ◦C for 4 h. After removal of supernatant, 100 µL of dimethylsulf-
oxide (DMSO, Sigma-Aldrich) was added to each well so that the formed
formazan crystals get dissolved. Absorbance was measured at 540 nm in
a microplate reader. Data were normalized following the equation: Cell
viability (%) = (sample value - blank control)/(cell control - blank
control) × 100. The cytotoxic concentration 50% (CC50) was calculated
as the concentration at which 50% cells remain viable. It was calculated
in GraphPad Prism 5 from a dose response curve obtained using a non-
linear regression (curve fit).
4.2.3.6. Enzyme-based NA inhibition assay. NA inhibitory activity was
determined using the NA-Star® influenza neuraminidase inhibitor
resistance detection kit (Applied Biosystem) as per previously reported
method [13]. Succinctly, 25 μL of candidate molecules (conc. ranging
from 100 µM to 0.001 µM) or quercetin (QR, conc. ranging from 100 µM
to 0.001 µM) or oseltamivir carboxylate (OMVC, conc. ranging from
1000 nM to 1 nM) at two times the desired concentration was added in
duplicate to a 96-well microtiter plate. H1N1 virus was diluted 5-fold
4.2.3.3. Cytopathic effect (CPE) inhibition assay. The virus (100 µL) was
inoculated onto near confluent MDCK cell monolayers for 1 h to allow
for viral adsorption in 24-well plates after removing the media at 37 ◦C
under 5% CO2 atmosphere. 2 mL of molecules prepared at different
concentrations (500, 250, 200, 125, 100, 10, 1, 0.1 µM) in MEM con-
taining 2 µg/mL TPCK-trypsin was added in the allotted wells. The
cultures were incubated for 3–4 days at 37 ◦C under 5% CO2 atmosphere
to develop CPE if any, checked every day. Controls were set consisting of
only cells (i.e. no virus, no drug), referred as Cell Control (CC); and cells
with virus only (i.e. virus but no drug), referred as Virus Control (VC).
The candidate molecule was said to have antiviral activity if there was
absence of viral CPE. The antiviral effect of the molecules was deter-
mined by grading system developed by Kudi and Myint mentioned in our
previous work [59]. After 3–4 days, when VC showed 95–100% CPE, the
supernatant from each well was removed and tested for hemagglutina-
tion (HA) titer of virus. The reduction of HA titer of virus in molecule
treated wells was compared to HA titer of virus in VC well. The HA titer
was determined by means of hemagglutination inhibition (HAI) assay.
with the assay buffer. To the plate, 25
μL of the diluted virus was
mixed with the molecules and incubated at 37 ◦C for 20 min. The sub-
strate was diluted at 1:1000 in assay buffer immediately before use.
Then 10 μL of the diluted substrate were added to each well. The reac-
tion mixtures were kept for incubation at RT for 15 min and then acti-
vated by adding 60 L of accelerator. The chemiluminescent signal was
μ
quantified immediately by microplate reader [63]. A 50% inhibitory
concentration (IC50), relative to the activity in positive control i.e., the
reaction mixture well containing virus but no test molecule, was
determined to measure inhibitory activity of test molecules using
GraphPad Prism 5.
4.2.3.7. Enzyme kinetics assay. The mechanism of NA inhibition was
determined by kinetics assay with previously reported method [13]. To
96-well microtiter plate, 25 µL of OMVC or QR or candidate molecules
(two conc. bracketing IC50, along with I0) 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 catalysed by H1N1-NA was carried out by
measuring the chemiluminescent signal of hydrolysis product. The
parallel control experiment was implemented without molecules in the
mixture. Km and Vmax values were obtained from GraphPad Prism 5.
4.2.3.4. Hemagglutination inhibition (HAI) assay. Serial two-fold di-
lutions of supernatant of infected cells (100 μL) were prepared using 1X
PBS in a 96-well U-bottom plate. A 50 μL of 1% chicken red blood cells
was added to each well. After 30 min incubation at 22 ◦C, hemaggluti-
nation (reddish mesh across entire well) and precipitation of red blood
cells (red dot in the centre of well) was noted. The highest dilution factor
that caused hemagglutination is the HA titer of the virus. [60]. HA titer
of the VC and molecule treated wells was compared to ascertain the
reduction in %HA viral titer treated by molecules.
4.2.4. Statistical analyses
Mean ± standard deviation (std dev, SD) of at least three indepen-
dent biological replicates (n ≥ 3) was considered for analyses. For
antiviral assays, p < 0.05 (Mann-Whitney U test and Student’s t test) was
believed to be statistically significant. All the statistical analyses were
performed using GraphPad Prism 5 and the R statistical environment.
4.2.3.5. Cell-based screening of hit molecules using crystal violet (CV)
assay. CV assay was executed by reported method [61,62], with few
modifications. MDCK cells were seeded in 48-well tissue culture plates
and incubated for 24 h at 37 ◦C under 5% CO2 atmosphere. The media
was aspirated. Controls were set i.e., CC and VC (as discussed above)
along with DC (Drug control i.e., only drugs and cells). 30 µL of virus
dilution (256 HA units/mL) was added in each well except for CC and
DC. The plates were incubated for 1 h for maximum adsorption of virus.
The dilutions of candidate molecules at different concentrations (100
µM to 0.001 µM) were then added in the treatment cells while serum free
media was added in CC, DC and VC. The plates were kept for incubation
at 37 ◦C under 5% CO2 atmosphere for 24–36 h till the CPE was
observed. Further, the media was aspirated from all wells and washings
were given twice with 1X PBS. Plates were inverted on filter paper and
tapped to remove remaining fluid. 100 µL of crystal violet (0.5%) was
added to each well and incubated for 20 min at room temperature (RT)
on a bench rocker with frequency of 20 oscillations/min. The plates
were washed with tap water to remove the unbound dye. Plates were
inverted on filter paper and tapped to remove remaining fluid. The
plates were air dried at RT without the lid on. 300 µL of absolute
methanol was added to each well and incubated with lid on for 20 min at
RT on bench rocker with a frequency of 20 oscillations/min. Optical
Funding sources
The biological evaluation was funded by Indian Council of Medical
Research (ICMR), New Delhi, India (58/36/2013-BMS).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
K. Malbari and M. Kanyalkar are grateful to ICMR (58/27/2007-
BMS) for funding computational facility at Department of Pharmaceu-
tical Chemistry, Prin K M Kundnani College of Pharmacy. K. Malbari is
indebted to ICMR for Senior Research Fellowship (58/36/2013-BMS)
¨
and acknowledges Schrodinger Inc. for the trial license with OPLS3e
forcefield. Authors also acknowledge the infrastructure funding pro-
vided by Department of Science and Technology’s Fund for
15

K. Malbari et al.
Bioorganic Chemistry 114 (2021) 105139
[18] Y.B. Ryu, J.H. Kim, S.-J. Park, J.S. Chang, M.-C. Rho, K.-H. Bae, K.H. Park, W.
S. Lee, Inhibition of neuraminidase activity by polyphenol compounds isolated
from the roots of Glycyrrhiza uralensis, Bioorg. Med. Chem. Lett. 20 (2010)
971–974, https://doi.org/10.1016/j.bmcl.2009.12.106.
Improvement of S&T Infrastructure in Higher Educational Institutions
(DST-FIST), New Delhi (SR/FST/College-264/2015(C)) at Prin KM
Kundnani College of Pharmacy, Mumbai, India. K. Malbari and M. Joshi
acknowledge National NMR Facility provided by Tata Institute of
Fundamental Research (TIFR), Colaba, Mumbai, India.
[19] T.N.A. Nguyen, T.T. Dao, B.T. Tung, H. Choi, E. Kim, J. Park, S.I. Lim, W.K. Oh,
Influenza A (H1N1) neuraminidase inhibitors from Vitis amurensis., Food Chem.
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[20] G. Patrick, Drug design: optimizing target interactions, in: G. Patrick, An Introd. to
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Appendix A. Supplementary material
[21] H.D. Gravina, N.F. Tafuri, A. Silva Júnior, J.L.R. Fietto, T.T. Oliveira, M.A.N. Diaz,
M.R. Almeida, In vitro assessment of the antiviral potential of trans-cinnamic acid,
quercetin and morin against equid herpesvirus 1, Res. Vet. Sci. 91 (2011)
e158–e162, https://doi.org/10.1016/j.rvsc.2010.11.010.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105139.
[22] A.P. Stankyavichyus, L.M.M. Stankyavichene, M.S. Sapragonene, L.
V. Korobchenko, E.I. Boreko, G.V. Vladyko, Synthesis and antiviral activity of
cinnamic acid derivatives, Pharm. Chem. J. 22 (1988) 896–900, https://doi.org/
10.1007/BF00771641.
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