Synthesis, antitubercular activity and docking study of novel cyclic azole substituted diphenyl ether derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.04.013

The re-emergence of tuberculosis (TB) as a global health problem over the past few decades, accompanied by the rise of drug-resistant strains of Mycobacterium tuberculosis, emphasizes the need for discovery of new therapeutic drugs against this disease. The emerging serious problem both in terms of TB control and clinical management prompted us to synthesize a novel series of heterocyclic o/m/p substituted diphenyl ether derivatives and determine their activity against H37Rv strain of Mycobacterium. All 10 compounds inhibited the growth of the H37Rv strain of mycobacterium at concentrations as low as 1 μg/mL. This level of activity was found comparable to the reference drugs rifampicin and isoniazid at the same concentration. Molecular modeling of the binding of the diphenyl ether derivatives on enoyl-ACP reductase, the molecular target site of triclosan, indicated that these compounds fit within the binding domain occupied by triclosan. Hence the diphenyl ether derivatives tested in this study were docked to ENR and the binding of the diphenyl ether derivatives was also estimated using a variety of scoring functions that have been compiled into the single consensus score. As the scores ranged from 47.27% to 65.81%, these bioactive compounds appear to have a novel mechanism of action against M. tuberculosis, and their structural features should be studied further for their potential use as new antitubercular drugs.

Full text of DOI:10.1016/j.ejmech.2008.04.013

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 492e500
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antitubercular activity and docking study of novel
cyclic azole substituted diphenyl ether derivatives
b
c
Suvarna G. Kini a, , Anilchandra R. Bhat , Byron Bryant ,
*
John S. Williamson c, Franck E. Dayan d
a Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal e 576 104, Karnataka, India
b Department of Pharmaceutical Chemistry, K.L.E’s College of Pharmaceutical Sciences, Belgaum e 560 010, Karnataka, India
c Department of Medicinal Chemistry, University of Mississippi, University, MS 38677, USA
d USDA-ARS Natural Products Utilization Research Unit, University, MS 38677, USA
Received 17 October 2007; received in revised form 28 March 2008; accepted 8 April 2008
Available online 1 May 2008
Abstract
The re-emergence of tuberculosis (TB) as a global health problem over the past few decades, accompanied by the rise of drug-resistant strains
of Mycobacterium tuberculosis, emphasizes the need for discovery of new therapeutic drugs against this disease. The emerging serious problem
both in terms of TB control and clinical management prompted us to synthesize a novel series of heterocyclic o/m/p substituted diphenyl ether
derivatives and determine their activity against H37Rv strain of Mycobacterium. All 10 compounds inhibited the growth of the H37Rv strain of
mycobacterium at concentrations as low as 1 mg/mL. This level of activity was found comparable to the reference drugs rifampicin and isoniazid
at the same concentration. Molecular modeling of the binding of the diphenyl ether derivatives on enoyl-ACP reductase, the molecular target site
of triclosan, indicated that these compounds fit within the binding domain occupied by triclosan. Hence the diphenyl ether derivatives tested in
this study were docked to ENR and the binding of the diphenyl ether derivatives was also estimated using a variety of scoring functions that have
been compiled into the single consensus score. As the scores ranged from 47.27% to 65.81%, these bioactive compounds appear to have a novel
mechanism of action against M. tuberculosis, and their structural features should be studied further for their potential use as new antitubercular
drugs.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Tuberculosis; Enoyl-ACP reductase; Antitubercular activity; Docking
1. Introduction
These numbers however, are only a partial depiction of the
global TB threat. More than 80% of TB patients are in the
economically productive age of 15e49 years, which results
in tremendous economic and social problems.
Tuberculosis (TB) is a chronic infectious disease caused by
mycobacteria of the ‘‘tuberculosis complex’’, including
primarily Mycobacterium tuberculosis, but also Mycobacte-
rium bovis and Mycobacterium africanum [1,2]. In the last
decade, TB has re-emerged as one of the leading causes of
death worldwide (nearly 3 million deaths annually) [3]. The
estimated 8.8 million new cases every year correspond to
52,000 deaths per week or more than 7,000 each day [4,5].
It was estimated that nearly 1 billion more people will be
infected with TB in the next 20 years. About 15% of that
group (150 million) will exhibit symptoms of the disease,
and about 3.6% (36 million) will die from TB if new disease
prevention and treatment measures are not developed [6]. In
2005, the TB incidence rate was stable or in decline in all
six WHO regions, and had reached a peak worldwide.
However, the total number of new TB cases is still rising
slowly, because the case-load continues to grow in the African,
Eastern Mediterranean and South-East Asia regions [6]. The
* Corresponding author. Tel.: þ91 820 257 1201x22482; fax: þ91 820
2571998.
E-mail address: suvarna_kini@yahoo.com (S.G. Kini).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.04.013

S.G. Kini et al. / European Journal of Medicinal Chemistry 44 (2009) 492e500
493
dramatic increase in TB cases observed in the recent years is
a result of two major factors. First is the increased susceptibil-
ity of people infected with Acquired Immunodeficiency
Syndrome (AIDS) to TB, which augments the risk of develop-
ing the disease 100-fold [7]. Second is the increase in resistant
strains of the disease [8] with some showing cross-resistance
to as many as nine drugs [7].
Although one possible long term solution to the problem is
a better vaccine, in the short term, the major reliance will be
on chemotherapy [9] requiring the development of novel,
effective and non-toxic antitubercular agents [9e11]. The
identification of novel target sites will also be needed to
circumvent the problems associated with the increasing occur-
rence of multi-drug resistant strains. To do this, biochemical
pathways specific to the mycobacteria and related organisms’
disease cycle must be better understood. Many unique meta-
bolic processes occur during the biosynthesis of mycobacterial
cell wall components [12]. One of these attractive targets for
the rational design of new antitubercular agents are the
mycolic acids, the major components of the cell wall of M.
tuberculosis [13].
stabilized by the pep stacking interaction between the hy-
droxyl chloro phenyl ring (ring A in Fig. 1) and the hydroxyl
group of a tyrosine from hydrogen bonding interactions with
the hydroxyl group of triclosan. Ring B of triclosan makes
several hydrophobic contacts with ENR. The ether oxygen
of triclosan may also be critical in the formation of the stable
ENRetriclosaneNADþ complex, since the replacement of the
group by a sulfur atom abolishes the inhibitory activity [26].
2. Chemistry
Since diphenyl ethers (including triclosan) are well known
for their antitubercular activities [37] and that their mode of
action has been characterized, our work has focused on the
synthesis of antimycobacterial compounds based on the
diphenyl ether skeleton and the synthesized compounds were
tested then for their in vitro antitubercular activity. The syn-
thetic pathways are illustrated in Schemes 1 and 2. Molecular
modeling of their binding to ENR was also performed to study
whether this target was the ideal site to exhibit their mecha-
nism of antitubercular activity.
Mycolic acids are high molecular weight C74eC90 a-alkyl,
b-hydroxy fatty acids covalently linked to arabino-galactan
[13e15]. They represent the major lipid components of the
mycobacterial cell walls and are unique to mycobacteria and
related species [16]. Enzymes that comprise the fatty acid
synthetase (FAS) complex responsible for fatty acid biosynthe-
sis are considered ideal targets for designing new antibacterial
agents. Enoyl-ACP reductase (ENR) is a key regulatory enzyme
in fatty acid elongation, and it catalyses the NADH-dependent
stereospecific reduction of a,b-unsaturated fatty acids bound to
the acyl carrier protein [17e19]. Two studies have shown that
5-chloro-pyrazinamide (5-Cl-PZA) [20,21] and pyrazinamide
(PZA) [21] inhibit M. tuberculosis FAS I, indicating that FAS
I is also a good drug target.
The diphenyl ether, triclosan 5-chloro-2-(2,4-dichloro-phe-
noxy) phenol ether, is a broad-spectrum biocide that has been
used for over 30 years mainly as a component of antimicrobial
wash in consumer products such as toothpastes, mouthwashes,
deodarant soaps, lotions, children toys and cutting boards [22].
Until recently, it was thought that triclosan, being a rela-
tively small hydrophobic molecule, was absorbed via diffusion
into the bacterial cell wall and that its antibacterial activity
was the result of a non-specific disruption of the organism’s
cell wall [23,24]. However, the first evidence that this diphenyl
ether inhibits fatty acid biosynthesis came when a genetic
analysis of an Escherichia coli strain resistant to triclosan
linked the resistance to the FabI gene which encodes for
ENR [25]. Subsequently extensive biochemical and structural
studies have been performed to confirm that triclosan is a spe-
cific inhibitor of E. coli ENR [18,26e28]. Triclosan also
directly inhibits ENR from Staphylococcus aureus [29],
Haemophilus influenzae [30], M. tuberculosis and Mycobacte-
rium smegmatis (encoded by InhA) [31e33] and Plasmodium
falciparum, the malarial parasite [34e36]. The common theme
in the inhibition of ENRs by triclosan is the requirement of the
NADþ cofactor. The interaction of triclosan with ENR is
3. Antitubercular activity and docking studies
3.1. Antitubercular activity
Antitubercular activity of the set of diphenyl ether deriva-
tives was tested using the LowensteineJensen medium (L.J.
medium) method [47]. Briefly, eggs were broken aseptically
to obtain 200 mL of egg solution. The solution was filtered
through a sterile muslin cloth into a sterile conical flask
containing glass beads. Sterilized mineral salt solution (120
mL) (consisting of 4.0 g potassium phosphate (anhydrous), 0.4
g of magnesium sulfate, 1.6 g magnesium citrate, 6.0 g of
asparagine, 20 mL of glycerol, distilled water to fill to 1 L)
and 4 mL of sterilized malachite green solution (2.0%) were
added to the 200 mL of egg solution. The contents were mixed
well to form a uniform medium.
Compounds (10 mg) were dissolved in 10.0 mL of DMSO
and were diluted with DMSO to make 100 mg/mL and 10 mg/
mL stock solutions. A 0.8 mL aliquot of each concentration
was transferred into different McCartney bottles. To this,
7.2 mL of L.J. medium was added and mixed well.
INH and rifampicin (10 mg) were chosen as the standard
drugs for the comparison of antitubercular activity. The drug
was dissolved in DMSO and diluted and tested as described
above. The bottles were incubated at 75e80 ꢀC for 3 days
for solidification and sterilization.
Procedure for inoculation. A sweep from H37Rv strain of
M. tuberculosis culture was discharged with the help of 22
Cl
B
OH
A
O
Cl
Cl
Fig. 1. Structure of triclosan.

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S.G. Kini et al. / European Journal of Medicinal Chemistry 44 (2009) 492e500
[O]
CH3OH
Ar COOCH3
3
Ar COOH
2
Ar CHO
1
+
H
NH2NH2.H2O
CH3
N
NaOH
O
O
NH2
Ar
N
H
Ar
4
N
O
8
Phenyl
CS2
H
isothiocyanate
KOH
O
O
NH2
H
N
H
N
N
N
N
H
Ar
N
H
Ar
S
O
5
S
6
Ar
N
OXADIAZOLE THIONE
NaOH
H
N
N
O
9
N
N
Ar
S
PYRAZOLINE
N
TRIAZOLE THIONE
7
Scheme 1. Synthesis of the oxadiazole thiones, triazoles, and pyrazoline derivatives of diphenyl ethers, where Ar ¼ o-, m-, or p-phenoxy phenyl (a, b, c) in case of
oxadiazolethione (5a, 5b, and 5c) and triazole derivatives (7a, 7b, and 7c) and Ar ¼ m-phenoxy phenyl (9).
S.W.nichrome wire loop of 3 mm external diameter into
a sterile bijou bottle containing six 3 mm glass beads and
4 mL of sterile distilled water. Each loop of culture delivered
approximately 4 mg of bacilli cells. The bottle was shaken
with the help of a mechanical shaker for 2 min to constitute
the suspension. The suspension was inoculated on the surface
of each L.J. medium containing the test compounds using 27
S.W.G nichrome wire loop of 3 mm external diameter. L.J.
medium containing INH and rifampicin as well as the me-
dium containing DMSO (control) were inoculated with the
test organism for positive and negative controls. Medium
without any test compound/DMSO was also inoculated with
the test organism to check whether the media supports the
growth of the tubercle bacilli or not. The inoculated bottles
were incubated at 37 ꢀC for 6 weeks, at the end of which
readings were taken.
hydrogens were added. The model was checked for conforma-
tional problems using the module ProTable from Sybyl.
Ramachandran plot [48,49] of the backbone torsion angles
PHI and PSI, local geometry and the location of buried polar
residues/exposed non-polar residues were examined.
The structure was then subjected to an energy refinement
procedure. GasteigereHuckel charges [49] were calculated
for the ligand, while Kollman charges [50] were used for the
protein. The model was then subjected to energy minimization
following the gradient termination of the Powell method for
3000 iterations using Kollman united force field with non-
bonding cutoff set at 9.0 and the dielectric constant set at 4.0.
The 3-D structure of triclosan was obtained from its X-ray
crystal coordinate found in 1C14.pdb file. The 3-D structures
O
O
NaOH
Ar
3.2. Computer modeling of ENR and docking
of the diphenyl ether data set
Ar CHO
1
R
+
CH3
R
NH2.OH.HCl
CH3COONa
CH3COOH
The X-ray crystal coordinates of E. coli ENReNADþe
˚
triclosan complex at a resolution of 2.0 A from Qiu et al.
[39] were obtained from the Protein Data Bank (http://www.
rcsb.org) under the accession code 1C14.pdb. Using the
program Sybyl 6.9 (Tripos Inc., St. Louis, MO) on a Silicon
Graphics O2 250 MHz R10000 workstation, mislabeled atom
types from the pdb files were first corrected. Subsequently,
proline F angles were fixed at 70ꢀ, side chain amides were
checked to maximize potential H-bonding, side chains were
checked for close van der Waals contacts, and essential
R
Ar
N
O
10
ISOXAZOLE
Scheme 2. Synthesis of isoxazoles, where Ar ¼ m-phenoxy phenyl; R ¼ H
(10a), 4-chloro (10b), and 4-methoxy (10c).

S.G. Kini et al. / European Journal of Medicinal Chemistry 44 (2009) 492e500
495
of the diphenyl derivatives tested in this study were derived
from the coordinate of the triclosan backbone.
performed. The crystal structure of E. coli ENReNADþe
˚
triclosan complex (1C14.pdb) at a resolution of 2.0 A [39]
was optimized and minimized as shown in Fig. 2A with tri-
closan in ball and stick form and the amino acid residue of
NADþ below. It showed that triclosan bound non-covalently
in the location adjacent to the nicotinamide portion of
NADþ and formed a parallel stack with nicotinamide ring
3.3. Docking experiment
The diphenyl ether derivatives tested in this study were
docked to ENR using FlexX in Sybyl. This software uses an
incremental construction approach to place ligands into a bind-
ing domain of a protein [51]. The first phase of the FlexX
process consists of establishing the base selection where
a part of the ligand is selected as a base fragment based on its
placeability and specificity [52]. This fragment is then placed
into binding domain independently of the rest of the ligand
using the pose clustering pattern recognition technique, which
optimizes the number of favorable interactions that can occur
between the fragment and the protein. Finally, the rest of the
ligand is built into the site using the different placements of
the base fragment as starting points. The FlexX scoring function
˚
of the cofactor with an interplanar distance of 3.4 A. Analysis
of the Ramachandran plot shows a normal distribution of
points without any amino acid residues classified as outliers.
PHI angles were restricted to negative values, and PSI values
were clustered in two distinct regions. Several glycine resi-
dues had PHI angle values outside the recommended range,
but this amino acid is not subjected to the same steric
constraints as other residues due to their lack of a side chain.
Analysis of the local geometry of the structure, which mea-
sures deviations from acceptable bond lengths and bond
angles, indicated a robust model with all amino acids within
the core region, except 22 residues within the allowable
region. No amino acid had bond lengths or angles exceeding
the acceptable range.
uses a modified version of the Bohm function designed for the
¨
de novo design program LUDI that estimates the free binding
energy DG of the proteineligand complex [40].
The binding of the diphenyl ether derivatives was also
estimated using a variety of scoring functions that have been
compiled into the single consensus score (CScore). The
CScore module available in Sybyl includes the G_Score,
PMF_Score, D_Score, ChemScore, and the FlexX F_Score
scoring functions.
The binding domain identified to accommodate triclosan is
large enough to hold the diphenyl ether derivatives synthesized
in this study, although these molecules occupy a larger volume
than triclosan (Table 3). In particular, the rings with ortho sub-
stitutions are larger. The meta and para substituted structures
are also more voluminous than triclosan but could still fit
within the binding domain of E. coli ENR as observed from
the docking results. The potential binding of these structures
was determined by a virtual docking procedure using the pro-
gram FlexX [40,41] in combination with CScore commonly
used for virtual high-throughput screening projects [42,43]
(Table 2). The diphenyl ether with triazole thione at meta
position, compound 7b, had the highest docking score of
65.81% while the substituted pyrazoline ring at meta position,
compound 9, of diphenyl ether showed the least docking score
of 47.27% indicating that larger molecular volume might
result in low docking score.
The diphenyl ethers used in this study occupy a larger
molecular volume than triclosan (Table 3), therefore, their
cyclic azole substitutions tend to extend beyond the binding
domain when positions in order to retain the critical interac-
tions with NADþ. Fig. 2A shows the position of triclosan
(ball and stick) relative to NADþ, Fig. 2B shows the overlay
of highest scoring 3-triazole thione diphenyl ether derivative
(ball and stick), Fig. 2C, the overlay of least scoring pyrazo-
line derivative of diphenyl ether (ball and stick) and Fig. 2D
the overlap of all 10 derivatives of diphenyl ether 5a, 5b, 5c
in wire, 7a, 7b, 7c in stick, 9 in yellow highlighted and 10a,
10b, and 10c in ball and stick form within the binding
domain of triclosan diphenyl ether derivative. Changing the
spatial orientation of the derivatives to fit more correctly in
the pocket precludes or disrupts the pep stacking interaction
that is critical for sustaining the inhibitory activity of
diphenyl ethers [27,44]. The stabilization of the ENRe
diphenyl ethereNADþ complex obtained via the ether oxy-
gen is not possible.
4. Results and discussion
A series of cyclic azole substituted diphenyl ether deriva-
tives were synthesized using o-, m-, or p-phenoxy benzalde-
hydes with different reagents to form the desired heterocyclic
diphenyl ether derivatives. Compounds of type 5 possessed an
oxadiazolethione group; compounds of type 7 consisted of
a triazole ring, compound 9 with pyrazoline and compounds
of type 10 possessed isoxazole ring. Overall, the yields were
in the range of 60e88% and the compounds were easy to
crystallize. Their structures were confirmed by GCeMS,
1
LCeMS, H NMR and IR spectroscopies (given in synthetic
procedure).
The compounds listed in Table 1 were evaluated for antitu-
bercular activity. All 10 diphenyl ether derivatives were highly
active against the H37Rv strain M. tuberculosis (Table 2). The
compounds inhibited the growth of the human pathogen at
concentrations as low as 1 mg/mL. A concentration of 1 mg/
mL corresponds to 3.7 mM for the compounds in 5, 2.9 mM
for compounds in 7, and 2.4, 3.2, 2.9 and 2.9 mM for com-
pounds 9, 10a, 10b, and 10c, respectively. Such level of
antitubercular activity is comparable to other standard drugs
such as isoniazid and rifampicin, which have MICs at 0.01e
1.25 and 0.06e0.25 mg/mL, respectively [38]. The diphenyl
ether triclosan has an MIC of 5 mg/mL (17.4 mM) for M. tuber-
culosis [32].
To understand the basis of the mechanism of antitubercu-
lar activity of the diphenyl ether derivatives synthesized for
this study, molecular modeling and docking studies were

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S.G. Kini et al. / European Journal of Medicinal Chemistry 44 (2009) 492e500
Table 1
Structures of the compounds synthesized and some of their physical properties
Code
Structure
Mw
Yield (%)
87
M.p (ꢀC)
122
Elemental analysis
Calculated (found)
S
5a
270.31
C-62.21% (62.19%)
H-3.73% (3.53%)
N-10.36% (10.25%)
NH
N
O
O
S
O
NH
O
O
5b
5c
7a
270.31
270.31
345.42
85
70
88
154
190
270
C-62.21% (62.19%)
H-3.73% (3.63%)
N-10.36% (10.35%)
N
O
C-62.21% (62.19%)
H-3.73% (3.53%)
N-10.36% (10.25%)
S
N
NH
S
NH
N
N
C-69.54% (69.55%)
H-4.38% (4.36%)
N-12.17% (12.16%)
O
S
N
7b
7c
345.42
345.42
86
78
268
188
C-69.54% (69.55%)
H-4.38% (4.36%)
N-12.17% (12.16%)
NH
O
O
N
N
C-69.54% (69.55%)
H-4.38% (4.36%)
N-12.17% (12.16%)
S
N
NH
N
9
419.47
65
130
C-77.31% (77.11%)
H-5.05% (5.00%)
N-10.02% (10.00%)
N
O
O
N
O
N
N
N
O
O
O
10a
10b
10c
313.35
347.79
343.38
65
70
60
78
82
C-80.49% (80.39%)
H-4.82% (4.83%)
N-4.47% (4.45%)
O
O
Cl
C-72.52% (72.49%)
H-4.06% (4.03%)
N-4.03% (4.05%)
OCH3
100
C-80.75% (80.69%)
H-4.52% (4.53%)
N-4.48% (4.45%)

S.G. Kini et al. / European Journal of Medicinal Chemistry 44 (2009) 492e500
497
Table 2
Biological activity of the diphenyl ether derivatives against H37Rv strain of M.
tuberculosis and their docking scores
TLC analysis. Carried out on aluminum foil precoated with
silica gel 60 F254 (SigmaeAldrich Company, Bangalore
dealer).
Code
M. tuberculosis
inhibitiona (%)
Docking
scoreb
Equipments. Melting points were determined on Toshniwal
apparatus (Toshniwal Company, Bangalore, India) and are
uncorrected. IR spectra were taken on Shimadzu FTIR
8300 spectrometer. 1H NMR spectra were recorded on
a Brucker AMX-400 NMR spectrometer and were refer-
enced to TMS and all chemical shifts are reported as
d (ppm) values. Compounds were also analyzed by GCe
MS (QP 5010, Shimadzu Corporation, Japan), LCeMS,
FAB-MS (Joel SX-102, CDRI, Mumbai, India) and
EI-MS (Autospec-5, IICT, Hyderabad, India).
5a
5b
100
100
100
100
100
100
100
100
100
100
100
54.16
59.18
54.39
52.77
65.81
63.79
47.27
58.56
54.75
52.77
98.96
5c
7a
7b
7c
9
10a
10b
10c
Triclosan
a
At 1 mg/mL concentration, which corresponds to 2.4e3.7 mM.
Based on CScore values.
5.2. Synthesis of oxadiazolethione
b
Phenoxy benzaldehyde (o/m/p) 1 (0.01 mol) was oxidized to
phenoxy benzoic acid 2 (Scheme 1) using alkaline KMnO4
solution as per the procedure given in Vogel’s Text Book of
Practical Organic Chemistry [45]. (o/m/p) Phenoxy benzoic
acid (0.01 mol) was converted to the respective ester 3 (o/m/
p) using methanol, concentrated H2SO4, and (o/m/p) phenoxy
benzoic acid ester (0.01 mol) to hydrazide 4 (o/m/p) using hy-
drazine hydrate as per the procedure given in Ref. [46].
5. Experimental section
5.1. General
Reagents. All reagents were purchased from Sigma Chem-
icals (Bangalore, India) and were used without further
purification.
Fig. 2. Binding domain of E. coli ENR showing (A) the position of triclosan (ball and stick) relative to NADþ, (B) the overlay of highest scoring 3-triazole thione
diphenyl ether derivative (ball and stick), (C) the overlay of least scoring pyrazoline derivative of diphenyl ether (ball and stick) and (D) the overlap of all 10
derivatives of diphenyl ether 5a, 5b, 5c in wire, 7a, 7b, 7c in stick, 9 in yellow highlights and 10a, 10b, and 10c in ball and stick form within the binding domain
of triclosan (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

498
S.G. Kini et al. / European Journal of Medicinal Chemistry 44 (2009) 492e500
Table 3
Molecular properties of the diphenyl ethers used in this study
Code
Mw
Volume, A3
Dipole, Debye
Etotal, kcal/mol
IP, eV
eHOMO, eV
e
LUMO, eV
EPmin, eV
EPmax, eV
LPmin
LPmax
5a
5b
270.31
270.31
270.31
345.42
345.42
345.42
419.47
313.35
347.79
343.38
289.54
258.95
262.38
262.37
321.97
326.84
326.61
409.08
317.63
334.91
352.81
246.43
7.18
6.16
3.08
4.17
3.43
3.16
3.00
2.35
2.93
3.22
1.99
12.088
12.353
12.369
26.430
26.866
26.871
12.785
13.453
13.029
15.156
2.662
8.589
8.706
8.697
8.150
8.254
8.251
9.053
9.253
9.309
9.276
9.700
ꢁ8.793
ꢁ8.925
ꢁ8.902
ꢁ8.240
ꢁ8.357
ꢁ8.352
ꢁ9.429
ꢁ9.462
ꢁ9.702
ꢁ9.597
ꢁ9.251
ꢁ0.384
ꢁ0.387
ꢁ0.369
ꢁ0.308
ꢁ0.354
ꢁ0.355
ꢁ0.059
ꢁ0.136
ꢁ0.399
ꢁ0.275
ꢁ0.201
ꢁ26.253
ꢁ25.278
ꢁ25.746
ꢁ27.539
ꢁ26.529
ꢁ26.906
ꢁ40.750
ꢁ29.532
ꢁ28.267
ꢁ32.607
ꢁ22.831
28.637
32.249
30.978
24.782
28.016
26.570
29.375
16.845
18.111
20.167
32.121
0.106
0.109
0.111
0.114
0.116
0.117
0.067
0.113
0.123
0.094
0.167
0.178
0.182
0.181
0.170
0.172
0.172
0.126
0.154
0.198
0.148
0.263
5c
7a
7b
7c
9
10a
10b
10c
Triclosan
Note: volume ¼ connolly solvent accessible surface volume; Etotal ¼ total energy; IP ¼ ionization energy; eHOMO ¼ energy of highest occupied molecular orbital;
eLUMO ¼ energy of lowest unoccupied molecular orbital; EPmax and EPmin ¼ maximum and minimum electrostatic potentials; LPmax and LPmin ¼ maximum and
minimum lipophilicities.
A mixture of equimolar quantities (0.01 mol) of (o/m/p)
phenoxy benzoic acid hydrazide, CS2 and KOH were taken
in absolute ethanol in a round bottom flask. The solution
was refluxed until the evolution of H2S had nearly stopped
(about 18 h). The solvent was removed under reduced pres-
sure. The residue was dissolved in water and the solution
was acidified with dilute HCl (10%). The resulting product 5
was collected, washed with water, dried and recrytallized
from aqueous ethanol (80%). The purity of the compound
was ascertained by a single spot on the TLC plate. The phys-
ical and molecular data are given in Tables 1 and 3. The
solvent system for TLC was chloroform:methanol (8:2 v/v).
Anhydrous sodium acetate (0.01 mol) dissolved in a mini-
mum amount of hot acetic acid was added to the solution of
hydroxylamine hydrochloride (0.01 mol) in ethanol (15.0
mL). This solution was added to a solution of chalcone (0.01
mol) in ethanol (20.0 mL). The mixture was refluxed on an
oil-bath for 8 h, concentrated and neutralized with NaOH
solution (0.1%). The product 10 was crystallized from ethanol
(95%). The physical and molecular data are given in Tables 1
and 3.
5.5. Synthesis of pyrazoline derivatives
m/p Phenoxy benzaldehyde (0.01 mol) was dissolved in
15 mL of ethanol. 2-Methylpyridyl ketone dissolved in
15 mL of ethanol was added (Scheme 1). This mixture was
kept on a magnetic stirrer and slowly a stream of NaOH solu-
tion (40%, 10 mL) was added and stirred overnight. The
mixture was then acidified to pH 5.0 to obtain the chalcone.
The precipitate was filtered, dried and crystallized from
ethanol. This chalcone 8 was used to prepare different pyrazo-
lines. The hydrazide of the benzoic acid was synthesized by
the usual method.
The above-prepared chalcone (0.01 mol) was dissolved in
ethanol (15 mL). The hydrazide of the benzoic acid (0.01
mol) was dissolved in ethanol (20 mL) and added. To this mix-
ture few drops of glacial acetic acid was added and the mixture
was refluxed for 12 h. The solvent was then removed under
reduced pressure and the product was poured into ice-cold
water. The precipitate of pyrazoline (9) was obtained which
was filtered and dried. The product was crystallized from eth-
anol. The physical and molecular data are given in Tables 1
and 3. The solvent system for TLC was benzene:ethylacetate
(1:2 v/v).
5.3. Synthesis of triazole thione
Equimolar quantities (0.01 mol) of acid hydrazide and
phenyl isothiocyanate were dissolved in absolute ethanol in
a round bottom flask (Scheme 1). The solution was refluxed
for 3 h on a water bath. The solution was concentrated under
reduced pressure and the solid separated was collected and
recrystallized from appropriate solvent to get the isothiocya-
nate derivative 6. This was further used to prepare triazole
whereby it was suspended in 4% NaOH and refluxed for 1 h
(Scheme 1). The resulting solution was filtered and the filtrate
was cooled, acidified carefully with diluted acetic acid to pH
5.5. The precipitate formed was filtered, washed with water,
and the product 7 was crystallized from ethanol. The purity
of the compounds was established by single spot on the
TLC plate. The physical and molecular data are given in
Tables 1 and 3. The solvent system used consisted of chloro-
form:ethyl acetate (1:1 v/v).
5.4. Synthesis of isoxazole derivatives
Preparation of chalcone. m-Phenoxy benzaldehyde 1
(0.01 mol) in ethanol (95%, 15.0 mL) was added to the
mixture of acetophenone (0.01 mol), ethanol (95%, 20.0 mL)
and NaOH (40%, 8.0 mL) and stirred for 24 h (Scheme 2).
The content was poured into crushed ice. The product was iso-
lated by acidification and crystallization from ethanol (95%).
5.6. Spectral data
5.6.1. 5-(3-Phenoxyphenyl)-1,3,4-oxadiazole-2(3H )-thione
FTIR spectral data. KBr pellet: C]N (1583.4), C]S
(1180.4), CeO stretch (1693.4), CeOeC (oxadiazole)

S.G. Kini et al. / European Journal of Medicinal Chemistry 44 (2009) 492e500
499
(1336.6), CeOeC (diphenyl ether) (1232.4), Ar stretch
(688.5), NeH (3456.2).
References
1H NMR (DMSO). d 8.3 (s, 1H, NH of oxadiazole),
d 6.9e7.9 (m, 9H,Ar).
Mass calculated for C14H10N2O2S 270.0, found 270.0.
[1] E. Wolinsky, in: J.B. Wyangaarden, L.H. Smith Jr., J.C. Bennett (Eds.),
Tuberculosis, 19th ed., Cecil Textbook of Medicine, vol. 2 W.B.
Saunders Company, Philadelphia, PA, 1992, pp. 1733e1742.
[2] P. Sensi, I.G.G. Grass, in: A. Burger, M.E. Wolff (Eds.), Antimycobacte-
rial Agents, fifth ed., Burger’s Medicinal Chemistry and Drug Discovery,
vol. 2 John Wiley & Sons, New York, 1996, pp. 575e635.
[3] B.R. Bloom, C.J.L. Murray, Tuberculosis: commentary on a reemergent
killer, Science 257 (1992) 1055e1064.
5.6.2. 5-(2-Phenoxyphenyl)-4-phenyl-2,4-dihydro-
3H-1,2,4-triazole-3-thione
[4] M. Okada, K. Kobayashi, Recent progress in mycobacteriology, Kekkaku
(Japanese Journal) 82 (10) (2007) 783e799.
[5] World Health Organization Report on TB epidemic, Global TB
Programme, World Health Oraganization, Geneva, 1997.
FTIR spectral data. KBr pellet: C]N (1577.7), C]S
(1157.2), CeN stretch (1612.4), CeNeC (1394.4), Ce
OeC (diphenyl ether) (1242.1), Ar stretch (692.4), NeH
(3436.9).
[6] World Health Organisation, Tuberculosis, Fact Sheet No. 104, 2007 (site ac-
cessed: <www.who.int/mediacentre/factsheets/who104/en/index.html>).
[7] K.A. ElSayed, P. Bartyzel, X.Y. Shen, T.L. Perry, J.K. Zjawiony,
M.T. Hamann, Marine natural products as antituberculosis agents, Tetra-
hedron 56 (2000) 949e953.
1H NMR (DMSO). d 3.2 (s, 1H, triazole), d 6.7e7.7 (m,
14H, Ar).
Mass calculated for C20H15N3OS 345.1 and molecular ion
(m þ 1) peak from LCeMS was obtained at 346.14.
[8] M.J. Goldberg, Antituberculosis agents, Med. Clin. North. Am. 72 (1988)
661e668.
[9] H. Tomioka, K. Namba, Development of antitubercular drugs: current
status and future prospects, Kekkaku (Japanese Journal) 81 (12) (2006)
753e774.
5.6.3. 3-(4-Methoxyphenyl)-5-(3-phenoxyphenyl)isoxazole
FTIR spectral data. KBr pellet: C]N (1579.6), C]C (iso-
xazole) (1515.9), CeO stretch (1614.3.6), CH3 (1461.9),
CeOeC (diphenyl ether) (1245.9), Ar stretch (692.4).
1H NMR (DMSO). d 2.3 (s, 1H, CH), d 6.9e8.0 (m, 13H,
Ar), d 3.8 (s, 1H,CH3).
[10] S.E. Berning, The role of fluoroquinolones in tuberculosis today, Drugs
61 (2001) 9e18.
[11] V.M. Reddy, G. Nadadhur, D.D. Daneluzzi, J.F. Osullivan,
P.R.J. Gangadharam, Antituberculosis activities of clofazimine and its
new analogs B4154 and B4157, Antimicrob. Agents Chemother. 40
(1996) 633e636.
Mass calculated for C22H17NO3 is 343.37 and molecular
ion peak was obtained at 343 indicating that the compound
is formed.
[12] C.E. Barry, New horizons in the treatment of tuberculosis, Biochem.
Pharmacol. 54 (1997) 1165e1172.
[13] K.F.M. Pasquato, E.I. Ferreira, An approach for the rational design of
new antitubercular agents, Curr. Drug Targets 2 (2001) 427e437.
[14] E.K. Schroeder, N. de Souza, D.D. Santos, J.S. Blanchard, L.A. Basso,
Drugs that inhibit mycolic acid biosynthesis in Mycobacterium tubercu-
losis, Curr. Pharm. Biotechnol. 3 (3) (2002) 197e225.
5.6.4. 2-[5-(3-Phenoxyphenyl)-4,5-dihydro-1
(benzoyl)-pyrazol-3-yl] pyridine
[15] C.E. Barry III., R.E. Lee, K. Mdluli, A.E. Sampson, B.G. Schroeder,
R.A. Slayden, Y. Yaun, Mycolic acid structure, biosynthesis and physio-
logical functions, Prog. Lipid Res. 37 (1998) 143e179.
FTIR spectral data. KBr pellet: C]N (1442.7), C]O
(1685.7), CeOeC (diphenyl ether) (1242.1), Ar stretch
(694.3, 3055).
[16] P.E. Kolattukudy, N.D. Fernandes, A.K. Azad, A.M. Fitzmaurice,
T.D. Sirakova, Biochemistry and molecular genetics of cell-wall lipid
biosynthesis in mycobacteria, Mol. Microbiol. 24 (1997) 263e270.
[17] H. Bergler, S. Fuchsbichler, G. Hogenauer, F. Turnowsky, The enoyl-
[acyl-carrier-protein] reductase (FabI) of Escherichia coli, which
catalyzes a key regulatory step in fatty acid biosynthesis, accepts
NADH and NADPH as cofactors and is inhibited by palmitoyl-CoA,
Eur. J. Biochem. 242 (1996) 689e694.
1H NMR (DMSO). d 6.9e8.5 (m, 17H), d 3.2 and 4.0 (d,
CH2), d 4.2 and 5.0 (d, CH).
Mass calculated for C27H22 N3O3 is 435.23 and molecular
ion (m þ 1) peak was obtained at m/z 436.23. The other
fragment peaks were obtained at m/z 316.2, 269.2, 121.1.
[18] M.J. Stewart, S. Parikh, G. Xiao, P.J. Tonge, C. Kisker, Structural basis
and mechanism of enoyl reductase inhibition by triclosan, J. Mol. Biol.
290 (1999) 859e865.
Acknowledgements
[19] D.A. Rozwarski, C. Vilcheze, M. Sugantino, R. Bittman, J.C. Sacchettini,
Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reduc-
tase, InhA, in complex with NADþ and a C16 fatty substrate, J. Biol.
Chem. 274 (1999) 15582e15589.
We are thankful to the Microbiology Department of
Kasturba Medical College, Manipal for helping with the anti-
tubercular assay, and to the Central Drug Research Institute,
Lucknow; Indian Institute of Science, Bangalore and Indian
Institute of Chemical Technology, Hyderabad for providing
the elemental analysis, 1H NMR and mass spectra of the
compounds. Portions of molecular modeling work were
funded at Department of Medicinal Chemistry, University of
Mississippi, University, MS 38677, USA by Centers for
Diseases Control Cooperative Agreements U50/CCU18839
and UR3/CCU418652, and supported by National Institutes
of Health (NIH) MFGN INBRE RR016476 and NIH NCRR
Facilities Improvement grant CO6 RR1450301.
[20] H.I. Boshoff, V. Mizrahi, C.E. Barry III, Effects of pyrazinamide on fatty
acid synthesis by whole mycobacterial cells and purified fatty acid syn-
thase I, J. Bacteriol. 184 (2002) 2167e2172.
[21] O. Zimhony, J.S. Cox, J.T. Welch, C. Vilcheze, W.R. Jacobs Jr., Pyrazi-
namide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of
Mycobacterium tuberculosis, Nat. Med. 6 (2000) 1043e1047.
[22] H.P. Schweizer, Triclosan: a widely used biocide and its link to antibi-
otics, FEMS Microbiol. Lett. 202 (2001) 1e7.
[23] J. Regos, O. Zak, R. Solf, W.A. Vischer, E.G. Weirich, Antimicrobial
spectrum of triclosan, a broad-spectrum antimicrobial agent for topical
application. II. Comparison with some other antimicrobial agents,
Dermatologica 1158 (1979) 72e79.

500
S.G. Kini et al. / European Journal of Medicinal Chemistry 44 (2009) 492e500
[24] W.A. Vischer, J. Regos, Antimicrobial spectrum of triclosan, a broad-
spectrum antimicrobial agent for topical application, Zentralbl. Bakteriol.
[Orig A] 226 (1974) 376e389.
[39] X. Qiu, C.A. Janson, R.I. Court, M.G. Smyth, D.J. Payne, S.S. Abdel-
Meguid, Molecular basis for triclosan activity involves a flipping loop
in the active site, Protein Sci. 8 (1999) 2529e2532.
[25] L.M. McMurry, M. Oethinger, S.B. Levy, Triclosan targets lipid synthe-
sis, Nature 394 (1998) 531e532.
[26] R.J. Heath, Y.T. Yu, M.A. Shapiro, E. Olson, C.O. Rock, Broad spectrum
antimicrobial biocides target the FabI component of fatty acid synthesis,
J. Biol. Chem. 273 (1998) 30316e30320.
[40] M. Rarey, B. Kramer, T. Lengauer, G.A. Klebe, Fast flexible docking
method using an incremental construction algorithm, J. Mol. Biol. 261
(1996) 470e489.
[41] B. Kramer, M. Rarey, T. Lengauer, Evaluation of the FlexX incremental
construction algorithm for proteineligand docking. Proteins: structure,
function, and genetics, J. Mol. Biol. 37 (1999) 228e241.
[42] C. Bissantz, G. Folkers, D. Rognan, Protein-based virtual screening of
chemical databases. Part 1. Evaluation of different docking/scoring
combinations, J. Med. Chem. 43 (2000) 4759e4767.
[43] R.J. Heath, J.R. Rubin, D.R. Holland, E. Zhang, M.E. Snow, C.O. Rock,
Mechanism of triclosan inhibition of bacterial fatty acid synthesis, J.
Biol. Chem. 274 (1999) 11110e11114.
[27] C.W. Levy, A. Roujeinikova, S. Sedelnikova, P.J. Baker, A.R. Stuitje,
Molecular basis of triclosan activity, Nature 398 (1999) 383e384.
[28] A. Roujeinikova, C.W. Levy, S. Rowsell, S. Sedelnikova, P.J. Baker,
C.A. Minshull, A. Mistry, J.G. Colls, R. Camble, A.R. Stuitje, A.R. Slabas,
J.B. Rafferty, R.A. Pauptit, R. Viner, D.W. Rice, Crystallographic analysis
of triclosan bound to enoyl reductase, J. Mol. Biol. 294 (1999) 527e535.
[29] R.J. Heath, J. Li, G.E. Roland, C.O. Rock, Inhibition of the Staphylococ-
cus aureus NADPH-dependent enoyl-acyl carrier protein reductase by
triclosan and hexachlorophene, J. Biol. Chem. 275 (2001) 4654e4659.
[30] J. Marcinkeviciene, W. Jiang, L.M. Kopcho, G. Locke, Y. Luo, Enoyl-
ACP reductase (FabI) of Haemophilus influenzae: steady-state kinetic
mechanism and inhibition by triclosan and hexachlorophene, Arch.
Biochem. Biophys. 390 (2001) 101e108.
[44] S. Sivarana, T.J. Sullivan, F. Johnson, P. Novichenok, G. Cui,
C. Simmerling, P.J. Tonge, Inhibition of the bacterial enoyl reductase
FabI by triclosan: a structureereactivity analysis of FabI inhibition by
triclosan analogues, J. Med. Chem. 47 (2004) 509e518.
[45] B.S. Furniss, A.J. Hannaford, V. Rogers, P.W.G. Smith, A.R. Tatchell,
Aromatic carboxylic acids, Vogel’s Text Book of Practical Organic
Chemistry, fourth ed. Longman Group Limited, London, 1980, p.
824.
[31] L.M. McMurry, P.F. McDermott, S.B. Levy, Genetic evidence that InhA
of Mycobacterium smegmatis is a target for triclosan, Antimicrob. Agents
Chemother. 43 (1999) 711e713.
[46] R. Udupi, Studies on the Synthesis of Substituted Triazoles, Azetidi-
nones, Quinazolinones and Related Compounds for Possible Antituber-
cular Activity and other Pharmacological Profiles, Ph.D. Thesis, 1995.
[47] B. Watt, A. Rayner, G. Harris, Mackie, McCartney, in: J.G. Colle,
A.G. Fraser, B.P. Marmion, A. Simmons (Eds.), Practical Medical
Microbiology, Churchill Livingstone, New York, 1996, pp. 331e335
(Chapter18).
[32] S.L. Parikh, G. Xiao, P.J. Tonge, Inhibition of InhA, the enoyl reductase
from Mycobacterium tuberculosis, by triclosan and isoniazid, Biochemis-
try 39 (2000) 7645e7650.
[33] M.R. Kuo, H.R. Morbidoni, D. Alland, S.F. Sneddon, B.B. Gourlie, Targeting
tuberculosis and malaria through inhibition of enoyl reductase: compound
activity and structural data, J. Biol. Chem. 278 (2003) 20851e20859.
[34] N. Surolia, A. Surolia, Triclosan offers protection against blood stages of
malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum,
Nat. Med. 7 (2001) 167e173.
[48] J.T. Edsall, P.J. Flory, J.C. Kendrew, A.M. Liquori, G. Nemethy,
G.N. Ramachandran, H.A. Scheraga, A proposal of standard conventions
and nomenclature for the description of polypeptide conformations, J.
Biol. Chem. 241 (1966) 1004e1008.
[49] W. Purcell, J.A. Singer, Brief review and table of semiempirical param-
eters used in the Hueckel molecular orbital method, J. Chem. Eng. Data.
12 (1967) 235e246.
[35] M. Kapoor, M.J. Dar, N. Surolia, A. Surolia, Kinetic determinants of the
interaction of enoyl-ACP reductase from Plasmodium falciparum with its
substrates and inhibitors, Biochem. Biophys. Res. Commun. 289 (2001)
832e837.
[36] R. Perozzo, M. Kuo, A. bir Singh Sidhu, J.T. Valiyaveettil, R. Bittman,
W.R. Jacobs Jr., D.A. Fidock, J.C. Sacchettini, Structural elucidation of
the specificity of the antibacterial agent triclosan for malarial enoyl
ACP reductase, J. Biol. Chem. 277 (2002) 13106.
[50] P. Cieplak, W.D. Cornell, C. Bayly, P.A. Kollman, Application of the
multimolecule and multiconformational RESP methodology to biopoly-
mers: charge derivation for DNA, RNA, and proteins, J. Comput.
Chem. 16 (1995) 1357e1377.
[37] C.B. Vincent, D. Twomey, Derivatives of diploicin, Proc. Royal Irish
Acad. 53B (1950) 55e59.
[51] A.R. Leach, I. Kuntz, Conformational analysis of flexible ligands in
macromolecular receptor sites, J. Comput. Chem. 13 (1992) 730e748.
[52] M. Rarey, B. Kramer, T. Lengauer, Multiple automatic base selection:
proteineligand docking based on incremental construction without
manual intervention, J. Comput.-Aided Mol. Des. 11 (1997) 369e384.
[38] Dosages and Pharmacokinetics of Antituberculosis medications e A Re-
port in India, 13.06.2006 (site accessed: <http://www.angelfire.com/indie/
tbindia/attdrugs.html>).