In silico studies, synthesis and anticancer activity of novel diphenyl ether-based pyridine derivatives

Article abstract of DOI:10.1007/s11030-018-9889-1

Abstract: A series of novel 2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(4-substituted phenyl) nicotinonitriles were synthesized and evaluated against HepG2, A-549 and Vero cell lines. Compounds 3b (IC₅₀ 16.74 ± 0.45 μM) and 3p (IC₅₀ 10.

Full text of DOI:10.1007/s11030-018-9889-1

Molecular Diversity
https://doi.org/10.1007/s11030-018-9889-1
ORIGINAL ARTICLE
In silico studies, synthesis and anticancer activity of novel diphenyl
ether-based pyridine derivatives
Ruchi Verma1 · Indira Bairy2 · Mradul Tiwari3 · G. Varadaraj Bhat1 · G. Gautham Shenoy1
Received: 26 July 2018 / Accepted: 26 October 2018
© Springer Nature Switzerland AG 2018
Abstract
A series of novel 2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(4-substituted phenyl) nicotinonitriles were synthesized and
evaluated against HepG2, A-549 and Vero cell lines. Compounds 3b (IC50 16.74 0.45 µM) and 3p (IC50 10.57 0.54 µM)
were found to be the most active compounds against A-549 cell line among the evaluated compounds. Further 3b- and 3p-
induced apoptosis was characterized by AO/EB (acridine orange/ethidium bromide) nuclear staining method and also by DNA
fragmentation study. A decrease in cell viability and initiation of apoptosis was clearly evident through the morphological
changes in the A-549 cells treated with 3b and 3p when stained with this method. Fragmentation of DNA into nucleosomes
was observed which further confirmed the cell apoptosis in cells treated with compound 3b. Flow cytometry studies confirmed
the cell cycle arrest at G2/M phase in A549 cells treated with compound 3b. Further in silico studies performed supported
the in vitro anticancer activity of these compounds as depicted by dock score and binding energy values.
Graphical Abstract
Keywords Triclosan · Diphenyl ether derivatives · Human enoyl-acyl carrier protein reductase (hER)
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11030-018-9889-1) contains supplementary
material, which is available to authorized users.
1
Department of Pharmaceutical Chemistry, Manipal College of
Pharmaceutical Sciences, Manipal Academy of Higher
Education, Manipal 576 104, India
G. Gautham Shenoy
gautham.gs@manipal.edu
B
123

Molecular Diversity
Introduction
activity (Fig. 1a) [14]. Few 4-[2-amino-3-cyano-5-oxo-4-
substituted aryl-4H-indeno [1, 2-b] pyridin-1-(5H)-yl] ben-
zenesulfonamide derivatives were synthesised and subjected
to cytotoxicity studies against MCF-7 cell lines by Mostafa
and Mansour. Compound 6d was found to be more potent
than doxorubicin (Fig. 1b) [15]. 4,6-diaryl-2-imino-1,2-
dihydropyridine-3-carbonitriles were synthesized and tested
against human HT-29 colon adenocarcinoma tumor cells by
A.H. Abadi et al. The IC50 value for few substituents was
found to be 3, 4.17, 12 µM, respectively. Through the molec-
ulardockingstudies, thecyanoandiminogroupdisplayedthe
hydrogen-bonding interaction with the amino acid residues
present at the target site. Compound 1d was found to be most
potent (Fig. 1c) [16]. Literature survey indicated that there
is still more to explore in this area. Therefore, we sought
to develop molecules combining two moieties, i.e., diphenyl
ether-bearing hydroxyl group and 3-cyano-2-aminopyridine,
in order to develop potential cytotoxic agents targeting hER.
Triclosan is reported to adversely affect HepG2 cells by DNA
methylation dysregulation, and Diorcinol D (DD), a diphenyl
ether derivative, has also shown potent cytotoxicity against
A-549 cell lines [8, 17]. Hence, we chose A-549, HepG2
and Vero cells for carrying out the cytotoxicity studies of
the synthesized compounds (3a–q). In this study, we have
carried out computational studies to investigate the binding
mode of the molecules with the receptor. For this purpose,
we have docked the synthesized compounds with the crys-
tal structure of human enoyl-acyl carrier protein reductase
(PDB 4W82) obtained from the protein data bank [18]. Com-
putational studies were carried out using Schrodinger suite
2017-4. The present work is a preliminary work involving
computational study, and in vitro enzyme inhibition studies
have not been performed to support the work.
Cancer continues to be one of the leading causes of death
worldwide in spite of significant knowledge in the under-
standing of the pathophysiology, treatment, diagnosis and
discovery of drugs acting through novel mechanism [1, 2].
Cancer was responsible for 8.8 million deaths worldwide in
2015. It is estimated that the number of new cases will rise
by about 70% over the next two decades [3]. As far as the
Indian scenario is concerned, the estimated cancer cases for
the year 2020 in males are 534,353 and in females, the esti-
mation is approximately 614,404. The total number of cases
is expected to reach 1,148,757 in the year 2020 [4].
Novelanticanceragentsareassociatedwithsevereadverse
effects and tumors recurrence. These drawbacks reduce their
clinical efficacy. Thus, there is a need to develop new
anticancer agents with minimal adverse effects and drug
resistance related to chemotherapy [5, 6]. In the past few
years, triclosan (TCL) derivatives have gathered a lot of
attention as potent anticancer agents and hence we decided
to evaluate novel TCL derivatives as anticancer agents [7].
TCL, which is a diphenyl ether derivative, has been reported
to target bacterial enoyl-acyl protein reductase and inhibit
fatty acid synthase (FASN) through the enoyl-acyl reductase
domain [8]. It is evident through literature that TCL is cyto-
toxic to tumor cells [9]. Human fatty acid synthase is a large
multi-domain protein involved in the synthesis of long-chain
fatty acids that are usually expressed at very low levels but
gets highly upregulated in cancer [10]. Human enoyl-acyl
carrier protein reductase (hER) is one among the FAS cat-
alytic domains, and drugs similar to triclosan inhibit hER
producing cell toxicity and minimizing drug resistance in
cancer cells [8]. TCL as an anticancer agent has been reported
to inhibit FASN in various cancer cell lines that include breast
cancer cell lines MCF-7, SKBr-3, retinoblastoma Y79 RB
cell line, epithelial carcinoma KB cell line and choriocar-
cinoma JEG-3 cell line [11, 12]. 2-Amino-3-cyanopyridine
derivatives have been reported to possess various biological
activities such as antimicrobial, anti-parkinsonism, antioxi-
dant, anti-inflammatory, etc. [13]. 2-Amino-3-cyanopyridine
derivatives have also exhibited profound anticancer activ-
ity against several cell lines. Mohamed et al. synthesized
novel 3 cyano-aminopyridine derivatives by reacting 3-
indole carboxaldehyde and 3-aminoacetophenone and tested
them for anticancer activity in MCF-7 (breast cancer cell
line). Some of the compounds exhibited potent anticancer
Results and discussion
Synthesis and spectral analysis
Scheme (Fig. 2) outlines the synthesis of the target com-
pounds 3a–q. Initially, vanillin was condensed with phenyl
boronic acid to obtain 3-methoxy-4-phenoxybenzaldehyde
(1). The aldehyde (1) obtained was demethylated by treat-
ing with HBr giving 3-hydroxy-4-phenoxybenzaldehyde (2)
[19]. Compounds 3a–q were synthesized by the reaction
of aldehyde (2), aryl ketones, malononitrile and ammonium
acetate in dioxane. The reported mechanism involved in this
synthesis initially involves the formation of enamine deriva-
tive obtained by reaction of ketone with ammonium acetate
and formation of alkylidene malononitrile obtained by reac-
tion of aldehyde (2) with malononitrile. Further, the reaction
of enamine derivative with alkylidene malononitrile results
in the formation of the Michael adduct. The intermediate
2
Department of Pharmaceutical Biotechnology, Manipal
College of Pharmaceutical Sciences, Manipal Academy of
Higher Education, Manipal 576 104, India
3
Department of Microbiology, Melaka Manipal Medical
College, Manipal Academy of Higher
Education, Manipal 576104, India
123

Molecular Diversity
Fig. 1 2-Amino-3-cyanopyridine-related analogues
Fig. 2 Scheme
formed undergoes cycloaddition, isomerization and arom-
atization to get the desired compounds [20, 21]. Various
electron-donating/-withdrawing groups were substituted at
the D ring in order to understand the influence of various
groups and their position on the anticancer activity. The IR
spectra of the synthesized compounds indicated characteris-
tic C≡N peak around 2210–2220 cm−1 and NH2 asymmetric
and symmetric stretch between 3300 and 3500 cm−1 and
a broad peak above 3300 cm−1, indicating the presence of
hydroxyl group. 1HNMR spectra of all the synthesized com-
pounds showed OH proton as singlet around δ 9.5–10.0 ppm.
Aromatic protons and NH2 group were seen as a multiplet
around δ 8.0–6.5 ppm [21]. The 13C NMR of the syn-
thesized compounds showed a signal at 109 ppm due to
123

Molecular Diversity
C≡N group. Signals due to phenyl group were found in
Table 1 Effect of synthetic compounds treatment on proliferation of
A-549 and HepG2 cells after 48 h and Vero cells after 72 h
116–149.65 ppm range. The signal at 165 was attributed to
1
C–NH2 of the pyridine ring. HNMR spectra for the com-
Entry
Compound
IC50 (µM)*
pound 3p showed multiplet between 8.02 and 6.83 ppm
corresponding to 13 aromatic protons and two NH2 group
protons and a singlet for two protons of hydroxyl group at
ring B and ring D at 9.95 ppm. 13C NMR spectra for the same
compound displayed chemical shift at 161.33 ppm (C–NH2
of the pyridine ring), 160.04 ppm (C–OH of the D ring),
159.05 ppm (C–O of A ring), 157.86 ppm, 117.84 ppm,
154.44 ppm (C of pyridine ring), 149.58 ppm (C–OH of B
ring), 144.53 ppm (C–O of B ring), 130.23 ppm (two equiv-
alent carbon of D ring), 129.41 ppm (two equivalent carbon
of A ring), 128.80 ppm (C of D ring), 122.98 ppm (C of A
ring), 134.50 ppm, 121.87 ppm, 120.25 ppm, 117.54 ppm (C
of B ring), 117.28 ppm (two equivalent carbon of A ring),
115.89 ppm (two equivalent carbon of A ring), 108.48 ppm
(CN group) and 85.53 ppm (C–CN of pyridine ring). 1HNMR
spectra for the compound 3b showed multiplet between 8.13
and 6.62 ppm corresponding to 13 aromatic protons and two
NH2 group protons and a singlet for proton of hydroxyl
group at ring B (9.952 ppm) and a singlet for three pro-
tons of methoxy group at 3.87ppm. 13C NMR spectra for
the same compound displayed chemical shift at 161.36 ppm
(C–NH2 of the pyridine ring), 161.3 ppm (C–OCH3 of the D
ring), 158.68 ppm (C–O of A ring), 157.84 ppm, 154.59 ppm,
117.56 ppm (C of pyridine ring), 149.56 ppm (C–OH of B
ring), 144.58 ppm (C–O of B ring), 134.39 ppm (C of B ring),
130.35 ppm (C of D ring), 129.41 ppm (two equivalent car-
bon of A ring), 128.80 ppm (C of D ring), 122.99 ppm (C of
A ring), 121.84 ppm, 117.72 ppm (C of B ring), 120.28 ppm
(two equivalent carbon of D ring), 117.30 ppm (two equiv-
alent carbon of A ring), 114.49 ppm (two equivalent carbon
of D ring), 108.74 ppm (CN group), 85.95 ppm (C–CN of
pyridine ring) and 55.79 ppm (OCH3 group).
A-549
HepG2
Vero
1.
3a
38.25 0.23
16.74 0.45
34.22 0.62
29.33 0.13
36.46 0.54
41.47 0.24
35.62 0.60
22.46 0.90
27.43 1.20
36.09 0.00
60.45 0.35
38.41 0.43
22.11 0.30
33.29 0.88
44.43 0.30
10.57 0.54
25.316 0.51
23.96 0.70
33.99 0.90
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.
3b
3.
3c
4.
3d
5.
3e
6.
3f
7.
3g
8.
3h
9.
3i
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
3j
3k
3l
3m
3n
3o
3p
3q
Triclosan
Cisplatin
*Data presented as mean SEM, N ꢀ3
significantly cytotoxic (IC50 above 40 µM). Almost all the
compounds showed good selectivity to screened cell lines
over normal kidney epithelial cells (Vero). The compounds
were found to be safe with IC50 value>100 µM. Compounds
did not exhibit any notable cytotoxicity against HepG2 cell
line [IC50 above>100 µM] (Table 1). Among the tested
compounds, substitutions at the D ring of the test molecules
resulted in enhanced anticancer activity in comparison with
substitutions at meta- and ortho-position. Among the para
substituents, methoxy group substitution and hydroxyl group
substitution at para position of the phenyl ring were found to
be most active.
Cell viability study by MTT assay
MTT cell viability assay which involves the action of
mitochondrial dehydrogenase on MTT to produce dark-
blue formazan from MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) was used to evaluate the
anticancer activity of the synthesized compounds [22]. Syn-
thesized compounds 3a–q were evaluated for cell toxicity
activity against A-549, HepG2, and normal kidney epithe-
lial cells (Vero) using MTT assay. Compounds 3b (IC50 of
16.74 0.45 µM) and 3p (IC50 of 10.57 0.54 µM) were
found to be the most active against A-549 cell line and
were chosen for further studies. Eleven compounds showed
moderate cytotoxicity (IC50 between 20 µM-40 µM). The
positive control cisplatin showed cytotoxicity at IC50 of
33.99 0.90 µM, whereas triclosan exhibited cytotoxicity
at 23.96 0.70 µM. Remaining four compounds were not
AO/EB nuclear staining
Apoptosis is the programmed cell death phenomenon lead-
ing to characteristic morphological changes in the cell [23].
Cell staining with fluorescent acridine orange and ethidium
orange is a very common and precise method to detect, clas-
sify apoptosis and differentiate it from necrosis [24]. Based
on the IC50 values of 3b and 3p in A-549, these compounds
were selected for AO/EB nuclear staining method. The cell
line was treated with the synthesized compounds, incubated
for 48 h, stained with AO/EB dye and was observed under a
fluorescent microscope to visualize the changes in the mor-
123

Molecular Diversity
Fig. 3 Apoptosis induction by AO/EB stain after 48 h in A-549 cells: a normal control, b compound 3b, c compound 3p
phology of the cell nucleus (Fig. 3). From Fig. 3a, it is
evident that untreated normal control A-549 cells showed
normal green integral nuclei depicting normal cell morphol-
ogy. A-549 cells treated with 3b and 3p (Fig. 3b, c) showed
fluorescent green nuclei, condensed chromatin and smaller
diameter indicating initial apoptosis when observed under
a fluorescence microscope. Further, compounds 3b and 3p
were taken up for DNA fragmentation study.
DNA fragmentation assay
Nuclear DNA fragmentation is considered as one of the
biochemical hallmarks of apoptosis showing distinct DNA
ladder fragments of 180–200 base pairs and multiples on
agarose gel, whereas cell necrosis results in production of
diffused smear of DNA on electrophoresis [25]. DNA lad-
dering assay was performed to ensure that the cell death of
A-549 cells when exposed to the synthesized compounds
(3b, 3p, TCL and cisplatin) occurred due to apoptosis. DNA
fragmentation pattern was obtained for control cells and the
treated cells at their IC50 value concentration by agarose gel
electrophoresis method (Fig. 4). After 4 h of treatment of
A-549 cells with 3b, TCL and cisplatin, a distinct ladder was
seen (Fig. 4, lane 2–4) and untreated control did not show
any DNA ladder (Fig. 4, lane 5). For compound 3p (Fig. 4,
lane 1), characteristic DNA ladder was not seen. The results
obtained showed the cleavage of chromosomal DNA into
nucleosomal unit, thus confirming cell cytotoxicity [26] by
the compound (3b) due to apoptosis. For compound 3p, char-
Fig. 4 DNA fragmentation studies by agarose gel electrophoresis
acteristic DNA fragmentation was not seen, thus indicating
the possibility of cell toxicity through necrosis.
(1.5%); lane 1: A-549 cells+3p; lane 2: A-549 cells+3b; lane 3: A-
549 cells+TCL; lane 4: A-549 cells+cisplatin; lane 5: A-549 cells+
control; lane 6: DNA ladder
Cell cycle analysis
The cell distribution in various phases of its growth progres-
sion cycle depends on the underlying mechanism for the
cytotoxicity produced by the synthesized compounds [26,
27]. Based on the DNA fragmentation studies, flow cytome-
try study was done to understand the underlying mechanism
of the cell inhibition by the compounds 3b and TCL (Fig. 5).
The results were obtained as % cells distribution in G0/G1, S,
and G2/M phase. The untreated cells (control) exhibited the
123

Molecular Diversity
Fig. 5 Cell cycle phase distribution of A-549 cells after 48 h treatment;
the % cells enrichment specifically in G0/G1, S, and G2/M phase was
assessed by flow cytometry; a FSC A versus SSC plot for normal control
cells; b width versus FL2-A plot for 2n to 4n normal control gated cells;
(c) FL2-A versus count histogram plot for normal control (0.01%); (d,
e and f) FL2-A versus count histogram plots for 3b, TCL and cisplatin,
respectively. FSC forward scatter, SSC side scatter; TCL triclosan
cell enrichment of 67.8% in G0/G1, 11.1% in S and 20.3% in
G2/M phase, whereas in cisplatin-treated cells, the percent-
ageofcellsinSphaseandG2/Mphasewas22.9%and26.7%,
respectively, indicating cell cycle arrest. As mentioned in ear-
lier studies, TCL (IC50 23.96 0.7 µM)-treated A-549 cells
exhibited 70.6% of accumulation in G0/G1 phase. In com-
parison with cisplatin and TCL, significant increase in the
cell count was found in G2/M phase (45.0%) and a reduction
of cell count was found in S phase and G0/G1 phase when
treated with 3b (IC50 16.74 0.45 µM). Therefore, it may
be attributed that compound 3b produced cytotoxicity due
to cell cycle arrest at G2/M phase. The mechanism for cell
growth inhibition by compound 3b was found to be different
when compared to the triclosan through this study.
based on its site score (1.076) and site volume (569.037 Å3)
(supplementary data) with active residues. A grid was gen-
erated at site 1. After the grid was generated, the prepared
ligands were docked with the target active site using Glide
Xtra precision (XP) mode. Thirteen molecules showed dock
score above −7.00 kcal/mol when compared to triclosan
(dock score of −6.13 kcal/mol). Compound 3b showed dock
score of −7.098 kcal/mol and 3p of −7.85 kcal/mol. The lig-
ands showed H-bond interactions with amino acid residues
Glycine 1679, Asparagine 1572, Glycine 1846, and few lig-
ands showed π cation interaction with Lysine 1771 of the
protein 4W82 (supplementary data). Compound 3b showed
dock score above −7.0 kcal/mol and displayed one hydro-
gen bond interaction between OH group on the diphenyl
ether ring and Asparagine 1572, second hydrogen bond
interaction between NH2 group and Glycine 1846 and third
hydrogen bond interaction between CN group and Glycine
1679. The π cation interaction was found between A ring of
diphenyl ether moiety and Lysine 1771 (Fig. 6). Compound
3p exhibited maximum four hydrogen bonds interactions
involving OH group at D ring with Alanine 1844, NH2
group with Glycine 1846, CN group with Glycine 1679 and
OH group at the diphenyl ether moiety with Asparagine
1572 (Fig. 7). Triclosan showed one H-bond interaction with
Molecular docking studies
The in silico computational method is very useful to deter-
mine the types of interactions between the compounds and
the target receptor. All the synthesized compounds were
docked with 4W82 protein by using grid-based method. Site
map tool was used to determine the best binding site for the
ligands on the protein. A total of five sites were identified
by the site map. Site 1 was selected as the best binding site
123

Molecular Diversity
Fig. 6 a Graphical illustration of predicted binding mode in the active
site of 4W82 for compound 3b. The hydrogen bond interactions are rep-
resented by magenta arrow, and π cation interactions are represented
by red bond, b 3D interaction between human enoyl-acyl carrier protein
reductase (PDB: 4W82) and compound 3b
Fig. 7 a Graphical illustration of predicted binding mode in the active site of 4W82 for compound 3p. The hydrogen bond interactions are represented
by magenta arrow; b 3D interaction between human enoyl-acyl carrier protein reductase (PDB: 4W82) and compound 3p
Phenylalanine 1573 (Fig. 8). More number of interactions
particularly H-bond between ligand and receptor along with
dock score contributes toward its strong affinity toward the
receptor [28] which may be responsible for the potent activ-
ity of compounds 3b and 3p. This was further confirmed
through MMGBSA. The binding energy between receptor
and ligand is dependent on H-bond strength and π cation
interactions. The negative dG values correspond to stable
ligand–receptor complex and thermodynamic practicability
of binding [29]. The results (Table 2) show that compounds
3b, 3h, 3m, 3p and 3q showed binding energies more than
−56.500 k cal mol which is appreciable score depicting the
strong affinity of the ligands toward the receptor. Thus, more
number of interaction between ligand and receptor, better
binding energy, better glide g score and thermodynamically
stableligand-receptorcomplexofsynthesizedcompounds3p
and 3b would have contributed towards the potent anticancer
activity when compared to triclosan.
123

Molecular Diversity
Fig. 8 a Graphical illustration of predicted binding mode in the active site of 4W82 for triclosan. The hydrogen bond interactions are represented
by magenta arrow; b 3D interaction between human enoyl-acyl carrier protein reductase (PDB: 4W82) and triclosan
Drug-likeness property
iments would assist in structure modifications to increase the
anticancer activity of this scaffold. Hence, it could be inter-
preted that 3b compound-treated cells could produce damage
to the cancerous A549 cells, signifying the substantial poten-
tial of this compound in the treatment of lung cancer.
The results obtained through QikProp tool (Table 3) show
all the ligands following Lipinski’s rule of five; i.e., drug-
gable molecule has log P ≤5, molecular weight<500 g/mol,
hydrogen bond acceptors≤10, hydrogen bond donors≤5
and molar refractivity between 40 and 130. No ligand was
found to violate the Lipinski’s rule of five [29].
Experimental
Materials and methods
Reagents and chemicals
Conclusion
The synthesis of novel diphenyl ether analogues 3a–q has
been reported in the present research work showing anti-
cancer activity against cancer cell lines. Compounds 3b (IC50
value 16.74 0.45) and 3p (IC50 value 10.57 0.54 µM)
inhibited A-549 cells more effectively in comparison with the
other synthesized compounds. Compound 3b (para-methoxy
substituted) showed cell toxicity in A-549 cells due to apop-
tosis. This compound can serve as a promising lead for the
development of new anticancer drugs. It was found that TCL
arrested cell cycle growth at G0/G1 phase, whereas com-
pound 3b showed tremendous increase in the cells at G2/M
phase. In silico studies depicted good docking score of the
compounds 3b and 3p with the receptor 4W82, strong affinity
toward receptor target through H-bond interaction and bind-
ing energy score in comparison with triclosan. Detailed study
is required to study the proper mechanism of cell growth
inhibition in A549 cells by these compounds. Further exper-
The chemicals and solvents used in synthesis and cell line
studies were purchased commercially. Melting points were
obtained in open capillary tubes and are uncorrected. The 1H
NMR and 13C NMR spectra were obtained on NMR spec-
trometer (AV400—400 MHz high-resolution multinuclear
FT-NMR spectrometer, Bruker, USA). Mass spectroscopy
was performed on LC–MS (linear ion trap, APCI mode,
LC/MS, Thermo Fisher Scientific LTQ21532 series, USA)
and GC–MS (GCMS-QP5050A, Shimadzu, Japan).
Three cell lines such as HepG2 (human hepatocellular car-
cinoma cell line), A-549 (adenocarcinomic human alveolar
basal epithelial cells), Vero (normal African green monkey
kidney epithelial cells) used in this study were obtained
from the National Center for Cell Science, Pune, India,
and maintained in tissue and cell culture in high glucose
DMEM medium with 10% FBS and 1% antibiotic–antimy-
123

Molecular Diversity
Table 2 Binding energy calculation of the (4W82-ligands) docked complexes
Compound XP glide Prime MMGBSA dG MMGBSA dG bind
MMGBSA dG bind
covalent (k cal/mol)
MMGBSA dG bind
vdW (k cal/mol)
MMGBSA dG bind
Solv GB (k cal/mol)
score
bind (k cal/mol)
coulomb (k cal/mol)
3a
−8.001
−7.098
−7.63
−54.079
−56.634
−54.571
−53.833
−56.472
−55.037
−53.798
−58.924
−54.571
−56.055
−53.670
−54.199
−57.515
−53.798
−55.296
−57.314
−57.769
−43.275
−11.037
−13.559
−9.335
1.702
2.543
0.817
0.322
1.752
1.875
1.860
1.611
0.817
1.806
2.011
0.773
1.577
1.860
1.754
2.594
4.210
1.529
−50.724
−53.436
−50.325
−51.379
−52.779
−52.160
−50.214
−53.182
−50.325
−51.460
−51.228
−50.013
−53.853
−51.622
−52.026
−50.591
−49.603
−33.548
33.529
36.338
31.792
35.555
34.260
34.498
32.792
32.230
31.792
32.018
30.162
32.199
33.220
32.625
35.217
37.800
34.355
18.199
3b
3c
3d
3e
−7.513
−7.954
−6.785
−6.008
−7.5
−10.758
−11.243
−11.324
−10.669
−11.019
−9.335
3f
3g
3h
3i
−7.686
−7.712
−6.563
−7.839
−7.541
−5.446
−7.764
−7.851
−8.418
−6.13
3j
−10.748
−7.282
3k
3l
−9.721
3m
3n
3o
−10.955
−10.669
−15.619
−19.482
−18.812
−12.089
3p
3q
Triclosan
Table 3 Drug-likeness
Compd.
MW
Hda
Hbb
PSAc
QPlogPo/wd
Rule of
Oral abs
(%)
properties of the compounds as
per the QikProp software
application
fivee
3a
379
409
409
409
393
393
393
414
414
397
397
397
458
458
458
395
395
290
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
4.0
4.0
1.0
4.25
5.0
98.7
98.6
4.091
4.221
4.208
4.243
4.386
4.41
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
92.723
94.094
93.817
93.529
94.525
95.021
96.873
95.604
96.01
3b
3c
5.0
96.4
3d
5.0
90.41
90.4
3e
4.25
4.25
4.25
4.25
4.25
4.25
4.25
4.25
4.25
4.25
4.25
5.0
3f
88.5
3g
90.37
90.37
90.37
90.42
90.62
90.42
90.43
91.04
112.92
112.97
110.63
28.76
4.418
4.574
4.462
4.317
4.336
4.256
4.647
4.668
4.495
3.298
3.308
4.75
3h
3i
3j
94.099
94.612
93.661
96.029
96.553
94.907
78.859
79.133
100
3k
3 l
3 m
3n
3o
3p
3q
5.0
Triclosan
1.25
aHd hydrogen bond donor
bHa hydrogen bond acceptor
cPSA polar surface area
dQPlogPo/wꢀLogP in o/w
eRule of 5ꢀLipinski’s rule of five: 1ꢀviolation, 0ꢀwithin the limit
fHuman oral absorption (%)
123

Molecular Diversity
cotic solution, at 37 °C in an CO2 incubator under 5% CO2
and 95% air-humidified condition (CLS-170-B-8, Serial No.
201020569, Esco Micro Pte. Ltd.).
4-phenoxybenzaldehyde (2) (2.3 mmol), acetophenone
(2.3 mmol), ammonium acetate (1.5 g, 19.4 mmol) and
malononitrile (0.253 mL, 2.3 mmol) in dioxane (5.0 mL)
was refluxed with stirring for 24 h. Dioxane was evaporated
under vacuum, and the reaction mixture was poured into
water (30 mL) and the product was extracted with ethyl
acetate [20, 21]. The ethyl acetate layer was separated,
dried over anhydrous MgSO4 and evaporated under reduced
pressure. The crude mixture was purified by column chro-
matography (hexane: ethyl acetate; 8:2) to get the desired
compound.
Synthesis
3-methoxy-4-phenoxybenzaldehyde,
1 Vanillin
(32.89 mmol) was stirred in anhydrous dichloromethane
(60 mL) at 25–27 °C. Then, activated molecular sieves (4
A°, 2.5 g), phenyl boronic acid (49.33 mmol), copper (II)
acetate (40.58 mmol) and anhydrous pyridine (64.78 mmol,
5.28 mL) were added sequentially. The progress of the reac-
tion was checked by TLC (hexane/ethyl acetate; 8:2). After
the completion of the reaction (72 h), the reaction mixture
was filtered under vacuum. Further washing of filtrate was
done using a dilute aqueous hydrochloric acid solution
(2 M, 50 mL), dried over anhydrous MgSO4 and evaporated
under vacuum till dryness [19]. The purification of the
crude product was done using column chromatography on
silica 100-200 using hexane: ethyl acetate (8:2) to achieve
the desired compound. Yieldꢀ5 g (67%); mpꢀ40–42 °C;
Rf ꢀ0.83 (hexane: ethyl acetateꢀ8:2); λmax ꢀ275.2 nm
(MeOH).
Yield ꢀ0.46 g (53%); mpꢀ168–169 °C; Rf ꢀ0.78 (hex-
ane: ethyl acetateꢀ8:2); IR (KBr, cm−1)ꢀ3479 and 3360
(asym. and sym. stretching of –NH2), 3068 (Ar–H), 2214
1
(C≡N stretching); H NMR (400 MHz, DMSO-d6) δ 9.97
(s, 1H, OH), 8.17–6.78 (m, 16H, Ar–H+NH2); 13C NMR
(101 MHz, DMSO) δ 161.39, 159.01, 157.81, 154.83,
149.57, 144.68, 138.01, 134.22, 130.58, 130.23, 130.08,
129.13, 127.69, 123.02, 121.83, 120.33, 117.59, 117.33,
109.54, 86.89; LCMS(+APCI, m/z): 380 (M+H)+.
2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(4-
methoxyphenyl) nicotinonitrile (3b) Yield ꢀ0.53 g (46%);
mpꢀ178–179 °C; Rf ꢀ0.74 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3415 and 3360 (asym. and sym. stretching
3-hydroxy-4-phenoxybenzaldehyde,
2 3-Methoxy-4-
1
phenoxybenzaldehyde (1) (3 g, 13.15 mmol) was stirred
in glacial AcOH (50 mL), and HBr (48%v/v, 12 mL)
was added. Resulting reaction mixture was refluxed at
112–115 °C. The reaction progress was monitored by TLC
(hexane/ethyl acetate; 8:2). At the end of 14 h, the reaction
mixture was cooled to room temperature and was poured
into 50 mL of ice-cold water with continuous stirring. The
product was extracted using ethyl acetate (3×50 mL). The
ethyl acetate layers were combined and rinsed with saturated
NaHCO3 solution. Ethyl acetate layer was dried over anhy-
drous MgSO4, and the ethyl acetate was evaporated under
vacuum [19]. The crude product was purified by column
chromatography on silica 100–200 using hexane: ethyl
acetate (8:2) system to achieve the corresponding off-white
crystalline solid compound.
of –NH2), 3072 (Ar–H), 2212 (C≡N stretching); H NMR
(400 MHz, DMSO-d6) δ 9.952 (s, 1H, OH), 8.13–6.92
(m, 15H, Ar–H+NH2), 3.87 (s, 3H, OCH3); 13C NMR
(101 MHz, DMSO) δ 161.47, 161.32, 158.68, 157.84,
154.59, 149.56, 144.58, 134.39, 130.35, 130.22, 129.29,
122.99, 121.84, 120.28, 117.72, 117.56, 117.30, 114.49,
108.74, 85.95, 55.79; LCMS(+APCI, m/z): 410 (M+H)+.
2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(3-
methoxyphenyl) nicotinonitrile (3c) Yield ꢀ0.44 g (50%);
mpꢀ201–202 °C; Rf ꢀ0.73 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3473 and 3332 (asym. and sym. stretching
1
of –NH2), 3066 (Ar–H), 2220 (C≡N stretching); H NMR
(400 MHz, DMSO-d6) δ 9.95 (s, 1H, OH), 7.75–6.92 (m,
15H, Ar–H+NH2), 3.87–3.81 (s, 3H, OCH3); 13C NMR
(101 MHz, DMSO) δ 161.31, 160.07, 158.81, 157.83,
154.85, 149.57, 144.68, 139.56, 134.22, 130.23, 123.02,
121.83, 120.37, 120.11, 117.64, 117.54, 117.34, 116.34,
114.24, 112.92, 109.74, 87.06, 55.75; LCMS(+APCI, m/z):
410 (M+H)+.
Yield ꢀ1.6 g (60%); mpꢀ72–74 °C; Rf ꢀ0.59 (hex-
ane:ethyl acetateꢀ6:4); λmax ꢀ275 nm (MeOH); IR (KBr,
cm−1)ꢀ3452, 3057, 1680, 1527, 1483, 1440, 1245, 1132;
1H NMR (400 MHz, DMSO-d6): δ 10.08 (s, 1H, OH), 9.85
(s, 1H, CHO), 7.42 (d, J ꢀ1.6 Hz, 1H, Ar–H), 7.39–7.35
(m, 3H, Ar–H), 7.14–7.10 (m, 1H, Ar–H), 7.01–7.26 (d, J ꢀ
8 Hz, 1H, Ar–H), 6.98–6.96 (td, J ꢀ8 Hz and 1.6 Hz, 2H,
Ar–H); LCMS (+ESI, m/z): 215.069 (M+H)+.
2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(2-
methoxyphenyl) nicotinonitrile (3d) Yield ꢀ0.39 g (45%);
mpꢀ214–215 °C; Rf ꢀ0.71 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3446 and 3350 (asym. and sym. stretching
1
General procedure for the preparation of compounds, 3a–q
of –NH2), 3242 (Ar–H), 2210 (C≡N stretching); H NMR
(400 MHz, DMSO-d6) δ 9.987 (s, 1H, OH), 7.80–6.96
(m, 15H, Ar–H+NH2), 3.843 (s, 3H, OCH3); 13C NMR
(101 MHz, DMSO) δ 161.35, 158.41, 157.73, 157.62,
Synthesis of 2-amino-4-(3-hydroxy-4-phenoxyphenyl)-
6-phenylnicotinonitrile, 3a A solution of 3-hydroxy-
123

Molecular Diversity
153.39, 149.60, 144.70, 134.24, 131.47, 131.20, 130.25,
127.68, 123.06, 121.80, 120.94, 120.14, 117.59, 117.44,
117.39, 114.04, 112.54, 86.24, 56.20; LCMS(+APCI, m/z):
410 (M+H)+.
2-amino-6-(2-chlorophenyl)-4-(3-hydroxy-4-
phenoxyphenyl) nicotinonitrile (3i) Yield ꢀ0.48 g (55%);
mpꢀ179–180 °C; Rf ꢀ0.78 (hexane: ethyl acetateꢀ8:2);
(MeOH); IR (KBr, cm−1)ꢀ3485 and 3388 (asym. and sym.
stretching of –NH2), 3064 (Ar–H), 2216 (C≡N stretching);
1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H, OH),
7.65–6.93 (m, 15H, Ar–H+NH2);13C NMR (101 MHz,
DMSO) δ 161.47, 159.46, 157.70, 153.90, 149.64, 144.89,
138.60, 133.73, 131.74, 131.43, 131.02, 130.42, 130.24,
127.74, 123.08, 121.82, 120.23, 117.47, 117.43, 117.27,
113.57, 87.11.; LCMS(+APCI, m/z): 414 (M+H)+.
2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(p-tolyl)
nicotinonitrile (3e) Yield ꢀ0.57
g
(65%); mpꢀ
174–175 °C; Rf ꢀ0.77 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3495 and 3358 (asym. and sym. stretching
1
of –NH2), 3037(Ar–H), 2218 (C≡N stretching); H NMR
(400 MHz, DMSO-d6) δ 9.96 (s, 1H, OH), 8.05–6.97 (m,
15H, Ar–H+NH2), 2.38 (s, 3H);13C NMR (101 MHz,
DMSO) δ 161.36, 158.97, 157.83, 154.71, 149.57, 144.62,
140.39, 135.22, 134.30, 130.22, 129.74, 127.63, 123.00,
121.85, 120.30, 117.61, 117.57, 117.30, 109.19, 86.52,
21.38; LCMS(+APCI, m/z): 394 (M+H)+.
2-amino-6-(4-fluorophenyl)-4-(3-hydroxy-4-
phenoxyphenyl) nicotinonitrile (3j) Yield ꢀ0.53 g (60%);
mpꢀ152–153 °C; Rf ꢀ0.75 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3495 and 3358 (asym. and sym. stretching
1
of –NH2), 3066 (Ar–H), 2218 (C≡N stretching); H NMR
2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(m-
(400 MHz, DMSO-d6) δ 9.96 (s, 1H, OH), 8.21–6.83 (m,
15H, Ar–H+NH2); 13C NMR (101 MHz, DMSO) δ 165.11,
162.65, 161.33, 157.81, 154.94, 149.57, 144.70, 134.17,
130.23, 130.08, 123.03, 121.82, 120.35, 117.62, 117.33,
116.73, 116.13, 115.91, 109.35, 86.84; LCMS(+APCI, m/z):
398 (M+H)+.
tolyl)nicotinonitrile (3f) Yield ꢀ0.44 g (50%); mpꢀ
199–200 °C; Rf ꢀ0.76 (hexane: ethyl acetateꢀ8:2); IR
(KBr, cm−1)ꢀ3485 and 3365 (asym. and sym. stretching
1
of –NH2), 3234, 3059 (Ar–H), 2214 (C≡N stretching); H
NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H, OH), 8.02–6.98
(m, 15H, Ar–H+NH2), 2.45 (s, 3H).; 13C NMR (101 MHz,
DMSO) δ 161.31, 157.82, 157.14, 155.13, 149.57, 144.75,
140.29, 134.04, 133.18, 131.28, 130.26, 130.23, 126.65,
123.03, 122.72, 121.82, 120.44, 117.69, 117.37, 117.33,
109.74, 87.58, 21.24.; LCMS(+APCI, m/z): 394 (M+H)+.
2-amino-6-(3-fluorophenyl)-4-(3-hydroxy-4-
phenoxyphenyl) nicotinonitrile (3k) Yield ꢀ0.44 g (50%);
mpꢀ172–173 °C; Rf ꢀ0.74 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3473 and 3352 (asym. and sym. stretching
1
of –NH2), 3076 (Ar–H), 2218 (C≡N stretching); H NMR
(400 MHz, DMSO-d6) δ 9.96 (s, 1H, OH), 8.00–6.96 (m,
15H, Ar–H+NH2); 13C NMR (101 MHz, DMSO) δ 164.22,
161.80, 161.29, 157.80, 157.40, 155.10, 149.56, 144.75,
140.58, 134.05, 131.17, 130.24, 123.74, 123.04, 121.81,
120.42, 117.67, 117.34, 114.37, 114.14, 109.76, 87.52;
LCMS (+APCI, m/z): 398 (M+H)+.
2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(o-
tolyl)nicotinonitrile (3g) Yield ꢀ0.39 g (45%); mpꢀ
209–210 °C; Rf ꢀ0.74 (hexane: ethyl acetateꢀ8:2); IR
(KBr, cm−1)ꢀ3514, 3446 and 3313 (asym. and sym.
stretching of –NH2), 3089 (Ar–H stretching), 2214 (C≡N
stretching); 1H NMR (400 MHz,
DMSO-d6) δ 9.96 (s, 1H, OH), 7.49–6.78 (m, 15H,
Ar–H+NH2), 2.39(s, 3H, CH3).13C NMR (101 MHz,
DMSO) δ 162.58, 161.21, 157.75, 154.05, 149.59, 144.76,
139.67, 133.99, 131.17, 130.23, 129.94, 129.66, 126.23,
123.05, 121.78, 120.27, 117.53, 117.40, 117.37, 113.06,
86.28, 20.72; LCMS(+APCI, m/z): 394 (M+H)+.
2-amino-6-(2-fluorophenyl)-4-(3-hydroxy-4-
phenoxyphenyl) nicotinonitrile (3l) Yield ꢀ0.39 g (45%);
mpꢀ182–183 °C; Rf ꢀ0.72 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3491& 3456 (asym. and sym. stretching
1
of –NH2), 3066 (Ar–H), 2218 (C≡N stretching); H NMR
(400 MHz, DMSO-d6) δ 10.00 (s, 1H, OH), 7.96–6.90 (m,
15H, Ar–H+NH2); 13C NMR (101 MHz, DMSO) δ 161.37,
160.72, 157.73, 155.55, 154.49, 149.65, 145.47, 144.84,
134.76, 133.90, 132.31,132.22, 131.43, 130.24, 123.06,
121.87, 120.19, 119.49, 117.40, 113.14, 110.25, 87.37;
LCMS(+APCI, m/z): 398 (M+H)+.
2-amino-6-(4-chlorophenyl)-4-(3-hydroxy-4-
phenoxyphenyl) nicotinonitrile (3h) Yield ꢀ0.53 g (60%);
mpꢀ154–155 °C; Rf ꢀ0.79 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3496, 3361 (asym. and sym. stretching
1
of –NH2), 3062 (Ar–H), 2218 (C≡N stretching); H NMR
(400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.23–6.83 (m, 15H,
Ar–H+NH2). 13C NMR (101 MHz, DMSO) δ 161.35,
157.81, 157.66, 155.03, 149.58, 144.74, 136.81, 135.42,
134.11, 130.23, 129.47, 129.16, 123.03, 121.83, 120.37,
117.63, 117.46, 117.34, 109.52, 87.24; LCMS (+APCI,
m/z): 414 (M+H)+.
2-amino-6-(4-bromophenyl)-4-(3-hydroxy-4-
phenoxyphenyl) nicotinonitrile (3m) Yield ꢀ0.57 g (65%);
mpꢀ146–147 °C; Rf ꢀ0.82 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3496 and 3392 (asym. and sym. stretching
1
of –NH2), 3062 (Ar–H), 2218 (C≡N stretching); H NMR
(400 MHz, DMSO-d6) δ 9.96 (s, 1H, OH), 8.55–6.91 (m,
123

Molecular Diversity
15H, Ar–H+NH2); 13C NMR (101 MHz, DMSO) δ 161.35,
157.81, 157.73, 155.05, 149.57, 144.73, 137.17, 134.10,
132.10, 130.24, 129.73, 124.30, 123.03, 121.84, 120.38,
117.62, 117.44, 117.33, 109.47, 87.26; LCMS (+APCI,
m/z): 459 (M+H)+.
Biological evaluation
Cell viability assay
MTT assay was executed for the synthesized compounds as
per the standardized procedure. HepG2, A-549 and Vero cell
lines were used in this study. At first, stock solution of the test
compounds and standard compounds (positive control) was
prepared in DMSO, and then it was diluted with DMEM (1%
antibiotic–antimycotic solution was added) to get the desired
concentration before the experiment. The final DMSO con-
centration was 0.2% in each well which was found to be
non-toxic. The cells 5×103 cells/100 µL were plated in
96-well plates where each cell contained 100 µL DMEM
containing 10% FBS and were incubated. The final concen-
tration of the compounds was kept to be (400, 200, 100, 50,
25 µg/ml) starting from the first row. After 24 h, medium was
removed and varying concentrations of test compounds were
added in 96-well plates. The plates were again incubated for
48 h at 37 °C. After 48 h, sample solution was removed,
10 µl MTT reagent [5 mg/mL in PBS (phosphate-buffered
saline)] was added and the cells were incubated for 3 h. After
incubation, DMSO was added to solubilize the formed for-
mazan crystals and the sample absorbance was measured at
540 nm using microplate ELISA reader (ELx800, BioTek
Instruments Inc., Winooski, VT, USA [30, 31].
2-amino-6-(3-bromophenyl)-4-(3-hydroxy-4-
phenoxyphenyl) nicotinonitrile (3n) Yield ꢀ0.48 g (55%);
mpꢀ175–176 °C; Rf ꢀ0.80 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3468 and 3361(asym. and sym. stretching
1
of –NH2), 3068 (Ar–H), 2214 (C≡N stretching); H NMR
(400 MHz, DMSO-d6) δ 9.98 (s, 1H, OH), 8.38–6.96 (m,
15H, Ar–H+NH2); 13C NMR (101 MHz, DMSO) δ 161.30,
157.82, 157.14, 155.13, 149.56, 144.76, 140.29, 134.03,
133.18, 131.28, 130.23, 129.14, 126.65, 123.03, 122.71,
121.81, 120.43, 118.19, 117.69, 117.34, 109.74, 87.59;
LCMS(+APCI, m/z): 459 (M+H)+.
2-amino-6-(2-bromophenyl)-4-(3-hydroxy-4-
phenoxyphenyl) nicotinonitrile (3o) Yield ꢀ0.39 g (45%);
mpꢀ184–185 °C; Rf ꢀ0.79 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3388 and 3321 (asym. and sym. stretch-
1
ing of –NH2), 3230 (Ar–H), 2216 (C≡N stretching); H
NMR (400 MHz, DMSO-d6): 9.98 (s, 1H, OH), 7.75–6.86
(m, 15H, Ar–H+NH2); 13C NMR (100.64 MHz, DMSO-
d6):161.42, 160.93, 157.70, 153.80, 149.63 144.93, 140.67,
133.71, 133.51, 131.57, 131.08, 130.23, 128.20, 123.09,
121.77, 121.15, 120.22, 117.47, 117.26, 117.11, 113.50,
87.03; LCMS (+ESI, m/z): 459 (M+H)+.
AO/EB staining method
A-549 cells at the final concentration of 5×104 cells/well
were added to 24-well plates with DMEM containing 10%
FBS and were incubated for 24 h. Further, the synthe-
sized compounds were added to the cells at concentration
corresponding to their IC50 and incubated for 48 h. After
incubation, about 1–2 mL of PBS was added to wash the
cells followed by addition of 1 mL of ice-cold methanol to
fix the cells. After 20 min, the methanol was removed and
the cells were washed with PBS (2 mL). 300 µL of dual stain
AO/EB (20/30 µg/mL dissolved in PBS) was added to every
well, and the plates were incubated at 37 °C for 20 min.
Finally, PBS was used to wash the plates which was then
examined using a fluorescent microscope (Eclipse TS100-F,
Nikon Instruments Inc., Melville, NY, USA) to visualize the
changes in the morphology of the cell nucleus [32, 33].
2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(4-
hydroxyphenyl) nicotinonitrile (3p) Yield ꢀ0.57 g (65%);
mpꢀ158–159 °C; Rf ꢀ0.70 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3502 (Ar–OH stretching), 3387 and 3358
(asym. and sym. stretching of –NH2), 3076 (Ar–H), 2218
1
(C≡N stretching); H NMR (400 MHz, DMSO-d6): 9.95
(s, 2H, 2OH), 8.02–6.83 (m, 15H, Ar–H+NH2); 13C NMR
(100.64 MHz, DMSO-d6):161.33, 160.04, 159.05, 157.86,
154.44, 149.58, 144.53, 134.50, 130.23, 129.41, 128.80,
122.98, 121.87, 120.25, 117.84, 117.54, 117.28, 115.89,
108.48, 85.53; LCMS (+ESI, m/z): 396 (M+H)+.
2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(3-
hydroxyphenyl) nicotinonitrile (3q) Yield ꢀ0.63 g (63%);
mpꢀ178–179 °C; Rf ꢀ0.69 (hexane: ethyl acetateꢀ8:2);
IR (KBr, cm−1)ꢀ3464 and 3369 (asym. and sym. stretching
of –NH2), 3070 (Ar C–H), 2210 (C≡N stretching), 1H NMR
(400 MHz, DMSO-d6): 9.95 (s, 1H, OH), 9.61 (s, 1H, OH),
7.57–6.84 (m, 15H, Ar–H+NH2); 13C NMR (100.64 MHz,
DMSO-d6):161.32, 160.24, 159.18, 158.12, 157.81, 154.67,
149.56, 144.72, 139.46, 134.22, 130.23, 130.12, 123.04,
121.81, 120.28, 118.52, 117.63, 117.54, 117.36, 114.44,
109.61, 86.84; LCMS (+ESI, m/z): 396 (M+H)+.
DNA fragmentation assay
DNA fragmentation assay was carried out in vitro as per
the standardized protocol [34, 35]. In brief, A549 cell lines
were treated with compounds 3b and 3p. After incubation for
48 h, the cells were lysed with trypsin and proteinase K and
RNase were added to precipitate the DNA. The DNA pellet
obtained was dispersed in 100 µL TE buffer (Tris buffer).
123

Molecular Diversity
The DNA was analyzed on 1.5% agarose gel electrophore-
sis. The cleaved DNA pattern was visualized at 365 nm UV
transilluminator.
ADME properties and toxicity of the compounds in silico
[38].
Molecular mechanics: generalized born surface area
(MMGBSA)
Cell cycle analysis
MMGBSA is a very useful tool for predicting good binding
poses and estimation of binding free energies in three-
dimensional space. This was done using prime module of the
Schrodinger suite–molecular mechanics–generalized born
surface area (Prime MM–GBSA v 3.000–Schrodinger 2017-
4). The following formula is used for calculating binding free
energy [28, 38, 39]:
The mechanism for cytotoxicity by the synthesized com-
pounds on the human lung cancer A549 was determined
through flow cytometry. Briefly, the cells were treated with
synthesized compounds and incubated for 24 h. Further cells
were trypsinized, centrifuged, washed with PBS, ice-cold
methanol was added to fix the cell pellets and they were kept
at −20 °C for 24 h. Then, PBS was added to wash the cell pel-
lets followed by isotonic PI solution (25 µg/mL propidium
iodide, 0.03% NP-40 and 40 µg/mL RNase) for cell staining.
The cells analysis was done using Accuri C6 flow cytometer
(BD Biosciences, San Jose, CA, USA). For every sample, a
minimum of 10,000 events was achieved and data analysis
was carried out using BD Accuri™ C6 software [33, 36, 37].
ꢀGbin ꢀ ꢀEmm + ꢀGsol + ꢀGSA
,
where ꢀEmm is the difference in the minimized energy
between the 4W82 and ligand complex and the total of the
energies of the free 4W82 and the free ligands.ꢀGsol is the
difference in the GBSA solvation energy between the 4W82
and ligand complex and the total of the energies of the free
4W82 and the free ligands. ꢀGSA is the difference in the sur-
face energy between the 4w82 and ligand complex and the
total of the energies of the free 4W82 and the free ligands.
Molecular docking
Schrodinger small-molecule drug discovery suite with Mae-
stro 11.04 interface was used for the molecular docking
simulations. Here, X-ray crystal structure of enoyl-acyl
carrier protein-reductase domain from human fatty acid syn-
thase (PDB entry: 4W82) of resolution 1.7 A° was taken
from PDB (www.rscb.org). First, the protein preparation
was done using protein preparation wizard (Maestro 11.04,
Schrodinger LLC, USA 2017-4). The protein optimization
was done by adding hydrogens, deleting water molecules
with less than three H-bonds from the protein and assigning
the bond order. OPLS 2003 force field is used for the energy
minimization to root-mean-square deviation (RMSD) of 0.30
Å. The three-dimensional structures of ligands were drawn
by using the 2D sketcher application in maestro. The ligand
preparation was carried out by using LigPrep application
(version 44011) of Maestro 11.04 with default parameters
and ligand energy minimization by using OPLS 2003. The
binding site for the ligands on the prepared protein was gen-
erated by using site map tool (Schrödinger release 2017-4).
Glide module version 77011, schrodinger release 2017-4 was
utilized for ligands docking with xtra precision mode (XP)
and standard precision mode (SP) at the predicted binding
site of the target protein, and final score was obtained through
glide multi-ligand scoring function [27, 28, 38].
Acknowledgements The authors wish to thank Manipal College of
Pharmaceutical Sciences and Manipal Academy of Higher Education
for providing the infrastructure facility to carry out this work.
Compliance with ethical standards
Conflict of interest The authors declare that there is no conflict of inter-
est.
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