Chemical synthesis and molecular modeling of novel substituted N-1,3-benzoxazol-2yl benzene sulfonamides as inhibitors of inhA enzyme and Mycobacterium tuberculosis growth

Article abstract of DOI:10.1007/s13738-020-02080-0

Abstract: Tuberculosis (TB) is one of the major contagious diseases with high mortality which is caused by Mycobacterium tuberculosis (Mtb) pathogen. Due to the existing antibiotic resistance (MDR-TB) to tuberculosis, the demand for the development of new potential chemotherapy drugs is increasing. Herein, we report synthesis of two novel benzoxazole-based series, namely 2-phenyl benzoxazole sulfonamide and 2-piperidine-benzoxazole sulfonamides. These compounds were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv strain, using the microplate alamarBlue assay. Molecular docking studies were carried out to comprehend the binding mode of the compounds. It is evident from molecular docking studies and minimum inhibitory concentration assay (MIC) that 2-phenyl benzoxazole sulfonamide scaffold has a greater potential of antitubercular activity possibly by ENR inhibition (inhA inhibitors). In silico cytotoxicity studies using CLC-Pred tool database suggested that both the series were relatively safe. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13738-020-02080-0

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-02080-0
ORIGINAL PAPER
Chemical synthesis and molecular modeling of novel substituted
N‑1,3‑benzoxazol‑2yl benzene sulfonamides as inhibitors of inhA
enzyme and Mycobacterium tuberculosis growth
Narendra Singh Chundawat1 · Gajanan S. Shanbhag1 · Narendra Pal Singh Chauhan1
Received: 30 June 2020 / Accepted: 29 September 2020
© Iranian Chemical Society 2020
Abstract
Tuberculosis (TB) is one of the major contagious diseases with high mortality which is caused by Mycobacterium tubercu-
losis (Mtb) pathogen. Due to the existing antibiotic resistance (MDR-TB) to tuberculosis, the demand for the development
of new potential chemotherapy drugs is increasing. Herein, we report synthesis of two novel benzoxazole-based series,
namely 2-phenyl benzoxazole sulfonamide and 2-piperidine-benzoxazole sulfonamides. These compounds were evaluated for
their antimycobacterial activity against Mycobacterium tuberculosis H37Rv strain, using the microplate alamarBlue assay.
Molecular docking studies were carried out to comprehend the binding mode of the compounds. It is evident from molecular
docking studies and minimum inhibitory concentration assay (MIC) that 2-phenyl benzoxazole sulfonamide scafold has a
greater potential of antitubercular activity possibly by ENR inhibition (inhA inhibitors). In silico cytotoxicity studies using
CLC-Pred tool database suggested that both the series were relatively safe.
Graphic abstract
In silico CLC-Pred cell line
Mycobacterium tuberculosis (MTB)-MABA Assay
cytotoxicity prediction
N
N
O
N
O
Diversity 2
Diversity 1
O
S
O
N
O
NH
S
NH
S
O
O
Br
N
O
O
O
Br
Benzoxazole
Compound 8
1.6µg/mL
Compound 11
3.12µg/mL
Compound 20
Me
Me
HEK293 Cell line(Embryonic kidney ):
Pa 0.862 /Pi 0.004
MIC:
Keywords Mycobacterium tuberculosis · Benzoxazole sulfonamide · Antitubercular activity · inhA inhibitors · Molecular
docking · Cytotoxicity
Introduction
immunodefciency virus/AIDS. Ever since 1993, TB has
been recognized as a global health emergency by the World
Health Organization. TB causes more than nine million new
cases annually with a casualty of about 1.8 million people
worldwide every year. In modern era, numerous drugs have
emerged, for eradicating TB. However, resistance to those
drugs is an alarming matter. Due to recurring incidences
of multidrug-resistant (MDR-TB) and extensively drug-
resistant (XDR) strains of M. tuberculosis, there is a global
necessity to develop new age chemotherapeutics to battle
diferent forms of TB Rattan et al. [2].
Tuberculosis (TB) which is caused by Mycobacterium tuber-
culosis is one of the prime infectious diseases in human
beings Bogatcheva et al. [1]. It is responsible for the sec-
ond highest cause of death for humans following human
* Gajanan S. Shanbhag
gajushanbhag@yahoo.com
1
Department of Chemistry, Bhupal Nobles’ University,
Udaipur, Rajasthan, India
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
Mycolic acid is a basic component of mycobacterial
cell wall present in the FAS (fatty acid synthase) system
of human pathogen M. tuberculosis. For the survival and
pathogenesis of M. tuberculosis, “Mycolate biosynthesis”
is a key factor. In the biosynthesis of fatty acids Qureshi
et al. [3] in M. tuberculosis, two distinct enzymes FAS I
and II are involved. FAS I is involved in synthesis of the
C26 saturated straight-chain fatty acids to provide branch
of the mycolic acids. FAS II will provide the meromyco-
late backbone by synthesizing C56 fatty acids. Mycobacte-
rial FAS II utilizes the yields of FAS-I as the primers to
expand fatty acyl chain lengths even further Bloch et al. [4],
Kikuchi et al. [5]. For type II fatty acid synthase (FAS II),
from the M. tuberculosis, inhA, the enoyl acyl carrier protein
reductase (ENR) Maria et al. [6] is the major enzyme which
catalyzes the NADH-dependent reduction of trans double
bond of acyl carrier protein (2-trans-enoyl-ACP) to yield
NAD+and reduced enoyl-thioester-ACP substrate, which
ultimately contributes to the synthesis of mycolic acid (MA).
We could envisage the growing publications on the MTB
enoyl-ACP reductase (ENR/inhA) enzyme with various O-,
N- Desai et al. [7] and azole-containing heterocyclic moie-
ties Martínez-Hoyos et al. [8].
Recent report from Ertan-Bolelli et al. [17] has suggested
5-substituted phenyl sulfonamido benzoxazole derivatives
as inhA inhibitors. Joshi et al. [18, 19] demonstrated in
their study that the inhA inhibitor, 1-cyclohexyl-N-(3,5-
dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (pyrroli-
dinecarboxamide or 641), encompasses three hydrophobic
moieties, namely cyclohexyl, oxopyrrolidine, and 3,5-dichlo-
rophenyl, were swapped by the newly designed molecules
containing 1,3,4-thiadiazole, pyrrole, and substituted phenyl
ring which emulated the interactions of the former groups.
These previous reports prompted us to explore the possibil-
ity of inhA as the target for our newly planned benzoxazole
derivatives.
Cell toxicity is characterized by cytotoxicity. Early assess-
ment of the cytotoxicity of potential NCEs is an essential
part of the drug discovery, as it could be a potential derailer.
Various in silico approaches for phenotypic screening are
available to reduce the time and cost of in vivo experimental
screening of cytotoxic agents. Freely available alternatives,
such as ChEMBL Gaulton et al. [20] and PubChem Wang
et al. [21], are very useful in assessing cytotoxicity data for
tumor and normal cell line chemicals. These techniques
provide a prospect for the use of these data as a cell line
cytotoxicity assessment for newly synthesized compounds.
In our previous study, we have demonstrated that the syn-
thesis and in vitro antimicrobial activity of novel substituted
N-1,3-benzoxazol-2yl benzene sulfonamides. The present
work is based on a ligand-based drug design that uses ben-
zoxazole as its core. To speed up the drug discovery course,
the crystallographic 3D structural information of the bio-
molecular targets ofers unbelievable prospect for advancing
such novel drug design. To attain this, docking simulation
was executed to visualize the binding orientation pattern of
small molecules to protein targets to envisage their afnity
and activity.
Recent studies suggest that substituted benzoxazoles are
of great importance in the medicinal feld due to their wide
range of pharmaceutical activity. Benzoxazole nucleus is
very common motif among heterocyclic compounds exhib-
iting signifcant pharmacological activities, especially anti-
microbial activities Kamal et al. [9]. Many literature reports
suggest that benzoxazole scafolds have signifcant biologi-
cal activities such as antibacterial, antifungal, antiparasitic,
antihistaminic, antiviral and anthelmintic activities. Kumar
and Kumar [10], Katsura et al. [11], Haugwitz et al. [12],
Paget et al. [13], Ozlem et al. [14], Hitesh et al. [15]. Many
natural products contain benzoxazole moiety as major com-
ponent, for example boxazomycin A, nakijinol B (Fig. 1).
Recently, Lucie Brulíková et al. [16] published a review on
benzoxazole derivatives as promising antitubercular agents.
Refer Fig. 1 for representative benzoxazole derivatives as
potential antitubercular agents reported recently.
R1
HO
HO
N
O
H
H2N
HO
O
X
N
N
O
OH
S
HO
N
O
R1
H
N
N
O
O
N
O
NH
R2
N
NH
H
R2
O
HO
Benzoxazoles as component of natural products
Benzoxazole-based compounds to find a new treatment against tuberculosis
Fig. 1 Representative benzoxazole derivatives as potential antitubercular agents and natural products containing benzoxazole motif
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Journal of the Iranian Chemical Society
Results and discussion
Chemistry
1370–1335 cm−1, N–H stretch 3550–3000 cm−1 and S–N
stretch of 925–910 cm−1.
For the piperidine-benzoxazole sulfonamide derivatives
1
(compounds 15–20), H NMR confrmed the presence of
piperidine ring by showing the peaks in the ranges of δ
1.70–2.00 ppm (m, 2H), 2.07–2.23 ppm (m, 2H), 2.53–2.60
(m, 2H), 2.90–3.30 ppm (m, 1H) 3.56–3.80 ppm (m, 1H).
This was further confirmed by IR spectroscopy by the
presence of S=O stretch in the range of 1370–1335 cm−1,
1195–1168 cm−1 and S–N stretch of 925–910 cm−1. The
presence of the tert-butyl group in its penultimate inter-
mediate (3a–b, refer Scheme 2) was confrmed by singlet
obtained at 1.42 ppm. Synthesized compound details along
with physical data and corresponding yields are given in
Tables 1, 2 (Scheme 1) and Tables 3, 4 (Scheme 2)
Synthetic outlines for synthesizing N-1,3-benzoxazol-2yl
benzene sulfonamides (Compounds 1–14) are shown in
Schemes 1, and 2-piperidine-1,3-benzoxazole sulfonamides
(Compounds 15–20) are synthesized in Scheme 2. These
compounds have been characterized by advanced spectral
means. Synthesized compound structures using Schemes 1
and 2 were confrmed using 1H NMR, IR spectroscopy and
mass spectroscopic analysis.
Broad singlet at δ 12.7 to 13.0 ppm corresponding to one
of –SO2NH (D2O exchangeable) in 1H NMR confrms the
formation of benzoxazol-2yl benzene sulfonamide deriva-
tives (1–14). Structures were further confrmed by IR spec-
troscopy for the presence of S=O stretch in the range of
R3
O
S
O
R3
R1
R2
H
R1
NH2
OH
R1
R3
Cl
N
O
R1
Br
N
NH2
H
CH3
Br
O
H
H
H
Cl
N
S
CH3
Pyridine,DCM, RT
Ethanol, 60oC
Step 1
O
N
H
R2
R2
Br
Cl
O
Step 2
R2
2a-d
1a-d
60-85 %
70-95 %
Compound 1-14
Scheme 1 Synthesis of N-1,3-benzoxazol-2yl benzene sulfonamides
H
O
R1
R1
N
O
R1
NH2
OH
O
O
N
O
ZnBr2
TFA
NH
N
+
Toluene, 110oC
Step 1
N
DCM, RT
Step 2
4a-b
O
R2
R2
R2
O
O
3a-b
Pyridine
DCM, RT
1a-b
Step 3
2
S
Cl
O
R1
R1=H,Cl,
R2=H,Cl
R4= H,Br,CH3
O
N
O
N
S
R4
O
R2
Compound 15-20
Scheme 2 Synthesis of 2-piperidine-1,3-benzoxazole sulfonamides
Table 1 Reaction time and
Intermediate R1 R2
Reaction Yield (%) LCMS characterization
time (h)
yields for step 1 are given below
2a
2b
2c
2d
H
H
H
H
CH3
Br
8
6
10
12
75
80
93
86
MS (ESI): m/z=found 135.14 [M+]; calcd. 134.05
MS (ESI): m/z=found 149.16 [M+]; calcd. 148.06
MS (ESI): m/z=found 214.03 [M++2]; calcd. 211.96
MS (ESI): m/z=found 203.02 [M++1]; calcd. 201.97
Cl Cl
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Journal of the Iranian Chemical Society
Table 2 Reaction time and yields for step 2 are given below along
with fnal compound details. Spectral analysis is provided in the
Spectral analysis section
Table 2 (continued)
Compound Compound structure
no.
Reaction Yield
time (h)
2
(%)
69
Compound Compound structure
no.
Reaction Yield
7
time (h)
(%)
N
O
NH
S
O
O
1
2.5
56
Cl
N
O
NH
S
Br
Br
O
O
O
O
Cl
8
3
4
60
70
N
O
NH
S
2
3
4
59
67
Cl
N
O
NH
S
O
O
Cl
Me
Br
9
N
O
Me
Br
NH
S
O
3
Cl
O
N
O
Br
NH
S
O
O
Cl
10*
11*
3
3
62
67
N
O
NH
S
O
O
4
5
2.5
4
60
69
O
N
NH
O
O
O
S
O
Me
N
O
NH
S
O
N
O
NH
S
O
Me
Me
Me
Br
12
13
2
3
69
72
N
O
NH
S
Me
Br
O
O
6
3
66
O
N
O
NH
S
O
O
N
O
NH
S
O
NO2
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Journal of the Iranian Chemical Society
Table 2 (continued)
available inhA ‚ NAD+‚ pyrrolidine carboxamide structures
He et al. [23]. Benzoxazole derivatives have similar hydro-
gen-bonding arrangement with the substrate NAD+and side
chain of Tyr158, which is a part of the enzyme’s catalytic
residues (Fig. 2a and b).
Compound Compound structure
no.
Reaction Yield
time (h)
4
(%)
55
14
N
O
NH
S
O
Two key H-bonding interactions were shown by the pyr-
rolidinecarboxamide (4TZK ligand). The oxygen atom on
the carbonyl group of fve-membered ring lactam is con-
nected via hydrogen bond to the hydroxyl group of amino
acid residue Tyr158 and to 2′-hydroxyl moiety of the nico-
tinamide ribose. The lactam ring of the pyrrolidinecar-
boxamide (4TZK ligand) also interacted with the cofactor
NAD+(Fig. 5a–c).
O
NH2
Table 3 Reaction time and yields for step 1 are given below. Reaction
We focused our attention on the highly active compounds
from series 1, namely compound 8 (MIC of 1.6 μg/mL) and
compound 11 (MIC 3.12 μg/mL), for the characteristic
receptor–ligand interactions projected by the Surfex-Dock
binding mode. In our fndings, compounds 8 and 11 dis-
played a relative type of conformation and orientation as that
of standard crystallographic ligand, i.e., pyrrolidinecarboxa-
mide, along with additional hydrogen bonding interactions
with surrounding amino acids.
yields for step 2 were quantitative
Intermediate R1 R2 Reaction Yield (%) LCMS characterization
time (h)
3a
H
H
2
70
83
MS (ESI): m/z=found
303.37 [M+]; calcd.
302.16
MS (ESI): m/z=found
372.26 [M++1];
calcd. 370.09
3b
Cl Cl 12
As illustrated in Fig. 3a–c, at the enoyl acyl carrier pro-
tein reductase (ENR) enzyme (PDB ID: 4TZK) active site,
the benzoxazole Sulfonamide compound 8 makes the three
key hydrogen bonding interactions. Among these three
interactions, benzoxazole ring nitrogen (N) atom formed
two hydrogen bonding interactions, one with the co-factor
NAD+(2.33 Å) and other with TYR158 (2.32 Å). Another
hydrogen bonding interaction rose from the hydrogen atom
of NH of sulfonamide group (SO2NH), with oxygen atom
of TYR158 (2.48 Å).
In silico molecular docking studies
Docking study of benzene sulfonamide (series 1)- and
phenylsulfonyl-piperidine (series 2)-based benzoxazole
derivatives:
In order to examine the mechanism of antitubercular
activity and to understand intermolecular drug–receptor
interactions of the newly synthesized 20 benzoxazole deriva-
tives from both the series, molecular docking studies were
executed using the crystal structures of inhA, complexed
with 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-
3-carboxamide (i.e., PDB ID 4TZK, 1.62 Å X-ray resolu-
tion) [22] exercising the Surfex-Dock program of SYBYL-X
2.0 software. Because of the growing resistance to isonia-
zid [INH] against inhA, enoyl acyl carrier protein reductase
(ENR) from M. tuberculosis was used as an active site for
all the inhibitors for docking experiment as shown in Fig. 2a
and b. The predicted binding energies of the compounds
are recorded in Table 5. The docking study revealed good
docking score against M. tuberculosis for all the synthesized
compounds.
As depicted in Fig. 4a–c, in the active site of the enoyl
acyl carrier protein reductase (ENR) enzyme (PDB ID:
4TZK), compound 11 also demonstrated the similar bind-
ing pattern with three key hydrogen bonding interactions.
Among those, two interactions are coming from the nitrogen
atom (N) of the benzoxazole ring with the hydrogen atoms of
NAD+(2.37 Å) and TYR158 (2.34 Å). Remaining hydrogen
bonding interaction outstretched from the hydrogen atom of
N–H of sulfonamide group (SO2NH) with oxygen atom of
TYR158 (2.50 Å).
Overall outcome of the docking studies using Surfex-
Dock revealed that all the synthesized compounds engaged
the similar binding interactions as those of pyrrolidinecar-
boxamide derivative (Fig. 5).
Figure 6a, b represents the other hydrophilic and hydro-
phobic amino acids surrounded by the studied compounds
8 &11.
Docking studies with Surflex-Dock assessment have
confrmed that the binding patterns of all the synthesized
benzoxazole analogues are identical in comparison with the
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Journal of the Iranian Chemical Society
Table 4 Reaction time and yields for step 3 are given along with fnal compound details below
Compound no.
15
Compound structure
Reaction time (h)
2
Yield (%)
63
Cl
Cl
Cl
O
N S
O
N
O
Cl
Cl
16
17
3
4
67
68
O
N S
O
N
O
Me
Br
O
N S
O
N
O
Cl
N
18
19
20
3
3
2
68
69
80
O
N S
O
O
O
N S
O
N
O
N
O
Me
Br
O
N S
O
Spectral analysis is shown in the Spectral analysis section
Fig. 2 Docked view of all the
compounds at the active site of
the enzyme PDB ID: 4TZK
N
N
O
NH
O
O
NH
S
O
O
S
O
Br
Compound-8
Compound-11
Me
Me
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Journal of the Iranian Chemical Society
Table 5 Surfex Docking score
(kcal/mol) of the benzoxazole
derivatives
Compounds C scorea Crash scoreb Polar scorec D scored
PMF scoree G scoref
Chem scoreg
4TZK ligand 8.73 −168.11 −49.19
−1.39
−1.04
−1.49
−1.03
−0.81
−0.82
−0.87
−1.71
−0.68
−1.57
−0.85
−0.82
−0.79
−1.40
−2.72
−0.78
−1.98
−2.01
−1.15
−0.76
−0.90
1.18
0.11
0.98
0.98
0.66
0.95
1.06
0.47
0.74
1.38
0.62
0.66
0.61
1.06
1.11
0.04
0.00
1.12
0.00
0.52
0.68
−285.29 −37.47
19
8
6.94
6.77
6.29
6.18
5.97
5.76
5.35
5.26
5.23
5.19
5.17
4.99
4.91
4.90
4.61
4.53
4.13
4.06
3.61
3.07
−106.075 −44.224
−103.226 −49.423
−89.633 −54.975
−90.276 −57.459
−93.268 −57.501
−92.905 −58.247
−111.421 −58.163
−99.342 −49.206
−75.614 −43.630
−93.250 −57.971
−96.735 −46.708
−97.764 −49.148
−89.627 −54.161
−92.221 −45.364
−96.986 −53.485
−103.641 −43.298
−101.788 −58.955
−89.580 −36.472
−89.695 −48.189
−93.992 −50.518
−262.286 −32.535
−243.558 −32.250
−220.471 −30.480
−228.287 −30.226
−214.938 −28.347
−221.634 −32.385
−262.986 −33.821
−200.760 −30.075
−204.957 −31.873
−215.132 −32.988
−205.388 −29.770
−217.801 −30.921
−200.000 −30.145
−227.320 −30.666
−194.166 −27.674
−213.708 −29.488
−234.130 −30.815
−183.882 −29.195
−181.627 −29.795
−183.842 −30.201
11
18
13
20
15
9
14
16
6
17
12
10
7
5
1
4
2
3
aCScore (Consensus Score) integrates a number of popular scoring functions for ranking the afnity of
ligands bound to the active site of a receptor and reports the output of total score
bCrash score reveals the inappropriate penetration into the binding site. Crash scores close to 0 are favora-
ble. Negative numbers indicate penetration
cPolar indicates the contribution of the polar interactions to the total score. The polar score may be useful
for excluding docking results that make no hydrogen bonds
dD score for charge and van der Waals interactions between the protein and the ligand
ePMF score indicates the Helmholtz free energies of interactions for protein–ligand atom pairs (potential of
mean force, PMF)
fG score shows hydrogen bonding, complex (ligand–protein) and internal (ligand–ligand) energies
gChem-score points for H-bonding, lipophilic contact and rotational entropy, along with an intercept term
The scoring functions, viz. C Score, Crash Score, Polar
Score, D Score, PMF Score, Chem Score, G Score, from the
Surfex-Dock are shown in Table 5 to understand the bind-
ing afnity between benzoxazole derivatives and the ENR
protein receptor.
compared to the reference ligand reference 4TZK ligand
(pyrrolidinecarboxamide). Compounds from both the series
are efciently binding to the substrate (ligand) binding site
of ENR. Ligand–receptor complex was stabilized by the key
H-bonding interactions with cofactor NAD + and Tyr158,
along with additional surrounding hydrophobic and hydro-
philic amino acid residues. These observations conclude that
these benzoxazole sulfonamide derivatives are tight binders
of the ENR and can be further developed as the superior
ENR inhibitors. Rational correlation between predicted and
experimental results was established for the synthesized
compounds. With this, we conclude that the molecular dock-
ing with the support of in vitro assay confrmation could
serve as an efcient preselection method for recognizing new
enoyl acyl carrier protein reductase (ENR) inhibitors.
All the compounds displayed a Consensus Score (C
Score) in the range of 7.00–3.07, signifying the summary
of all forces of interaction between the ligands and the enoyl
acyl carrier protein reductase (ENR) inhA. Helmholtz free
energies of interactions for protein–ligands atom pairs
ranged from 45 to 50. Electrostatic and van der Waals inter-
actions between the protein and benzoxazole hits were found
around 100. Compounds H-bonding, complex (ligand–pro-
tein) and internal (ligand–ligand) energies are around 250.
These scores indicate that the molecules preferably bind to
the enoyl acyl carrier protein reductase (ENR) inhA enzyme
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Journal of the Iranian Chemical Society
Fig. 3 Docked view of com-
pound 8 at the active site of the
enzyme PDB: 4TZK
Fig. 4 Interaction of compound
11 at the binding site of the
enzyme (PDB ID: 4TZK)
microplate alamarBlue assay (MABA) technique with cip-
rofoxacin, pyrazinamide and streptomycin as the internal
standards. All the synthesized compounds exhibited signif-
cant, moderate even negligible activity at diferent concen-
trations. Among all the assessed molecules, compound 8
from Scheme 1 was exceptionally active with the MIC of
Biology
Antimycobacterial Activity
In vitro evaluation of compounds for antimycobacterial
activity against M. tuberculosis H37Rv was performed using
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Journal of the Iranian Chemical Society
Fig. 5 a Docked mode of
4TZK_ligand; b Inside the pro-
posed binding pocket of InhA; c
3D docked view of the 4TZK_
ligand. Binding site residues;
red-colored Try158 amino acid,
blue-colored cofactor NAD+;
the molecule is colored accord-
ing to atom type
Fig. 6 a Hydrophobic amino
acids surrounded to compounds
8 (green color) and 11 (cyan
color). b Hydrophilic amino
acids surrounded to compounds
8 and 11
1.6 μg/mL, followed by compound 11 from Scheme 1 with
3.12 μg/mL. The present study suggested that the structural
substitution of synthesized compounds could have better
impact on changing the efcacy of antimycobacterial activ-
ity when further screened for MIC.
cell lines. Compounds from piperidine-based benzoxazole
series specifcally target the HEK293 cell line, which is pre-
sent in human embryonic kidney. We strongly believe that
piperidine-based benzoxazole type of compounds may be the
best starting points for renal fbroblasts and myofbroblasts
of Chronic Kidney cancer research. Compilation of cell line
toxicity study results is shown in Table 6.
Human cell line cytotoxicity (in silico Model)
In silico prediction of benzoxazole derivatives
Conclusion
All the 20 benzoxazole derivatives from both the series were
selected and taken for cytotoxicity prediction in diferent
cell lines by making use of CLC-Pred tool Lagunin et al.
[24]. Almost all the compounds from the phenyl sulfona-
mide benzoxazole series showed aspirated outcome of prob-
ability of being lesser cytotoxic to any of the mentioned
In the present study, we have developed a novel benzo-
xazole sulfonamide-based chemical series, which can be
developed as possible antitubercular agents. Two distinct
benzoxazole sulfonamide scafolds were synthesized and
evaluated for their antimycobacterial potential. Among all,
N-phenyl benzoxazole compounds, 8 (N-(7-bromobenzo[d]
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Journal of the Iranian Chemical Society
oxazol-2-yl)-4-methylbenzenesulfonamide) and 11
(N-(benzo[d]oxazol-2-yl)-4-methylbenzenesulfonamide)
showed prominent antimycobacterial activities. Utilizing
In-silico ligand- and structure-based approaches, we have
successfully demonstrated the preferential binding of these
benzoxazole derivatives to inhA protein, which was further
supported by the biochemical M. tuberculosis inhibition
assay. Cytotoxicity profle prediction, using the CLC-Pred
tool, indicated cleaner profle for N-phenyl benzoxazole
compounds. Piperidine-based benzoxazole compounds are
specifcally active in embryonic human kidney HEK293 cell
line. In conclusion, we believe that phenyl benzoxazole sul-
fonamide compounds can be developed as new lead antitu-
bercular agents with preferential binding to the enoyl acyl
carrier protein reductase (ENR) enzyme. Benzoxazole-based
piperidine compounds may also be an appropriate chemo-
type for kidney cancer research.
To a stirred solution of substituted 1 mmol of amino phe-
nol (1a–d) in 20 ml of ethanol, a 3 mmol of cyanogen
bromide has been added slowly at room temperature.
Reaction mixture was heated to 60 °C for 12 h. The reac-
tion was monitored by TLC. After the disappearance of
starting material (1a–d), reaction mass was evaporated
and the residue was treated with aqueous sodium bicarbo-
nate solution (20 mL) till pH of 8. Solid obtained was fl-
tered and recrystallized with ethanol. The yields obtained
ranged from 70 to 90%.
Step 2: Synthesis of N-1,3-benzoxazol-2yl benzene sul-
fonamides (Compounds 1–14)
Sulfonyl chloride (1.1 mmol) has been added to a stirred
solution of 1 mmol step 1 product (benzo[d]oxazol-
2-amine) in dry DCM and 10 mmol pyridine. The reac-
tion mixture has been stirred at room temperature for
1–4 h. Reaction was monitored by TLC. After the disap-
pearance of the starting material (2a–d), reaction mass
was evaporated under vacuum. Residue obtained was
treated with water, to obtain a solid which was purifed
either by recrystallization using ethyl alcohol or by col-
umn chromatography (eluent: ethyl acetate/hexane, 3:7).
The yields obtained during the transformation ranged
between 54 and 75%.
Materials and methods
Experimental
All reagents and solvents for the present study were pur-
chased from commercial sources and used without puri-
1
fcation. Both H and proton-decoupled 13C spectra were
A total of 14 sulfonamides and four intermediates were
prepared, and the details of the compounds 1–15 are given
in Tables 1 and 2.
recorded on Bruker Supercon Magnet Avance DRX-300
with 300 MHz and 50 MHz spectrometer, in deuterated sol-
vents with TMS as an internal reference. Chemical shifts δ
were given in ppm and coupling constant J in Hz. Multiplici-
ties obtained in NMR spectra are reported as follows: singlet
(s), doublet (d), triplet (t), multiplet (m) and broad singlet
(br s). IR spectra were recorded using the PerkinElmer BX
series FTIR 5000 spectrometer using KBr pellet (Vector-22,
Bruker, France). Mass spectra were recorded on 70 eV on
VG-Micromass 7070H spectrometer, and elemental analy-
sis was carried out using FLASH EA 1112 CHN analyzer
(Thermo finnigan, Italy). Progress of the reactions was
monitored by thin-layer chromatography (TLC) on precoated
silica gel plates using UV light as a visualizing agent. All
the compounds were purifed by either recrystallization
using suitable solvents or performing column chromatog-
raphy over Merck silica gel (230–400 fash). Melting points
were measured on an open capillary tube in melting point
apparatus and are uncorrected. All compounds have been
characterized by TLC, 1H NMR, MS and IR spectroscopy.
Synthetic outlines are shown in Scheme 2.
Step 1: Synthesis of substituted tert-butyl 4-(benzo[d]
oxazol-2-yl)piperidine-1-carboxylate (3a-b)
To a mixed solution of piperidine aldehyde 2 (1.1 mmol)
and o-aminophenol 1a-b (1.0 mmol) in 10 mL of toluene,
zinc bromide (0.01 mmol) catalyst has been added. The
reaction mixture was stirred for 2 h at 111 °C. Reaction
was monitored by TLC. Upon completion of the reac-
tion, the reaction mixture was fltered through the Celite
bed. The catalyst was washed with ethyl acetate. Solvent
was removed under vacuum, and the residue was puri-
fed either by recrystallization or by the use of column
chromatography to isolate the pure compound. The yields
obtained were between 70 and 85%.
Step 2: Synthesis of 2-(piperidin-4-yl)benzo[d]oxazole
(4a–b)
To a stirred solution of 1 mmol of substituted tert-butyl
4-(benzo[d]oxazol-2-yl)piperidine-1-carboxylate (3a–b)
was added DCM, trifuoroacetic acid (TFA) (40 eq) was
added and the reaction mass was stirred at RT for 2 h.
TLC showed the absence of starting material. The reac-
tion mass was evaporated in order to obtain a crude prod-
uct which was taken directly for the next step without
further workup.
General procedures and spectral data
Synthetic outlines are shown in Scheme 1.
Step 1: Synthesis of 1, 3-benzoxazol-2-amines (2a-d)
1 3

Journal of the Iranian Chemical Society
Table 6 In silico CLC-Pred cell line cytotoxicity prediction of benzoxazole derivatives from the study area
S. no Compound name
Cell line
Cell line model type
Afecting parts
Tumor type
Pa
Pi
1
N-(5,7-dichlorobenzo[d]
HT-29
HeLa
T47D
5637
Colon adenocarcinoma
Cervical adenocarcinoma
Breast carcinoma
Colon
Cervix
Breast
Urinary tract
Adenocarcinoma 0.465 0.039
Adenocarcinoma 0.421 0.036
oxazol-2-yl)benzenesul-
fonamide
Carcinoma
Carcinoma
0.374 0.040
0.338 0.017
Urothelial bladder carci-
noma
Hs 683
Oligodendroglioma
Brain
Glioma
Leukemia
0.410 0.110
0.488 0.015
2
N-(5,7-dichlorobenzo[d]
oxazol-2-yl)-4-methylben-
zenesulfonamide
CCRF-CEM Childhood T acute lympho- Blood
blastic leukemia
Colon adenocarcinoma
Cervical adenocarcinoma
HT-29
HeLa
5637
Colon
Cervix
Urinary tract
Adenocarcinoma 0.402 0.057
Adenocarcinoma 0.378 0.046
Carcinoma
Urothelial bladder carci-
0.333 0.022
noma
C8166
T47D
Hs 683
HEK293
Leukemic T cells
Breast carcinoma
Oligodendroglioma
Embryonic kidney
Blood
Breast
Brain
Leukemia
Carcinoma
Glioma
fbroblast
Leukemia
0.319 0.039
0.327 0.056
0.323 0.190
0.494 0.024
0.469 0.017
Kidney
3
4
4-bromo-N-(5,7-
CCRF-CEM Childhood T acute lympho- Blood
dichlorobenzo[d]oxazol-
2-yl)benzenesulfonamide
blastic leukemia
HeLa
HT-29
T47D
Cervical adenocarcinoma
Colon adenocarcinoma
Breast carcinoma
Cervix
Colon
Breast
Kidney
Adenocarcinoma 0.380 0.045
Adenocarcinoma 0.335 0.086
Carcinoma
carcinoma
0.304 0.066
0.486 0.014
N-(7-methylbenzo[d]oxazol- ACHN
Papillary renal
2-yl)benzenesulfonamide
C8166
5637
Leukemic T cells
Blood
Urinary tract
Leukemia
Carcinoma
0.454 0.003
0.443 0.003
Urothelial bladder carci-
noma
G-361
Raji
Melanoma
B-lymphoblastic cells
Skin
Melanoma
Leukemia
0.421 0.004
0.342 0.014
Hematopoietic and lym-
phoid tissue
NCI-H522
Nonsmall cell lung carci-
noma
Lung
Carcinoma
0.332 0.055
MCF7
HEK293
Breast carcinoma
Breast
Kidney
Carcinoma
fbroblast
0.334 0.118
0.473 0.026
Embryonic kidney fbro-
blast
5
4-methyl-N-(7-
ACHN
Papillary renal
Kidney
carcinoma
0.476 0.015
methylbenzo[d]oxazol-
2-yl)benzenesulfonamide
C8166
5637
Leukemic T cells
Blood
Urinary tract
Leukemia
Carcinoma
0.450 0.003
0.439 0.003
Urothelial bladder carci-
noma
G-361
Raji
Melanoma
B-lymphoblastic cells
Skin
Melanoma
Leukemia
0.415 0.004
0.339 0.014
Hematopoietic and lym-
phoid tissue
NCI-H522
HEK293
ACHN
Nonsmall cell lung carci-
noma
Lung
Carcinoma
fbroblast
carcinoma
0.324 0.060
0.485 0.024
0.439 0.021
Embryonic kidney fbro-
Kidney
Kidney
blast
6
4-bromo-N-(7-
Papillary renal
methylbenzo[d]oxazol-
2-yl)benzenesulfonamide
C8166
Leukemic T cells
Blood
Leukemia
0.419 0.003
1 3

Journal of the Iranian Chemical Society
Table 6 (continued)
S. no Compound name
Cell line
5637
Cell line model type
Afecting parts
Tumor type
Carcinoma
Pa
Pi
Urothelial bladder carci-
noma
Urinary tract
0.410 0.004
G-361
Raji
Melanoma
B-lymphoblastic cells
Skin
Melanoma
Leukemia
0.387 0.004
0.304 0.023
Hematopoietic and lym-
phoid tissue
MCF7
Breast carcinoma
Colon adenocarcinoma
Breast
Colon
Carcinoma
Adenocarcinoma 0.466 0.039
0.362 0.102
7
8
N-(7-bromobenzo[d]oxazol- HT-29
2-yl)benzenesulfonamide
T47D
5637
Breast carcinoma
Breast
Urinary tract
Carcinoma
Carcinoma
0.413 0.031
0.310 0.049
Urothelial bladder carci-
noma
C8166
Leukemic T cells
Blood
Leukemia
Leukemia
0.310 0.054
0.484 0.016
N-(7-bromobenzo[d]oxazol- CCRF-CEM Childhood T acute lympho- Blood
2-yl)-4-methylbenzenesul-
fonamide
blastic leukemia
HeLa
HT-29
T47D
C8166
5637
Cervical adenocarcinoma
Colon adenocarcinoma
Breast carcinoma
Cervix
Colon
Breast
Blood
Adenocarcinoma 0.454 0.030
Adenocarcinoma 0.390 0.061
Carcinoma
Leukemia
Carcinoma
0.355 0.046
0.312 0.050
0.308 0.053
Leukemic T cells
Urothelial bladder carci-
Urinary tract
noma
9
4-bromo-N-(7-
HT-29
Colon adenocarcinoma
Colon
Adenocarcinoma 0.433 0.048
bromobenzo[d]oxazol-
2-yl)benzenesulfonamide
T47D
C8166
5637
Breast carcinoma
Leukemic T cells
Breast
Blood
Urinary tract
Carcinoma
Leukemia
Carcinoma
0.377 0.039
0.302 0.069
0.382 0.005
10
N-(benzo[d]oxazol-2-yl)
benzenesulfonamide
Urothelial bladder carci-
noma
C8166
G-361
YAPC
Hs 683
5637
Leukemic T cells
Melanoma
Pancreatic carcinoma
Oligodendroglioma
Blood
Skin
Pancreas
Brain
Urinary tract
Leukemia
Melanoma
Carcinoma
Glioma
0.372 0.005
0.304 0.017
0.380 0.130
0.350 0.161
0.370 0.005
11
N-(benzo[d]oxazol-2-yl)-
4-methylbenzenesulfon-
amide
Urothelial bladder carci-
Carcinoma
noma
C8166
YAPC
A2058
C8166
Leukemic T cells
Pancreatic carcinoma
Melanoma
Blood
Pancreas
Skin
Leukemia
Carcinoma
Melanoma
Leukemia
0.364 0.006
0.367 0.151
0.303 0.148
0.348 0.011
12
13
N-(benzo[d]oxazol-2-yl)-
4-bromobenzenesulfon-
amide
Leukemic T cells
Blood
5637
Urothelial bladder carci-
noma
Pancreatic carcinoma
Urinary tract
Pancreas
Carcinoma
Carcinoma
0.348 0.012
0.454 0.029
N-(benzo[d]oxazol-2-yl)-
4-nitrobenzenesulfona-
mide
YAPC
5637
Urothelial bladder carci-
noma
Urinary tract
Carcinoma
0.388 0.004
C8166
U-266
RKO
Leukemic T cells
Plasma cell myeloma
Colon carcinoma
Blood
Blood
Colon
Urinary tract
Leukemia
Myeloma
Carcinoma
Carcinoma
0.364 0.006
0.317 0.112
0.310 0.140
0.370 0.005
14
4-amino-N-(benzo[d]
oxazol-2-yl)benzenesul-
fonamide
5637
Urothelial bladder carci-
noma
1 3

Journal of the Iranian Chemical Society
Table 6 (continued)
S. no Compound name
Cell line
Cell line model type
Leukemic T cells
Afecting parts
Tumor type
Pa
Pi
C8166
MOLT-4
Blood
Blood
Leukemia
Leukemia
0.363 0.006
0.316 0.074
Acute T-lymphoblastic
leukemia
YAPC
U-266
Hs 683
Pancreatic carcinoma
Plasma cell myeloma
Oligodendroglioma
Pancreas
Blood
Brain
Carcinoma
Myeloma
Glioma
0.369 0.148
0.303 0.135
0.311 0.204
15
16
17
18
5,7-dichloro-2-(1-
(phenylsulfonyl)piperidin-
4-yl)benzo[d]oxazole
5,7-dichloro-2-(1-
HEK293
HEK293
HEK293
Embryonic kidney
Embryonic kidney
Embryonic kidney
Kidney
Kidney
Kidney
Fibroblast
Fibroblast
Fibroblast
0.764 0.005
0.789 0.005
0.790 0.005
(phenylsulfonyl)piperidin-
4-yl)benzo[d]oxazole
2-(1-((4-bromophenyl)sul-
fonyl)piperidin-4-yl)-5,7-
dichlorobenzo[d]oxazole
2-(1-(phenylsulfonyl)
piperidin-4-yl)benzo[d]
oxazole
19
20
2-(1-tosylpiperidin-4-yl)
HEK293
HEK293
Embryonic kidney
Embryonic kidney
Kidney
Kidney
Fibroblast
Fibroblast
0.823 0.005
0.862 0.004
benzo[d]oxazole
2-(1-((4-bromophenyl)
sulfonyl)piperidin-4-yl)
benzo[d]oxazole
Step 3: Synthesis of 2-piperidine-1,3-benzoxazole sul-
fonamides (Compound 15–20)
DMSO d6): δ 7.32 (d, 1H, Ar–H), 7.52–7.54 (m, 2H, Ar–H),
7.62–7.64 (m, 1H, Ar–H), 7.72–7.74 (m, 2H, Ar–H), 7.79
(d, 1H, Ar–H), 12.7 (s, 1H, 1×NH); MS (ESI): m/z=found
342.96 [M++1]; calcd. 341.96.
Sulfonyl chloride (1.1 mmol) has been added to a stirred
solution of 1 mmol step 2 product (2-(piperidin-4-yl)
benzo[d]oxazole) in dry DCM and 10 mmol pyridine.
The reaction mixture has been stirred at room tempera-
ture for 1–4 h. The reaction mass was evaporated under
vacuum, after the disappearance of the starting material
(4a–b). Residue obtained was treated with water to obtain
a solid that was purifed either by recrystallization with
ethyl alcohol or by column chromatography (eluent: ethyl
acetate/hexane, 4:6). The yields obtained during the trans-
formation ranged between 60 and 80%.
N‑(5,7‑dichlorobenzo[d]
oxazol‑2‑yl)‑4‑methylbenzenesulfonamide (2)
Yield (%): 59; white solid; m.p.:194–198 °C; Rf: 0.52 (ethyl
acetate/hexane, 5:5); IR (KBr, cm−1): 3140 cm−1 (Ar–H
stretching), 1630, 1410 cm−1 (benzoxazole stretching),
1380,1160 cm−1 (–SO2–NH– stretching), 925 cm−1 (S–N
stretch), 860 (C–Cl stretching); 1H NMR (300 MHz, DMSO
d6): δ 2.33 (s, 3H, 1×CH3), 7.29–7.35 (m,3H, Ar–H), 7.71
(ddd, 2H Ar–H), 7.79 (1H,Ar–H), 12.8 (s, 1H, 1×NH). MS
(ESI): m/z=found 357.86 [M++1]; calcd. 355.98.
A total of six piperidine-based sulfonamides were pre-
pared, and the details of the compounds (15–20) are listed
in Tables 2 and 3.
4‑bromo‑N‑(5,7‑dichlorobenzo[d]oxazol‑2‑yl)benzene
sulfonamide (3)
Spectral analysis of all the synthesized compounds
N‑5,7‑dichlorobenzo[d]oxazol‑2‑yl)benzene sulfonamide
(1)
Yield (%): 67; gray solid; m.p.:184–188 °C; Rf: 0.62 (ethyl
acetate/hexane, 5:5); IR (KBr, cm−1): 3145 cm−1 (Ar–H
stretching), 1620, 1430 cm−1(benzoxazole stretching),
1400,1180 cm−1 (–SO2–NH– stretching), 925 cm−1 (S–N
Yield (%): 56; white semisolid; m.p:187–189 °C; Rf: 0.62
(ethyl acetate/hexane, 5:5); IR (KBr, cm−1): 3145 cm−1
(Ar–H stretching), 1620, 1400 cm−1 (benzoxazole stretch-
ing), 1370, 1120 cm−1 (–SO2–NH– stretching), 920 cm−1
(S–N stretch), 850 (C–Cl stretching); 1H-NMR (300 MHz,
1
stretch), 775 (C–Cl stretching), 690 (C–Br stretching); H
NMR (300 MHz, DMSO d6): δ 7.32 (d,1H, Ar–H), 7.57
(ddd,2H, Ar–H), 7.71 (ddd,2H,Ar–H), 7.79 (d,1H,Ar–H),
1 3

Journal of the Iranian Chemical Society
12.7 (s, 1H, 1×NH). MS (ESI): m/z=found 421.89 [M++1];
N‑(7‑bromobenzo[d]
oxazol‑2‑yl)‑4‑methylbenzenesulfonamide (8)
calcd. 419.87.
N‑(7‑methylbenzo[d]oxazol‑2‑yl)benzene sulfonamide (4)
Yield (%): 60; white crystalline solid; m.p.:193–198 °C; Rf:
0.54 (ethyl acetate/hexane, 5:5); IR (KBr, cm−1): 3160 cm−1
(Ar–H stretching), 1630, 1420 cm−1 (benzoxazole stretch-
ing), 1370, 1120 cm−1 (–SO2–NH– stretching), 920 cm−1
(S–N stretch), 680 (C–Br stretching); 1H NMR (300 MHz,
DMSO d6): δ 2.33 (s,3H, 1 × CH3), 7.32 (ddd,2H, Ar–H),
7.45 (dd, 1H,Ar–H), 7.66–7.78 (m,4H,Ar–H). 12.6 (s, 1H,
1 × NH). MS (ESI): m/z = found 367.22 [M++1]; calcd.
365.97.
Yield (%): 60; yellow crystalline solid; m.p.:174–178 °C; Rf:
0.58 (ethyl acetate/hexane, 5:5); IR (KBr, cm−1): 3150 cm−1
(Ar–H stretching), 1620, 1420 cm−1 (benzoxazole stretch-
ing), 1400, 1170 cm−1 (–SO2–NH– stretching), 925 cm−1
1
(S–N stretch); H NMR (300 MHz, DMSO d6): δ 2.21 (s,
3H, 1×CH3), 7.07 (dd,1H,Ar–H), 7.46–7.59 (m,3H,Ar–H),
7.64–7.66 (m,1H,Ar–H), 7.68–7.77 (m,3H,ArH), 12.8 (s,
1H, 1 × NH). MS (ESI): m/z = found 289.06 [M+]; calcd.
288.06.
4‑bromo‑N‑(7‑bromobenzo[d]oxazol‑2‑yl)benzene
sulfonamide (9)
4‑methyl‑N‑(7‑methylbenzo[d]oxazol‑2‑yl)benzene
sulfonamide (5)
Yield (%): 70; pale-green solid; m.p:192–196 °C; Rf: 0.68
acetate/hexane, 5:5); IR (KBr, cm−1): 3150 cm−1 (Ar–H
stretching), 1640, 1420 cm−1 (benzoxazole stretching), 1370,
1120 cm−1 (–SO2–NH– stretching), 930 cm−1 (S–N stretch),
675 (C–Br stretching); 1H NMR (300 MHz, DMSO d6): δ
7.45 (dd,1H,Ar–H), 7.57 (ddd,2H,Ar–H), 7.66–7.78 (m,4H,
Ar–H), 12.2 (s, 1H, 1×NH). MS (ESI): m/z=found 432.09
[M++2]; calcd. 429.86.
Yield (%): 69; white crystalline solid; m.p.:184–188 °C; Rf:
0.63 (ethyl acetate/hexane, 5:5); IR (KBr, cm−1): 3160 cm−1
(Ar–H stretching), 1620, 1420 cm−1(benzoxazole stretch-
ing), 1400,1150 cm−1 (–SO2–NH– stretching), 920 cm−1
1
(S–N stretch); H NMR (300 MHz, DMSO d6): δ 2.21 (s
3H, 1 × CH3), 2.33 (s 3H, 1 × CH3), 7.07 (dd, 1H,Ar–H),
7.32 (ddd, 2H,Ar–H), 7.50 (dd, 1H,Ar–H), 7.68–7.74
(m,3H,Ar–H), 12.8 (s, 1H, 1×NH). MS (ESI): m/z=found
303.10 [M+]; calcd. 302.07.
N‑(benzo[d]oxazol‑2‑yl)benzene sulfonamide (10)*
Yield (%): 62; white crystalline solid; m.p: 211–212 °C
◦
4‑bromo‑N‑(7‑methylbenzo[d]oxazol‑2‑yl)benzene
sulfonamide (6)
(Lit. [35] 210–211 C); Rf: 0.60 (ethyl acetate/hexane,
5:5); IR (KBr, cm−1): 3160 cm−1 (Ar–H stretching), 1640,
1420 cm−1 (benzoxazole stretching), 1370, 1120 cm−1
(–SO2–NH– stretching), 930 cm−1 (S–N stretching);
1H NMR (300 MHz, DMSO d6): δ 7.21–7.96 (m, 9H,
Ar–H),12.7 (s, 1H, 1×NH); MS (ESI): m/z=found 275.29
[M+]; calcd. 274.04.
Yield (%): 66; white solid; m.p.:194–198 °C; Rf: 0.64
(ethyl acetate/hexane, 5:5); IR (KBr, cm−1): 3160 cm−1
(Ar–H stretching), 1630, 1420 cm−1(benzoxazole stretch-
ing), 1370, 1120 cm−1 (–SO2–NH– stretching), 920 cm−1
(S–N stretch) 670(C–Br stretching); 1H NMR (300 MHz,
DMSO d6): δ 2.21 (s,3H, 1 × CH3), 7.07 (dd,1H, Ar–H),
7.46–7.60 (m,3H,Ar–H), 7.68–7.74 (m,3H,Ar–H), 12.7 (s,
1H, 1×NH). MS (ESI): M/Z=: 369.2 ([M+ +2], 100%); MS
(ESI): m/z=found 367.87 [M++1]; calcd. 365.97.
N‑(benzo[d]oxazol‑2‑yl)‑4‑methylbenzenesulfonamide
(11)*
Yield (%): 67; white crystalline solid; m.p: 214–216 °C; Rf:
0.64 (ethyl acetate/hexane, 5:5); IR (KBr, cm−1): 3160 cm−1
(Ar–H stretching), 1640, 1420 cm−1 (benzoxazole stretch-
ing), 1370, 1120 cm−1 (–SO2–NH– stretching), 930 cm−1
N‑(7‑bromobenzo[d]oxazol‑2‑yl)benzene sulfonamide (7)
1
Yield (%): 69; white crystalline solid; m.p.:200–201 °C; Rf:
0.65 (ethyl acetate/hexane, 5:5); IR (KBr, cm−1): 3150 cm−1
(Ar–H stretching), 1630, 1420 cm−1 (benzoxazole stretch-
ing), 1370, 1120 cm−1 (–SO2–NH– stretching), 930 cm−1
(S–N stretch), 670 (C–Br stretching); 1H NMR (300 MHz,
DMSO d6): δ 7.41–7.59 (m, 3H,Ar–H), 7.61–7.77
(m,4H,Ar–H), 7.75 (dd,1H,Ar–H), 12.7 (s, 1H, 1×NH). MS
(ESI): m/z=found 352.93 [M++1]; calcd. 351.95.
(S–N stretching); H NMR (300 MHz, DMSO d6) δ: 2.35
(s, 3H 1 × CH3), 7.16–7.40 (m, 5H, Ar–H), 7.49 (d,1 H,
Ar–H), 7.81 (d, 2H,Ar–H), 12.70 (s, 1H, 1×NH); MS (ESI):
m/z=found 289.06 [M+]; calcd. 288.06.
N‑(benzo[d]oxazol‑2‑yl)‑4‑bromobenzenesulfonamide (12)
Yield (%): 69; gray crystalline solid; m.p:201–202 °C; Rf:
0.61(ethyl acetate/hexane, 5:5); IR (KBr, cm−1): 3170 cm−1
(Ar–H stretching), 1650, 1420 cm−1 (benzoxazole
1 3

Journal of the Iranian Chemical Society
stretching), 1370, 1120 cm−1 (–SO2–NH– stretching),
stretching), 1108 cm−1(C–N–C stretching), 760 (C–Cl
1
1
930 cm−1 (S–N stretching), 635 (C–Br stretching); H
stretching); H NMR (300 MHz, DMSO d6): δ 1.40–1.49
NMR (300 MHz, DMSO d6) δ 7.27–7.38 (m,2H, Ar–H),
7.54–7.62 (m,3H, Ar–H), 7.68–7.75 (m,3H,Ar–H), 12.70 (s,
1H, 1×NH). MS (ESI): m/z=found 353.19 [M++1]; calcd.
351.95.
(m, 2H, piperidine CH2), 1.72–1.78 (m,2H piperidine CH2),
2.00–2.31 (m,2H piperidine CH2), 2.40(s, 3H, 1 × CH3),
2.78–3.00 (m, 1H, piperidine CH), 3.44–3.67 (m, 2H,
piperidine CH2), 7.40–7.42 (m, 2H, Ar–H), 7.61–7.70 (m,
4H, Ar–H); MS (ESI): m/z=found 426.32 [M++1]; calcd.
424.04.
N‑(benzo[d]oxazol‑2‑yl)‑4‑nitrobenzenesulfonamide (13)
Yield (%): 72; white crystalline solid; m.p:189–191 °C;
Rf: 0.44 (ethyl acetate/hexane, 5:5); IR (KBr, cm−1): 3170
(Ar–H stretching), 1650, 1420 cm−1 (benzoxazole and
NO2 stretching), 1370, 1120 cm−1 (–SO2–NH– stretching),
930 cm−1 (S–N stretching); 1H NMR (300 MHz, DMSO d6)
δ 7.27–7.38 (m,2H,Ar–H), 7.59 (ddd, 1H, Ar–H), 7.72 (ddd,
1H, Ar–H), 8.03 (ddd, 2H, Ar–H), 8.10 (ddd, 2H, Ar–H),
12.70 (s, 1H, 1×NH). MS (ESI): m/z=found 320.03 [M+];
calcd. 319.03.
2‑(1‑((4‑Bromophenyl)sulfonyl)
piperidin‑4‑yl)‑5,7‑dichlorobenzo[d]oxazole (17)
Yield (%): 68; white solid; m.p: 209–211 °C; Rf: 0.46 (ethyl
acetate/hexane, 6:4); IR (KBr, cm−1): 3260 cm−1 (Ar–H
stretching), 2900–2800 cm−1 (Piperidine ring stretching),
1650, 1420 cm−1(benzoxazole stretching), 1170 cm−1 (–SO2
stretching), 1108 cm−1(C–N–C stretching), 750 (C–Cl
1
stretching), 620 (C–Br stretching); H NMR (300 MHz,
DMSO d6): δ 1.42–1.49 (m, 2H, piperidine CH2), 1.72–1.76
(m,2H piperidine CH2), 1.98–2.21 (m,2H piperidine CH2),
2.78–3.00 (m, 1H, piperidine CH), 3.34–3.62 (m, 2H, piperi-
dine CH2), 7.35 (d, 1H, Ar–H), 7.56–7.61 (m, 2H, Ar–H),
7.69–7.72 (m, 2H, Ar–H), 7.69(m, 1H, Ar–H); MS (ESI):
m/z=found 491.19 [M++3]; calcd. 487.94.
4‑amino‑N‑(benzo[d]oxazol‑2‑yl)benzene sulfonamide (14)
Yield (%): 55; white crystalline solid; m.p:189–191 °C;
Rf: 0.44 (ethyl acetate/hexane, 5:5); IR (KBr, cm−1):
3170 cm−1 (Ar–H stretching), 1650, 1420 cm−1(benzoxazole
stretching), 1570 (NH2 stretching), 1370, 1120 cm−1
1
(–SO2–NH– stretching), 930 cm−1 (S–N stretching); H
2‑(1‑(phenylsulfonyl)piperidin‑4‑yl)benzo[d]oxazole (18)
NMR (300 MHz, DMSO d6) δ 6.07(2H, br,s Ar-NH2),
7.02 (ddd, 2H,Ar–H), 7.27–7.38 (m,2H,Ar–H), 7.59 (ddd,
1H,Ar–H), 7.72 (ddd,1H,Ar–H), 7.83 (ddd,2H, Ar–H),
12.70 (s, 1H, 1×NH). MS (ESI): m/z=found 290.31 [M+];
calcd. 289.05.
Yield (%): 68; gray solid; m.p: 185–187 °C; Rf: 0.54 (ethyl
acetate/hexane, 6:4); IR (KBr, cm−1): 3260 cm−1 (Ar–H
stretching), 2900–2800 cm−1 (Piperidine ring stretching),
1650, 1420 cm−1(benzoxazole stretching), 1160 cm−1
(–SO2 stretching), 1108 cm−1(C–N–C stretching); 1H NMR
(300 MHz, DMSO d6): δ 1.69–1.99 (m, 2H, piperidine CH2),
2.10–2.30 (m,2H piperidine CH2), 2.53–2.60 (m,2H piperi-
dine CH2), 2.70–2.75 (m, 1H, piperidine CH), 3.57–3.74(m,
2H, piperidine CH2), 7.17–7.46 (m, 1H, Ar–H), 7.52–7.91
(m, 8H, Ar–H); MS (ESI): m/z=found 343.41 [M+]; calcd.
342.10.
5,7‑dichloro‑2‑(1‑(phenylsulfonyl)piperidin‑4‑yl)benzo[d]
oxazole (15)
Yield (%): 63; yellow solid; m.p:191–194 °C; Rf: 0.45
(ethyl acetate/hexane, 6:4): IR (KBr, cm−1): 3170 cm−1
(Ar–H stretching), 2900–2800 cm−1 (piperidine ring stretch-
ing), 1650, 1420 cm−1(benzoxazole stretching), 1182 cm−1
(–SO2 stretching), 1108 cm−1(C–N–C stretching), 750 (C–Cl
2‑(1‑Tosylpiperidin‑4‑yl)benzo[d]oxazole (19)
1
stretching); H NMR (300 MHz, DMSO d6) δ 1.67–2.00
(m, 2H, piperidine CH2), 2.07–2.21 (m,2H piperidine CH2),
2.53–2.60 (m,2H piperidine CH2), 3.00–3.21 (m, 1H, piperi-
dine CH), 3.56–3.79 (m, 2H, piperidine CH2), 7.55–7.64
(m, 5H, Ar–H), 7.85–7.88 (m, 2H, Ar–H); MS (ESI):
m/z=found 412.30 [M++1]; calcd. 410.03.
Yield (%): 69; gray solid; m.p:194–200 °C; Rf: 0.64 (ethyl
acetate/hexane, 6:4); IR (KBr, cm−1): 3250 cm−1 (Ar–H
stretching), 2900–2800 cm−1 (Piperidine ring stretching),
1650, 1420 cm−1(benzoxazole stretching), 1170 cm−1
(–SO2 stretching), 1108 cm−1(C–N–C stretching); 1H NMR
(300 MHz, DMSO d6): δ 1.73–1.94 (m, 2H, piperidine CH2),
2.08–2.26 (m,2H piperidine CH2), 2.48(s, 3H, 1 × CH3)
2.55–2.61 (m,2H piperidine CH2), 2.98–3.18 (m, 1H, piperi-
dine CH), 3.59–3.72 (m, 2H, piperidine CH2), 7.09–7.18 (m,
1H, Ar–H), 7.18–7.27 (m, 1H, Ar–H), 7.39–7.50 (m, 1H,
Ar–H), 7.63–7.71 (m, 2 H, Ar–H) 7.71–7.75 (m, 1 H, Ar–H)
5,7‑Dichloro‑2‑(1‑tosylpiperidin‑4‑yl)benzo[d]oxazole (16)
Yield (%): 67; white solid; m.p:201–204 °C; Rf: 0.44 (ethyl
acetate/hexane, 6:4); IR (KBr, cm−1): 3270 cm−1 (Ar–H
stretching), 2900–2800 cm−1 (piperidine ring stretching),
1650, 1420 cm−1(benzoxazole stretching), 1170 cm−1 (–SO2
1 3

Journal of the Iranian Chemical Society
7.75–7.83 (m, 2 H, Ar–H); MS (ESI): m/z=found 357.44
resolution) was extracted from the Brookhaven Protein Data-
base (PDB http://www.rcsb.org/pdb). [22].
[M+]; calcd. 356.12.
In our docking procedure, water molecules and other
ligands in the crystal structures were removed (except cofac-
tor NAD+). Polar hydrogen atoms with Gasteiger-Huckel
charges Gasteiger et al. [31] were added. Then, the ligand-
based mode was adopted to generate the “protomol,” by fx-
ing the threshold and bloat parameters with their default
values of 0.50 and 0 Å. After these procedures, benzoxazole
derivatives were docked within the prepared protein. In the
crystal structure of 1-cyclohexyl-N-(3, 5-dichlorophenyl)-
5-oxopyrrolidine-3-car-boxamide-inhA (4TZK PDB)
domain, the mode of collaboration of the relative ligands has
been used as the standard docked template. While perform-
ing the molecular docking studies, the maximum number of
orientations per ligand was fxed to 20 without restraints.
Based on three selection criteria, the docking complex
was acknowledged,
2‑(1‑((4‑Bromophenyl)sulfonyl)piperidin‑4‑yl)benzo[d]
oxazole (20)
Yield (%): 80; gray solid; m.p: 201–203 °C; Rf: 0.54 (ethyl
acetate/hexane, 6:4); IR (KBr, cm−1): 3250 cm−1 (Ar–H
stretching), 2900–2800 cm−1 (piperidine ring stretching),
1650, 1420 cm−1(benzoxazole stretching), 1160 cm−1 (-SO2
stretching), 1108 cm−1(C–N–C stretching), 630 (C–Br
1
stretching); H NMR (300 MHz, DMSO d6): δ 1.67–1.99
(m, 2H, piperidine CH2), 2.07–2.31 (m,2H piperidine CH2),
2.46–2.51 (m,2H piperidine CH2), 3.00–3.25 (m, 1H, piperi-
dine CH), 3.56–3.79 (m, 2H, piperidine CH2), 7.48–7.56 (m,
2H, Ar–H), 7.62–7.70 (m, 4H, Ar–H), 7.76 (d, 1H, Ar–H),
7.94 (d, 1H, Ar–H); MS (ESI): m/z=found 422.31 [M++1];
calcd. 420.01.
*Compounds known in the literature [25, 26]
(i) Highest docking score should represent docking
score of the best orientation
(ii) Alignment of the aromatic/aliphatic rings of the our
molecules (ligands) into the active site in a compa-
rable fashion with the cocrystallized ligands orienta-
tion
Molecular docking studies
It will provide up-front information that can also be used for
structural optimization. Molecular modeling is a computer-
based process for the derivation, demonstration and manipu-
lation of molecular entities and their reactions. It has been
used to understand many of the three-dimensional structure-
dependent properties.
(iii) Conservation of two major key interactions, i.e.,
H-bonds with cofactor NAD+and amino acid resi-
due Tyr158
In order to predict the binding afnity of the designed
molecules in the relative analysis, score functions, i.e., D
Score Kuntz et al. [32], PMF score Muegge and Martin [33],
Chem score Eldridge et al. [34] and Score Jones et al. [35],
were estimated using C-Score module in SYBYL-X 2.0.
The 3D structures were created by using the SKETCH
module employed in the SYBYL-X 2.0 program (Tripos
Associates, St. Louis, MO, USA) [27]. Using the standard
Tripos force feld, Clark et al. [28] the geometry optimiza-
tion was applied with a distance-dependent dielectric func-
tion at the convergence gradient of 0.001 kcal/mol. Utilizing
MMFF94 (Merck Molecular Force Field) the electrostatic
charges were calculated.
Antimycobacterial assay
Using microplate alamarBlue assay (MABA), antimyco-
bacterial activity of the synthesized compounds was evalu-
ated against M. tuberculosis Lourenço et al. [36]. In order
to reduce background efect, assay was executed in 96-well
microplates with black and clear bottom. In this assay, 200 μl
of autoclaved deionized water was supplemented to all exter-
nal perimeter wells of sterile 96-well plates, to reduce evapo-
ration of culture medium in the test wells during incubation.
In the 96-well plates, 100 μl of the middle brook 7H9 culture
broth was added and serial dilutions of compounds were per-
formed in the microplate and fnally 0.2 μg/ml to 100μg/ml
drug concentrations were selected for the assay. Microplates
were incubated at 37ºC for fve days. Meanwhile, 25 μl of
fresh 1:1 mixture of alamarBlue reagent and 10% Tween 80
was added to the plate and incubated for 24 h. Blue color in
the well indicates no bacterial growth, and pink color was
Utilizing the in-built program of SYBYL-X 2.0, struc-
tures were subjected to molecular dynamic simulation, to
perform conformational analysis. Using the repeated molec-
ular dynamics, molecules were heated to 1000 K for 2000 fs
(2 ps) and annealed slowly to 0 K in a step of 100 K for
1000 fs at each temperature, utilizing an exponential anneal-
ing function. By following this process, 100 conformations
were sampled out throughout 100 cycles which accounted
for conformational fexibility with the least energy and stable
conformations.
Surfex-Dock, a patented search engine, which is coupled
with a unique scoring function, was adopted Jain et al. [29,
30] for molecular docking. The crystal structure of M. tuber-
culosis inhA, the enoyl acyl carrier protein reductase (ENR)
complexed with 1-cyclohexyl-N-(3, 5-dichlorophenyl)-5-ox-
opyrrolidine-3-carboxamide (PDB ID 4TZK, 1.62 Å X-ray
1 3

Journal of the Iranian Chemical Society
3. N. Qureshi, N. Sathyamoorthy, K. Takayama, Biosynthesis of C30
to C56 fatty acids by an extract of Mycobacterium tuberculosis
H37Rv. J. Bacteriol. 157, 46–52 (1984)
interpreted as bacterial growth. Pyrazinamide (3.125 μg/ml),
ciprofoxacin (3.125 μg/ml) and streptomycin (6.25 μg/ml)
were used as standards. The MIC was defned as the lowest
drug concentration which prevented the color change from
blue to pink. The vaccine strain Mycobacterium tuberculosis
H37Rv (ATCC No-27294) was used in this assay.
4. K. Bloch, Control mechanisms for fatty acid synthesis in
Mycobacterium Smegmatis. Science (1977). https://doi.
org/10.1002/9780470122907.ch1
5. S. Kikuchi, D.L. Rainwater, P.E. Kolattukudy, Purifcation and
characterization of an unusually large fatty acid synthase from
Mycobacterium tuberculosis var. bovis BCG. Arch. Biochem.
Biophys. 295, 318–326 (1992). https://doi.org/10.1016/0003-
9861(92)90524-z
In silico prediction of human cell line cytotoxicity
for benzoxazole derivatives
6. M.C. Lourenco, M.V. de Souza, A.C. Pinheiro, M.D.L. Ferreira,
R.S. Gonçalves, T.C.M. Nogueira, M.A. Peralta, Evaluation of
anti-Tubercular activity of nicotinic and isoniazid analogues.
Arkivoc 15, 181–191 (2007)
CLC-Pred (cell line cytotoxicity predictor) is a freely avail-
able web service which was utilized for in silico cytotoxic
efect prediction of chemical compounds in non-transformed
and cancer cell lines Lagunin et al. [24]. CLC-Pred is a
very useful tool in the prediction of cytotoxicity of organic
compounds to evaluate the signifcance of the chemical
substance’s inclusion in cytotoxic experimental screening
cascade.
7. N.C. Desai, A. Trivedi, H. Somani, K.A. Jadeja, D. Vaja, L.
Nawale, D. Sarkar, Synthesis, biological evaluation, and molecu-
lar docking study of pyridine clubbed 1, 3, 4-oxadiazoles as poten-
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doi.org/10.1080/00397911.2017.1410892
8. M. Martínez-Hoyos, E. Perez-Herran, G. Gulten, L. Encinas, D.
Álvarez-Gómez, E. Alvarez, J. Rullas-Trincado, Antitubercular
drugs for an old target: GSK693 as a promising inhA direct inhibi-
tor. EbioMedicine 8, 291–301 (2016). https://doi.org/10.1016/j.
ebiom.2016.05.006
The prediction relies on the technology, viz. Prediction of
Activity Spectra for Substances (PASS) (http://www.way2d
rug.com/PASSonline). Training set was produced on the
basis of cytotoxicity data salvaged from ChEMBLdb (ver-
sion 23) (https://www.ebi.ac.uk/chembldb/).
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90.0008.e12
The prediction results were displayed in two tables, one
for tumor and another for nontumor (normal) cell lines.
Table contains details of cytotoxicity with short or full
names of the cell lines, along with corresponding names of
the tissue or organs. Each table includes the Pa (probability
“to be active”) and Pi (probability “to be inactive”) values.
The predictive parameter generated, viz. the Pa values (prob-
ability “to be active”), is categorized as “active” if the val-
ues are>0.5 with the projected cancer cell line. If Pa value
is<0.5 for the set compounds, then the probabilities of being
cytotoxic to the mentioned cell lines are less.
11. Y. Katsura, Y. Inoue, S. Nishino, M. Tomoi, H. Itoh, H. Takasugi,
Studies on antiulcer drugs. III. Synthesis and antiulcer activi-
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benzoxazoles and benzothiazoles. J. Med. Chem. 25, 969–974
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Additional investigations on benzoxazole scafolds are
under active investigations and will be communicated in the
future.
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Acknowledgements The authors thankfully acknowledge Dr KG Bhat,
Maratha Mandal’s Dental College, Hospital and Research Centre,
Belgaum, India, for antitubercular tests. The authors are grateful to
Shrinivas D. Joshi, Professor and Head, Department of Pharmaceutical
Chemistry, S.E.T’s College of Pharmacy, Dharwad, India, for docking
studies.
15. Z.A. Bhavsar, P.T. Acharya, D.J. Jethava, H.D. Patel, Recent
advances in development of anthelmintic agents: synthesis and
biological screening. Synth. Commun. 50, 917–946 (2020). https
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antitubercular agents. Chem. Select 3, 4653–4662 (2018). https://
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