Molecular dynamics simulation, synthesis and topoisomerase inhibitory actions of vanillin derivatives: a systematic computational structural integument

Article abstract of DOI:10.1080/07391102.2021.1961867

A series of 4-hydroxy-3-methoxy benzaldehyde (vanillin) derivatives (3a–3r) was designed for the principle of Schiff base condensation with several individual sulfanilamide analogues. The inhibitory potencies of the designed compounds were evaluated through molecular docking simulation studies against the targets, breast cancer-topo isomerase-IIα and estrogen receptor-α; and the top scoring poses with higher binding energy were selected to assess the mode of binding and stability of each complex through molecular dynamics simulations. Compounds that remained stable in the active sites of the both target receptors through a number of strong H-bonds and hydrophobic contacts were selected. Based on the computational results, these selected compounds, 3b, 3e and 3f were synthesized and were followed up for structural elucidation attempts, by FT/ATR, ¹H NMR and ¹³C NMR. From the experimental in vitro studies on 3b, 3e and 3f, the following remarkable activities against breast cancer cell line were done; IC₅₀ values of 3b, 3e and 3f were noted, 6.7, 4.3 and 11 ng/mL, respectively. These newly synthesized compounds may be used as novel inhibitors of nuclear receptors with potential therapeutic applications in control of cancer. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2021.1961867

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Molecular dynamics simulation, synthesis and
topoisomerase inhibitory actions of vanillin
derivatives: a systematic computational structural
integument
Chita Ranjan Sahoo, Sudhir Kumar Paidesetty, Subbarayan Sarathbabu,
Budheswar Dehury, Nachimuthu Senthil Kumar & Rabindra Nath Padhy
To cite this article: Chita Ranjan Sahoo, Sudhir Kumar Paidesetty, Subbarayan Sarathbabu,
Budheswar Dehury, Nachimuthu Senthil Kumar & Rabindra Nath Padhy (2021): Molecular
dynamics simulation, synthesis and topoisomerase inhibitory actions of vanillin derivatives: a
systematic computational structural integument, Journal of Biomolecular Structure and Dynamics,
DOI: 10.1080/07391102.2021.1961867
To link to this article: https://doi.org/10.1080/07391102.2021.1961867
Published online: 06 Aug 2021.
Article views: 25
View supplementary material
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=tbsd20

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2021.1961867
Molecular dynamics simulation, synthesis and topoisomerase inhibitory actions
of vanillin derivatives: a systematic computational structural integument
Chita Ranjan Sahooa,b, Sudhir Kumar Paidesettyb, Subbarayan Sarathbabuc, Budheswar Dehuryd , Nachimuthu
ꢀ
Senthil Kumarc and Rabindra Nath Padhya
aCentral Research Laboratory, Institute of Medical Sciences & Sum Hospital, Siksha ‘O’ Anusandhan (Deemed to be University),
b
Bhubaneswar, Odisha, India; Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan (Deemed to
c
d
be University), Bhubaneswar, Odisha, India; Department of Biotechnology, Mizoram University, Aizawl, Mizoram, India; Biomedical
Informatics Centre, ICMR-Regional Medical Research Centre, Bhubaneswar, Odisha, India
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 6 April 2021
Accepted 24 July 2021
A series of 4-hydroxy-3-methoxy benzaldehyde (vanillin) derivatives (3a–3r) was designed for the prin-
ciple of Schiff base condensation with several individual sulfanilamide analogues. The inhibitory poten-
cies of the designed compounds were evaluated through molecular docking simulation studies
against the targets, breast cancer-topo isomerase-IIa and estrogen receptor-a; and the top scoring
poses with higher binding energy were selected to assess the mode of binding and stability of each
complex through molecular dynamics simulations. Compounds that remained stable in the active sites
of the both target receptors through a number of strong H-bonds and hydrophobic contacts were
selected. Based on the computational results, these selected compounds, 3b, 3e and 3f were synthe-
sized and were followed up for structural elucidation attempts, by FT/ATR, 1H NMR and 13C NMR.
From the experimental in vitro studies on 3b, 3e and 3f, the following remarkable activities against
breast cancer cell line were done; IC50 values of 3b, 3e and 3f were noted, 6.7, 4.3 and 11 ng/mL,
respectively. These newly synthesized compounds may be used as novel inhibitors of nuclear recep-
tors with potential therapeutic applications in control of cancer.
KEYWORDS
Vanillin; sulfanilamide;
synthesis; molecular
docking; molecular
dynamics; breast
cancer; caspase
1. Introduction
methoxy benzaldehyde is a phenolic aldehyde (a form of
glycoside, glucovanillin), and it is isolated from pods of
Vanilla planifolia (family, Orchidaceae); it has potent anti-
microbial and antioxidant activities (Bezerra et al., 2017).
Alternately, synthetic VA could be obtained from the natural
guaiacol by Vilsmaier formylation; the synthetic VA acted as
an intermediate subtract for the synthesis of several pharma-
ceuticals, papaverine, L-methyl dopa and Trimethoprim in
industries (Hocking, 1997). Earlier, VA had been in use as
food additives in flavoring chocolates, biscuits, confectionery
and a few more edibles; additionally, for the destruction of
pathogens from vegetables and fruits it is too in use.
Moreover, its synthetic analogues, ethylvanillin and butylhy-
droxytoluene have been used as food preservatives
(Bamforth et al., 1993).
Chemo-preventive effects of VA in carcinogenic rats in
causing moratorium of metastatic invasion of tumor cell lines
through the activation NF-KB was demonstrated (Akagi et al.,
1995; Cheng et al., 2008). Additionally, it was used in the
treatment of sickle cell anemia (Zhang et al., 2004). Anti-
mutagenic activities against chromosomal mutations in
human cells were exhibited by natural VA and cinnamalde-
Breast cancer is the most frequent cancer–type reported glo-
bally. Anti-cancer drugs have been developing in continual
efforts through the medicinal chemistry approach using the
advanced computational tools (Sahoo et al., 2019a, 2019b).
Indeed, the treatment for breast cancer involves a member
of nuclear receptors of cancer cells, with the estrogen recep-
tor-a (ERa) as a target; since, the over-expression and pre-
dominant involvement of ERa lead to the anomaly (Luo et
al., 2017). Concomitantly, topoisomerase II-alpha (topo IIa) is
used as a marker for breast cancer, which has a link to cell
proliferation with the HER2/neu gene (Depowski et al., 2000).
It had been established that a drug-protein-DNA ternary
complex would have an enormous approaches for the study
of drug-molecular interactions with ERa and topo IIa
(Nitiss, 2009).
Phytochemical-derived effective anti-cancer drugs are lim-
ited due to the inherent dearth in knowledge of properties
including host-toxicity of natural compounds; but there is a
need of semi-synthetic drugs with lesser toxicity. When such
a chemical is from the plethora of antioxidants or food-addi-
tives, the host-toxicity would be far less. Indeed, the naturally
occurring phyto-constituent vanillin (VA) or 4-hydroxy, 3- hyde (CA). Furthermore, these two natural aldehydes had
CONTACT Rabindra Nath Padhy
rnpadhy54@yahoo.com; rnpadhy54@gmail.com
ꢀ
Present address: Budheswar Dehury, Department of Chemistry, Technical University of Denmark, Lyngby, Denmark.
Supplemental data for this article can be accessed online at http://doi.org/10.1080/07391102.2021.1961867.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
C. R. SAHOO ET AL.
Figure 2. Rational synthetic approach for development new anticancer vanil-
lin congeners.
Figure 1. Structure of Schiff base derived antibacterial drugs.
2. Material and methods
inhibitory activity on the spontaneous mutation in bacteria.
VA was a non-toxic product seen in both oral administrations
and intraperitoneal injections; ethereal-linked VA with cou-
marin derivatives were reported as potent anti-inflammatory
and antioxidant agent (Ho et al., 2011).
Compounds obtained through Schiff-base condensation
generally contain the azomethine (CH ¼ N-) group formed by
the condensation of the primary amine and an aliphatic
2.1. Molecular docking
Structures of compounds 3a–3r were drawn virtually by the
principle of Schiff base condensations, which occurs between
4-hydroxy-3-methoxy benzaldehyde (VA) and Sulfa drugs; the
structures were drawn with ChemDraw Ultra tool-v.12.0
assigned with appropriate 2D orientations with appropriate
geometrical optimizations. Consequently, the designed indi-
vidual 2D structures were optimized with ChemSketch-2015,
and minimizations of energy were executed by OpenBabel
GUI-2.4.1. The target-protein structures cited with PDB IDs,
1AD1 (Dihydropteroate synthetase of the Gram-positive bac-
terium, Staphylococcus aureus) and 1AJ0 (dihydropteroate
synthase of Gram-negative bacterium Escherichia coli), as well
as 4COX (Cyclooxygenase-2), 1ZXN (Human DNA topoisomer-
ase IIa of human) and 3ERT (human estrogen receptor alpha);
which were obtained from RCSB-Protein Data Bank. The
€
long-chain/aromatic aldehyde in the presence of Bornsted-
Lowry or Lewis acid; and those have enormous pharmaco-
logical properties (Sahoo et al., 2021). A few of these
products, viz., Fumoxicillin, Furoxone and Nitrofurantoin are
established as antibacterial drugs for the treatment of several
infectious diseases (Figure 1). Additionally, Sulfanilamide (4-
amino benzene sulfonamide) had been seen as an important
isosteric group of 4-amino benzoic acid (PABA), required in
the bacterial substrate for syntheses dihydropteroate and
Tetrahydro folic acid (THF). Moreover, Sulfanilamide deriva-
tives have been in use as adjunct to other antibacterial selection of target receptors in the present study is based on
agents, viz., Trimethoprim and Pyrimethamine for synergistic
actions against pathogenic uropathogenic bacteria, which
resist this sulfa-drug due to mutations in the bacterial DHPS.
Additionally, VA derivatives had been reported having anti-
inflammatory activity against Cyclo-oxygenase (COX), which
was produced from a series of prostaglandin and other
inflammatory mediator pathways (Figure 2) (J. Sahoo et al.,
2020). This study was aimed at conjugation of VA with sulf-
anilamide (SA) analogues by the principle of Schiff base con-
densation and subsequent in silico assessment of the
compounds for drug-likeness properties by RO5 and ADMET.
The mode of inhibition of these compounds at the active
sites of the cancer nuclear receptor structures, that is isomer-
ase-IIa and estrogen receptor-a was evaluated through
molecular docking and molecular dynamics (MD) simulations
studies. Binding poses and predicted binding energies of the
designed compounds at the binding pockets of targets were
determined. The highest docking scored poses were further
evaluated through MD simulations in aqueous solution for
100 ns. After obtaining promising results from in silico studies
with compounds, 3b, 3e and 3f were synthesized and char-
acterized by spectral analyses viz. FT/ATR, 1H NMR and 13C
NMR. Finally, in vitro studies to test the inhibitory activity of
these molecules against breast cancer lines and assay of cas-
pase enzymes were performed (Figure 3). Overall, this study
successfully designed and synthesized 4-hydroxy-3-methoxy
benzaldehyde (VA) derivatives as potent small molecule with
nuclear receptor inhibitors using both in silico and in
vitro studies.
the evidences cited in published literature on their role in
development of therapeutics against cancer. The heteroa-
toms and water molecules were removed using BIOVIA
Discovery Studio Visualizer (DSV) Version 4.5 and the missing
sidechains were assigned by Server-What-If (Hekkelman et
al., 2010; Sahoo et al., 2020). All ligands and the PTP1B were
prepared for docking using AutoDockToolsv1.5.6. Prediction
of binding affinity and the intermolecular interaction were
done against individual dihydropteroate synthetase, cycloox-
ygenase-2, topo IIa and ERa. The default Kollman charges
and solvation parameters were allocated to the protein
atoms; whereas, Gasteiger charges were added to each lig-
and atom. Based on the co-crystallized ligand, a grid box
was defined. The parameters of the Lamarckian algorithm
and individual population methods were adopted from pub-
lished studies (Dehury et al., 2013; Girdhar et al., 2019; Sahoo
et al., 2020). The resultant docked conformations were clus-
tered using a root-mean-square deviation (RMSD) tolerance
of 2.0 Å. The ligand conformation with the lowest free energy
of binding (DG) and a greater number of hydrogen-bonds
(H-bonds), chosen from the most favored cluster was
selected for the protein-ligand studies using DSV, and
PyMOL (Baral et al., 2019; Dehury et al., 2013; Girdhar et al.,
2019; Sahoo et al., 2019c) To cross-check the accuracy of the
docked poses generated using AutoDock, we also docked
the co-crystallized ligand to these receptors in the ligand
binding pocket. The top scoring poses from docking simula-
tion were finally subjected to 100 ns molecular dynamics

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
Figure 3. Synthesized the potent anticancer molecules of vanillin derivative against MCF-7.
simulation to understand the dynamics of receptor-inhibitor
interaction at the molecular scale.
2.3.1. Synthesis of Schiff base VA-sulfur drugs (3a-3r)
An equimolar mixture of VA derivative 1a–1f individually in
lots of 1 mmole and substituted sulfanilamide 2a–2c in etha-
nolic solution were stirred together and refluxed for 2–3 h at
60–70 ꢂC; thereafter, the reaction mixture was poured into
ice-cold water and it was kept overnight in 15 ꢂC. Finally, the
obtained precipitate was filtered and was dried at room tem-
perature and recrystallized with hot ethanol.
2.2. Molecular dynamics simulation
The screened docked complexes were subjected to molecu-
ꢀ
lar dynamics simulation using Amber-99SB -ILDN force field
in GROMACS version2018.4 (Abraham et al., 2015). Ligand
topology was partaken by the interface of ACPYPE as done
previously (Dehury et al., 2017; Sahoo et al., 2020). Each com-
plex was solvated in the TIP3P water model and the electro-
neutralization was done by addition of 0.15 M NaCl. After
electro-neutralization, each system was subjected to energy
minimization using the steepest descent algorithm and was
terminated upon reaching a maximum force of 1000 kJ
molꢁ1 nmꢁ1. Furthermore, following energy minimization, all
systems were equilibrated through NVT and NPT ensemble
for 2 ns, each. The atoms forming H-bonds were constrained
by LINC algorithm. The Particle-mesh Ewald (PME) electro-
statics method was employed to treat all the long-range
electrostatic interactions. Finally, all systems were subjected
to production MD simulations for 100,000 ps (100 ns) at 300 K
and 1 bar pressure, as reported elsewhere (Dehury et al.,
2014, 2017). Trajectory files were collected after every 20 ps
and were used to gauge the dynamics stability of each sys-
tem through backbone RSMD, radius of gyration (Rg) and
Ca-RMSF. The change in the secondary structures were moni-
tored and mapped by VMD tool. The generated 2-D struc-
tures were sketched by Xmgrace and rendered using Adobe
Illustrator.
2.3.2. Synthesis of VA acetate 1e
VA was dissolved in a solution of dichloromethane (DCM)
and acetic anhydride in a conical flask, to which solution dry
pyridine in the under anhydrous condition was poured
(Yadav et al., 2018). The mixture was stirred for a few hours
at the ambient temperature. The reaction mixture was moni-
tored by TLC using an ‘ethyl acetate: n-hexane’ mixture.
Finally, DCM was evaporated from the mixture and the rest
contents were poured into ice-cold water to get vanillin acet-
ate. The obtained product was structurally confirmed in
terms of the found frequencies (d, cm-1); 3016 (OH str.), 2944
(ArCH), 2845 (CHO). 1747 (C ¼ Ostr. Carboxylate), 1677
(C ¼ Ostr.aldehyde), 1596(C ¼ Cstr.), 1276 (C-Ostr.),861 (tri-
subst. phenyl) that appeared in FTIR, which structure was fur-
1
ther confirmed by HNMR and DSC.
2.3.3. Synthesis of VA isobutyrate 1f
VA was dissolved in dichloromethane (DCM) and isobutyric
anhydride in a conical flask, to which solution of dry pyridine
was poured the under anhydrous condition. The mixture was
stirred for a few h at the ambient temperature. The reaction
mixture was monitored by TLC using ‘ethyl acetate: n-hex-
ane’ mixture. Finally, DCM was evaporated from the mixture
and the rest contents were poured into ice-cold water to get
vanillin isobutyrate. The obtained product was structurally
confirmed by the found frequencies (d, cmꢁ1); 3073 (OH
str,),2975 (ArCH), 2877 (CHO). 1759(C ¼ Ostr.carboxylate),
1697(C ¼ Ostr.aldehyde), 1599(C ¼ Cstr.), 1265(C-Ostr.), 862
(trisubs.phenyl) appeared in FTIR; which structures were fur-
2.3. Chemistry
All the chemicals were used herein Synthetic grade chemi-
cals from Merck company Ltd. The synthesized compounds
were
characterized
by
ATR/IR
(JASCO
FT/IR4600
Spectrophotometer); and 1H,13C-NMR spectra were recorded
on 400 MHz JEOL NMR spectrophotometer with deuterated
chloroform CDCl3, as the solvent and in comparison to the
standard TMS. Consequently, chemical shifts were reported
in ppm with d scale and melting points were measured by
using the Elico Melting point apparatus. Elemental analyses
of synthesized derivatives were studied by Perkin Elmer
Analyser-2400 CHNO/S.
1
ther confirmed by HNMR.

4
C. R. SAHOO ET AL.
doses 50 (LD50) were assessed. It was observed that 4-
hydroxy-3-methoxy benzaldehyde derivatives, 3b, 3e and 3f
had good absorption and cell permeability (Table 1).
2.4. In vitro studies
2.4.1. MTT assays
Cytotoxic effects of compounds 3b, 3e and 3f were exam-
ined by a non-radioactive, quantitative assay (colorimetric)
using tetrazolium salt, MTT (3-[4] [4,5-dimethylthiazol-2yl]-2,
5-diphenyl-tetrazolium bromide), Sigma-Aldrich, KGaA on
human-breast cancer cell-lines. Those viable cells were
seeded at 1 ꢃ 105 per mL level and were incubated at 37ꢂC,
5% CO2 and 100% relative-humidity. The compounds were
added at concentrations of 1, 2, 4, 8, 12, 16, 32, 64, 128, 256,
512 mg for 72 h. As the maximum, volumes in aliquots 20 mL
solvent were added to cells, and the similar volume of buffer
was added to control wells. The cytotoxicity assay was eval-
uated by MTT assay; consequent absorbance was measured
by a microplate-reader at 570 nm (wavelength). The viability
of cell (%) was calculated by formula; cell viability (%) ¼ OD
of treated sample/OD of the untreated sample ꢃ 100. The
IC50 values were obtained by nonlinear regression (a curve
fit) method, GraphPad Prism6.0 (Sarathbabu et al., 2019;
Subbarayan et al., 2016).
Moreover, the designed vanillin derivatives 3b, 3e and 3f
were docked against bacterial dihydropteroate synthetase,
inflammatory COX-2 (Table S1; Figures S16–S18); breast cancer-
topo IIa and Era were obtained binding affinity score with
binding interactions (Table 2; Figures S13–S14) and compared
with standard drug, Doxorubicin (Figure S15). The binding
affinity of 3a–3r of breast cancer-associated proteins were
within the range of ꢁ8.9 to ꢁ7.5 kcal/mol. Based on the mag-
nitudes of binding affinity, these derivatives were further con-
sidered structural for analysis and synthesis. Top scoring poses
from the docked complex structures of topoisomerase IIA with
3b, 3e and 3f revealed that ligands prefer to bind the central
position of the b-strand region being surrounded by adjoining
N-terminal residues (Figure 4). Val127 formed one H-bond with
3b, while Ser127 formed a single H-bond with 3e and 3f,
respectively. In addition, Arg70 and Lys126 formed a number
of hydrophobic contacts. In case of 3ERT, Cys225 formed a
strong hydrogen-bond consistently with three compounds,
where Thr42 formed a single hydrogen bond with 3b alone.
Leu41, Ala45 and Leu220 formed crucial hydrophobic (pi-alkyl)
contacts with tested compounds. However, esterified VA ana-
logue was interacting with ASN188,191, HIS194 of mushroom
tyrosine kinase inhibitor (Ashraf et al., 2015).
2.4.2. Caspase assay
The caspase-3, 8 and 9 assays were measured by the colori-
metric assay-kit of caspase, the standard protocol was pro-
vided by the manufacturer (Sigma-USA). The breast cancer
cell-lines were treated with individual compounds for 72 h;
Cell pellets (1 ꢃ 105 cells) were suspended in 100 mL of
chilled Cell Lysis Buffer, and cells were incubated on ice for
15-20min. Thereafter, it was centrifuged and the cells were
lysed at 16,000 to 20,000 ꢃ g for 10–15 min at 4 ꢂC. The
supernatants were transferred to a fresh tube, to which an
aliquot of 980 mL of 1ꢃ assay buffer was added to 10 mL of
cell lysates, and the reaction was initiated by addition of
10 mL of 20 mM caspase-3, 8 and 9 substrates and mixture,
gradually. Then, the tubes were covered and incubated at
37 ꢂC for 2 h; the incubation was continued for overnight as
an observation, if the color intensity was too low and the
absorbance was read at 405 nm. The activities of caspase-3, 8
and 9 in mol of pNA released per min/mL of cell lysate was
determined by formula: mol/PNPA/min/mL ¼ OD ꢃ d/e
mM ꢃ t ꢃ v, in comparison to the level of untreated control
(Sarathbabu et al., 2019).
3.2. MD Simulation
Dynamics stabilities of six selected docked complexes were
assessed through molecular dynamics information in aque-
ous solution for 100 ns, each. To determine stability of six
MD systems, backbone RMSD was computed with respect to
the initial structure used for production MD simulation.
During 100 ns simulations, all systems achieved stability after
ꢄ50 ns, where the average RMSD ranged from ꢄ0.31 to
0.34 nm, for the 1ZXN complexes (Figure 5). In contrast, a
lower RMSD was observed in case of 3ERT complexes with
an average RMSD of ꢄ0.21 nm (Figure 6). Looking at the
RMSD profiles, most of the complexes were attained equilib-
rium states could be observed. Furthermore, the RMSD value
each compound was monitored, where the average RMSD
values of 3b, 3e and 3f was 0.26, 0.23 and 0.21 nm, respect-
ively, in 1ZXN complexes. Similarly, in case of 3ERT com-
plexes, the RMSD value was below 0.2 nm. The radius of
gyration (Rg) profile of each system to understand the overall
conformational shape and compactness of each system dur-
ing MD were computed. The compactness of 1ZXN-com-
plexes by plotting their Rg with respect to time was
witnessed (Figure 5). The Rg values of 1ZXN-3b, 3e and 3f
complexes were as, 2.44, 2.42 and 2.41 nm, respectively. Like
RMSD, the average Rg values of 3ERT complexes were found
to be lower within the range of ꢄ1.85–1.91 nm, which indi-
cate the more compactness of these systems during MD. To
provide more detailed information of flexibility in 1ZXN and
3ERT complexes upon binding of selected compounds, the
3. Results and discussion
3.1. In silico screening of the compounds
The in silico Lipinski rule of five or Pfizer rule of five (RO5)
and the ADMET prediction approach offered a cost-effective
alternative to standardize prudent molecules based on struc-
ture linked to drug-likeness properties, toxicity classes and
pharmacokinetics parameters as preliminary approaches. In
RO5, the calculated molecular mass, H-bond donor, H-bond
acceptor, octanol-water partition coefficient and topological
polar-surface area with a percentage of absorption were
evaluated individually. Consequently, blood-brain-barrier, root mean squared fluctuations (RMSFs) as function of the
human-intestinal-absorption Caco-2-permeability and lethal residue number for the last 50 ns trajectories were measured

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
Table 1. Computational validation of 4-hydroxy-3-methoxy benzaldehyde analogue.
Lipinski rule of five (RO5)
HBA HBD clogP
Toxicity
Class LD50 (mg/kg) Blood-brain barrier Human intestinal absorption Caco-2 permeability
AB
(%)
Compound
MW
tPSA
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
383.09
428.08
461
6
8
6
6
7
7
7
9
7
7
8
8
7
9
7
7
8
8
2
2
2
1
1
1
2
2
2
1
1
1
2
2
2
1
1
1
2.95
82.81 80.43
6
5
6
6
6
6
6
5
6
6
5
5
5
5
5
5
5
5
15,800
2500
15,800
15,800
15,800
15,800
6000
2500
6000
6000
3000
3000
3500
3471
3471
3500
3471
3471
0.6751
0.7456
0.7940
0.5460
0.6029
0.6579
0.7705
0.8136
0.8702
0.5508
0.6777
0.7066
0.7721
0.7259
0.8380
0.5507
0.7001
0.7493
0.8599
0.8053
0.8370
0.9678
0.9365
0.9488
0.8497
0.7928
0.8253
0.9652
0.9314
0.9445
0.9640
0.9189
0.9584
0.9937
0.9872
0.9905
0.5170
0.5581
0.5310
0.5154
0.6221
0.6143
0.5534
0.5683
0.5504
0.5427
0.6121
0.6055
0.5825
0.5818
0.5542
0.5764
0.6232
0.6167
2.56 119.70 67.70
3.68
3.30
2.95
3.67
2.40
81.74 80.80
73.89 83.51
87.10 78.95
397.11
425.10
453.14
384.09
429.07
462.00
398.10
426.10
454.13
387.09
432.07
465
87
78.99
93.76 76.65
2.00 130.66 63.92
3.13
2.75
2.39
3.11
2.91
92.69 77.02
84.85 79.73
98.05 75.17
97.95 75.21
94.99 76.23
2.52 131.88 63.50
3.64
3.26
2.91
3.63
93.92 76.60
86.07 79.31
99.28 74.75
99.18 74.78
401.10
429.10
457.13
MW: molecular weight, HBA: number of hydrogen bond acceptor, HBD: number of hydrogen bond donor, clog P: logarithm of the octanol-water partition coeffi-
cient, tPSA: topological polar surface area, AB: absorbance, LD50: lethal doses, Blood-brain barrier; Human intestinal absorption; Caco-2 permeability (0 ¼ high
and 3 ¼ low).
Table 2. Molecular docking studies on breast cancer-associated receptors.
Docking score
(kcal/mol)
Topo-IIa
Docking score
(kcal/mol)
ERa
Compound
PDB: 1ZXN
Binding interactions with PDB: 1ZXN
PDB: 3ERT
Binding interactions with PDB: 3ERT
3a
3b
ꢁ7.6
ꢁ8.5
Arg70, Asn92, Thr194, Ile186, Ala64
Val127, Arg70, Ser120, Ile113, Lys126
ꢁ7.9
ꢁ8.7
Cys225, Leu220, Met223, Thr42, Leu41, Met116, Ala45
Cys225, Thr42, Leu220, Ala45, Leu41, Glu48, Leu82,
Leu44, Leu86, Thr42
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
ꢁ7.5
ꢁ7.7
ꢁ8.8
ꢁ8.5
ꢁ7.9
ꢁ7.9
ꢁ8.2
ꢁ8.0
ꢁ8.0
ꢁ7.4
Ser121, Ala136, Ile113, Arg70, Lys126
Asn92, Asn63, Arg70, Asn122, Lys126, Ser121
Ser121, Lys126, Arg70
ꢁ7.6
ꢁ7.8
ꢁ8.9
ꢁ8.5
ꢁ8.0
ꢁ7.8
ꢁ8.1
ꢁ8.2
ꢁ7.9
ꢁ7.6
Leu231, Asp46, Leu49, Leu41, Leu86, Leu220, Leu45
Leu220, Thr42, Met116, Ala45
Cys225, Leu220, Thr42, Met116, Ala45
Val228, Lys224, Cys225, Leu220, Thr42, Met116, Leu41, Ala45
Thr42, Leu220, Met116, Ala45, Thr42
Cys225, Thr42, Leu220, Glu48, Leu41, Phe99, Leu86, Ala45
Leu220, Leu79, Ile119, Met116, Ala45, Leu41, Met38
Leu220, Thr42, Met116, Ala45
Lys126, Ser121, Ile113, Arg70
Lys126, Asp66, Asn63, Ala136, Ile113, Arg70
Thr184, Asn92, Asn122, Arg70,
Lys137, Ala136, Asn122, Arg70
Arg70, Asn63, Ile186, Asn92, Thr184
Asn63, Asn122, Lys126, Arg70
Ala64, Ile186, Ile60, Phe114, Arg70,
Ser121, Lys126
Leu79, Leu220, Cys225, Ala45
Val228, Lys224, Cys225, Leu220, Met38, Ala45
3m
3n
3o
3p
ꢁ8.1
ꢁ8.0
ꢁ7.8
ꢁ7.6
Lys137, Ala136, Asn63, Arg70
Phe114, Ile99, Ala64, Asn92, Thr184, Arg70
Lys126, Arg70, Ser121, Ile113,
Asn122, Arg70, Phe114, Asn63, Ala136,
Lys137, Thr134, Gly135
ꢁ8.1
ꢁ8.2
ꢁ7.6
ꢁ7.7
Leu79, Ile119, His219, Leu220, Met38, Thr42, Leu41, Ala45
Asp46, Leu41, Met38, Ile119, His219, Met116
His219, Met116, Ala45, Leu220, Thr42
Asp46, Leu231, Trp78, Leu49, Leu220, Ala45, Met38, Leu41
3q
3r
Standard
ꢁ7.9
ꢁ8.3
ꢁ8.8
Asn63, Pro98, Val2, Ile5, Arg70
Thr184, Ile113, Phe114, Ser121, Arg70
Ser121, Ile113, Arg70
ꢁ8.0
ꢁ8.4
ꢁ7.2
Cys225, Thr42, Leu220, His219, Met116, Ala45
Met116, Ile119, Val228, Lys224, Cys225, Leu220
Leu220, Met116, Leu41, Ala45
PDB: protein data bank, topo IIa: topoisomerase II-alpha, ERa: Estrogen receptor-a.
(Figures 5 and 6). The RMSF values of 3b, 3e and 3f with vital roles in molecular recognition, stability and maintenance
1ZXN complexes were observed to be 0.193, 0.187 and of the overall conformation. The number of H-bonds were
0.188 nm, respectively, with least fluctuation of ligand bind- analyzed to get insight into the protein-ligand interaction
ing sites. However, in case of 3ERT complexes, slightly RMSF and stability. The average number of H- bonds in the 1ZXN
with three flopping regions with high degree of fluctuations complexes of with 3b, 3e and 3f were observed to 1.7, 2.3
as shown in RMSF plot were observed (Figure 6). The solvent and 1.67 per frame, respectively. In case 3ERT complexes, the
accessible surface area of each complex during 100 ns MD average numbers of H-bonds were computed to 3.21, 1.73
was too measured. The average solvent accessible area for and 1.13 for 3b, 3e and 3f systems. Comparative analysis of
1ZXN complexes was 208.8, 209.1 and 213.4 nm2 for 3b, 3e the docked conformation and MD simulated conformation
and 3f complexes, respectively. For the 3ERT complexes the revealed that these compounds gained more hydrophobic
SASA was computed to be 142.7, 141.9 and 140.8 nm, contacts with 1ZXN and 3ERT during MD. The post-MD inter-
respectively, for systems of the compounds 3b, 3e and 3f. actions of the compounds with 1ZXN and 3ERT have been
The intermolecular H-bonds in protein-ligand complexes play illustrated (Figure S1).

6
C. R. SAHOO ET AL.
Figure 4. Mode of binding of 3b, 3e and 3f against the target receptor DNA topoisomerase II (1ZXN) and Estrogen receptor alpha (3ERT) of the top ranked docked
conformations, obtained from AutoDock. (A–C): Interaction of 3b, 3e and 3f with 1ZXN. (D–F): Interaction of 3b, 3e and 3f with 3ERT. The interacting amino acids
are labeled where H-bonds are displayed in green dotted lines.
Figure 5. Dynamics stabilities of vanillin-topoisomerase-II complexes during 100 ns MD simulations. (A) Time evolution RMSD of the backbone atoms. (B) Radius of
gyration of the 1ZXN complexes. (C) RMSF of Ca atoms of complexes during the last 50 ns MD. (D) RMSD of each ligand over the 100 ns time scale. (E) Solvent
accessible surface area of each complex during MD. (F) The number of hydrogen bond interactions for during simulation between 1ZXN and compounds 3b, 3e
and 3f. Different color schemes have been used to present each complex system.
compared to the 3b (45.30 nm2) and 3e (35.72 nm2) com-
3.3. Principal component analysis
plexes were obtained that signifies this complex to have
occupied more conformational space with higher values.
Since the first few eigenvectors play an important role in
overall motions, the first two eigenvectors from each com-
plex for a clear depiction of the movements were consid-
ered. Similarly, in case of 3RT complexes, 3f ligand bound
PCA was used to investigate the total expansion or global
motion of protein during the MD simulation. The collective
motions and dynamics of each complex were exemplified
utilizing gmx covar and gmx anaeig modules of GROMACS
with reference to the Ca-atoms through PCA. The first few
eigenvectors for 1ZXN complexes with 3b, 3e and 3f complex displayed high degree motion with more conform-
accounted for more than 80%, of the motions observed for ational space indicates that 3f behaves differently as com-
the last 50 ns, respectively (Figure 7). In case of 3f complex, pared to other compounds. The principal component graph
a higher trace value of the covariance matrix 62.87 nm2 as of PC1 versus PC2 showed that both the systems showed a

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 6. Molecular dynamics of the vanillin- estrogen receptor-a complex at 100 ns scale. (A) Time evolution RMSD of the backbone atoms. (B) Radius of gyration
of the 3ERT complexes. (C) RMSF of Ca atoms of complexes during the last 50 ns MD. (D) RMSD of each ligand over the 100 ns time scale. (E) Solvent accessible sur-
face area of each complex during MD. (F) The number of hydrogen bond interactions for during simulation between 3ERT and compounds 3b, 3e and 3f. Different
color schemes have been used to present each complex system.
much defined and stable cluster for 3b and 3e as compared indicates the existence of azomethine, sulfonyl radical and
to 3f ligands; whereas, 3f did not show any stable cluster in
phase space. So, the two-dimensional PCA result analysis
suggested that the 3f somehow behaved differently than 3b
and 3e for recognizing drug targets.
phenolic ether, respectively.
1H/C13NMR spectral data indicate that the number of pro-
tons/carbons and environment were quantitively detected in
the structures and was confirmed in each, in related pro-
duced signals as either sharp singlet or doublet or multiplets.
1
The HNMR of compound 3f had appeared three sharp sin-
3.4. Chemistry
glets peaks with respect to azomethine proton(-CH ¼ N),
methoxy (OCH3) proton and isopropyl methyl -CH(CH3)2 pro-
ton at d 8.35, 3.85 and 1.35 ppm, respectively; and seven aro-
matic protons includes three protons in vanillin ring and four
in phenyl ring residue of the structure were appeared at
range of d 7.22–7.86 ppm whereas the same compound, in
13CNMR spectra found carbonyl of the carboxylate and car-
bon of azomethine group reflect the peak at d 174.89 and
161.20 ppm, respectively.
The desired Schiff base VA-sulfanilamide derivatives were
synthesized by re-fluxing of equimolar mixtures of alcoholic
solution of an individual vanillin analogue and Sulfanilamide
in glacial acetic acid (Scheme 1; Table S2) and finally recrys-
tallized from hot ethanol. The structures of synthesized com-
pounds were confirmed using different spectral studies
(Figures S2–S12). However, VA acetate 1e and VA isobutyrate
1f was prepared by the esterified the vanillin with acetic
anhydride and isobutyryl chloride in DCM solution and dry
pyridine, respectively (Yadav et al., 2018).
FTIR spectral data reveal the presence and identification
of functionality in the structure was confirmed in terms of
frequencies as strong, moderate or weak vibration stretching.
FTIR spectra of all these structures compound had been
3.4.1.
(E)-4-((4-hydroxy-3-methoxy-5-nitrobenzylidene)a-
mino)-N-(pyridin-2-yl)benzenesulfonamide (3b)
The obtained product was prepared by the condensation of
5-nitro vanillin and Sulfapyridine in glacial acetic acid. IR
(ATR, c,cmꢁ1): 2938 (CH2str.), 1684(CH ¼ N), 1379,
1159(SO2str.), 1269(C-Ostr.), 868 (ArCH bend.); 1H NMR
(DMSO-d6dppm, 400 MHz):9.89(s,1H, SO2NH) 8.23 (s,1H,
CH¼N), 7.26-7.65 (m, 7H, PhenylH), 7.92(m.1H.Pyridyl H-6),
6.72 (m.1H.Pyridyl H-5), 7.65(m.1H.Pyridyl H-4), 7.26
(m.1H.Pyridyl H-3), 4.04 (s,3H,OCH3); 13C NMR: 174.89, 160.00,
151.99, 151.05, 140.00, 138.11, 127.90, 121.34, 134.31, 114.01,
113.46, 56.95; ESI-HRMS (m/z):Anal. Calcd. for C19H16N4O6S
[M þ H] þ 428.08; found: 428.38(M þ 1); Anal. Calcd. % for
C19H16N4O6S Found: C, 53.27 (53.29); H, 3.76 (3.75); N,
observed strong vibration stretching at
a
range of
1693–1671 cmꢁ1, 1277–1269 cmꢁ1 and 1379–1351 cmꢁ1, that
indicated the presence of different functional groups such
as, azomethine -CH ¼ N- str., C-Ostr. of phenolic ether and
SO2str. of asymmetrical of sulfonamide, respectively. In add-
ition to that the intermediate precursors vanillin acetate 1e
and vanillin isobutyrate 1f had shown strong vibrational
stretching at 1747, 1759 cmꢁ1 with respect to carbonyl
stretching (C ¼ Ostr.) of carboxylate, respectively; whereas,
the appeared board stretching -OH frequencies of vanillin in
both compounds was absent. In compound 3b had shown
vibration stretching at 1684, 1378 and 1269 cmꢁ1, that 13.08(13.12); S, 7.48(7.53).

8
C. R. SAHOO ET AL.
Figure 7. Principal component analyses of 1ZXN and 3ERT complexes during the last 50 ns MD. (A) Plot of eigenvalues versus eigenvector index of vanillin-topoisomer-
ase-II complexes. (B) Projection of the motion of the vanillin-topoisomerase-II complexes in phase space along the EV1 and EV2. (C) Plot of eigenvalues versus eigenvector
index of vanillin-estrogen receptor-a complex. (D) Projection of the motion of the vanillin-estrogen receptor-a complexes in phase space along the EV1 and EV2.
Scheme 1. The designed strategies of 4-hydroxy-3-methoxy benzaldehyde (VA) derivatives.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Table 3. MTT assay of synthesized VA derivatives.
Concentration (ug/mL)
3b
3e
3f
1
2
4
8
12
16
32
64
128
256
512
90.55245
73.81143
62.9067
48.32062
33.34293
25.9243
18.07764
10.20265
9.748524
9.411892
8.592091
87.66775
67.54575
55.75987
35.74834
16.03693
15.36273
14.49673
14.37222
13.23229
11.39415
9.273656
84.61115
75.1765
57.77847
56.05938
48.84411
39.38866
32.44044
28.90762
23.89105
18.82097
11.88936
Represent in graphical concentration vs % cell viability.
Figure 8. Cytotoxic activity of compounds 3b, 3e and 3f on breast cancer cell
lines. Each value is the Mean SD of six replications.
step reactions, initially aryloxy ethyl bromide was converted
corresponding azide derivative by treatment of NaN3 in
DMF. Finally, the desired target 1,2,3-triazole was synthesized
by mixing o-propargylated vanillin with respective aryloxy
azide congener in the presence of CuSO4 5H2O and sodium
ascorbate in DMF. The obtained compounds had good bio-
logical action (Hussain et al., 2019). Furthermore, Schiff base
sulfonamides congener had good antimicrobial and cytotox-
icity activity. These Schiff base ligands derived from sulfa-
methoxazole (SMZ) and their complexes had been reported
possessing octahedral geometry configuration. The schiffbase
ligand was prepared by the reaction of 5-bromo salicylalde-
hyde with SMZ in the presence of ethanol and liberated lig-
and coordinated with transitional metal ions (Chohan et al.,
2012). Whereas, cupper-vanillin complexes were chemically
prepared by the treatment with o-vanillin and corresponding
amines, produce ligands-LH (Schiffbase), which further coord-
inate with the Cu (II) metal ions by the treatment with cup-
per acetate that acting as metal-based co-ligand. From these
intermediates, these co-ligands were chelated with respect-
ive imidazole analogues, 2-trifluromethyl benzimidazole,
2,4,5-triphenyl imidazole and 1,10-phenanthroline; conse-
quently, the developed compounds were mixed with ligand
Cu(II)-vanillin-schiff base complexes, which had good inhibi-
tory action against human carcinoma cell lines (Tabassum et
al., 2013). Thus, SARs indicate that the presence of azome-
thine and sulfonamide groups in individual structures would
be responsible for lending coveted synergistic anti-
cancer action.
3.4.2. (E)-2-methoxy-4-(((4-(N-(pyridin-2-yl)sulfamoyl)phe-
nyl)imino)methyl)phenyl acetate (3e)
The obtained product was prepared by the mixing of vanillin
acetate and Sulfapyridine in glacial acetic acid. IR (ATR,
c,cmꢁ1): 2937 (CH2str.), 1760 (C ¼ Ostr.),1675 (CH ¼ N), 1377,
1157 (SO2str.), 1268 (C-Ostr.), 954(S-Nstr.), 866 (ArCH bend.);
1H NMR (DMSO-d6dppm, 400 MHz): 9.95(s,1H, SO2NH), 8.29
(s,1H, CH¼N), 7.17-7.91 (m, 7H, PhenylH), 7.92(m.1H.Pyridyl
H-6), 6.80 (m.1H.Pyridyl H-5), 7.73(m.1H.Pyridyl H-4), 6.83
(m.1H.Pyridyl H-3), 3.90 (s,3H,OCH3) . 2.32 fs, 3H, CH3); 13C
NMR: 169.53, 161.20, 155.35, 151.71, 152.33, 148.78, 144.33,
142.11, 140.86, 138.51, 138.85, 135.61, 128.14, 128.18, 124.86,
122.56, 122.54, 121.19, 111.28, 117.30, 110.62, 56.05, 19.96;
ESI-HRMS
(m/z):
Anal.
Calcd.
for
C21H19N3O5S
[M þ H] þ 425.10; found: 425.13(M þ 1); Anal. Calcd. % for
C21H19N3O5S Found: C, 59.28 (59.25); H, 4.50 (4.56); N,
9.88(10.02); S, 7.54(8.53).
3.4.3. (E)-2-methoxy-4-(((4-(N-(pyridin-2-yl) sulfamoyl)phe-
nyl)imino)methyl)phenyl isobutyrate (3f)
The obtained product was prepared by the mixing of vanillin
isobutyrate and Sulfapyridine in glacial acetic acid. IR (ATR,
c,cmꢁ1): 2937 (CH2str.), 1750(C ¼ Ostr.),1693(CH ¼ N), 1379,
1
1163(SO2str.), 1270(C-Ostr.), 910(S-Nstr.), 858(ArCH bend.); H
NMR (DMSO-d6dppm, 400 MHz):9.94(s,1H, SO2NH) 8.35 (s,1H,
CH¼N), 7.22-7.86 (m, 7H, PhenylH), 7.95 (m.1H.Pyridyl H-6),
6.62 (m.1H.Pyridyl H-5), 7.63(m.1H.Pyridyl H-4), 7.13
(m.1H.Pyridyl H-3), 3.89 (s,3H,OCH3), 2.85fm, 1H, CH(CH3)2)g
1.35 fs, 6H, CH(CH3)2)g; 13CNMR: 174.89, 161.20, 155.35,
151.81, 143.33, 142.11, 140.86, 138.51, 134.31, 128.14, 123.57,
121.16, 115.28, 114.30, 110.62, 56.05, 34.00, 18.96; ESI-HRMS
(m/z):Anal. Calcd. for C23H23N3O5S [M þ H 1] þ 453.18; found:
453.39(M þ 1); Anal. Calcd. % for C23H23N3O5S Found: C,
60.91 (59.91); H, 5.11 (5.06); N, 9.27(9.72); S, 7.07(7.53).
However, Schiff base o-vanillin-sulfonamide with metal
complexes and the OH-proton was deprotonated as chelated
with metal ions and appearance of azomethine proton at d
8.62–8.93 ppm (Maurya & Patel, 1999). Moreover, o-propargy-
lated vanillin was synthesized from vanillin by the treatment
with propargyl bromide in the presence of K2CO3 and DMF.
3.5. In vitro studies
3.5.1. MTT assays
The synthetic compounds 3b, 3e and 3f were significantly
inhibited the viability and proliferation of MCF-7 cells in a
time and dose-dependent manner with an active IC50 of 6.7,
4.3 and 11 ng/mL, respectively (Figure 8; Table 3). The IC50
values of synthetic compounds 3b, 3e and 3f were showed
significant evidence of nuclear fragmentation (karyorrhexis),
chromatin condensation (pyknosis), loss of adhesion and
other symptomatic morphological modifications associated
with apoptosis in human breast cancer cell lines. These fea-
Thereafter, aryloxy azide derivatives were synthesized by two tures were not visually perceived in untreated control cells.

10
C. R. SAHOO ET AL.
acknowledge SPS, IMS & SUM Hospital of Siksha O Anusandhan Deemed
to be University, Bhubaneswar-3, for necessary facilities.
Disclosure statement
The authors declared no conflict of interest.
Funding
This study was supported by Indian Council of Medical Research,
Government of India (ICMR-SRF to CR Sahoo, File No. 45/3/2020-
DDI/BMS).
Figure 9. Caspase activity (Units/100 mg of total protein) of the breast cancer
cell lines with the treatment IC50 concentration of compounds 3b, 3e and 3f
for 72 h. Each value is the Mean SD of six replications.
References
ꢀ
Abraham, M. J., Murtola, T., Schulz, R., Pall, S., Smith, J. C., Hess, B., &
3.5.2. Caspase enzyme assay
Lindah, E. (2015). Gromacs: High performance molecular simulations
through multi-level parallelism from laptops to supercomputers.
SoftwareX, 1–2, 19–25. https://doi.org/10.1016/j.softx.2015. 06.001
Akagi, K., Hirose, M., Hoshiya, T., Mizoguchi, Y., Ito, N., & Shirai, T. (1995).
Modulating effects of ellagic acid, vanillin and quercetin in a rat
medium term multi-organ carcinogenesis model. Cancer Letters, 94(1),
113–121. https://doi.org/10.1016/0304-3835(95)03833-I
Ashraf, Z., Rafiq, M., Seo, S. Y., Babar, M. M., & Zaidi, N. U. S. S. (2015).
Synthesis, kinetic mechanism and docking studies of vanillin deriva-
tives as inhibitors of mushroom tyrosinase. Bioorganic & Medicinal
Chemistry, 23(17), 5870–5880. https://doi.org/10.1016/j.bmc.2015.06.
068
Bamforth, K. J., Jones, A. L., Roberts, R. C., & Coughtrie, M. W. H. (1993).
Common food additives are potent inhibitors of human liver 17a-ethi-
nyloestradiol and dopamine sulphotransferases. Biochemical
Pharmacology, 46(10), 1713–1720. https://doi.org/10.1016/0006-
2952(93)90575-H
Baral, N., Mohapatra, S., Raiguru, B. P., Mishra, N. P., Panda, P., Nayak, S.,
Pandey, S. K., Kumar, P. S., & Sahoo, C. R. (2019). Microwave-assisted
rapid and efficient synthesis of new series of Chromene-Based 1,2,4-
Oxadiazole derivatives and evaluation of antibacterial activity with
molecular docking investigation. Journal of Heterocyclic Chemistry,
56(2), 552–565. https://doi.org/10.1002/jhet.3430
The activation of the Caspase-8 enzyme was observed as
high when compared with caspases 3 and 9 in breast cancer
cell lines with the treatment of IC50 concentration of com-
pounds 3b, 3e and 3f for 72 h (Figure 9). These results were
confirmed that the synthetic compounds were induced
apoptotic cell death based on the activation of caspase-8
mediated pathway in breast cancer cell lines.
The individual VA was treated as an anti-mutagenic agent
and inhibitor of chemical carcinogenesis of mouse breast
cancer cells in vitro and in vivo (Lirdprapamongkol et al.,
2005). Consequently, the combination of VA and Doxorubicin
was reported as non-toxic for the treatment of enzymatic
and cytotoxic assays, particularly required for breast cancer
(Elsherbiny et al., 2016). Likewise, VA complexed with Mn(II)
and Co(II) was shown effective against human breast cancer
cell-line MC-7 at IC50 value 6.35 mmol/L (Yousef et al., 2018),
while the present compound 3e was notably effective at
4.3 ng/mL the same cancer cell lines.
Bezerra, C. F., Camilo, C. J., do Nascimento Silva, M. K., de Freitas, T. S.,
Ribeiro-Filho, J., & Coutinho, H. D. M. (2017). Vanillin selectively modu-
lates the action of antibiotics against resistant bacteria. Microbial
Pathogenesis, 113, 265–268. https://doi.org/10.1016/j.micpath.2017.10.
052
Cheng, W. Y., Wu, S. L., Hsiang, C. Y., Li, C. C., Lai, T. Y., Lo, H. Y., Shen,
W. S., Lee, C. H., Chen, J. C., Wu, H. C., & Ho, T. Y. (2008). Relationship
between San-Huang-Xie-Xin-Tang and its herbal components on the
gene expression profiles in HepG2 cells. The American Journal of
4. Conclusion
In this study, synthesis and characterization of novel Schiff
Base-vanillin-Sulfa drug derivatives were established through
in silico and in vitro studies. Primarily, the derivatives of 4-
hydroxy-3-methoxy benzaldehyde were validated through in
silico RO5, ADMET and molecular docking and dynamics
approaches at 100 ns run time, the ligand-receptor binding
affinity at least binding energy score were confirmed by H-
bonding interaction at CYS225 of 3ERT and VAL127, SER121
of 1ZXN in epitome docking poses. The compounds, 3b and
3e had promising ligand efficiency, absorbance and cell
membrane permeability and permeable toxicity profiles.
Indeed, SAR studies revealed that the compounds with sulfo-
moyl moiety, phenyl carboxylate system with azomethine
functionality would have shown anticancer activities.
Chinese
Medicine,
36(4),
783–797.
https://doi.org/10.1142/
S0192415X08006235
Chohan, Z. H., Shad, H. A., & Supuran, C. T. (2012). Synthesis, character-
ization and biological studies of sulfonamide Schiff’s bases and some
of their metal derivatives. Journal of Enzyme Inhibition and Medicinal
Chemistry, 27(1), 58–68. https://doi.org/10.3109/14756366.2011.574623
Dehury, B., Behera, S. K., & Mahapatra, N. (2017). Structural dynamics of
Casein Kinase I (CKI) from malarial parasite Plasmodium falciparum
(Isolate 3D7): Insights from theoretical modelling and molecular simu-
lations. Journal of Molecular Graphics
& Modelling, 71, 154–166.
https://doi.org/10.1016/j.jmgm.2016.11.012
Dehury, B., Patra, M. C., Maharana, J., Sahu, J., Sen, P., Modi, M. K.,
Choudhury, M. D., & Barooah, M. (2014). Structure-based computa-
tional study of two disease resistance gene homologues (Hm1 and
Hm2) in maize (Zea mays L.) with implications in plant-pathogen
interactions. PLoS One, 9(5), e97852 https://doi.org/10.1371/journal.
pone.0097852
Acknowledgments
Authors are thankful to the Department of Chemistry, Ravenshaw
University, Cuttack for 1H, 13C-spectral facilities; Department of
Biotechnology, Mizoram Central University, Aizawl; and we gratefully

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Dehury, B., Sahu, M., Patra, M. C., Sarma, K., Sahu, J., Sen, P., Modi, M. K.,
Choudhury, M. D., & Barooah, M. (2013). Insights into the structure-
function relationship of disease resistance protein HCTR in maize (Zea
mays L.): A computational structural biology approach. The Journal of
Molecular Graphics and Modelling, 45, 50–64. https://doi.org/10.1016/j.
jmgm.2013.08.011
Depowski, P. L., Rosenthal, S. I., Brien, T. P., Stylos, S., Johnson, R. L., &
Ross, J. S. (2000). Topoisomerase IIa expression in breast cancer:
Correlation with outcome variables. Modern Pathology, 13(5), 542–547.
https://doi.org/10.1038/modpathol.3880094
Elsherbiny, N. M., Younis, N. N., Shaheen, M. A., & Elseweidy, M. M.
(2016). The synergistic effect between vanillin and doxorubicin in
Ehrlich ascites carcinoma solid tumor and MCF-7 human breast cancer
cell line. Pathology, Research and Practice, 212(9), 767–777. https://doi.
org/10.1016/j.prp.2016.06.004
Girdhar, K., Dehury, B., Kumar Singh, M., Daniel, V. P., Choubey, A.,
Dogra, S., Kumar, S., & Mondal, P. (2019). Novel insights into the
dynamics behavior of glucagon-like peptide-1 receptor with its small
Sahoo, C. R., Paidesetty, S. K., Dehury, B., & Padhy, R. N. (2020). Molecular
dynamics and computational study of Mannich-based coumarin deriv-
atives: Potent tyrosine kinase inhibitor. Journal of Biomolecular
Structure
& Dynamics, 38(18), 5419–5428. https://doi.org/10.1080/
07391102.2019.1701554
Sahoo, C. R., Paidesetty, S. K., & Padhy, R. N. (2019a). Norharmane as a
potential chemical entity for development of anticancer drugs.
European Journal of Medicinal Chemistry, 162, 752–764. https://doi.org/
10.1016/j.ejmech.2018.11.024
Sahoo, C. R., Paidesetty, S. K., & Padhy, R. N. (2019b). Nornostocine con-
geners as potential anticancer drugs: An overview. Drug Development
Research, 80(7), 878–892. https://doi.org/10.1002/ddr.21577
Sahoo, C. R., Patro, R., Sahoo, J., Padhy, R. N., & Paidesetty, S. K. (2019c).
Design, molecular docking of synthesized Schiff-based thiazole/pyri-
dine derivatives as potent antibacterial inhibitor. Indian Drugs, 56(11),
20–25.
Sahoo, C. R., Sahoo, J., Mahapatra, M., Lenka, D., Sahu, P. K., Dehury, B.,
Padhy, R. N., & Paidesetty, S. K. (2021). Coumarin derivatives as prom-
ising antibacterial agent (s). Arabian Journal of Chemistry, 14(2),
102922. https://doi.org/10.1016/j.arabjc.2020.102922
Sahoo, J., Sahoo, C. R., Nandini Sarangi, P. K., Prusty, S. K., Padhy, R. N., &
Paidesetty, S. K. (2020). Molecules with versatile biological activities
bearing antipyrinyl nucleus as pharmacophore. European Journal of
Medicinal Chemistry, 186, 111911 https://doi.org/10.1016/j.ejmech.
2019.111911
molecule agonists. Journal of Biomolecular Structure
& Dynamics,
37(15), 3976–3986. https://doi.org/10.1080/07391102.2018.1532818
Hekkelman, M. L., Te Beek, T. A. H., Pettifer, S. R., Thorne, D., Attwood,
T. K., & Vriend, G. (2010). WIWS: A protein structure bioinformatics
web service collection. Nucleic Acids Research, 38(suppl Web Server
issue), W719–723. https://doi.org/10.1093/nar/gkq453
Ho, K. L., Yazan, L. S., Ismail, N., & Ismail, M. (2011). Toxicology study of
vanillin on rats via oral and intra-peritoneal administration. Food and
Chemical Toxicology : An International Journal Published for the British
Industrial Biological Research Association, 49(1), 25–30. https://doi.org/
10.1016/j.fct.2010.08.023
Hocking, M. B. (1997). Vanillin: Synthetic flavoring from spent sulfite
liquor. Journal of Chemical Education, 74(9), 1055. https://doi.org/10.
1021/ed074p1055
Hussain, M., Qadri, T., Hussain, Z., Saeed, A., Channar, P. A., Shehzadi,
S. A., Hassan, M., Larik, F. A., Mahmood, T., & Malik, A. (2019).
Synthesis, antibacterial activity and molecular docking study of vanil-
lin derived 1,4-disubstituted 1,2,3-triazoles as inhibitors of bacterial
DNA synthesis. Heliyon, 5(11), e02812 https://doi.org/10.1016/j.heliyon.
2019.e02812
Lirdprapamongkol, K., Sakurai, H., Kawasaki, N., Choo, M. K., Saitoh, Y.,
Aozuka, Y., Singhirunnusorn, P., Ruchirawat, S., Svasti, J., & Saiki, I.
(2005). Vanillin suppresses in vitro invasion and in vivo metastasis of
mouse breast cancer cells. European Journal of Pharmaceutical
Sciences, 25(1), 57–65. https://doi.org/10.1016/j.ejps.2005.01.015
Luo, G., Chen, M., Lyu, W., Zhao, R., Xu, Q., You, Q., & Xiang, H. (2017).
Design, synthesis, biological evaluation and molecular docking studies
of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERa
as anti-breast cancer agents. Bioorganic & Medicinal Chemistry Letters,
27(12), 2668–2673. https://doi.org/10.1016/j.bmcl.2017.04.029
Maurya, R. C., & Patel, P. (1999). Synthesis, magnetic and special studies
of some novel metal complexes of Cu(II), Ni(II), Co(II), Zn(II), Nd(III),
Sarathbabu, S., Marimuthu, S. K., Ghatak, S., Vidyalakshmi, S.,
Gurusubramanian, G., Ghosh, S. K., Subramanian, S., Zhang, W., &
Kumar, N. S. (2019). Induction of apoptosis by pierisin-6 in HPV posi-
tive HeLa and HepG2 cancer cells is mediated by the caspase-3
dependent mitochondrial pathway. Anticancer Agents in Medicinal
Chemistry,
19(3),
337–346.
https://doi.org/10.2174/
1871520619666181127113848
Subbarayan, S., Marimuthu, S. K., Nachimuthu, S. K., Zhang, W., &
Subramanian, S. (2016). Characterization and cytotoxic activity of
apoptosis-inducing pierisin-5 protein from white cabbage butterfly.
International Journal of Biological Macromolecules, 87, 16–27. https://
doi.org/10.1016/j.ijbiomac.2016.01.072
Tabassum, S., Amir, S., Arjmand, F., Pettinari, C., Marchetti, F., Masciocchi,
N., Lupidi, G., & Pettinari, R. (2013). Mixed-ligand Cu(II)-vanillin Schiff
base complexes; Effect of coligands on their DNA binding, DNA cleav-
age, SOD mimetic and anticancer activity. European Journal of
Medicinal Chemistry, 60, 216–232. https://doi.org/10.1016/j.ejmech.
2012.08.019
Yadav, R., Saini, D., & Yadav, D. (2018). Synthesis and evaluation of vanil-
lin derivatives as antimicrobial agents. Turkish Journal of
Pharmaceutical Sciences, 15(1), 57–62. https://doi.org/10.4274/tjps.
97752
Yousef, T. A., El-Reash, G. M. A., & El-Tabai, M. N. (2018). Comparative
studies on P-vanillin and O-vanillin of 2-hydrazinyl-2-oxo-N-phenylace-
tamide and their Mn(II) and Co(II) complexes. Journal of Molecular
Structure, 1159, 246–258. https://doi.org/10.1016/j.molstruc.2018.01.
003
ꢀ
Th(IV), and UO2(VI) with Schiff bases derived from sulfa drugs, vız.,
sulfanilamide/sulfamerazine and o-vanillin. Spectroscopy Letters, 32(2),
213–236. https://doi.org/10.1080/00387019909349979
Nitiss, J. L. (2009). Targeting DNA topoisomerase II in cancer chemother-
apy. Nature Reviews. Cancer, 9(5), 338–350. https://doi.org/10.1038/
nrc2607
Zhang, C., Li, X., Lian, L., Chen, Q., Abdulmalik, O., Vassilev, V., Lai, C. S.,
& Asakura, T. (2004). Anti-sickling effect of MX-1520, a prodrug of
vanillin: An in vivo study using rodents. British Journal of
Haematology, 125(6), 788–795. https://doi.org/10.1111/j.1365-2141.
2004.04892.x