Design, synthesis and biological evaluation of substituted flavones and aurones as potential anti-influenza agents

Article abstract of DOI:10.1016/j.bmc.2019.115191

We designed a series of substituted flavones and aurones as non-competitive H1N1 neuraminidase (NA) inhibitors and anti-influenza agents. The molecular docking studies showed that the designed flavones and aurones occupied 150-cavity and 430-cavity of H1N1-NA. We then synthesized these compounds and evaluated these for cytotoxicity, reduction in H1N1 virus yield, H1N1-NA inhibition and kinetics of inhibition. The virus yield reduction assay and H1N1-NA inhibition assay demonstrated that the compound 1f (4-methoxyflavone) had the lowest EC₅₀ of 9.36 nM and IC₅₀ of 8.74 μM respectively. Moreover, kinetic studies illustrated that compounds 1f and 2f had non-competitive inhibition mechanism.

Full text of DOI:10.1016/j.bmc.2019.115191

Journal Pre-proofs
Design, Synthesis and Biological Evaluation of Substituted Flavones and Au-
rones as Potential Anti-Influenza Agents
Anand S. Chintakrindi, Devanshi J. Gohil, Abhay S. Chowdhary, Meena A.
Kanyalkar
PII:
S0968-0896(19)30823-5
https://doi.org/10.1016/j.bmc.2019.115191
BMC 115191
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
18 May 2019
16 September 2019
29 October 2019
Please cite this article as: A.S. Chintakrindi, D.J. Gohil, A.S. Chowdhary, M.A. Kanyalkar, Design, Synthesis and
Biological Evaluation of Substituted Flavones and Aurones as Potential Anti-Influenza Agents, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115191
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Design, Synthesis and Biological Evaluation of Substituted Flavones and Aurones as
Potential Anti-Influenza Agents
Anand S. Chintakrindi1 Devanshi J. Gohil2 Abhay S. Chowdhary2 Meena A. Kanyalkar1*
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
*Corresponding Author:
Dr (Mrs.) Meena Atul Kanyalkar
Plot 23, Jote Joy Building,
Rambhau Salgaonkar Marg, Cuffe Parade,
Mumbai 400005, India
Email:
meenatul@gmail.com
Tel no:
Fax no.:
91-22-22164368, 22164387
91-22-22165282
1

ABSTRACT
We designed a series of substituted flavones and aurones as non-competitive H1N1 neuraminidase
(NA) inhibitors and anti-influenza agents. The molecular docking studies showed that the designed
flavones and aurones occupied 150-cavity and 430-cavity of H1N1-NA. We then synthesized these
compounds and evaluated these for cytotoxicity, reduction in H1N1 virus yield, H1N1-NA
inhibition and kinetics of inhibition. The virus yield reduction assay and H1N1-NA inhibition
assay demonstrated that the compound 1f (4-methoxyflavone) had the lowest EC50 of 9.36 nM and
IC50 of 8.74 μM respectively. Moreover, kinetic studies illustrated that compounds 1f and 2f had
non-competitive inhibition mechanism.
Keywords: Flavone, Aurones, H1N1, Neuraminidase, Non-competitive, Influenza.
2

1. INTRODUCTION
Influenza A virus (IAV), a single-stranded (−) RNA virus that triggers annual seasonal
influenza epidemics and occasional global pandemics, is the leading cause of morbidity and
mortality worldwide. IAV is classified according to the antigenicity of their envelope
glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA regulates viral binding to host
cell sialic acid (SA) receptors and aids the fusion of viral and endosomal membranes to allow entry
of the viral genome into host cells. NA mediates release of the newly formed virus particle from
the host cell by cleaving the glycosidic linkage between SA-linked host cell-surface receptor and
HA, facilitating the spread of infection to other host cells.[1] The distinct antigenic properties of
different HA and NA molecules are used to classify influenza type A viruses into subtypes:
eighteen for HA (H1-H18) and eleven for NA (N1-N11).[2] Among various circulating subtypes
of IAV, influenza A (H1N1) virus has repeatedly caused pandemics in 1918, 1977 and 2009.[3]
In 1918, the most lethal pandemic in history, resulted in around 50 million deaths worldwide.[4]
The pandemic form of influenza is expected to reappear.[5]
The current anti-influenza drugs, neuraminidase (NA) inhibitors such as oseltamivir
(OMV) and zanamivir (ZMV) , are transition state analogs of sialic acid (SA) [6], and both the
drugs have comparable structures and hence similar binding interactions with NA. However,
mutations in the NA enzyme have conferred resistance to these drugs.[7] Thus, search for
molecules with newer scaffolds and different binding pattern in NA is imperative. Non-
competitive inhibitors inhibit the enzyme by binding allosterically to the target enzyme. Thus, non-
competitive inhibitors by their alternate binding mode could avoid the problem of resistance
caused due to mutations. The alternate binding sites in NA could be 150-cavity and 430-cavity that
are present adjacent to SA/OMV binding site. Compounds binding to 150-cavity have shown
3

antiviral activity against OMV resistant strains.[8] Furthermore, compounds projecting towards
430-cavity have demonstrated potent inhibition of various NA subtypes.[9] In our endeavour to
search for NA inhibitors with alternate binding mode, we had reported a series of chalcones
binding to 150-cavity as non-competitive inhibitors of H1N1-NA and anti-influenza agents. [10]
In our attempt to further optimize the activity, we have selected flavone and aurone scaffolds that
are the cyclized derivatives of chalcone, since rigidification by cyclization has been a common
approach to increase the activity of a lead compound.[11] Interestingly, several plant-based
flavones and aurones are reported to show H1N1-NA inhibition.[12]
Considering the above facts, we have designed a series of substituted flavones and aurones
[nine flavones (1a-1i) and nine aurones (2a-2i)] and studied their binding mode using
computational studies. Furthermore, we have synthesized sixteen (eight flavones and eight
aurones) compounds and assessed nine (six flavones and three aurones) of these compounds in-
vitro for cytotoxicity, reduction of H1N1 virus yield, H1N1-NA inhibition. Additionally, the
binding mechanism of the compounds to NA has been explored by kinetic experiments.
2. RESULTS AND DISCUSSION
2.1. Computational studies
The docking validation studies could acceptably replicate (RMSD 0.5236) the binding pose
of the co-crystallized ligand viz. ZMV in the H1N1-NA X-ray crystal structure (PDB ID:
3B7E)[13]. We designed several substituted flavones and aurones (Table 1) by varying
substituents on the phenyl ring that provide diverse lipophilic and electronic properties to the
designed molecules.
4

Table 1: Designed substituted flavones (2-(substitutedphenyl)-4H-chromen-4-one) and aurones
((2Z) 2-(1-(substitutedphenyl)methylene)benzofuran-3(2H)-one)
O
4
O
3
5
8
4
7
3
6
7
5
6
R1
2'
1'
R1
3''
2
R2
R3
3'
4'
2''
2
O
1
O
1
1''
1'
6'
R2
6''
4''
R3
5'
5''
Substituted Flavones (1a-1i)
Substituted Aurones (2a-2i)
Sr. No.
1.
Compound
R1
Cl
R2
H
R3
H
1a
1b
1c
1d
1e
1f
2.
H
Cl
H
3.
H
H
Cl
4.
OCH3
H
H
H
5.
OCH3
H
H
6.
H
OCH3
H
7.
1g
1h
1i
NO2
H
H
8.
NO2
H
H
9.
H
NO2
H
10.
11.
12.
13.
14.
15.
16.
17.
18.
2a
2b
2c
2d
2e
2f
Cl
H
H
Cl
H
H
H
Cl
OCH3
H
H
H
OCH3
H
H
H
OCH3
H
2g
2h
2i
NO2
H
H
NO2
H
H
H
NO2
5

These designed flavones and aurones were then docked in H1N1-NA catalytic site with a view to
examine the effect of various substituents on the binding mode. Docking studies with the designed
flavones and aurones revealed that all compounds dwelled in 150-cavity and 430-cavity (fig. 1) of
the catalytic site comparable to previously reported chalcones.[10] This showed that the binding
pattern was not altered due to cyclization of chalcone to flavone and aurone that had alternate
binding mode similar to chalcones.
6

7

Fig. 1 Docked conformation of compounds (a) 1c-1g, (b) 1a, 1b & 1h, (c) 1i, (d) 2b-2e, 2g & 2h,
(e) 2f & 2i, (f) 2a in H1N1-NA
The docked conformation of compounds 1c-1g revealed that the chromenone ring of these
compounds had hydrophobic interactions with the sidechains of Trp403, Ile427, Pro431, Lys432
(fig. 1a). However, the compounds 1a, 1b, 1h had reverse orientation compared to compounds 1c-
1g owing to the electrostatic interaction and hydrophobic interaction of chromenone ring with
Arg118 and Val149, respectively (fig. 1b). The compound 1i had different docked pose than other
designed flavones, in which the p-nitro group on its phenyl ring had electrostatic interaction with
Trp403 and Arg428, while its chromenone ring had hydrophobic interaction with Pro326 (fig. 1c).
The compounds 2b-2e, 2g and 2h are docked (fig. 1d) such that the benzofuranone ring of
these compounds occupied hydrophobic hole (Trp403, Ile427, Pro431, Lys432). Whereas, the
binding conformations of compounds 2f and 2i had an opposite orientation compared to
compounds 2b-2e, 2g and 2h in which the p-methoxy group on the phenyl ring of 2f occupied
hydrophobic hole (Trp403, Ile427, Pro431, Lys432) while the p-nitro group on the phenyl ring of
2i had electrostatic interaction with Trp403 and Arg428 (fig. 1e). Compound 2a had an altered
docked conformation in comparison with remaining designed aurones due to electrostatic
interaction of its benzofuranone ring with Arg118 (fig. 1f). The detailed study of these non-bonded
interactions of designed flavone and aurones with catalytic site residues is summarized in Table
S1 (see supplementary data).
2.2. Chemistry
The target compounds 1a-1i (substituted flavones) and 2a-2i (substituted aurones) were
synthesized via oxidative cyclisation of substituted 2′-hydroxychalcone using iodine in DMSO
8

[14] and mercuric acetate in pyridine [15] respectively using Scheme 1 (see supplementary data).
The suitable 2′-hydroxychalcones were formerly prepared via the classical Claisen–Schmidt
condensation [16] reaction between 2′-hydroxyacetophenone and appropriately substituted
benzaldehydes in basic conditions using NaOH (20 % w/v aqueous solution, 5 ml).[17] The
presence of methoxy / chloro group on B-ring of 2′-hydroxychalcone is favoured as compound 1a-
1f and 2a-2f were obtained in comparatively higher yield than compounds 1h-1i and 2h-2i which
have nitro substituent on the B-ring. The structures of the synthesized compounds were
characterized by nuclear magnetic resonance spectroscopy (1H NMR), infrared spectroscopy (IR)
and mass spectroscopy (MS). In the 1H NMR spectra of the synthesized compounds, chromenone
ring proton (H-3) of flavone and vinylic proton (H-1′) of aurone appeared as singlets in the range
of δ 6.95-7.28 and δ 6.91-7.20 respectively. The cyclic C-O stretch in flavone and aurone was
observed near 1370 cm-1 and 1300 cm-1 respectively. The carbonyl C=O stretch in flavone was
characterised by a band near 1700 cm-1, while in aurone the band was near 1650 cm-1. The IR band
for endocyclic C=C stretch in flavone was near 1600 cm-1, whereas the exocyclic C=C was near
1660 cm-1. 13C-NMR chemical shift for vinylic carbon (C-1′) of the synthesized aurones were in
range of 107.29 - 113.23 [18] which confirmed the formation of thermodynamically more stable
Z form of aurone. Mass spectra of all synthesized flavones and aurones gave the predicted [M+H]+
peak corresponding to their estimated molecular mass.
2.3. In-vitro evaluation
The MTT-Formazan assay was used to evaluate the cytotoxicity of the synthesized
compounds[19]. The concentration that led to a 50% reduction in cell viability (CC50) was used to
ascertain the cytotoxicity. Compounds 2b and 2e with CC50 values of 26.90 and 7.81 µM
respectively, showed cytotoxicity. The anti-influenza effect of non-cytotoxic compounds was
9

tested for reduction of H1N1 virus yield in MDCK cells. Oseltamivir acid (OMVA) and quercetin
(QR, reported as the most potent natural non-competitive inhibitor) [20] were used as the standard
for competitive inhibition and non-competitive inhibition respectively. The concentration that
resulted in 50% reduction in log2HA titre (EC50) was measured and used to determine the antiviral
effect, fig. 2.
Fig. 2 Effects of Oseltamivir acid (OMVA), Quercetin (QR), compounds 1f and 2f on H1N1 virus
yield
The virus yield reduction assay demonstrated that the designed flavones and aurones
appreciably decreased the viral titer of H1N1 (Table 2).
10

Table 2: In-vitro evaluation of tested compounds.
Sr. No. Compound CC50 (µM) EC50 (nM)
388±1.03 Not tested
SI
IC50 (µM)
1.
1a
1b
1c
-
Not tested
32.60±1.15
27.65±1.16
Not tested
10.76±1.09
8.74±1.15
2.
174.3±1.08 47.7±1.06
115.4±1.07 35.9±1.12
170.9±1.06 Not tested
263.4±1.21 12.57±1.05
191.8±1.08 9.36±1.04
3654
3214
-
3.
4.
1d
1e
5.
20955
20491
6.
1f
7.
1g
Not synthesized
8.
1h
1i
269.1±1.12 14.12±1.16
19058
15.36±1.20
13.20±1.21
Not tested
Not tested
45.36±1.13
Not tested
Not tested
37.50±1.12
9.
162.5±1.09 11.93±1.07
338.7±1.06 Not tested
13621
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
2a
-
2b
2c
26.9±1.05
Not tested
-
272.4±1.10 51.39±1.18
273.2±1.09 Not tested
5301
2d
2e
-
7.81±1.01
Not tested
-
2f
169.4±1.07 35.28±1.10
4802
2g
Not synthesized
2h
2i
396.3±1.06 Not tested
-
Not tested
41.00±1.19
7.75±1.13
213.0±1.10 46.32±1.12
253.8±1.08 84.46±1.03
713.4±1.07 7.1±1.16
4598
3007
QR
OMVA
100478 6.48±1.13 x 10-4
All compounds were examined in a set of duplicated experiment; CC (mean ±std error of mean) values represent the concentration that showed
50
50 % cytotoxicity; EC (mean ±std error of mean) values represent the concentration that resulted in 50 % log HA viral titre reduction; SI =
50
2
Selectivity Index was generated by the ratio of CC and EC ; IC (mean ±std error of mean) values of compounds represent the concentration
50 50 50
that caused 50% enzyme activity loss.
Compound 1f (flavone with p-methoxy group on phenyl ring) had highest activity with EC50 value
of 9.36 nM, comparable with the commercially used drug, OMVA (EC50 = 7.1 nM) while the
compound 2c (aurone with p-chloro group on phenyl ring) had the lowest activity with EC50 of
51.39 nM. The high selectivity index values of these compounds indicated that they decreased the
11

virus replication without adverse effect on the living host cells. H1N1-NA inhibition by the
compounds was determined by calculating the concentration required to inhibit 50% of the enzyme
activity (IC50). The IC50 values of the evaluated flavones ranged from 8.74 to 32.60 µM, while
aurones ranged from 37.50 to 45.36 µM (Table 2), fig. 3. A detailed analysis of H1N1-NA
inhibition indicated that among tested flavones, para-substitution on the phenyl ring is preferred
over meta-substitution since compounds 1c, 1f, 1i showed relatively better H1N1-NA inhibition
compared to their meta-isomers 1b, 1e, 1h. In view of this, we tested only para-substituted aurones
for H1N1 viral titre reduction and H1N1-NA inhibition. Furthermore, methoxy substitution on
phenyl rings of flavone and aurone is favoured over nitro and chloro substitution as methoxy
substituted compounds 1e, 1f and 2f showed higher H1N1-NA inhibition than the nitro substituted
(1h, 1i, 2i) and chloro substituted (1b, 1c, 2c) compounds. A similar trend in SAR is observed for
H1N1 viral titre reduction assay. Overall, the substituted flavones exhibited better anti-influenza
activity over substituted aurones.
12

Fig. 3 Effects of Oseltamivir acid (OMVA), compounds 1b-1c, 1e-1f, 1h-1i, 2c, 2f, 2i and
Quercetin (QR) on H1N1-NA for the hydrolysis of substrate
The virus yield reduction assay and NA inhibition studies showed that the tested flavonoids
were more active in suppressing viral replication than inhibiting the NA enzyme alone. These
results indicate that our tested flavonoids may have supplementary unknown mechanism
controlling virus replication.
Enzyme kinetics study was performed on compounds 1f and 2f, the most active (based on
H1N1-NA inhibition) substituted flavone and aurone, respectively, to verify the non-competitive
inhibition of designed flavonoids, as exhibited by docking studies. OMVA and QR were used as
13

standard for competitive and non-competitive inhibition, respectively. OMVA, structurally similar
to SA, showed competitive inhibition.
Fig. 4 Lineweaver–Burk plots for the inhibition of Oseltamivir acid (OMVA), 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
Fig. 4 shows the Lineweaver-Burk plot of 1/V versus 1/[S], one obtained in the absence of inhibitor
and at two different concentrations of inhibitor. Increasing concentrations of OMVA resulted in a
family of lines with common y-axis intercept but with a higher value of x-axis intercept, indicating
its competitive inhibition. Whereas, increasing concentrations of QR, compounds 1f and 2f
14

resulted in families of lines having the same x-axis intercepts along with an increase in the y-axis
intercept values, demonstrating their non-competitive inhibition.
3. CONCLUSION
The advent of resistance to the existent anti-influenza agents demands the pursuit of a
different strategy in influenza therapy. To achieve this goal, eighteen substituted flavones and
aurones were designed by varying substituent on phenyl ring and then docked in H1N1-NA. All
the docked compounds occupied the 150-cavity and 430-cavity of the catalytic site. Sixteen of
these designed compounds were then synthesised and their structures were confirmed by IR, 1H-
13
NMR, C-NMR and mass spectroscopy. Compounds 2b and 2e were cytotoxic. The designed
compounds suppressed the H1N1 viral titre. Compound 1f exhibited the highest activity
comparable with oseltamivir. Compound 1f also demonstrated the highest H1N1-NA inhibition
however, the activity is lower than oseltamivir. The virus yield reduction and H1N1-NA inhibition
studies demonstrated that our designed flavones and aurones are more active in decreasing the
H1N1 viral titer than inhibiting the H1N1-NA enzyme alone hinting that our compounds may have
an additional unknown mechanism in controlling virus replication. Compounds 1f and 2f, the most
active substituted flavone and substituted aurone, respectively, displayed non-competitive
inhibition.
4. CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of interest.
15

5. ACKNOWLEDGEMENTS
M. A. Kanyalkar 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. Chintakrindi also thanks ICMR, New Delhi for Senior
Research Fellowship (58/27/2007-BMS). A. S. Chintakrindi acknowledges Bombay College of
Pharmacy, Mumbai for providing HPLC chromatograms.
D. J. Gohil, and A. S. Chowdhary 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.
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FIGURE CAPTIONS
Fig. 1 Docked conformation of compounds (a) 1c-1g, (b) 1a, 1b & 1h, (c) 1i, (d) 2b-2e, 2g & 2h,
(e) 2f & 2i, (f) 2a in H1N1-NA
Fig. 2 Effects of Oseltamivir acid (OMVA), Quercetin (QR), compounds 1f and 2f on H1N1 virus
yield
Fig. 3 Effects of Oseltamivir acid (OMVA), compounds 1b-1c, 1e-1f, 1h-1i, 2c, 2f, 2i and
Quercetin (QR) on H1N1-NA for the hydrolysis of substrate
Fig. 4 Lineweaver–Burk plots for the inhibition of Oseltamivir acid (OMVA), 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
18

Highlights




Substituted flavones and aurones are docked in 150-cavity and 430-cavity of H1N1 neuraminidase
Substituted flavones and aurones reduce H1N1 viral titre
Substituted flavones and aurones are non-competitive inhibitors of H1N1 neuraminidase
Substituted flavones and aurones may be useful as potential anti-influenza agents
19

Graphical abstract
20