Design, synthesis, in silico and in vitro evaluation of novel diphenyl ether derivatives as potential antitubercular agents

Article abstract of DOI:10.1007/s11030-019-09990-z

Abstract: Diphenyl ether derivatives inhibit mycobacterial cell wall synthesis by inhibiting an enzyme, enoyl-acyl carrier protein reductase (InhA), which catalyses the last step in the fatty acid synthesis cycle of genus Mycobacterium. To select and validate a protein crystal structure of enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis for designing inhibitors using molecular modelling, a cross-docking and correlation study was performed. A series of novel 1-(3-(3-hydroxy-4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) ethan-1-ones were synthesized from this model and screened for their antitubercular activity against M. tuberculosis H37Rv. Compound PYN-8 showed good antitubercular activity on M. tuberculosis H37Rv (MIC = 4–7?μM) and Mycobacterium bovis (% inhibition at 10?μM = 95.91%). Cytotoxicity of all the synthesized derivatives was assessed using various cell lines, and they were found to be safe. Structure of PYN-8 was also confirmed by single-crystal X-ray diffraction. The molecular modelling studies also corroborated the biological activity of the compounds. Further, in silico findings revealed that all these tested compounds exhibited good ADME properties and drug likeness and thus may be considered as potential candidates for further drug development. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-019-09990-z

Molecular Diversity
https://doi.org/10.1007/s11030-019-09990-z
ORIGINAL ARTICLE
Design, synthesis, in silico and in vitro evaluation of novel diphenyl
ether derivatives as potential antitubercular agents
Ashutosh Prasad Tiwari1 · B. Sridhar2 · Helena I. Boshof3 · Kriti Arora3 · G. Gautham Shenoy1 · K. E. Vandana4 ·
G. Varadaraj Bhat1
Received: 4 June 2019 / Accepted: 24 August 2019
© Springer Nature Switzerland AG 2019
Abstract
Diphenyl ether derivatives inhibit mycobacterial cell wall synthesis by inhibiting an enzyme, enoyl-acyl carrier protein
reductase (InhA), which catalyses the last step in the fatty acid synthesis cycle of genus Mycobacterium. To select and vali-
date a protein crystal structure of enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis for designing inhibi-
tors using molecular modelling, a cross-docking and correlation study was performed. A series of novel 1-(3-(3-hydroxy-
4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) ethan-1-ones were synthesized from this model and screened for
their antitubercular activity against M. tuberculosis H37Rv. Compound PYN-8 showed good antitubercular activity on M.
tuberculosis H37Rv (MIC=4–7 µM) and Mycobacterium bovis (% inhibition at 10 µM=95.91%). Cytotoxicity of all the
synthesized derivatives was assessed using various cell lines, and they were found to be safe. Structure of PYN-8 was also
confrmed by single-crystal X-ray difraction. The molecular modelling studies also corroborated the biological activity of
the compounds. Further, in silico fndings revealed that all these tested compounds exhibited good ADME properties and
drug likeness and thus may be considered as potential candidates for further drug development.
Graphic abstract
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-019-09990-z) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Molecular Diversity
Keywords TB · Diphenylether · InhA · Molecular docking · Correlation study · Antitubercular
component of mycobacterial cell wall and are responsible
for virulence of the microorganism [5]. InhA catalyses the
reduction of the double bond of 2-trans-enoyl-[acyl carrier
protein] in a NADH-dependent manner, which is the last
and a crucial step of the FAS-II pathway, involved in the
fatty acid elongation cycle. Isoniazid (INH), a frst line
antitubercular drug, targets this enzyme. InhA is inhibited
by an active form of isoniazid which is formed by react-
ing with the cofactor NAD(H), bound to the active site of
the enzyme. It forms a covalent adduct (isonicotinic acyl
NADH), which binds to InhA with high afnity [6]. The
activation is carried out by the catalase peroxidase enzyme
encoded by katG, and mutation in this gene is the cause
of resistance to isoniazid in 64% of clinical isolates [7].
Therefore, molecules which directly inhibit InhA without
any bio-activation circumvent this mechanism of resist-
ance and are promising candidates. Such a direct inhibi-
tor of InhA is triclosan (a diphenyl ether derivative) [8].
However, it is a weak inhibitor of InhA [9]. Therefore, in
the present work, a series of diphenyl ether derivatives
were designed and synthesized by studying the nature of
the substrate binding pocket and the interactions of potent
Introduction
Tuberculosis (TB) is the major cause of death from a single
infectious agent (above HIV/AIDS). It is also one among
leading 10 causes of death [1]. About 1.3 million deaths
among HIV-negative and an additional 0.3 million in HIV-
positive people in 2017 were due to TB. Though this indi-
cates the overall results of all countries and age groups, 90%
of them were adults (age≥15 years), in the most productive
years of their life. Millions of deaths are prevented each
year, about 54 million in 2000–2017, with timely diagnosis
and treatment [2]. The success rates of treatment for the
new cases are around 82% globally, as indicated by latest
data of the treatment outcome in 2016. TB treatments take
too long to cure and can be toxic. They are also complicated
to administer which results in poor patient compliance [3].
New treatments that are faster, simpler and afordable are
urgently needed.
A validated target for Mycobacterium is enoyl-acyl
carrier protein reductase (InhA) [4]. It is an enzyme in
the type II fatty acid synthase system, contributing to the
biosynthesis of mycolic acids. Mycolic acids are a chief
Fig. 1 Design strategy and
active site of InhA showing
diphenyl ether derivative sub-
stituted at 5th chlorine position
protruding into the isonicotinoyl
binding pocket containing InhA
complexes of isoniazid (red col-
our) and ethionamide (yellow
colour). (Color fgure online)
1 3

Molecular Diversity
Table 1 RMSD of the extracted ligands after docking on the respective crystal structures with poses in their native crystal structure
Co-crystallized ligand 2X23 4OYR 4OXY 4OXK 4OHU 2X22 3FNH 3FNG 3FNF 3FNE 1P45
Average
RMSD
(Å)
Outliers
Protein crystal structure
2X23 with water
0.893 0.955 0.631
0.849 0.954 0.595
1.393 0.254 1.516 10.689 1.298 1.365 10.415 2.2
1.391 0.226 0.817
1.192 1.334 1.237
1.166 1.066 1.08
1.164 1.118 0.301 10.313 2.043 1.784 1.591
1.637 1.09 0.921 10.443 1.382 1.785 1.589
1.941 1.715
1.004 1.982
1.001 2.022
9.422 3.837
2
1
4
3
2
2
0
0
6
6
6
6
3
3
0
0
0
0
0
0
3
2
2X23 without water
4OYR with water
4OYR without water
4OXY with water
4OXY without water
4OXK with water
4OXK without water
4OHU with water
2.011 1.336 1.363
1.985 1.512 1.111
2.362 1.367 1.069
1.324 1.401 1.151
1.328 1.453 1.169
1.979 8.909 1.941 1.715 10.116 2.891
2.028 2.021 1.725 1.678
2.125 1.212 1.059 1.696
0.76
1.508
0.726 1.357
0.599 1.165
0.649 1.070
6.168 4.866
6.168 4.748
9.091 5.466
8.988 5.614
0.571 1.618
0.571 1.618
0.592 1.187
0.591 1.288
0.486 1.174
0.659 1.299
0.442 1.003
0.97
0.971 0.778 1.232
1.72
1.195 1.232
1.79
1.891 0.622 0.637
0.713
1.739 0.985 0.75
0.847 1.109 11.336 0.452 1.669
9.724 2.181 9.467 8.857
9.723 2.186 9.197 8.857
9.866 8.854 8.816 9.038
9.872 8.851 8.828 9.054
1.667 1.645 0.663 1.318
1.667 1.645 0.663 1.318
1.451 1.744 0.611 0.534
1.481 1.735 0.515 1.911
4OHU without water 1.046 0.866 1.108 11.757 0.306 1.009
2X22 with water
0.893 1.271 0.629
1.011 0.959 0.984 11
9.51
1.306 0.848
1.308 0.896
2X22 without water
3FNH with water
3FNH without water
3FNG with water
3FNG without water
3FNF with water
3FNF without water
3FNE with water
3FNE without water
1P45 with water
2.321 1.703 1.709
2.321 1.703 1.709
1.179 1.435 1.178
1.273 1.483 0.918
1.128 1.582 1.144
1.411 1.538 1.51
1.126 1.197 1.292
1.549 1.239 1.045
2.001 1.356 1.643
1.207 1.369 1.641
1.428 2.398 2.375
1.428 2.398 2.375
1.427 1.648 1.262
1.209 1.705 1.345
1.164 1.789 1.272
1.452 1.835 1.557
1.501 1.325 1.183
1.445 1.315 1.647
1.987 1.512 2.04
2.248 1.418 1.214
1.91
0.737 0.763 0.943
2.233 0.672 0.713 0.706
1.095 0.962 0.608 0.302
1.604 0.508 0.351 0.298
2.178 1.879 0.839 1.576
2.331 0.697 0.839 1.592
0.45
1.041
0.572 1.598
0.609 1.379
1P45 without water
inhibitors with the amino acid residues of the active site.
The design used in this work is shown in Fig. 1.
in the protein data bank for the protein, enoyl-acyl carrier
protein reductase, of Mycobacterium tuberculosis of strain
H37Rv. It was thus essential to select a right protein crystal
structure, for the design and docking studies of the inhibi-
tor molecules. The primary prerequisite for selection of the
protein crystal structure is the presence of a co-crystallized
ligand in it, which is similar to the inhibitor molecule scaf-
fold. The protein crystal structure should be able to accom-
modate all the analogues of the scafold molecule without
much deviation, so as to corroborate the in vitro activity
results. Thus, a cross-docking study involving docking of the
co-crystallized ligands extracted from the selected protein
crystal structures on each of the protein crystal structures
was performed. The protein crystal structures were selected
based on screening parameters mentioned in the experimen-
tal section.
Physicochemical properties of the inhibitor molecules are
a major factor for drug efcacy. Therefore, Lipinski’s rule
was followed while designing the molecules to fulfl these
requirements. The synthesized compounds were screened
against H37Rv strain of Mycobacterium tuberculosis and
Mycobacterium bovis. The safety profles of the molecules
were also determined using various cell lines. To study the
plausible mechanism of inhibition and for determination
of ADME profle of the synthesized molecules, molecular
modelling study was carried out.
Results and discussion
Protein crystal structure selection
Cross‑docking study
To select and validate a protein crystal structure of enoyl-
acyl carrier protein reductase of Mycobacterium tuberculo-
sis for designing of the inhibitor molecules using molecular
modelling, a cross-docking and correlation study was per-
formed. There are 87 crystal structures available hitherto
All the ligands extracted from the 11 proteins were docked
on every protein. Root-mean-square deviation (RMSD) of
every ligand with its docked pose in all the proteins, rela-
tive to its pose in the aligned ligand set, was calculated. The
1 3

Molecular Diversity
Table 2 Binding free energy of diphenyl ether analogues (with
respective codes as in the reference) from the literature with their IC50
and pIC50 values
values of the RMSDs are listed in Table 1. Ligands with
RMSD values more than 2 Å were considered as outliers.
The protein crystal with the least number of outliers and the
least average RMSD value was thought to be appropriate for
use to study the docking of a variety of diphenyl ether deriv-
atives. The protein with PDB entry code 3FNE had the least
average RMSD value and no outliers. It had a resolution of
1.98 Å and a triclosan derivative, 2-(2,4-dichlorophenoxy)-
5-(pyridin-2-ylmethyl) phenol as its co-crystallized ligand.
Molecules
IC50
pIC50
ΔG Bind (Kcal/mol)
8PP
5
10
12.1
17
29.7
44.6
48
48
50.7
55
61.9
79.7
80
86
100
150
236
238.8
650
8.30103
8
−90.4033
−91.3701
−86.8914
−82.6221
−82.4981
−86.2859
−90.9673
−86.7372
−90.5046
−88.0505
−88.3477
−86.73
−80.3218
−83.2069
−87.3732
−73.6168
−80.5168
−80.0489
−81.2368
−67.8625
−77.4535
−71.675
PT092
PT113
5PP
PT095
PT096
PT114
13b
PT070
14c
PT013
PT133
4PP
PT109
PT111
14PP
3b
PT134
3e
10a
PT108
2PP
7.917215
7.769551
7.527244
7.350665
7.318759
7.318759
7.294992
7.259637
7.208309
7.098542
7.09691
7.065502
7
6.823909
6.627088
6.621966
6.187087
5.809668
5.8041
Correlation study
To validate the outcome of the cross-docking study and
therefore the applicability of the protein 3FNE, a correlation
study was done. Twenty-fve molecules from the literature
with their experimental IC50 values were collected [10–12].
These molecules were then docked on the protein 3FNE.
Molecular mechanics/generalized born surface area (MM/
GBSA) was used to calculate the binding free energy of
the ligand–protein complex. Prime MM/GBSA calculations
were performed for the complexes obtained after docking
of the molecules on 3FNE, and the pIC50 values were cor-
related with the free binding energy of the ligand–protein
complexes. The MM/GBSA values and the pIC50 values as
listed in Table 2. A graph was plotted between the MM/
GBSA values and the pIC50 values, and a Pearson’s correla-
tion coefcient value of 0.73 was obtained (Fig. 2). This
validates the protein crystal structure 3FNE chosen after
the screening of the available crystal structures of InhA. It
also hints that the mechanism of action of the synthesized
diphenyl ether derivatives could plausibly be the inhibition
of InhA.
1550
1570
2000
2360
3200
100,000
5.69897
5.627088
5.49485
4
11a
12a
2d
−58.3266
−64.9083
−62.3432
InhA–triclosan complex gives clues for structural modi-
fcations in diphenyl ether scafold for increasing afnity
towards InhA [15]. Hydrophobic substitutions in place of
the chloro group at 5 position of A ring in diphenyl ether
scafold might help in occupying and increasing interactions
with isonicotinoyl binding pocket which is important for
improved activity [16]. Additionally for the better position-
ing of the aryl group (hydrophobic substituent) into the tar-
get INH binding pocket, a heterocyclic linker was designed
between the hydrophobic moiety and the 5th carbon position
on A ring.
Drug design strategy
Molecules were designed by studying the nature of the
substrate binding pocket of InhA. Also, the interactions of
various potent direct inhibitors (diphenyl ether analogues)
with the amino acid residues, as exhibited in their protein
crystallographic structures, were studied. Sullivan et al.
reported the crystal structures of diphenyl ether derivatives
complexed with InhA, giving detailed insight into the bind-
ing pocket interactions with the direct inhibitors. A basic
scafold was then assigned which had the attributes required
for interactions in a favourable way with the binding pocket.
Hydrogen bonding interaction between amino acid residue
tyrosine 158 and the hydroxyl group of the diphenyl moi-
ety was considered essential for activity [13]. Also, the
π–π stacking interaction of the phenyl ring of the diphe-
nyl ether moiety with the pyridyl moiety of the NAD 300
cofactor InhA protein was observed in potent inhibitors [14].
Comparison of the crystal structures of InhA complexes
of isoniazid-NAD and ethionamide-NAD adducts with
Induced ft docking
Induced ft docking (IFD) of the synthesized PYN-8 and tri-
closan generated good poses with RMSD of 0.093 Å. PYN-8
showed hydrogen bonding interaction indicated by yellow
dotted lines, with the tyrosine 158 residue. A π–π stacking
interaction with the pyridyl ring of NAD 300 was observed
(indicated by blue dotted line). A cation–π interaction (indi-
cated by green dotted line) with the pyridine nitrogen of
1 3

Molecular Diversity
0
-10
-20
-30
-40
-50
-60
-70
-80
-90
-100
Fig. 2 Binding free energy
versus pIC50 correlation graph
with correlation value R2
0
1
2
3
4
5
6
7
8
9
R² = 0.7339
pIC50
Fig. 3 Induced ft docked pose of PYN-8 on 3FNE
Fig. 4 Induced ft docked pose of triclosan on 3FNE
NAD 300 was also observed. The induced ft docking score
of synthesized PYN-8 (−541.09) was better than triclosan
(−536.48) supporting its better activity. The interactions are
shown in Figs. 3 and 4.
of 2.24 kcal/mol, entropy (−T∆S) 1.61 kcal/mol and Gibbs
free energy (∆G) 3.85 kcal/mol. These values indicate that
it is favourable to displace this hydration site. The meta-nitro
group on the phenyl ring of the PYN-8 helps to increase
the hydrophobicity of the phenyl ring. The displacement of
the unstable water by this hydrophobic group might be the
reason for better activity of PYN-8.
Water map analysis
To understand the better activity of meta-nitro-substituted
derivative (PYN-8) compared to other analogues, a water
map analysis was performed. Water map shows the hydra-
tion sites in a protein along with their thermodynamic prop-
erties. Based on the disorderliness of the water molecules in
the hydration sites, it can be assessed if a hydration site has
to be displaced or eschewed [17]. It was observed that mol-
ecule PYN-8 displaced a hydration site with site number 68
as shown in Fig. 5. This hydration site has an enthalpy (∆H)
Chemistry
The diphenyl ether pyrazoline derivatives were synthesized
as depicted in Scheme 1. The synthesis of 1-(3-methoxy-
4-phenoxyphenyl)ethanone was done via Chan–Lam cou-
pling. Phenyl boronic acid and acetovanillone were reacted
in the presence of copper acetate monohydrate, pyridine in
anhydrous dichloromethane with activated molecular sieves
1 3

Molecular Diversity
Fig. 5 Water map of 3FNE pro-
tein showing PYN-8 displacing
water site number 68
Table 3 In vitro MIC values of synthesized derivatives determined against Mycobacterium tuberculosis H37Rv strain in two diferent media
Compound code
R group
(7H9/ADC/Tw) MIC
Day 7 (µM)
(GAST-Fe) MIC
Day 7 (µM)
Day 14 (µM)
Day 14 (µM)
PYN-1
PYN-2
PYN-3
PYN-4
PYN-5
PYN-6
PYN-7
PYN-8
PYN-9
PYN-10
PYN-11
Triclosan
Isoniazid
4-chloro
4-nitro
4-bromo
4-fuoro
4-methyl
4-methoxy
3-chloro
3-nitro
3-bromo
3-fuoro
3-methyl
–
123
240
28–55
128
>259
31
61
3
111–222
64
259
21
>246
>240
>222
128
>239
>249
61
4–7
111–222
64
61
120
28
128
32
31
61
15
111
64
129
43
123
>239
>222
128
32
62
61
15
111
64
259
43
0.2–0.4
259
43
1
–
0.8
0.2
at room temperature. 1-(3-methoxy-4-phenoxyphenyl)etha-
none was then deprotected using BBr3 in anhydrous dichlo-
romethane at −78 °C [18]. 1-(3-hydroxy-4-phenoxyphenyl)
ethan-1-one thus obtained was reacted with various substi-
tuted benzaldehydes using lithium hydroxide monohydrate
as catalyst for Claisen–Schmidt condensation in ethanol to
give various substituted 1-(3-hydroxy-4-phenoxyphenyl)-3-
phenylprop-2-en-1-ones [19].
The 1-(3-hydroxy-4-phenoxyphenyl)-3-phenylprop-
2-en-1-ones were then converted to 1-(3-(3-hydroxy-
4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)
ethan-1-ones by refuxing them with hydrazine hydrate in
acetic acid for a duration of 3 h. The synthesized com-
pounds were purifed by crystallization and column chro-
matography. Structures of all the synthesized fnal deriva-
tives were confrmed by 1H, 13C NMR and HRMS.
1 3

Molecular Diversity
1) Phenyl boronic acid 2) 3-hydroxy-4-phenoxy 3) 1-(3-methoxy-4-phenoxyphenyl)ethan-1-one 4) 1-(3-hydroxy-4-
phenoxyphenyl)ethan-1-one 5) (E)-1-(3-hydroxy-4-phenoxyphenyl)-3-phenylprop-2-en-1-one 6) 1-(3-(3-hydroxy-4-
phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one acetophenone a) Copper acetate
monohydrate, Pyridine, anhydrous dichloromethane, 4Å Molecular Sieves b) BBr3, anhydrous dichloromethane, -
78°C c) Substituted benzaldehydes, LiOH.H2O, Ethanol d) Hydrazine hydrate, Glacial acetic acid
Scheme 1 Synthesis of pyrazoline derivatives of diphenyl ethers (PYN series)
Fig. 6 ORTEP diagram of
PYN-8. Displacement ellipsoids
are drawn at the 30% probability
level
1
A typical H NMR spectrum of 1-(3-(3-hydroxy-
4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-
1-yl)ethan-1-ones showed protons of the pyrazoline moi-
ety at three diferent positions. The methylene protons in
the pyrazoline ring were observed at δ 3.01–3.11 ppm
and δ 3.65–3.73 ppm both with a coupling constant
of 17.6–17.8 Hz. The stereogenic proton appeared
between δ 5.46 and 5.59 ppm with a coupling constant of
11.8–12.0 Hz. A singlet or a small hump at δ 5.7–5.8 ppm
indicated the OH protons. The methyl protons of the acetyl
group on the nitrogen of pyrazoline ring were observed
at δ 2.3–2.42 ppm as a singlet. The HRMS (ESI/Q-TOF)
showed [M + H]+ peaks corresponding to their molecular
formula weight.
Further, the structure of compound PYN-8 was con-
frmed by single-crystal X-ray difraction study as shown
in Fig. 6.
Antitubercular study
All the synthesized fnal derivatives (1–11) were screened
for their antitubercular activity against Mycobacterium
1 3

Molecular Diversity
Table 4 Percentage inhibition values of cancer cells and Mycobacterium bovis at 10 µM concentration of the compounds
Compound code
% inhibition of cells at 10 µM concentration
MCF7
PANC-1
PC-3
SKOV-3
HeLa
M. bovis
PYN-1
PYN-2
PYN-3
PYN-4
PYN-5
PYN-6
PYN-7
PYN-8
6.77
4.75
6.16
4.07
4.04
1.65
5.15
4.88
4.68
4.80
5.65
81.84
–
4.29
5.42
5.17
3.20
1.07
2.17
3.27
4.06
2.69
2.68
2.27
60.82
–
17.29
18.10
−2.67
11.41
−9.88
12.01
0.04
2.71
3.05
4.12
4.32
4.79
0.97
4.50
4.32
3.92
4.92
3.67
77.37
–
4.45
4.78
4.10
3.31
3.06
4.40
3.66
3.52
3.32
3.25
2.99
47.91
–
10.15
23.00
−22.14
−19.60
11.04
38.00
−35.36
95.91
29.55
32.17
−35.31
–
5.06
PYN-9
−4.23
−4.02
0.82
76.52
–
PYN-10
PYN-11
Doxorubicin (2 µM)
Rifampicin
Isoniazid
99.3
99.1
–
–
–
–
–
tuberculosis, strain H37Rv. They were also tested for their
inhibitory efect against Mycobacterium bovis. The deriva-
tive with meta-nitro substitution PYN-8 showed better activ-
ity than the other analogues. Similar trend of activity in both
Mycobacterium tuberculosis and Mycobacterium bovis sig-
nifes the activity values. The activity values are reported
in Table 3.
evaluated at 10 µM concentration for their cytotoxic effect
on MCF-7, PANC-1, PC-3, SKOV-3 and HeLa cell lines.
Additionally, they were screened against BD-11 and HEK
cell lines where their cytotoxic concentration was greater
than 100 µM. Therefore, these studies indicate that these
compounds do not have any cytotoxic effect. The cyto-
toxicity data are shown in Table 4.
Cytotoxicity assay
Drug likeness properties
For the treatment of tuberculosis, it is essential to take
antitubercular drugs for a long period of time (at least
6 months). Thus, it is necessary to study cytotoxic effects
of the drug candidates. The compounds were therefore
In the advanced drug discovery phases, many hits often
don’t succeed [20]. Therefore, it is vital to evaluate lead
likeness and drug likeness of hits obtained after a compre-
hensive screening of molecules. Lipinski’s rule of fve was
Table 5 In silico ADME properties as predicted by Qikprop
Compound
Mol MW
HD
HA
QPlogPo/w
QPPCaco
QPlog BB
%Human oral
absorption
Rule of fve
PSA
69.6
PYN-1
PYN-2
PYN-3
PYN-4
PYN-5
PYN-6
PYN-7
PYN-8
PYN-9
PYN-10
PYN-11
406.8
417.4
451.3
390.4
386.4
402.4
406.8
417.4
451.3
390.4
386.4
1
1
1
1
1
1
1
1
1
1
1
4.75
5.75
4.75
4.75
4.75
5.5
4.75
5.75
4.75
4.75
4.75
5.291
4.084
5.367
5.036
5.107
4.928
5.335
4.127
5.412
5.075
5.148
1605.4
192.5
−0.305
−1.564
−0.293
−0.358
−0.484
−0.5
−0.254
−1.516
−0.244
−0.304
−0.431
100
91.747
100
100
100
100
100
92.936
100
100
1
0
1
1
1
0
1
0
1
1
1
114.7
70.4
69.7
70.3
78.1
69.6
113.1
69.6
69.9
69.8
1609.9
1596.5
1609.4
1782.8
1802.7
217.1
1803.4
1801.3
1814.6
100
HD hydrogen bond donor, HA hydrogen bond acceptor, PSA polar surface area
1 3

Molecular Diversity
formulated to ascertain drug likeness of a chemical com-
pound with properties that would probably make it an orally
active drug. Molecules which comply with these rules expe-
rience lower attrition and failure in clinical trials in the drug
discovery process. The probability of reaching the market for
such molecules increases because the parameters of this rule
were set after observing that most of the orally administered
drugs are moderately lipophilic and small molecules [21].
Thus, we also determined the ADME properties of the syn-
thesized compounds in silico, and all the compounds showed
desirable ADME profle (Table 5).
relative to internal standard, tetramethylsilane (TMS). The
mass spectra were recorded on Agilent Technologies 6545
Q-TOF LC/MS.
Synthesis of 1‑(3‑methoxy‑4‑phenoxyphenyl)
ethan‑1‑one
1-(3-methoxy-4-phenoxyphenyl)ethan-1-one was synthe-
sized as described in the previously reported procedure [22].
The compound was used without further purifcation in the
next step with a crude yield of 81%.
Synthesis of 1‑(3‑hydroxy‑4‑phenoxyphenyl)
ethan‑1‑one
Conclusion
A protein crystal structure, 3FNE, was selected after screen-
ing all the available protein X-ray crystal structures from
protein data bank. The selection was made after cross-dock-
ing and validation study of the protein crystal structures. The
molecules were designed using insights from the interaction
patterns of known diphenyl ether derivatives. The synthe-
sized compounds were then evaluated for their antituber-
cular activity against Mycobacterium tuberculosis H37Rv
strain and Mycobacterium bovis. Compound PYN-8 was
found to be most active among the synthesized derivatives
with MIC=4–7 µM in 7H9/ADC/Tw medium and showed
a 95.91% inhibition of Mycobacterium bovis at 10 µM con-
centration. All the synthesized compounds were subjected
to cytotoxicity evaluation against various cell lines and were
found to be noncytotoxic. The ADME properties calculated
in silico predicted good pharmacokinetic profle of the syn-
thesized molecules. These analogues can be further devel-
oped to improve their InhA inhibitory activity.
1-(3-hydroxy-4-phenoxyphenyl)ethan-1-one was synthe-
sized from 1-(3-methoxy-4-phenoxyphenyl)ethanone as
described in the previously reported procedure [22]. The
crude compound was purifed using column chromatography
using ethyl acetate: hexane (3:7) to give 76% yield.
Synthesis of 1‑(3‑hydroxy‑4‑phenoxyphenyl)‑3‑phe‑
nylprop‑2‑en‑1‑ones
1-(3-hydroxy-4-phenoxyphenyl)ethan-1-one (0.66 mmol)
in EtOH (1 mL) and LiOH·H2O (1.4 mmol) was stirred
for 10 min at room temperature. Then, various substituted
benzaldehydes (0.79 mmol) were added and stirred at
room temperature until complete consumption of the start-
ing materials. A yellow precipitate was usually obtained
after completion of the reaction. EtOH was removed under
reduced pressure. The residue was diluted with water (5 mL)
and neutralized with 2% dilute HCl. It was then extracted
with ethyl acetate (10 mL). The organic layer was washed
with saturated NaCl solution (5 mL), dried with MgSO4 and
concentrated under reduced pressure. It was then re-crystal-
lized from ethanol to aford various phenylprop-2-en-1-ones
in 80–89% yield.
Experimental
General chemistry
General method for the synthesis of substituted
1‑(3‑(3‑hydroxy‑4‑phenoxyphenyl)‑5‑phenyl‑4,5‑di‑
hydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one
All chemicals and reagents for synthesis were procured from
Sigma-Aldrich (St. Louis, MO, USA) and Spectrochem Pvt.
Ltd. (Bengaluru, India). LR-grade solvent was used in the
reaction. Distilled solvents were used for column chromatog-
raphy (CC). Silica gel 60 F254-precoated TLC (thin-layered
chromatography) plates were used for monitoring the reac-
tions and visualized in UV chamber. The compounds were
purifed by CC or recrystallization. Column chromatography
was performed on silica gel 60120 (100–200 mesh). Silica
gel for CC and TLC plate both was purchased from Merck
(Germany). The synthesized compounds were character-
1-(3-hydroxy-4-phenoxyphenyl)-3-phenylprop-2-en-1-ones
(0.47 mmol) and hydrazine hydrate (1.41 mmol) in glacial
acetic acid (2 mL) were refuxed for 2 h. Upon completion
of the reaction, the mixture was poured into ice cold water
(20 mL). The precipitate was then fltered and washed with
cold water (10 mL) and re-crystallized from ethanol to give
pure product.
1
1
ized using H and 13C NMR and mass spectrometry. H-
and 13C-NMR spectra were recorded using Bruker Ascend
400 MHz; chemical shift values (δ) are expressed in ppm
1‑(5‑(4‑chlorophenyl)‑3‑(3‑hydroxy‑4‑phenoxyphenyl)‑4,5‑di‑
hydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑1) %Yield: 88%, 1H
1 3

Molecular Diversity
NMR (400 MHz, Chloroform-d) δ 8.16–7.95 (m, 2H), 7.52
(d, J=7.6 Hz, 1H), 7.49–7.37 (m, 2H), 7.32 (t, J=7.7 Hz,
2H), 7.00 (d, J=8.1 Hz, 2H), 6.79 (d, J=8.4 Hz, 1H), 5.77
(s, 1H), 5.59 (dd, J=12.0, 5.0 Hz, 1H), 3.73 (dd, J=17.8,
12.0 Hz, 1H), 3.05 (dd, J=17.8, 5.0 Hz, 1H), 2.36 (s, 3H);
13C NMR (101 MHz, Chloroform-d) δ 168.92, 155.89,
153.16, 147.36, 145.98, 140.35, 133.48, 130.11, 129.09,
127.53, 127.13, 124.44, 119.34, 118.74, 117.94, 114.15,
59.46, 42.28, 21.92. HRMS (ESI/Q-TOF) m/z: [M + H]+
Calcd for C23H19ClN2O3+ 407.1157; found 407.1148.
1‑(3‑(3‑hydroxy‑4‑phenoxyphenyl)‑5‑(p‑tolyl)‑4,5‑dihy‑
dro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑5) %Yield: 84%, H
1
NMR (400 MHz, Chloroform-d) δ 7.47 (d, J=2.0 Hz, 1H),
7.43–7.34 (m, 2H), 7.22–7.13 (m, 2H), 7.06 (d, J=7.7 Hz,
2H), 6.86 (d, J = 8.4 Hz, 1H), 5.55 (dd, J = 11.8, 4.5 Hz,
1H), 3.69 (dd, J = 17.6, 11.8 Hz, 1H), 3.11 (dd, J = 17.6,
4.5 Hz, 1H), 2.40 (s, 3H), 2.30 (s, 3H); 13C NMR (101 MHz,
Chloroform-d) δ 167.02, 154.20, 151.54, 145.61, 143.96,
137.05, 135.47, 128.16, 127.67, 125.94, 123.68, 122.35,
117.38, 116.74, 116.30, 112.41, 57.95, 40.55, 20.06, 19.22.
+
HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C24H22N2O3
1‑(3‑(3‑hydroxy‑4‑phenoxyphenyl)‑5‑(4‑nitrophenyl)‑4,
5‑dihydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑2) %Yield:
91%, 1H NMR (400 MHz, Chloroform-d) δ 8.16–8.10 (m,
2H), 7.40 (d, J=2.0 Hz, 1H), 7.37–7.28 (m, 4H), 7.19 (s,
1H), 7.17–7.07 (m, 2H), 7.00 (d, J=8.0 Hz, 2H), 6.79 (d,
J=8.5 Hz, 1H), 5.73 (s, 1H), 5.58 (dd, J=12.0, 5.0 Hz, 1H),
3.72 (dd, J=17.7, 12.0 Hz, 1H), 3.03 (dd, J=17.7, 5.0 Hz,
1H), 2.35 (s, 3H); 13C NMR (101 MHz, Chloroform-d) δ
155.76, 153.07, 148.82, 147.44, 147.37, 146.24, 130.14,
127.09, 126.70, 124.55, 124.34, 119.38, 118.83, 117.85,
114.15, 59.53, 42.12, 21.84, 1.02. HRMS (ESI/Q-TOF) m/z:
[M+H]+ Calcd for C23H19N3O5+ 418.1397; found 418.1391.
387.1703; found 387.1701.
1‑(3‑(3‑hydroxy‑4‑phenoxyphenyl)‑5‑(4‑methoxyphenyl)‑4,
5‑dihydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑6) %Yield: 87%,
1H NMR (400 MHz, Chloroform-d) δ 7.48 (d, J=2.0 Hz,
1H), 7.43–7.34 (m, 2H), 7.22–7.12 (m, 4H), 7.10–7.02 (m,
2H), 6.85 (t, J=8.1 Hz, 3H), 5.82 (s, 1H), 5.54 (dd, J=11.7,
4.5 Hz, 1H), 3.77 (s, 3H), 3.68 (dd, J=17.6, 11.7 Hz, 1H),
3.11 (dd, J = 17.6, 4.5 Hz, 1H), 2.39 (s, 3H); 13C NMR
(101 MHz, Chloroform-d) δ 168.87, 159.04, 156.04, 153.35,
147.44, 145.82, 134.09, 130.06, 127.85, 126.96, 124.29,
119.28, 118.64, 118.11, 114.24, 59.52, 55.29, 42.34, 21.97.
+
HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C24H22N2O4
1‑(5‑(4‑bromophenyl)‑3‑(3‑hydroxy‑4‑phenoxyphenyl)‑4,
403.1652; found 403.1641.
5‑dihydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑3) %Yield:
1
87%, H NMR (400 MHz, Chloroform-d) δ 7.42–7.35
1‑(5‑(3‑chlorophenyl)‑3‑(3‑hydroxy‑4‑phenoxyphenyl)‑4,
5‑dihydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑7) %Yield: 82%,
1H NMR (400 MHz, Chloroform-d) δ 7.47 (d, J=2.0 Hz,
1H), 7.38 (t, J = 7.9 Hz, 2H), 7.30–7.03 (m, 8H), 6.86 (d,
J=8.5 Hz, 1H), 5.54 (dd, J=11.9, 4.7 Hz, 1H), 3.71 (dd,
J=17.9, 11.6 Hz, 1H), 3.09 (dd, J=17.7, 4.7 Hz, 1H), 2.42
(s, 3H); 13C NMR (101 MHz, Chloroform-d) δ 168.98,
155.93, 153.20, 147.41, 146.00, 143.81, 134.80, 130.23,
130.09, 127.93, 127.47, 125.73, 124.39, 123.92, 119.35,
118.72, 118.01, 114.21, 59.54, 42.32, 21.91. HRMS (ESI/Q-
TOF) m/z: [M + H]+ Calcd for C23H19ClN2O3+ 407.1157;
found 407.1141.
(m, 3H), 7.32 (t, J=7.8 Hz, 2H), 7.11 (t, J=9.3 Hz, 2H),
7.04 (d, J=8.0 Hz, 2H), 6.99 (d, J=8.0 Hz, 2H), 6.79 (d,
J=8.5 Hz, 1H), 5.73 (s, 1H), 5.46 (dd, J=12.5, 4.5 Hz, 1H),
3.65 (dd, J=17.7, 11.9 Hz, 1H), 3.01 (dd, J=17.3, 4.7 Hz,
1H), 2.33 (s, 3H); 13C NMR (101 MHz, Chloroform-d) δ
168.94, 155.91, 153.20, 147.39, 146.00, 140.85, 132.03,
130.09, 127.49, 127.46, 124.41, 121.58, 119.33, 118.73,
117.98, 114.18, 59.52, 42.23, 21.90. HRMS (ESI/Q-TOF)
m/z: [M+H]+ Calcd for C23H19BrN2O3+ 451.0652; found
451.0636.
1‑(5‑(4‑fuorophenyl)‑3‑(3‑hydroxy‑4‑phenoxyphenyl)‑4,5‑di‑
hydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑4) %Yield: 85%, 1H
NMR (400 MHz, Chloroform-d) δ 7.48 (d, J=2.1 Hz, 1H),
7.43–7.34 (m, 2H), 7.25–7.14 (m, 4H), 7.06 (dd, J = 7.7,
1.5 Hz, 2H), 7.04–6.96 (m, 2H), 6.86 (d, J= 8.5 Hz, 1H),
5.77 (s, 1H), 5.56 (dd, J = 11.8, 4.6 Hz, 1H), 3.72 (dd,
J=17.6, 11.8 Hz, 1H), 3.10 (dd, J=17.7, 4.6 Hz, 1H), 2.40
(s, 3H); 13C NMR (101 MHz, Chloroform-d) δ 167.88,
154.89, 152.16, 146.34, 144.91, 129.08, 126.59, 126.43,
126.35, 123.39, 118.30, 117.70, 116.94, 114.86, 114.64,
113.13, 58.36, 41.34, 20.92, − 1.02. HRMS (ESI/Q-TOF)
1‑(3‑(3‑hydroxy‑4‑phenoxyphenyl)‑5‑(3‑nitrophenyl)‑4,5‑di‑
hydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑8) %Yield: 88%,
1H NMR (400 MHz, Chloroform-d) δ 8.09–7.99 (m, 2H),
7.52 (d, J = 7.6 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.40 (d,
J = 2.0 Hz, 1H), 7.32 (t, J = 7.7 Hz, 2H), 7.16–7.08 (m,
2H), 7.00 (d, J=8.1 Hz, 2H), 6.79 (d, J=8.4 Hz, 1H), 5.77
(s, 1H), 5.59 (dd, J=12.0, 5.0 Hz, 1H), 3.73 (dd, J=17.8,
12.0 Hz, 1H), 3.05 (dd, J=17.8, 5.0 Hz, 1H), 2.36 (s, 3H);
13C NMR (101 MHz, Chloroform-d) δ 169.16, 155.85,
153.09, 148.71, 147.42, 146.21, 143.84, 132.08, 130.11,
129.97, 127.12, 124.46, 122.81, 120.84, 119.41, 118.77,
117.97, 114.24, 59.44, 42.22, 21.88. HRMS (ESI/Q-TOF)
+
m/z: [M + H]+ Calcd for C23H19FN2O3 391.1452; found
391.1439.
+
m/z: [M + H]+ Calcd for C23H19N3O5 418.1397; found
418.1386.
1 3

Molecular Diversity
1‑(5‑(3‑bromophenyl)‑3‑(3‑hydroxy‑4‑phenoxyphenyl)‑4,5‑di‑
hydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑9) %Yield: 79%, 1H
NMR (400 MHz, Chloroform-d) δ 7.47 (d, J=2.1 Hz, 1H),
7.44–7.32 (m, 4H), 7.24–7.13 (m, 4H), 7.10–7.02 (m, 2H),
6.86 (d, J=8.5 Hz, 1H), 5.53 (dd, J=11.9, 4.7 Hz, 1H), 3.72
(dd, J=17.7, 11.9 Hz, 1H), 3.09 (dd, J=17.7, 4.7 Hz, 1H),
2.42 (s, 3H); 13C NMR (101 MHz, Chloroform-d) δ 169.00,
155.93, 153.22, 147.41, 146.02, 144.07, 130.87, 130.52,
130.09, 128.63, 127.45, 124.39, 123.01, 119.36, 118.71,
118.02, 114.23, 59.49, 42.34, 21.91. HRMS (ESI/Q-TOF)
m/z: [M+H]+ Calcd for C23H19BrN2O3+ 451.0652; found
451.0634.
wells before addition of the assay components. 10 μM fnal
concentration of the compounds in the microtiter plate wells
was obtained by adding 98 μL of inoculum. Inoculum was
an overnight culture with 0.6 optical density diluted with
7H9 broth in 1:1000 ratio. Checks such as DMSO as solvent
control, media control (blank) and rifampicin and isoniazid
as positive controls to inhibit the growth of Mycobacterium
bovis in each plate were added for the purpose of better
determining the activity of compounds. To prevent evapo-
ration from the wells during assay, sterile distilled water was
flled in the peripheral wells. Plates were then incubated at
37 °C and at 80% relative humidity for 4 days. Growth of the
bacteria was studied after the incubation period using tur-
bidometry, by measuring the absorbance at 600 nm using a
Multi-Mode Reader (PerkinElmer). Percentage growth inhi-
bition was determined against DMSO control. The inhibition
of growth of bacteria due to the compounds was calculated
as:
1‑(5‑(3‑fuorophenyl)‑3‑(3‑hydroxy‑4‑phenoxyphenyl)‑4,5‑di‑
hydro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑10) %Yield: 81%,
1H NMR (400 MHz, Chloroform-d) δ 7.46 (d, J=2.0 Hz,
1H), 7.38 (t, J=7.9 Hz, 2H), 7.29 (td, J=7.6, 5.5 Hz, 1H),
7.22–7.14 (m, 2H), 7.09–6.99 (m, 3H), 6.99–6.88 (m, 2H),
6.86 (d, J = 8.4 Hz, 1H), 5.57 (dd, J = 11.8, 4.6 Hz, 1H),
3.71 (dd, J=17.7, 11.9 Hz, 1H), 3.09 (dd, J=17.7, 4.6 Hz,
1H), 2.42 (s, 3H); 13C NMR (101 MHz, Chloroform-d) δ
167.97, 154.95, 152.26, 146.42, 144.98, 129.55, 129.47,
129.05, 126.47, 123.32, 120.26, 120.24, 118.29, 117.66,
117.05, 113.73, 113.52, 113.22, 111.67, 111.45, 58.53,
41.29, 20.86. HRMS (ESI/Q-TOF) m/z: [M + H] + Calcd
for C23H19FN2O3 +391.1452; found 391.1439.
Percentage Inhibition = 100
[
]
× OD with compound − OD of Negative control
[
]
∕ OD of Positive control − OD of Negative control .
M. tuberculosis (H37Rv) inhibition assay
All the fnal synthesized substituted diphenyl ether deriva-
tives were evaluated for in vitro antitubercular activity
against Mycobacterium tuberculosis H37Rv strain. Mini-
mum inhibitory concentrations (MIC) were determined in
two diferent media: iron-supplemented GAST (pH 6.6) and
in Middlebrook 7H9 broth base supplemented with 0.2%
glycerol, 0.4% glucose, 0.5% BSA fraction V, 0.08% NaCl
and 0.05% Tween 80. The assay was performed using the
serial dilution technique where the Mycobacterium tubercu-
losis inoculum was added to the assay plate containing serial
twofold dilutions of the compounds in the medium of choice.
A fnal cell concentration of 1x105 cfu/mL and fnal com-
pound concentration series in the range of 100–0.78 µg/mL
were obtained. Plates were read after completion of week
1 and week 2 of incubation period at 37 °C using inverted
enlarging mirror plate reader and graded for growth and no
growth. The lowest concentration of compound that com-
pletely inhibited growth of the bacterium was reported as
MIC [24–26].
1‑(3‑(3‑hydroxy‑4‑phenoxyphenyl)‑5‑(m‑tolyl)‑4,5‑dihy‑
dro‑1H‑pyrazol‑1‑yl)ethan‑1‑one (PYN‑11) %Yield: 84%,
1H NMR (400 MHz, Chloroform-d) δ 7.48 (d, J=2.0 Hz,
1H), 7.38 (t, J=7.8 Hz, 2H), 7.19 (dd, J=8.6, 5.9, 2.6 Hz,
3H), 7.04 (dd, J = 17.6, 6.5 Hz, 5H), 6.86 (d, J = 8.4 Hz,
1H), 5.55 (dd, J=11.8, 4.5 Hz, 1H), 4.78 (s, 8H), 3.70 (dd,
J=17.6, 11.8 Hz, 1H), 3.11 (dd, J=17.7, 4.6 Hz, 1H), 2.42
(s, 3H), 2.32 (s, 3H); 13C NMR (101 MHz, Chloroform-d)
δ 168.98, 156.03, 153.47, 147.44, 145.86, 141.79, 138.59,
130.06, 128.82, 128.48, 127.79, 126.17, 124.29, 122.55,
119.31, 118.64, 118.11, 114.27, 60.04, 42.52, 21.94, 21.47.
+
HRMS (ESI/Q-TOF) m/z: [M+H]+ Calcd for C24H22N2O3
387.1703; found 387.1691.
Antitubercular activity
Antimycobacterial assay
Mycobacterium bovis (BCG) Pasteur is a surrogate model
for high-throughput screening of chemical compounds to
identify newer antimycobacterial agents [23]. Primary
screening of the compounds at 10 μM concentration against
Mycobacterium bovis was performed in fat-bottomed poly-
styrene microtiter 96-well sterile plates (Nunc). Test com-
pounds prepared in DMSO were dispensed into triplicate
Cytotoxicity
Resazurin assay
All cell lines used in this study were purchased from
the American Type Culture Collection (ATCC). MCF7,
1 3

Molecular Diversity
PANC-1, PC-3, SKOV-3 and HeLa were cultured in Dul-
becco’s Modifed Eagle’s Medium (containing 10% FBS
in a humidifed atmosphere of 5% CO2 at 37 °C). PC-3
cells were grown in RPMI-1640 medium containing 10%
FBS, 1 mM sodium pyruvate 10 mg/mL bovine insulin and
non-essential amino acids. The synthesized test compounds
were evaluated for their in vitro antiproliferative activity in
fve diferent human cancer cell lines by resazurin assay.
Cells were seeded in 100-µL aliquots into 96-well micro-
titer plates at plating densities depending on the duplica-
tion time of individual cell lines. Prior to the addition of
test compounds, microtiter plates were incubated at 37 °C,
5% CO2, 95% air, and 100% relative humidity for 24 h.
Aliquots of 1 µL of the test compounds were added to the
wells in triplicates containing 100 µL of cells, furnish-
ing fnal drug concentrations. Plates were then incubated
further for 48 h. Assay was terminated by adding 50 µL
of resazurin solution and then incubating for 60 min at
37 °C. Using a multimode plate reader (Tecan M200) at
a wavelength of 560-nm excitation/590 nm, the emission
fuorescence intensity was read. The cell growth is directly
proportional to the measure of absorbance and is used to
calculate the IC50 values.
(Tecan Spectra, Crailsheim, Germany). IC50 was calculated
using GraphPad Prism software.
Drug design strategy
Data set for cross‑docking study
A total of 87 crystal structures of enoyl-acyl carrier protein
reductase of Mycobacterium tuberculosis available in RCSB
protein data bank were selected for the present study. How-
ever, only the crystal structures containing co-crystallized
inhibitors similar to triclosan were selected for the study.
Of the numerous chains of InhA, only the A-chain was con-
sidered for the comparative study of the crystal structures.
Out of 15 selected crystal structures, 4 were not considered
appropriate for the study because they either had a missing
inhibitor ligand in the A-chain or had missing coordinates
for the substrate binding loop.
Protein preparation
Selected 11 protein crystal structures were prepared using
protein preparation module of Schrodinger. Bond orders of
the protein structure were assigned, and hydrogens were
added. The hydroxyl, asparagine, glutamine and histidine
states were optimized using ProtAssign. The complex was
subjected to restrained minimization using OPLS_2005
force field, which was terminated when the root-mean-
square deviation of the heavy atoms relative to their initial
location reached a maximum value of 0.3 Å.
Percentage Inhibition = 100
[
]
× OD with compound − OD of Negative control
[
]
∕ OD of Positive control − OD of Negative control .
Sulforhodamine B assay
The inhibitor ligands from respective crystal structures
were extracted out and were aligned using fexible shape-
based alignment in the fexible ligand alignment module as
shown in Fig. 7. The prepared protein structures were aligned
on the protein PDB entry code 2X23 [27] according to the
Cytotoxicity of the compounds was also tested on BD-11
and HEK cell lines using sulforhodamine B (SRB) (Sigma-
Aldrich, Germany) microculture colorimetric assay. On day
zero, cells were seeded in 96-well plates, at appropriate cell
densities. They were treated with serial dilutions of the com-
pounds (6.25 to 100 1M) after 24 h. The plates were incu-
bated at 37 °C in a humidifed incubator with 5% CO2 for
96 h. Care was taken to ensure that the fnal concentration
of DMSO does not exceed 0.5%, which was shown to be
non-toxic to the cells. Ninety-six hours after drug exposure,
percentage of surviving cells was determined in compari-
son with untreated controls. The cells were fxed with 10%
trichloroacetic acid after discarding the supernatant medium
from the 96-well plates. The plates were then allowed to
stand at 4 °C. Then, the cells were washed in a strip washer
four times with water using alternate dispensing and aspi-
ration procedures. 100 µL of 0.05% SRB was incubated at
room temperature for about 30 min. After dying, the plates
were washed with 1% acetic acid to remove the dye and
allowed to air dry overnight. Then, 50 µL of 10 mM Tris
base solution (pH-10) was added to each well and absorb-
ance was measured at 520 nm using a 96-well plate reader
Fig. 7 All the extracted ligands aligned using fexible ligand align-
ment
1 3

Molecular Diversity
Molecular docking
All the 25 inhibitors were docked on protein 3FNE which
was prepared by using protein preparation wizard module
keeping all the default settings and by removing explic-
itly all the water molecules from the crystal structure. The
selected molecules were drawn using build panel in Maes-
tro, Schrödinger. Bond orders of all the structures were cor-
rected, and structures were energy minimized using Lig-
Prep, Schrödinger. Grid was generated by using centroid
of the co-crystallized ligand in the protein–ligand com-
plex crystal as centre. Extra precision (XP) mode (Glide,
Schrödinger) was applied for analysing the binding modes
of the inhibitors.
Prime MM/GBSA calculations
Fig. 8 All 11 crystal structures aligned over 2X23 crystal structure
The MM/GBSA binding free energy was calculated (Prime,
Schrödinger) using the receptor ligand complex obtained
from molecular docking done in extra precision (XP) mode.
VSGB2.0 solvation model and OPLS_2005 force feld were
used for the simulation.
protein back bone using protein structure alignment as shown
in Fig. 8. In order to test the signifcance of water molecules
in the study, another set of protein structures were created by
removing all the water molecules from the crystal structures.
Induced ft docking
Receptor grid generation
The receptor protein and the ligands were prepared as per
the protocol mentioned above. Triclosan and PYN-8 were
docked into the active site of 3FNE using the induced ft
docking protocol (IFD) (Glide, Schrödinger) [29]. First, the
van der Waals radii of protein and ligand atoms are scaled
down by a factor of 0.5, and ligands are then docked into the
fxed receptor using the Glide SP docking protocol. Then,
optimal orientation of the side chains of the binding site
residues is predicted by Prime. Finally, the optimized bind-
ing site was used for re-docking of the ligand and poses were
scored with Glide XP. Following IFD, twenty top-scoring
poses were obtained.
Receptor grid was then generated on the chain-A of all the
prepared protein structures using glide grid generation with
centre of the grid as centroid of the co-crystallized ligand.
Docking
Docking was performed using glide dock on each of the
protein crystal structures using all the extracted ligands. The
docking was performed in extra precision (XP) mode. Every
ligand was docked on all the 11 protein crystal structures.
RMSD calculation
Water map
The aligned ligands were chosen individually and superposi-
tioned over their docked poses in diferent protein structures.
Root-mean-square deviations (RMSD) of all the docked
poses on proteins containing crystal waters and the ones
without the crystal water were then determined with respect
to the aligned ligand structures.
Water map (WaterMap, Schrödinger) was generated using
the protein 3FNE after preparation of the protein using
protein preparation wizard (Epik, Schrödinger) [30]. The
calculation was run for 9 ns (300 K, 1 atm) which is a three-
step process. It involves running a molecular dynamics
simulation of protein–ligand complex with explicit water
molecules. Clustering algorithm then locates the hydration
sites, and fnally, thermodynamic properties of the waters
are calculated.
Data set for correlation study
Twenty-fve diphenyl ether derivatives collected from the
literature [28] were used for this correlation study. The mol-
ecules had wide variation in their range of biological activ-
ity. The IC50 values were converted to −log IC50 (pIC50).
1 3

Molecular Diversity
X‑ray difraction
References
1. WHO (2018) Tuberculosis (TB). WHO, Geneva
2. World Health Organization (2018) BCG vaccine: WHO position
paper, February 2018 – Recommendations. Vaccine 73–96. https
://doi.org/10.1016/j.vaccine.2018.03.009
X-ray data of compound PYN-8 were collected at room
temperature using a Bruker Smart Apex CCD diffrac-
tometer with graphite monochromated MoKα radiation
(λ=0.71073 Å) with ω-scan method [31]. Preliminary lat-
tice parameters and orientation matrices were obtained from
four sets of frames.
3. WHO (2017) Guidelines for treatment of drug-susceptible tuber-
culosis and patient care. WHO, Geneva
4. Rožman K, Sosič I, Fernandez R, Young RJ, Mendoza A, Gobec
S, Encinas L (2017) A new ‘golden age’ for the antitubercular
target InhA. Drug Discov Today 22(3):492–502
Integration and scaling of intensity data were accom-
plished using SAINT program [31]. The structure was
solved by direct methods using SHELXS [32], and refne-
ment was carried out by full-matrix least-squares technique
using SHELXL [32]. Anisotropic displacement parameters
were included for all non-hydrogen atoms. Oxygen bound
hydrogen atoms were located in diferent Fourier maps,
and their positions and isotropic displacement parameters
were refned. All hydrogen atoms were positioned geo-
metrically and treated as riding on their parent carbon
atoms [C–H = 0.93–0.97 Å and Uiso(H) = 1.2 Ueq(C) for
other hydrogen atoms]. All attempts to model a disordered
ethylacetate solvent (used for crystallization) failed. There-
fore, the solvent contributions have been removed using the
SQUEEZE procedure in PLATON [33]. SQUEEZE calcu-
lated a void volume of approximately 334 Å3 occupied by
73 electrons per unit cell.
5. Forrellad MA, Klepp LI, Giofré A et al (2013) Virulence factors
of the Mycobacterium tuberculosis complex. Virulence. https://
doi.org/10.4161/viru.22329
6. Zhang Y, Heym B, Allen B et al (1992) The catalase-peroxidase
gene and isoniazid resistance of Mycobacterium tuberculosis.
Nature. https://doi.org/10.1038/358591a0
7. Narang A, Giri A, Gupta S et al (2017) Contribution of putative
efux pump genes to isoniazid resistance in clinical isolates of
Mycobacterium tuberculosis. Int J Mycobacteriol 6:177. https://
doi.org/10.4103/ijmy.ijmy_26_17
8. McMurry LM, Oethinger M, Levy SB (1998) Triclosan targets
lipid synthesis. Nature 394:531–532
9. Parikh SL, Xiao G, Tonge PJ (2000) Inhibition of InhA, the enoyl
reductase from Mycobacterium tuberculosis, by triclosan and
isoniazid. Biochemistry 39:7645–7650. https://doi.org/10.1021/
bi0008940
10. Sullivan TJ, Truglio JJ, Boyne ME et al (2006) High afnity InhA
inhibitors with activity against drug-resistant strains of Mycobac-
terium tuberculosis. ACS Chem Biol. https://doi.org/10.1021/
cb0500042
Crystal data for PYN-8: C23H19N3O5 (M = 417.41 g/
mol): triclinic, space group P-1 (no. 2), a =11.2865(7) Å,
b = 11.4236(7) Å, c = 11.9381(7) Å, α = 115.8430(10)°,
β = 93.3520(10)°, γ = 111.2500(10)°, V = 1247.36(13) Å3,
Z = 2, T = 294.15 K, μ(MoKα) = 0.080 mm-1,
Dcalc = 1.111 g/cm3, 15426 reflections measured
(3.924° ≤ 2Θ ≤ 56.71°), 6041 unique (Rint = 0.0259,
Rsigma = 0.0341) which were used in all calculations. The
fnal R1 was 0.0573 (I > 2σ(I)) and wR2 was 0.1671 (all
data). CCDC 1883097 contains supplementary crystallo-
graphic data for the structure. These data can be obtained
free of charge at www.ccdc.cam.ac.uk/conts/retrieving
.html [or from the Cambridge Crystallographic Data Centre
(CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax:
+44(0) 1223 336 033; email: deposit@ccdc.cam.ac.uk].
11. Pan P, Knudson SE, Bommineni GR et al (2014) Time-depend-
ent diaryl ether inhibitors of InhA: structure-activity relationship
studies of enzyme inhibition, antibacterial activity, and in vivo
efcacy. ChemMedChem. https://doi.org/10.1002/cmdc.20130
0429
12. am Ende CW, Knudson SE, Liu N et al (2008) Synthesis and
in vitro antimycobacterial activity of B-ring modifed diaryl ether
InhA inhibitors. Bioorganic Med Chem Lett 18:3029–3033. https
://doi.org/10.1016/j.bmcl.2008.04.038
13. Luckner SR, Liu N, Am Ende CW et al (2010) A slow, tight
binding inhibitor of InhA, the enoyl-acyl carrier protein reduc-
tase from Mycobacterium tuberculosis. J Biol Chem. https://doi.
org/10.1074/jbc.M109.090373
14. Stewart MJ, Parikh S, Xiao G et al (1999) Structural basis and
mechanism of enoyl reductase inhibition by triclosan. J Mol Biol.
https://doi.org/10.1006/jmbi.1999.2907
15. Freundlich JS, Wang F, Vilchèze C et al (2009) Triclosan deriva-
tives: towards potent inhibitors of drug-sensitive and drug-resist-
ant Mycobacterium tuberculosis. ChemMedChem 4:241–248.
https://doi.org/10.1002/cmdc.200800261
Acknowledgements The authors are thankful to Manipal Academy of
Higher Education and Manipal College of Pharmaceutical Sciences
for providing necessary supports and facilities to carry out the present
research work. Authors are thankful to D’IICT and National Mol Bank
(NMB) facility of CSIR-IICT, Hyderabad, India, for the help in biologi-
cal evaluation of compounds. Authors also thank Manipal-Schrödinger
Centre for molecular simulations. This work was funded in part by the
Intramural Research Program of the NIH, NIAID.
16. Chollet A, Maveyraud L, Lherbet C, Bernardes-Génisson V
(2018) An overview on crystal structures of InhA protein: apo-
form, in complex with its natural ligands and inhibitors. Eur J Med
Chem 146:318–343. https://doi.org/10.1016/j.ejmech.2018.01.047
17. Cappel D, Sherman W, Beuming T (2017) Calculating water
thermodynamics in the binding site of proteins—applications of
WaterMap to drug discovery. Curr Top Med Chem. https://doi.
org/10.2174/1568026617666170414141452
18. Cheraïti N, Brik ME (1999) Synthesis of a new triprotonated
ligand and selective O-demethylation of methyl aryl ether by
boron tribromide. Tetrahedron Lett 40:4327–4330. https://doi.
org/10.1016/S0040-4039(99)00786-8
Compliance with ethical standards
Conflict of interest The authors confrm that this article content has no
confict of interest.
19. Bhagat S, Sharma R, Sawant DM, Sharma L, Chakraborti AK
(2006) LiOH · H
2O as a novel dual activation catalyst for highly
1 3

Molecular Diversity
efficient and easy synthesis of 1, 3-diaryl-2-propenones by
ADME characterization. Bioorganic Med Chem 23:7240–7250.
https://doi.org/10.1016/j.bmc.2015.10.021
Claisen—Schmidt condensation under mild conditions. J Mol
Catal A: Chem 244(1–2):20–24. https://doi.org/10.1016/j.molca
ta.2005.08.039
27. Kumar V, Sobhia ME (2014) Insights into the bonding pattern for
characterizing the open and closed state of the substrate-binding
loop in Mycobacterium tuberculosis InhA. Future Med Chem.
https://doi.org/10.4155/fmc.14.27
20. Wei E, Liu B, Lin S, Liang F (2014) Multicomponent reaction of
chalcones, malononitrile and DMF leading to γ-ketoamides. Org
Biomol Chem 12:6389–6392. https://doi.org/10.1039/c4ob00971a
21. Lipinski CA (2004) Lead- and drug-like compounds: the rule-of-
fve revolution. Drug Discov Today Technol 1(4):337–341
22. Kar SS, Bhat VG, Shenoy VP et al (2018) Design, synthesis and
evaluation of novel diphenyl ether derivatives against drug suscep-
tible and resistant strains of Mycobacterium tuberculosis. Chem
Biol Drug Des. https://doi.org/10.1111/cbdd.13379
28. Pan P, Tonge J, Tonge P (2012) Targeting InhA, the FASII enoyl-
ACP reductase: SAR studies on novel inhibitor scafolds. Curr
Top Med Chem 12:672–693. https://doi.org/10.2174/1568026127
99984535
29. Sherman W, Day T, Jacobson MP et al (2006) Novel procedure
for modeling ligand/receptor induced ft efects. J Med Chem
49:534–553. https://doi.org/10.1021/jm050540c
23. Khan A, Sarkar D (2008) A simple whole cell based high through-
put screening protocol using Mycobacterium bovis BCG for
inhibitors against dormant and active tubercle bacilli. J Microbiol
Methods. https://doi.org/10.1016/j.mimet.2008.01.015
30. Robinson D, Bertrand T, Carry JC et al (2016) Diferential water
thermodynamics determine PI3 K-beta/delta selectivity for sol-
vent-exposed ligand modifcations. J Chem Inf Model 56:886–
894. https://doi.org/10.1021/acs.jcim.5b00641
24. Barot KP, Jain SV, Gupta N et al (2014) Design, synthesis and
docking studies of some novel (R)-2-(4′- chlorophenyl)-3-(4′-
nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5] imidazo [1,2-c]pyrimi-
din-4-ol derivatives as antitubercular agents. Eur J Med Chem
83:245–255. https://doi.org/10.1016/j.ejmech.2014.06.019
25. Kaniga K, Cirillo DM, Hofner S et al (2016) A multilabora-
tory, multicountry study to determine MIC quality control ranges
for phenotypic drug susceptibility testing of selected First-Line
Antituberculosis Drugs, Second-Line Injectables, Fluoroquinolo-
nes, Clofazimine, and Linezolid. J Clin Microbiol. https://doi.
org/10.1128/JCM.01138-16
31. Solutions BAX BRUKER ADVANCED X-RAY SOLUTIONS
SMART APEX User’s Manual, https://www.ndsu.edu/chemistry/
fles/mcl/smart5_UserManual.pdf
32. Sheldrick GM (2015) Crystal structure refnement with SHELXL.
Acta Crystallogr Sect C 71:3–8. https://doi.org/10.1107/S2053
229614024218
33. Spek AL (2015) PLATON SQUEEZE: a tool for the calculation
of the disordered solvent contribution to the calculated struc-
ture factors. Acta Crystallogr Sect C Struct Chem. https://doi.
org/10.1107/S2053229614024929
26. Candice SDM, Feng TS, Van Der Westhuyzen R et al (2015)
Aminopyrazolo[1,5-a]pyrimidines as potential inhibitors of
Mycobacterium tuberculosis: structure activity relationships and
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
Afliations
Ashutosh Prasad Tiwari1 · B. Sridhar2 · Helena I. Boshof3 · Kriti Arora3 · G. Gautham Shenoy1 · K. E. Vandana4 ·
G. Varadaraj Bhat1
3
* G. Varadaraj Bhat
Tuberculosis Research Section, Laboratory of Clinical
Immunology and Microbiology, National Institute of Allergy
and Infectious Diseases, National Institutes of Health,
Bethesda, MD 20892, USA
varad.g@manipal.edu
1
Manipal College of Pharmaceutical Sciences, Manipal
Academy of Higher Education, Manipal 576104, India
4
Department of Microbiology, Kasturba Medical College,
Manipal Academy of Higher Education, Manipal 576104,
India
2
X-ray Crystallography Division, CSIR – Indian Institute
of Chemical Technology, Hyderabad 500607, India
1 3