Synthesis, evaluation, molecular docking, and molecular dynamics studies of novel N-(4-[pyridin-2-yloxy]benzyl)arylamine derivatives as potential antitubercular agents

Article abstract of DOI:10.1002/ddr.21623

A new series of novel triclosan (2,4,4′-trichloro-2′-hydroxydiphenylether) analogues were designed, synthesized, and screened for their in vitro antimycobacterial and antibacterial activities. Most of the compounds showed significant activity against Myco

Full text of DOI:10.1002/ddr.21623

Received: 15 June 2019
DOI: 10.1002/ddr.21623
Revised: 16 October 2019
Accepted: 24 October 2019
R E S E A R C H A R T I C L E
Synthesis, evaluation, molecular docking, and molecular
dynamics studies of novel N-(4-[pyridin-2-yloxy]benzyl)
arylamine derivatives as potential antitubercular agents
Ruchi Verma1 | Helena I. M. Boshoff2 | Kriti Arora2 | Indira Bairy3
Mradul Tiwari4 | Varadaraj Bhat G.1 | G. Gautham Shenoy1
1Department of Pharmaceutical Chemistry,
|
Abstract
Manipal College of Pharmaceutical Sciences,
A new series of novel triclosan (2,4,40-trichloro-20-hydroxydiphenylether) analogues
were designed, synthesized, and screened for their in vitro antimycobacterial and
antibacterial activities. Most of the compounds showed significant activity against
Mycobacterium tuberculosis H37Rv strain with minimum inhibitory concentration
(MIC) values in 20–40 μM range in GAST/Fe medium when compared with triclosan
(43 μM) in the first week of assay, and after additional incubation, seven compounds,
that is, 2a, 2c, 2g, 2h, 2i, 2j, and 2m, exhibited MIC values at the concentration of
20–40 μM. The compounds also showed more significant activity against Bacillus sub-
tilis and Staphylococcus aureus. The synthesized compounds showed druggable prop-
erties, and the predicted ADME (absorption, distribution, metabolism, and excretion)
properties were within the acceptable limits. The in silico studies predicted better
interactions of compounds with target protein residues and a higher dock score in
comparison with triclosan. Molecular dynamics simulation study of the most active
compound 2i was performed in order to further explore the stability of the protein–
ligand complex and the protein–ligand interaction in detail.
Manipal Academy of Higher Education,
Manipal, Karnataka, India
2Tuberculosis Research Section, Laboratory of
Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious
Diseases, National Institutes of Health,
Bethesda, Maryland
3Department of Microbiology, Melaka Manipal
Medical College, Manipal Academy of Higher
Education, Manipal, Karnataka, India
4Department of Pharmaceutical
Biotechnology, Manipal College of
Pharmaceutical Sciences, Manipal Academy of
Higher Education, Manipal, Karnataka, India
Correspondence
Dr. G. Gautham Shenoy, Professor,
Department of Pharmaceutical Chemistry,
Manipal College of Pharmaceutical Sciences,
Manipal 576 104, Karnataka, India.
Email: gautham.gs@manipal.edu
Funding information
K E Y W O R D S
Department of Biotechnology
molecular dynamics, mycobacterial enoyl-reductase (InhA), pyridine, triclosan
95% of people suffering from DS-TB. MDR-TB needs longer treat-
ment and involves treatment with second-line drugs, which are
1
| INTRODUCTION
According to the World Health Organization (WHO), tuberculosis
(TB), a contagious disease mainly caused by Mycobacterium tuberculo-
sis (MTb), is one of the oldest and biggest killers. As per WHO report,
2018, globally TB was responsible for death of 1.3 million HIV-
negative and additional 0.3 million HIV-positive people. Emergence of
multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB
has further complicated the scenario (Gandhi et al., 2006;
WHO, 2018).
expensive, less effective, and less convenient with more adverse
effects in comparison with the first-line drugs (Migliori, De Iaco,
Besozzi, Centis, & Cirillo, 2007; Udwadia, Amale, Ajbani, & Rodrigues,
2012; Velayati et al., 2009). Global TB status presents an urgent need
to develop safe and effective antitubercular drugs with alternate tar-
gets to avoid drug resistance.
The biosynthesis of the unique cell wall of Mycobacterium pre-
sents various drug targets providing significant potential for novel
drug discovery (Barry, Crick, & McNeil, 2007). MTb utilizes the type II
pathway fatty acid synthesis (FAS-II) pathway for extension of
the fatty acid chains that are used for mycolic acid biosynthesis.
The current drug regimen for the drug susceptible (DS)-TB
treatment involves
a combination of isoniazid (INH), rifamycin,
pyrazinamide, and ethambutol (Johnson et al., 2006), which can cure
Drug Dev Res. 2019;1–14.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

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VERMA ET AL.
The FAS-II system is absent in higher eukaryotes and hence FAS-II
enzymes are an attractive target as novel drugs with selective toxicity
toward MTb could potentially be developed (Bloch, 1977). INH (pyri-
dine derivative) inhibits the mycobacterial enoyl-reductase (InhA), but
MDR and XDR MTb isolates have developed resistance toward INH
primarily due to the KatG mutations. INH is a prodrug and is activated
by the enzyme catalase peroxidase to form reactive species, which
further reacts with NAD+ to form an INH-NAD adduct. This activated
form of INH inhibits InhA, resulting in inhibition of mycolic acid bio-
synthesis and consequently mycobacterial cell death (Zhao et al.,
2006). KatG encodes catalase peroxidase and is the prime reason for
INH resistance. Mutation in katG activates INH and specifically the
KatG variant, and S315T is found in about 94% of INH-resistant clini-
cal isolates responsible for resistance (Nguyen, Claparols, Bernadou, &
Meunier, 2001). Also mutation in the inhA (c-15t) promoter region
results in inhA overexpression, thus resulting in the titration of the
drug. Expression changes of the drug's activators, redox variation,
inactivation of drug, and activation of efflux pump are the other
mechanisms of INH resistance (Vilchèze & Jacobs, 2014). Inhibitors of
the essential InhA component of the FAS-II system have been shown
to have in vivo efficacy, and thus, there is precedence to support stud-
ies aimed at discovering InhA inhibitors (Levy et al., 1999; Nguyen
et al., 2001; Rozwarski, Grant, Barton, Jacobs, & Sacchettini, 1998;
Zhang, Heym, Allen, Young, & Cole, 1992; Zhao et al., 2006).
Triclosan (2,4,40-trichloro-20-hydroxydiphenyl ether) has been
reported to inhibit InhA and thus inhibit fatty acid biosynthesis. Unlike
INH, it directly inhibits InhA without further activation, but its role as
an antitubercular agent is limited by its poor solubility (McLeod et al.,
2001) and suboptimal bioavailability (Wang, Falany, & James, 2004).
Despite these challenges, this small organic molecule provides a ratio-
nal starting point for structure-based drug discovery to produce effec-
tive InhA inhibitors. Encouraged by the antitubercular activity of the
2-phenoxy-5-([arylamino]methyl)phenol derivatives synthesized by
our research group, we decided to explore further these analogues by
doing modification (Thomas et al., 2015). We synthesized novel pyri-
dine ether derivatives with improved druggability, mimicking the
structural features of triclosan, and assessed them for antitubercular
activity. Since INH is an inhibitor of inhA, it was decided to introduce
pyridine pharmacophore in the newly designed molecules, thus com-
bining the features of triclosan and INH. Triclosan has two chlorine
atoms in the structure, which contributes to higher lipophilicity. In
order to reduce the lipophilicity of the molecules, the chlorine atoms
were not retained. Furthermore, replacement of one of the phenyl
rings with pyridine resulted in further reduction of lipophilicity of the
molecules. Based on the literature data as well as the work carried out
in our laboratory, a linker with two atoms was introduced between
the aryl substituent and B-ring (Freundlich et al., 2009; Hartkoorn
et al., 2012; Lu & Tonge, 2008).
discovery is essential to reduce the cost of drug discovery. Computa-
tional techniques are being used for the identification of lead mole-
cules among the several thousands of molecules designed and their
optimization. Advanced computational techniques have made lead
identification and optimization process rapid (Taft, da Silva, & da Silva,
2008). These techniques help in identifying most promising molecules
among several thousands of molecules that are initially designed.
Ligand-based drug design (LBDD) and structure-based drug design
(SBDD) are the two types of computer-aided dug design (CADD)
approaches that are being used for drug design (Ece, 2019). Molecular
docking study is an important study that helps to understand the
nature of interaction between target residues and the designed mole-
cule. The understanding of the nature of interactions helps in bringing
about structural modifications and establish structure–activity rela-
tionship (Tahtaci, Karacık, Ece, Er, & Seker, 2018). Hence, supporting
computational studies were carried out to explore the plausible inter-
action of the synthesized molecules with the target protein residue
and to gather information related to the structural requirements of
this scaffold to enhance their antitubercular potential. For this pur-
pose, the published X-ray crystal structure of MTb InhA (pdb 3FNE)
was used. This pdb consists of the crystal structure of InhA bound to
a triclosan derivative. As most of the novel compounds have ADME
(absorption, distribution, metabolism, and excretion) properties that
remain to be experimentally validated, these properties and other
drug-like properties were determined in silico for the synthesized
compounds. Molecular dynamics (MD) studies give an insight into the
protein dynamics, provide the possible binding sites, and also give
clear information on the stable interaction over the simulation time
period. The Desmond module of maestro was used for this purpose.
Desmond can compute energies and forces for the standard fixed-
charged force fields used in biomolecular simulations. Desmond sup-
ports algorithms normally used to perform fast and accurate
MD. Long-range electrostatic energy and forces are calculated using
particle-mesh-based Ewald techniques (Desmond, 2015). MD studies
were performed for the most active compound and were compared
with the triclosan.
2
| EXPERIMENTAL
2.1 | Material and methods
2.1.1 | Reagents and chemicals
All commercially available materials and solvents were used without
any further purification. Media components for 7H9 and glycerol-ala-
nine-salts-Tween-80 with iron (GAST-Fe) were purchased from Difco
and Sigma, respectively. HepG2 and Vero cell lines used in this study
were procured from the National Center for Cell Science, Pune, India,
and maintained in high-glucose DMEM medium with 10% FBS and
1% antibiotic–antimycotic solution, at 37ꢀC in a CO2 incubator under
5% CO2 (CLS-170-B-8, Serial No. 201020569, Esco Micro Pte. Ltd.).
Column chromatography was performed for the isolation of the pure
compounds from the crude reaction mixture on 100–200 mesh silica
Drug discovery process is time-consuming and very expensive. It
involves contribution by experts from various disciplines. The success
rate in drug discovery is extremely low. Out of the several thousands
of molecules that are synthesized, only a few make into the clinical tri-
als. Eliminating the unsuitable molecules in the early phase of drug

VERMA ET AL.
3
gel purchased from Merck. All reactions were monitored, and purity
of the compounds was checked by thin-layer chromatography (TLC,
aluminum-coated sheets of silica gel 60F24, Merck, India). Melting
points were determined with a laboratory melting point apparatus and
are uncorrected. 1H NMR and 13C NMR spectra were recorded on a
nuclear magnetic resonance (NMR) spectrometer (AV400—400 MHz,
high-resolution multinuclear FT-NMR spectrometer, Bruker) in
DMSO-d6 (Dimethyl sulfoxide) as a solvent. Chemical shifts are
reported here in parts per million (ppm). Mass spectrometric analysis
was performed using LC–MS (linear ion trap, APCI mode, LC/MS,
Thermo Fisher Scientific LTQ21532 series). δIR spectral data were
recorded on an FT-IR spectrophotometer (IR Affinity-1, Shimadzu,
Japan) in KBr pellets. The characteristic IR absorption frequencies are
presented as wave numbers (cm−1).
N-(4-(pyridin-2-yloxy)benzyl)aniline (2a)
Yield = 0.50 g (72%); mp = 78–80ꢀC; Rf = 0.80 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) = 3,390.86, 3,047.53, 3,018.60, 1,465.90,
1,506.41, 1,595.13, 1,249.87, 1,197.79, 1,151.50; 1H NMR (400 MHz,
DMSO-d6): δ 8.13 (s, 1H), 7.84 (d, J = 5.2 Hz, 1H), 7.39 (s, 2H), 7.06 (d,
J = 4.4 Hz, 6H), 6.59–6.50 (m, 3H), 6.25 (d, J = 4.4 Hz, 1H), 4.26 (d,
J = 8.0 Hz, 2H); 13C NMR (100.62 MHz, DMSO-d6): 163.61, 152.99,
149.11, 147.86, 140.56, 136.92, 129.32, 128.89, 121.53, 119.36,
116.24, 112.76, 111.86, 46.38; calculated for C18H16N2O [M+]:
276.34.43, found LC–MS (-APCI, m/z): 275.90 (M–H)−.
4-Methoxy-N-(4-(pyridin-2-yloxy)benzyl)aniline (2b)
Yield = 0.52 g (67.6%); mp = 52–54ꢀC; Rf = 0.82 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) =3,342.64, 3,057.17, 3,022.45, 2,997.38,
1,593.20, 1,512.19, 1,465.90, 1,253.73, 1,109; 1H NMR (400 MHz,
DMSO-d6): δ 8.14 (t, J = 5.0, 1H), 7.85 (ddd, J = 8.8, 7.3, 2.0 Hz, 1H),
7.39 (d, J = 8.2 Hz, 2H), 7.16–7.03 (m, 3H), 7.01 (d, J = 8.3 Hz, 1H),
6.74–6.66 (m, 2H), 6.61–6.50 (m, 2H), 5.85 (t, J = 6.2 Hz, 1H), 4.22 (d,
J = 6.1 Hz, 2H), 3.63 (s, 3H); 13C NMR (100.62 MHz, DMSO-d6):
163.61, 152.93, 151.19, 147.86, 143.33, 140.57, 137.19, 128.93,
121.48, 119.35, 115.05, 113.80, 111.85, 55.75, 47.18; calculated for
C19H18N2O2 [M+]: 306.37, found LCMS (-APCI, m/z): 305.13 (M–H)−.
2.1.2 | Synthesis
General procedure for the synthesis of 4-(pyridin-2-yloxy)
benzaldehyde, 1
Solution of 4-hydroxybenzaldehyde (2.0 mmol), 2-bromopyridine
(0.95 g, 6.0 mmol), and potassium carbonate (0.87 g, 6.0 mmol) in
dimethylacetamide (5 ml) were refluxed for 24 hr. The progress of the
reaction was monitored by TLC using the hexane:ethyl acetate (8:2)
solvent system. After reaction completion (24 hr), the reaction mixture
was diluted using water (100 ml) by continuous stirring and the resi-
due obtained was extracted using dichloromethane (3 × 50 ml). The
combined organic layers were separated, dried over anhydrous
MgSO4, and evaporated under vacuum. The crude compound
obtained was purified by column chromatography over silica 100–200
with hexane:ethyl acetate (8:2) as the mobile phase to afford
4-(pyridin-2-yloxy)benzaldehyde (Zheng et al., 2018).
3-Methoxy-N-(4-(pyridin-2-yloxy)benzyl)aniline (2c)
Yield = 0.48 g (62.4%); mp = 48–50ꢀC; Rf = 0.81 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) =3,340, 3,089, 2,997, 1,608.63, 1,593.20,
1,247, 1,205, 1,163, 1,240.23; 1H NMR (400 MHz, DMSO-d6): 8.13 (t,
J = 4.9 Hz, 1H), 7.84 (ddd, J = 8.8, 7.2, 2.0 Hz, 1H), 7.37 (d, J = 8.2 Hz,
2H), 7.15–7.03 (m, 3H), 7.00 (d, J = 8.3 Hz, 1H), 6.94 (t, J = 8.0 Hz,
1H), 6.28 (t, J = 6.1 Hz, 1H), 6.20 (dd, J = 8.1, 2.1 Hz, 1H), 6.16–6.07
(m, 2H), 4.24 (d, J = 6.1 Hz, 2H), 3.64 (s, 3H); 13C NMR (100.62 MHz,
DMSO-d6): 163.61, 160.75, 152.97, 150.44, 147.86, 140.58, 136.93,
130.01, 128.88, 121.54, 119.36, 111.86, 105.93, 101.67, 98.59,
55.05, 46.37; calculated for C19H18N2O2 [M+]: 306.37, found LC–MS
(-APCI, m/z): 305.26 (M–H)−.
Yield = 0.20 g (24%); mp = 35–36ꢀC; Rf = 0.80 (hexane:ethyl ace-
tate = 8:2); 1H NMR (400 MHz, DMSO-d6): δ 9.98 (s, 1H), 8.22 (s, 1H),
7.97–7.92 (m, 3H), 7.32 (d, J = 8.224 Hz, 2H), 7.32–7.24 (m, 1H), 7.16
(d, J = 7.6 Hz, 2H).
General procedure for the synthesis of N-(4-(pyridin-2-yloxy)benzyl)
arylamine (2a–2m)
4-Methyl-N-(4-(pyridin-2-yloxy)benzyl)aniline (2d)
Yield = 0.569 g (73.9%); mp = 56–58ꢀC; Rf = 0.84 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) =3,390.86, 3,053.32, 3,024.38, 1,583.56,
1,516.05, 1,465.90, 1,246.02, 1,201.65, 1,153.43; 1H NMR (400 MHz,
DMSO-d6): δ 8.14 (t, J = 5.0 Hz, 1H), 7.84 (ddd, J = 8.3, 7.2, 2.0 Hz,
1H), 7.38 (d, J = 8.3 Hz, 2H), 7.16–7.03 (m, 3H), 7.01 (dt, J = 8.3,
0.9 Hz, 1H), 6.87 (d, J = 8.0 Hz, 2H), 6.55–6.48 (m, 2H), 6.05 (t,
J = 6.2 Hz, 1H), 4.24 (d, J = 6.1 Hz, 2H), 2.14 (s, 3H); 13C NMR
(100.62 MHz, DMSO-d6): 163.61, 152.92, 147.85, 146.82, 140.56,
137.11, 129.76, 128.86, 124.57, 121.49, 119.34, 112.90, 111.84,
46.61, 20.54; calculated for C19H18N2O [M+]: 290.37, found LC–MS
(-APCI, m/z): 289.13 (M–H)−.
To
a solution of 4-(pyridin-2-yloxy)benzaldehyde (1) (0.2 g,
0.93 mmol) in methanol (10 ml), amine (1.12 mmol) was added and
the mixture was refluxed until reaction completion. After the reaction
was over, the reaction mixture was cooled and NaBH3CN (1.86 mmol)
was added. The reaction mixture was refluxed. The progress of the
reaction was monitored by TLC, using hexane:ethylacetate (8:2) as the
mobile phase. When the reaction completed (8 hr), the solvent from
the reaction mixture was evaporated under vacuum. Ice-cold water
was added to the residue obtained and the product was extracted
with ethyl acetate (3 × 25 ml). The combined organic layer was
washed with water, dried using anhydrous MgSO4, and evaporated
using vacuum. The crude product obtained was purified using column
chromatography over silica 100–200 with hexane:ethylacetate (9:1)
as the mobile phase to get the target compound N-(4-(pyridin-
2-yloxy)benzyl)arylamine (Thomas et al., 2015).
3-Methyl-N-(4-(pyridin-2-yloxy)benzyl)aniline (2e)
Yield = 0.50 g (68.6%); mp = 55–57ꢀC; Rf = 0.82 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) = 3,344.57, 3,041.74, 2,927.94, 1,597.06,

4
VERMA ET AL.
1,512.19, 1,469.76, 1,421.54, 1,274.95, 1,201.65, 1,159.22; 1H NMR
(400 MHz, DMSO-d6): δ 8.13 (d, J = 4.6 Hz, 1H), 7.87–7.78 (m, 1H),
7.37 (dd, J = 8.4, 2.0 Hz, 2H), 7.14–6.87 (m, 5H), 6.43 (s, 1H), 6.36 (dd,
J = 18.6, 7.8 Hz, 2H), 6.13 (t, J = 6.1 Hz, 1H), 4.24 (d, J = 6.0 Hz, 2H),
2.15 (d, J = 2.0 Hz, 3H); 13C NMR (100.62 MHz, DMSO-d6): 163.61,
152.95, 149.12, 147.86, 140.57, 138.23, 137.04, 129.20, 128.86,
121.51, 119.35, 117.21, 113.46, 111.85, 109.96, 46.34, 21.86; calcu-
lated for C19H18N2O [M+]: 290.37, found LC–MS (-APCI, m/z):
289.12 (M–H)−.
J = 6.3 Hz, 1H), 4.41 (d, J = 6.2 Hz, 2H); 13C NMR (100.62 MHz,
DMSO-d6): 163.56, 153.04, 147.85, 144.32, 140.58, 136.37, 129.47,
128.57, 128.36, 121.60, 119.38, 118.31, 116.97, 112.10, 111.89,
45.88; calculated for C18H15ClN2O [M+]: 310.78, found LC–MS
(-APCI, m/z): 309.68 (M–H)−.
4-Fluoro-N-(4-(pyridin-2-yloxy)benzyl)aniline (2j)
Yield = 0.60 g (81.0%); mp = 75–77ꢀC; Rf = 0.79 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) = 3,315.63, 3,089.96, 3,057.17, 3,030.17,
1,597.061512.19, 1,274.95, 1,207.44, 1,151.50; 1H NMR (400 MHz,
DMSO-d6): δ 8.13 (t, J = 4.9 Hz, 1H), 7.89–7.79 (m, 1H), 7.42–7.34
(m, 2H), 7.16–6.96 (m, 4H), 6.95–6.84 (m, 2H), 6.63–6.53 (m, 2H),
6.20 (t, J = 5.6 Hz, 1H), 4.23 (d, J = 5.1 Hz, 2H); 13C NMR
(100.62 MHz, DMSO-d6): 163.59, 155.89, 153.02, 147.85, 145.80,
140.59, 136.71, 128.95, 121.55, 119.37, 115.80, 115.58, 113.48,
113.40, 111.87, 46.88; calculated for C18H15CFN2O [M+]: 294.33,
found LC–MS (-APCI, m/z): 293.30 (M–H)−.
2-Methyl-N-(4-(pyridin-2-yloxy)benzyl)aniline (2f)
Yield = 0.52 g (71.3%); mp = 51–53ꢀC; Rf = 0.81 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) =3,340.71, 3,014.74, 2,951.09, 1,595.13,
1,510.26, 1,495.90, 1,274.95, 1,201.65, 1,153.43; 1H NMR (400 MHz,
DMSO-d6): δ 8.13 (dd, J = 5.0, 2.0 Hz, 1H), 7.87–7.78 (m, 1H),
7.45–7.35 (m, 2H), 7.16–7.01 (m, 3H), 7.03–6.87 (m, 3H), 6.52–6.38
(m, 2H), 5.63 (t, J = 6.1 Hz, 1H), 4.35 (d, J = 6.0 Hz, 2H), 2.16 (s, 3H);
13C NMR (100.62 MHz, DMSO-d6): 163.61, 152.88, 147.85, 146.57,
140.56, 137.13, 130.21, 128.59, 127.08, 122.24, 121.50, 119.34,
116.18, 111.85, 110.02, 46.28, 18.25; calculated for C19H18N2O [M
+]: 290.37, found LC–MS (-APCI, m/z): 289.10 (M–H)−.
2-Fluoro-N-(4-(pyridin-2-yloxy)benzyl)aniline (2k)
Yield = 0.57 g (77.0%); mp = 68–70ꢀC; Rf = 0.77 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) = 3,323.35, 2,881.65, 2,843.07, 1,606.70,
1,583.56, 1,274.95, 1,195.87,1,155.36; 1H NMR (400 MHz, DMSO-
d6): 8.13 (t, J = 4.9 Hz, 1H), 7.83 (t, J = 8.3 Hz, 1H), 7.43–7.36 (m, 2H),
7.14–6.96 (m, 5H), 6.89 (t, J = 7.8 Hz, 1H), 6.62 (m, 3H), 4.35 (d,
J = 6.3 Hz, 2H); 13C NMR (100.62 MHz, DMSO-d6): δ 163.57, 153.01,
150.20, 147.85, 140.58, 136.63, 128.68, 125.10, 121.54, 119.37,
115.98, 115.92, 114.69,112.68, 111.88, 45.77; calculated for
C18H15CFN2O [M+]: 294.33, found LC–MS (-APCI, m/z): 293.09
(M–H)−.
4-Chloro-N-(4-(pyridin-2-yloxy)benzyl)aniline (2g)
Yield = 0.55 g (70%); mp = 94–96ꢀC; Rf = 0.87 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) = 3,321, 3,059.10, 3,024.38, 1,597.06,
1,510.26, 1,465.90, 1,273.02, 1,207.44, 1,151.50; 1H NMR (400 MHz,
DMSO-d6): δ 8.14 (dd, J = 4.9, 2.0 Hz, 1H), 7.85 (ddd, J = 8.8, 7.3,
2.1 Hz, 1H), 7.42–7.33 (m, 2H), 7.25–7.02 (m, 5H), 7.02 (d, J = 8.2 Hz,
1H), 6.65–6.56 (m, 2H), 6.49 (t, J = 6.1 Hz, 1H), 4.26 (d, J = 6.0 Hz,
2H); 13C NMR (100.62 MHz, DMSO-d6): 163.58, 153.07, 147.99,
147.94, 140.58, 136.41, 130.86, 129.03, 128.91, 121.59, 119.20,
114.09, 111.87, 46.34; calculated for C18H15ClN2O [M+]: 310.78,
found LC–MS (-APCI, m/z): 309.06 (M–H)−.
4-Bromo-N-(4-(pyridin-2-yloxy)benzyl)aniline (2l)
Yield = 0.65 g (73%); mp = 96–98ꢀC; Rf = 0.79 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) = 3,321.42, 3,059.10, 3,022.45, 1,595.13,
1,510, 1,465.90, 1,273.02, 1,205.51, 1,151.50; 1H NMR (400 MHz,
DMSO-d6): δ 8.14 (dd, J = 5.0, 1.9 Hz, 1H), 7.85 (ddd, J = 8.6, 7.3,
2.0 Hz, 1H), 7.41–7.34 (m, 2H), 7.24–7.15 (m, 2H), 7.18–7.04 (m, 3H),
7.02 (d, J = 8.3 Hz, 1H), 6.61–6.48 (m, 3H), 4.26 (d, J = 6.0 Hz, 2H);
13C NMR (100.62 MHz, DMSO-d6): 163.58, 153.07, 148.34, 147.86,
140.59, 136.36, 131.83, 128.89, 121.60, 119.39, 114.68, 111.88,
106.84, 46.23; calculated for C18H15BrN2O [M+]: 355.24 found LC–
MS (-APCI, m/z): 354.32 (M–H)−.
3-Chloro-N-(4-(pyridin-2-yloxy)benzyl)aniline (2h)
Yield = 0.489 g (63%); mp = 88–90ꢀC; Rf = 0.85 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) =3,323.35, 3,061.03, 3,022.45, 1,589.34,
1,508.33, 1,477.47, 1,276.88, 1,203.58, 1,153.43; 1H NMR (400 MHz,
DMSO-d6): δ 8.17–8.10 (m, 1H), 7.88–7.79 (m, 1H), 7.41–7.34 (m,
2H), 7.15–7.00 (m, 4H), 7.04–6.97 (m, 1H), 6.65–6.57 (m, 2H), 6.54
(ddd, J = 13.6, 7.9, 2.1 Hz, 2H), 4.27 (d, J = 6.0 Hz, 2H); 13C NMR
(100.62 MHz, DMSO-d6): 163.57, 153.11, 150.60, 147.86, 140.59,
136.24, 134.06, 130.84, 128.93, 121.63, 119.39, 115.62, 111.89,
111.87, 111.42, 46.10; calculated for C18H15ClN2O [M+]: 310.78,
found LC–MS (-APCI, m/z): 309.17 (M–H)−.
3-bromo-N-(4-(pyridin-2-yloxy)benzyl)aniline (2m)
Yield = 0.63 g (70.6%); mp = 92–94ꢀC; Rf = 0.78 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) = 3,325.28, 3,059.10, 3,020.53, 1,587.42,
1,506.41, 1,473.62, 1,246.02, 1,205.51, 1,153.43; 1H NMR (400 MHz,
DMSO-d6): δ 8.13 (ddd, J = 5.0, 2.1, 0.9 Hz, 1H), 7.84 (ddd, J = 8.2,
7.2, 2.0 Hz, 1H), 7.41–7.28 (m, 2H), 7.21–7.05 (m, 3H), 7.09–6.95 (m,
2H), 6.76 (t, J = 2.1 Hz, 1H), 6.65 (ddd, J = 7.9, 1.9, 0.9 Hz, 1H),
6.63–6.55 (m, 2H), 4.27 (d, J = 6.0 Hz, 2H); 13C NMR (100.62 MHz,
DMSO-d6): 163.57, 153.12, 150.78, 147.86, 140.59, 136.22, 131.18,
128.93, 122.79, 121.63, 119.39, 118.51, 114.82, 111.89, 111.68,
2-Chloro-N-(4-(pyridin-2-yloxy)benzyl)aniline (2i)
Yield = 0.531 g (68%); mp = 86–88ꢀC; Rf = 0.84 (hexane:ethyl ace-
tate = 8:2); IR (KBr, cm−1) = 3,419.79, 3,064.89, 3,016.67, 1,591.27,
1,508.33, 1,463.97, 1,274.95, 1,197.79, 1,153.43; 1H NMR (400 MHz,
DMSO-d6): δ 8.13 (dd, J = 5.0, 2.0 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H),
7.41–7.34 (m, 2H), 7.25 (dd, J = 7.8, 1.4 Hz, 1H), 7.07 (qd, J = 11.0,
9.7, 6.5 Hz, 4H), 7.00 (d, J = 8.2 Hz, 1H), 6.64–6.51 (m, 2H), 6.14 (t,

VERMA ET AL.
5
46.08; calculated for C18H15BrN2O [M+]: 355.24 found LC–MS
(-APCI, m/z): 354.07 (M–H)−.
dilutions were added to each well of 96-well plate containing Vero cells
(104 cells/well) and incubated at 37ꢀC for 72 hr. Inoculum was kept as
positive control and media as negative control. After 72 hr of incuba-
tion, to each well, 50 μl of MTT was added and the plate was again
incubated for 4 hr at 37ꢀC. After 4 hr, 50 μl of sterile DMSO was added
to dissolve any formazan crystals that had formed. Furthermore, the
absorbance was recorded by using an ELISA plate reader at 540 nm,
and the CC50 value was determined using the curve-fitting program
(Ammerman, Beier-Sexton, & Azad, 2008; Guo et al., 2010).
2.1.3 | Biological activities
Antitubercular activity
All the synthesized compounds were assayed by microtiter dilution
assay in a 96-well plate protocol using two different media, that is,
regular 7H9-based medium and GAST-Fe (pH 6.6) medium for mini-
mum inhibitory concentration (MIC) determination against MTb
H37Rv. The MTb inoculum at approximately 2 × 105 CFU/well was
used for this assay. Sterile stock solution of synthesized compounds
and standard compounds was prepared in DMSO. The assay was per-
formed using the serial dilution technique where the MTb inoculum
was added to the assay plate containing serial twofold dilutions of the
compounds in the medium of choice attaining a final cell concentra-
tion of 1 × 105 cfu/ml and the final compound concentration series
range of 100 to 0.048 μM. INH and triclosan were taken as positive
control. Plates were sealed in a ziplock bag and incubated at 37ꢀC for
14 days. Plates were read after completion of Weeks 1 and 2 using
inverted enlarging mirror plate reader and graded for growth and no
growth. The MIC was defined as the lowest drug concentration that
prevented growth of bacteria (Barot et al., 2014; Kaniga et al., 2016;
Soares de Melo et al., 2015).
As antitubercular drugs are associated with hepatotoxicity (Ajay
et al., 2010), in vitro cell line studies were performed using MTT assay
in HepG2 cells to assess the hepatotoxicity of the synthesized com-
pounds. The protocol used was the same as that for Vero cell lines.
Here, the concentration of HepG2 cells was kept as 5 × 103 cells/well
and incubated for 24 hr before the addition of MTT. The following
equations were used to calculate the percentage cell viability and per-
centage cell inhibition:
% Cell viability = (optical density of the test/optical density of con-
trol)/100% cell inhibition = 100−% cell viability.
2.1.4 | In silico studies
Molecular modeling
Molecular docking was performed to understand the binding mode of
the compounds. The crystal structure of InhA complexed with triclosan
derivative (PDB 3FNE) was downloaded from the Protein Data Bank.
The protein preparation wizard module of Schrodinger 2017-4 was used
to process the protein. The multistep process involved bond order
assignment, hydrogens addition, treatment of disulfides, removal of
unimportant water molecules within 5 Å of the binding site, hydrogen
bond assignment via exhaustive sampling, and energy minimization to
root mean square deviation (RMSD) 0.30 Å by OPLS-2003. A grid was
generated at the center of the cocrystallized ligand (triclosan derivative)
to include the cofactor and substrate-binding sites by utilizing default
parameters of the software. The ligand was excluded from the protein
and was limited to the enclosing box at the centroid. The docking proce-
dure was validated by removing the cocrystallized ligand from the bind-
ing site and redocking it at the same binding site. The obtained RMSD
score was 0.4163 Å. A 2D sketch tool of Schrodinger 2017-4 was used
to draw the designed molecules, and the possible conformers (through
Epik) were generated using the LigPrep module. Optimized potential for
liquid simulation (OPLS) force field was used to obtain low-energy con-
formers of the designed molecules. Then single low-energy 3D con-
formers were docked using a flexible docking method with the catalytic
site of the receptor using the extra precision (XP) Glide algorithm of
Schrodinger suite 2017-4 (Bari & Haswani, 2017; Friesner et al., 2006;
Harder et al., 2015; Malikanti et al., 2017; Schrödinger Release 2017-4,
2017; Sharma et al., 2018).
Antibacterial activity
All the synthesized compounds were tested for antibacterial activity
by resazurin-based microtiter dilution assay in 96-well plates in the
concentration range of 50 to 1.56 μg/ml. Four bacterial species were
used, that is, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus
aureus, and Escherichia coli. At first, 100 μl of the test compound in
10% (v/v) DMSO was added to the first row of the microtiter plate
and 50 μl of the nutrient broth was added to the remaining rows. The
compounds were serially diluted using a multichannel pipette. Then,
10 μl of resazurin dye and 30 μl of 3.3× strength broth were added to
each well to achieve the concentration of single strength. Finally,
10 μl of bacterial suspension (5 × 106 cfu/ml) was added to each well.
The plates were sealed in a ziplock bag and placed in the incubator at
37ꢀC for 18–24 hr. Ciprofloxacin and triclosan were used as positive
controls. After 24 hr, any color change from violet to pink was
recorded as positive. The lowest concentration where color change
occurred was considered as the activity of the compound (Sharma,
Kothapalli, Dongen, & Swaminathan, 2012).
Cytotoxicity
The cytotoxicity of the synthesized compounds was assessed using the
MTT [3-(4, 5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide]
assay on Vero cells (from the kidneys of African Green monkey) and
HepG2 cells (human liver cancer cell lines). This is a colorimetric assay
involving the reduction of MTT to insoluble formazan by cellular
enzymes (Guo et al., 2010). The test samples were prepared in various
concentrations using DMEM and added to the 96-well plates. DMSO
concentration was maintained at ≤0.1%. Then, 100 μl of sample
MD simulations
Protein dynamics changes during docking and with the help of MD
simulation study, and the affinity of a molecule on various conforma-
tions of the target receptor can be measured. Hence, we performed

6
VERMA ET AL.
MD study using the Desmond module from Schrodinger (Desmond,
2015). MD simulations were carried out using Maestro version 11.4
(Schrodinger Inc.) on a HP computer with a Linux Ubuntu 18.04.1 LTS
platform, Intel Haswell graphics card, 8 GB RAM, and Intel Core
i3-4160 processor. First, a system builder panel was used for prepar-
ing the orthorhombic simulation box using an SPC explicit water
model. The minimum distance between the protein surface and the
solvent surface was 10 Å, and the charge was neutralized by adding
sodium and chloride ions. The simulation was run for 20 ns, using
NPT ensemble at a temperature of 300 K and atmospheric pressure
(1.013 bar) using default setting before simulation. During MD simula-
tion, the trajectory was written with 1,000 frames and a frame was
captured at every 15 ps. Postmolecular dynamics analysis was per-
formed using a simulation interaction diagram tool of the Schrodinger
software.
The IR spectra of compounds 2a–2m showed NH peak between
3,300 and 3,000 cm−1. The 1H NMR spectra of the compound 2a–2m
exhibited aromatic protons in between 6.00 and 8.00 ppm and a dou-
blet around 4.00 ppm representing two protons of NH─CH2 group,
thus confirming the formation of reduced Schiff bases (Additional
spectral details are given in supplementary file).
3.2 | Antitubercular activity
All the synthesized compounds were evaluated for their antitubercular
activity in vitro against MTb H37Rv (ATCC-27294) using the standard
microbroth dilution minimum inhibitory concentration (MIC) protocol
in two different media; regular 7H9-based medium; and GAST-Fe
(pH 6.6) medium. The compounds were screened at 12 different con-
centrations, ranging from 100 to 0.048 μM. The results of in vitro
antitubercular activity are presented in Table 1.
With GAST-Fe medium for minimum inhibitory concentration,
compounds tested (series 2a–2m) showed potent MIC values. All
13 compounds showed better or comparable activity with respect to
triclosan, and among them, 5 compounds showed MIC ranging from
19 to 25 μM. The most potent compound was found to be 4-fluoro-
substituted derivative with an MIC of 19 μM. After additional incuba-
tion (second week), except for the compound 2k (MIC 70 μM), all the
compounds exhibited activity equal to or better than triclosan,
suggesting that the compounds maintained their ability to inhibit
mycobacterial growth over time. The substantial activity of com-
pounds after additional incubation suggests that the compounds
maintained their ability to inhibit mycobacterial growth over time.
In 7H9-based medium, among the compounds tested (series
2a–2m), compounds 2b, 2f, 2g, and 2l showed moderate MIC values
of 41 μM, 40 μM, 40 μM, and 35 μM at the end of Week 1, but none
of the compounds showed antitubercular activity better than triclosan
(MIC 22 μM). At the end of Week 2, none of the compounds were
found to display activity better than triclosan (MIC 43 μM). The lower
MIC in GAST-Fe medium as compared to regular 7H9-based medium
is likely driven by protein binding of the compounds to the BSA com-
ponent of 7H9-based broth.
In silico drug-likeliness and ADME prediction
The drug-like properties of the synthesized compounds were calculated
using the QikProp module (v 5.4, Schrodinger release 2017-4). The
properties such as molecular weight, hydrogen bond donor, hydrogen
bond acceptor, log p, QPlog S, QPPCaco, QPlogBB, QPPMDCK, Percent
Human Oral Absorption, CNS activity, and QPlogKhsa (Lipinski,
Lombardo, Dominy, & Feeney, 1997; Sharma et al., 2012; Veber et al.,
2002) were predicted using the QikProp module.
3
| RESULTS AND DISCUSSION
3.1 | Synthesis and spectral analysis
The final compounds were prepared as per the steps described in
Scheme 1. 4-Hydroxy benzaldehyde was condensed with
2-bromopyridine to obtain 4-(pyridin-2-yloxy)benzaldehyde (Zheng
et al., 2018). The aldehyde obtained (compound 1) was reacted with
various aromatic amines to obtain the target compounds 2a–2m
(Thomas et al., 2015). The mechanistic approach for the compounds
(2a–2m) formation involves attack of nucleophilic amines on the elec-
trophilic carbon atoms of aldehyde resulting in replacement of C=O
bond with C=N bond, which is an imine or Schiff's base (Schiff base,
2014). Furthermore, the C=N bond was reduced with sodium
cyanoborohydride to get the final compounds. The products were
obtained in moderate yield (62–82%). The molecular characterization
through NMR, IR, and mass spectroscopy confirmed their formation.
3.3 | Antibacterial activity
The synthesized compounds were screened against P. aeruginosa,
S. aureus, E. coli, and B. subtilis for antibacterial activity. The
SCHE ME 1 Synthesis of
N-(4-(pyridin-2-yloxy)benzyl) arylamine
derivatives

VERMA ET AL.
7
TABLE 1 In vitro antimycobacterial
activity and cytotoxicity of
compounds (2a–2m)
b
MICa (μM)
(GAST/Fe)
MIC (μM)
(7H9/ADC/Tween)
CC50
(μM)
Compounds
Week 1
25
Week 2
37
Week 1
>50
41
Week 2
cVero
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
–
dHepG2
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
–
2a
>50
82
2b
41
41
2c
25
37
>50
86
>50
86
2d
22
43
2e
43
43
>100
>100
40
>100
>100
80
2f
43
43
2g
40
40
2h
20
40
40
80
2i
20
20
>100
>50
>100
70
>100
>50
>100
70
2j
19
25
2k
43
43
2l
35
70
2m
35
35
35
70
Triclosan
Isoniazid
43
43
22
43
0.2
0.2–0.4
0.78
1.5
aMIC is defined as the minimal drug concentration required to stop the growth of Mycobacterium
tuberculosis H37Rv.
bCC50 is defined as the minimal drug concentration required for 50% death of viable cells.
cVero: African green monkey kidney cell line.
dHepG2: human liver cells.
TABLE 2 In vitro antibacterial activity of compounds 2a–2m
Bacillus subtilis
Pseudomonas aeruginosa
Staphylococcus
Escherichia coli
Compounds
activity (μg/ml)
activity (μg/ml)
aureus activity (μg/ml)
activity (μg/ml)
2a
1.6
>6.24
1.6
>25
1.8
>3.1
1.6
>25
>25
>50
>25
>50
>25
>50
>25
>25
>12.5
>25
>25
>50
>3.2
1.6
2b
>25
2c
>25
2d
1.8
>50
1.8
2e
>3.1
1.8
>25
>6.2
1.6
2f
>50
2g
>6.2
1.8
>25
>3.1
1.8
2h
>50
2i
1.8
>25
1.6
2j
1.6
>12.5
>25
1.8
2k
1.8
1.6
2l
>3.1
>6.2
1.8
>25
>6.2
>3.1
1.8
2m
>50
Triclosan
Ciprofloxacin
>25
1.6
1.6
1.6
preliminary screening of the compounds was conducted at concentra-
tions ranging from 50 to 1.56 μg/ml. The results are displayed in
Table 2. The compounds were found to be more active against B. sub-
tilis and S. aureus than those against P. aeruginosa and E. coli. The
results suggest that the compounds are mostly active against gram-
positive bacteria.
3.4 | In vitro cytotoxicity evaluation
The compounds were found to be safe when screened against normal
Vero cell lines as all the compounds showed activity >300 μM in cell
viability assay. The synthesized compounds were also found to be
nontoxic to liver cancer cell lines, that is, HepG2 cell lines, as all the

8
VERMA ET AL.
compounds showed activity >300 μM studied through cell viability
RMSD score of 0.4163 Å indicated a similar binding pattern of red-
ocked ligand compared with cocrystallized ligand, thereby confirming
the accuracy of the experiment. Furthermore, all the ligands and tri-
closan were docked at the same binding site of protein and the bind-
ing mode of the synthesized molecules was compared with triclosan.
Also the binding interaction of the molecules with the target protein
residue was studied. The compounds showed hydrogen bond interac-
tions with NAD 300 such as triclosan and additional interaction with
phenylalanine 149 active site residue. The predicted binding energies
of the compounds are listed in Table 3.
assay.
3.5 | Molecular docking studies
In silico computational studies have become an integral part of the
studies for identifying the targets for diverse ligands due to the non-
availability of the resources. Hence, the molecular docking studies of
the synthesized compounds in the active site of crystal structure of
InhA (pdb: 3FNE) bound to a triclosan derivative was performed. This
crystal structure was downloaded from Protein Data Bank, and it con-
sisted of two ligands, that is, triclosan derivative and NAD 300. The
protein was corrected structurally stepwise in silico using the protein
preparation wizard module of Schrodinger 2017-4. A Glide XP proce-
dure was used. The docking procedure was validated by splitting the
prepared protein into ligand and protein. The ligand that is a triclosan
derivative was redocked into the same binding site. The observed
The most active compound 2j (MIC 19 μM) showed a dock score
of −9.270 kcal/mol and a binding affinity of −61.84 kcal/mol. The
compound exhibited one hydrogen bonding interaction with NAD
300. One π–π stacking interaction was observed between ring B and
phenylalanine 149 (Figure 1a).
Compound 2i (MIC 20.069 μM) displayed
a dock score of
−9.563 kcal/mol and
a
binding energy of −71.64 kcal/mol,
TABLE 3 Molecular docking results of compounds (2a–2m)
Docking score
(kcal/mol)
Prime MMGBSA
Number of
Hydrogen bond
Compounds
2a
dG bind (kcal/mol)
interacting bonds
Interacting residues
NAD 300
distance (Å)
−9.066
−7.025
−8.798
−8.161
−8.211
−9.095
−66.60
−63.17
−62.37
−62.83
−61.09
−65.43
1 H-bond
1 π–π stack
1 H-bond
1 π–π stack
1 H-bond
1 π–π stack
1 H-bond
1 π–π stack
1 H-bond
1 π–π stack
2 H-bond
1 π–π stack
2.11
Phenylalanine 149
NAD 300
2b
2c
2d
2e
2f
1.90
2.29
1.92
1.46
2.17
Phenylalanine 149
NAD 300
NAD 300
Phenylalanine 149
NAD 300
NAD 300
Tyrosine 158
Phenylalanine 149
–
2g
2h
2i
−8.483
−8.909
−9.563
−48.91
−70.25
−71.64
–
–
1 π–π stack
1 H-bond
1 π–π stack
1 H-bond
1 π–π stack
1 H-bond
1 π–π stack
1 H-bond
1 π–π stack
Phenylalanine 149
NAD 300
–
1.84
Phenylalanine 149
NAD 300
2j
−9.270
−9.608
−7.102
−61.84
−66.21
−73.39
2.17
2.13
1.96
Phenylalanine 149
NAD 300
2k
2l
Phenylalanine 149
NAD 300
Tyrosine 158
Phenylalanine 149
2m
−9.029
−8.523
−69.43
−60.49
1 π–π stack
–
Phenylalanine 149
NAD 300
Triclosan
2 H-bond
2.47, 1.85
1 π–π stack
Phenylalanine 149
Abbreviations: H, hydrogen; NAD, nicotinamide adenine dinucleotide.

VERMA ET AL.
9
FIGURE 1 (a) Molecular docking interaction of compound 2j (green) with NAD 300 ligand and phenylalanine 149 residue of Mtb InhA (PDB
3FNE). (b) Molecular docking interaction of compound 2i (green) with NAD 300 ligand and phenylalanine 149 residue of Mtb InhA (PDB 3FNE).
(c) Molecular docking interaction of compound 2a (green) with NAD 300 ligand and phenylalanine residue of Mtb InhA (PDB 3FNE). (d) Molecular
docking interaction of compound 2c (green) with NAD 300 ligand of MTb Inh A (PDB 3FNE). Phe, phenylalanine
Note. Dotted yellow line shows the hydrogen bonding interaction
respectively. It showed similar hydrogen bonding and π–π stacking
interaction as compound 2j (Figure 1b).**
3.6 | MD analysis
Compounds 2i-InhA complex and triclosan-InhA complex were sub-
jected to MD simulation. Compound 2i was selected as it showed
potent antitubercular activity for both weeks, that is, 20 μM. The sta-
bility of the protein structural model and binding pattern of triclosan
and 2i with InhA enzyme was examined throughout the MD simula-
tions. This was evident through conformational changes occurring in
the structure and the interaction between ligand and protein. MD
simulation studies were carried out with an explicit solvent for 20 ns.
The complex stability during MD simulation was calculated by aligning
the protein backbone frames to the backbone of initial frame. The
overall deviation from the starting structure was measured by RMSD.
Figure 3 depicts that 2i-InhA is stabilized much better when com-
pared with triclosan-InhA during the simulation studies, which is visi-
ble through higher structural fluctuations of triclosan than 2i. The
RMSD plot for compound 2i suggests that the protein backbone
RMSD recorded during simulation showed high fluctuations for the
initial 5 ns due to the initial protein structural stabilization for the 2i
protein complex. The backbone RMSD observed for protein between
5 to 20 ns was in the range of 2.2 to 1.7 Å, compared with that of the
original structure and was within a range of 0.8 Å to 0.6 Å. The ligand
(2i) RMSD remained within the range of 2.9 to 1.9 Å in comparison
Compound 2d (MIC 21.55 μM), 2c (MIC 25 μM), and 2a (MIC
25 μM) displayed dock scores of −8.161, −8.978, and − 9.066 kcal/
mol individually and binding energies of −62.83, −62.37, and
− 66.60 kcal/mol, respectively. Compounds 2d and 2a showed hydro-
gen bonding interactions with NAD 300 and one π–π stacking interac-
tion between B ring and phenylalanine 149. Figure 1c depicts
molecular interactions between compound 2a and the catalytic target
residue. Compound 2c displayed hydrogen-bonding interactions with
NAD 300 (Figure 1d).
The molecular docking interactions of the synthesized compounds
were compared with triclosan. Triclosan showed a dock score of
−8.523 kcal/mol,
a binding energy of −60.49 kcal/mol, and two
hydrogen-bonding interactions with NAD 300. Figure 2 displays molec-
ular interactions between triclosan and catalytic site residues of protein.
The theoretical predictions through molecular docking studies
were found to be in agreement with the experimentally observed
antitubercular activity results. The most active compounds were
found to have good dock score, high binding affinities, and a greater
number of interactions with the protein residues in comparison with
triclosan.

10
VERMA ET AL.
with the original structure and was within the range of 1.0 Å to 0.6 Å,
suggesting the formation of the stable ligand–protein complex. Triclo-
san-InhA complex stabilized at 11.64 ns comparatively and exhibited
slightly higher structural fluctuations during MD simulation. The struc-
tural deviations observed for protein between 11 and 20 ns were in
the range of 2.3 to 0.72 Å compared with that of the original structure
and was within the range of 2.0 Å to 0.7 Å and the triclosan RMSD
remained in the range of 1.53 Å to 0.57 Å compared with the original
structure. The ligand fluctuations in MD trajectory observed to be
slightly higher for triclosan, compared with that of molecule 2i,
depicting that 2i is stabilizing the complex better than the triclosan.
Root mean square fluctuation (RMSF) values were used to depict
the fluctuations of each residue present in protein over the simulation
time period (Figure 4). Higher peaks represent the amino acid residues
that fluctuate the most. The highest fluctuating residue was observed
to be A:Leu 207 with RMSF value of 3.22 Å and A:Ala 252 with RMSF
value of 2.71 Å. The high fluctuations also represent maximum bind-
ing between the protein and the ligand. RMSF fluctuation for the
remaining protein was observed within the range of 0.64 Å to 2.20 Å.
For triclosan-InhA complex, protein RMSF fluctuation was observed
in the range of 1.14–2.3 Å. The most fluctuating amino acid residues
were A:Thr 259 and A:Ala 252 with RMSF values of 2.83 Å and
2.82 Å, respectively.
FIGURE 2 Molecular docking interaction of triclosan (blue) with
NAD 300 ligand of Mtb InhA (PDB 3FNE).
Note: Dotted yellow line shows the hydrogen bonding interaction
Ligand root mean square fluctuation (L-RMSF) depicts characteris-
tic changes in the ligand atom positions during the simulation time
interval (Figure 5). Carbon atoms at B ring of 2i compound were found
to fluctuate the most. The RMSF value was observed within the range
of 1.00–2.00 Å. The chloro group at the second position in ring A and
the OH group on ring B of triclosan displayed maximum fluctuation
with RMSF values of 1.11 and 0.96 Å, respectively.
FIGURE 3 The RMSD of protein backbone structure in
comparison with the original structure for molecule (a) 2i and
(b) Triclosan
The nonbonded intermolecular interactions between the InhA-
binding residue and the ligand 2i and triclosan observed during the
FIGURE 4 Protein RMSF plot during molecular dynamics simulation: (a) 2i-InhA complex; (b) Triclosan-InhA complex

VERMA ET AL.
11
FIGURE 5 Ligand RMSF plot during molecular dynamics simulation: (a) 2i-InhA complex; (b) Triclosan-InhA complex
FIGURE 6 Plot of protein interactions with ligand (a) 2i and (b) Triclosan as monitored throughout the simulation
FIGURE 7 The atomic interactions of ligand (a) 2i and (b) Triclosan with the key amino acid residues at the active site

12
VERMA ET AL.
TABLE 4 Predicted ADME properties and drug-likeness properties of the compounds (2a–2m) as per the QikProp software application
QPPMDCKb
nm/s
%
QPlogPo/
wg
Lipinskirule
of fivei
Compounds
QPlogBBa
−0.093
0.003
QPlogKhsac
0.541
0.582
0.578
0.706
0.706
0.666
0.662
0.662
0.605
0.585
0.582
0.686
0.687
0.394
QPlogSd
−4.972
−5.395
−5.382
−5.564
−5.558
−5.279
−5.724
−5.72
CNSe
0
HOAf
PSAh
2a
2b
2c
2d
2e
2f
2,503.828
3,733.112
3,767.527
2,505.132
2,511.431
4,120.574
6,169.814
6,188.495
5,741.128
4,520.428
4,666.815
6,636.604
6,658.051
10,000
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
3.69
3.79
3.79
4.18
4.18
4.18
4.7
30.07
38.37
38.36
30.07
30.07
28.37
30.08
30.09
28.81
30.07
29.5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.007
0
−0.104
−0.102
0.114
1
1
1
2g
2h
2i
0.074
1
0.075
1
4.7
0.085
−5.209
−5.342
−5.281
−5.841
−5.837
−4.52
1
4.7
2j
0.018
1
4.13
4.13
4.13
4.8
2k
2l
0.026
1
0.086
1
30.09
30.08
28.767
2m
TCL
0.088
1
0.396
1
4.75
aPredicted brain/blood partition coefficient (acceptable range: −3.0 to −1.2).
bPredicted apparent MDCK cell permeability (acceptable range: <25 poor to >500 great).
cPrediction of binding to human serum albumin (acceptable range: −1.5 to 1.5).
dPredicted aqueous solubility in mol/L (acceptable range: −6.5 to 0.5).
ePredicted central nervous system activity on a −2 (inactive) to +2 (active) scale.
fPercentage human oral absorption (<25% is poor and >80% is high).
gLog p in o/w (acceptable range: −2.0 to 6.5).
hPolar surface area (≤140 Å2).
iLipinski's rule of five: the number of violations to the Lipinski's rule of five.
MD simulation are shown in Figure 6. All the ligand–protein interactions
observed in the docking study were present throughout the MD simula-
tion. The molecule 2i exhibited hydrogen bonding and hydrophobic
interactions. The molecule also formed water bridge interaction with
lysine 165 amino acid residue. Most prominent hydrogen bonding inter-
action between target receptor and ligand was observed with A:Lysine
165 with 89.4% occupancy and A:Isoleucine 194 amino acid residues
with 91.5% occupancy. The hydrophobic interaction was mostly
observed with A:Phenylalanine 149 with 76.3% occupancy. The residues
A:Met 155, A-Tyr158 A:Met 161, A:Pro193, A:Ala 198, A:Met 199, A:Ile
215, A:Leu 218, and A:Met 232 also took part in hydrophobic interac-
tions but showed very less occupancy. These interactions during the sim-
ulation time period make stable protein–ligand complexes. Triclosan
showed hydrogen-bonding interaction with A:Tyr 158 and hydrophobic
interaction with A:Phe 149, A:Tyr 158, A: Met 161, A: Ala 198, and A:
Met 199. Among these interactions, only phenylalanine 149 occupancy
was observed to be 83.0% with triclosan. The other residues displayed
occupancy less than 30%. This depicts that during simulation time com-
pound 2i interacted and maximum interaction occurred with amino acid
residues, whereas triclosan interactions were found to be less.
π–π interaction with Phe 149. Triclosan displayed π–π stacking interac-
tion with phenylalanine 149 amino acid residue and its B ring.
The interaction observed between protein and 2i was stronger
than the triclosan–protein complex. Owing to this, 2i is showing a
higher potency than triclosan.
3.7 | Lipinski's rule for drug-likeliness and in silico
ADME prediction
The synthesized compounds were investigated for physical descrip-
tors as well as pharmaceutically significant properties using the
QikProp v 5.4 tool of Schrodinger release 2017-4. The synthesized
molecules followed the Lipinski's rule of five as given in Table 4 and
showed better partition coefficients than triclosan.
The parameters assessed were hydrogen bond acceptor, hydrogen
bond donors, partition coefficient between octanol and water (log p)
and polar surface area (PSA). In silico ADME prediction is very impor-
tant for selecting suitable drug candidates, hence these parameters
were studied using Schrodinger suite release 2017-4. The in silico
ADME properties of the synthesized compounds are given in Table 4.
The predicted values for QPlogBB (which predicts brain/blood
partition coefficient for drugs delivered orally) are from −3.0 to 1.2.
The QPPMDCK was analyzed to predict the apparent Madin–Darby
Canine Kidney (MDCK) cell permeability in nm/s. The permeability of
the test compounds to these cells is used to assess ability to permeate
The residues that interacted with ligands for more than 30% of the
simulation time in the trajectory are shown as ligand–protein contacts
(Figure 7). The residue A:Lys 165 displayed hydrogen-bonding interac-
tion with nitrogen of pyridine ring and A:Ile194 showed hydrogen-
bonding interaction with NH group. B ring of the ligand moiety formed

VERMA ET AL.
13
the blood–brain barrier. A value <25 indicates poor permeability and
value >500 indicates good permeability. The QPlogKhsa property pre-
dicts a given compound's binding affinity to human serum albumin. All
the synthesized compounds showed acceptable ADME properties.
The predicted aqueous solubility (QPlogS) of the synthesized com-
pounds was found to be above −4.972 mol dm−3 in comparison with
triclosan (−4.92 mol dm−3). The compounds were found to be inactive
for central nervous system (CNS) activity. All the synthesized mole-
cules showed acceptable ADME properties and followed the
Lipinski's rule.
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How to cite this article: Verma R, Boshoff HIM, Arora K,
et al. Synthesis, evaluation, molecular docking, and molecular
dynamics studies of novel N-(4-[pyridin-2-yloxy]benzyl)
arylamine derivatives as potential antitubercular agents. Drug
Dev Res. 2019;1–14. https://doi.org/10.1002/ddr.21623