Design and synthesis of sulfur cross-linked 1,3,4-oxadiazole-nitro(furan/thiophene)-propenones as dual inhibitors of inflammation and tuberculosis: molecular docking and Hirshfeld surface analysis

Article abstract of DOI:10.1007/s00706-019-02507-2

Abstract: A series of 3-[5-nitro(furan/thiophene)-2-yl]-1-aryl-3-(5-aryl-1,3,4-oxadiazol-2-ylthio)prop-2-en-1-one derivatives was synthesized and studied with the aim of developing dual inhibitors of multidrug-resistant tuberculosis and inflammation. The in vivo anti-inflammatory activity results showed excellent inhibition of rat paw edema. The methoxybenzene/nitrofuryl derivative of title compounds showed 83% inhibition of inflammation during 2–6?h after carrageenan injection. All compounds showed anti-tuberculosis activity at MIC of 50?μg/cm³. The molecular docking studies revealed that the oxadiazole and nitrofuran groups played a significant role in the inhibiting site of the enzymes COX1, COX2, 5-LOs, and InhA by forming hydrogen bonding with Tyr 385, Ser 530, Tyr 467, and Tyr 158 amino acid residues, respectively. The novel compounds are active antibacterial agents with potential inhibition on E. coli bacteria. The toxicity results showed good percentage viability of human kidney cell lines with IC₅₀ value greater than 100?μg/cm³ concentration. The Hirshfeld surface analysis and electrostatic potential map of compound showed good intermolecular contacts and hydrogen bonding donor and acceptor potential. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-019-02507-2

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-02507-2
ORIGINAL PAPER
Design and synthesis of sulfur cross‑linked
1,3,4‑oxadiazole‑nitro(furan/thiophene)‑propenones as dual
inhibitors of infammation and tuberculosis: molecular docking
and Hirshfeld surface analysis
Vishwanath Turukarabettu1,2 · Balakrishna Kalluraya1 · Monika Sharma3
Received: 15 July 2019 / Accepted: 8 October 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
A series of 3-[5-nitro(furan/thiophene)-2-yl]-1-aryl-3-(5-aryl-1,3,4-oxadiazol-2-ylthio)prop-2-en-1-one derivatives was
synthesized and studied with the aim of developing dual inhibitors of multidrug-resistant tuberculosis and infammation.
The in vivo anti-infammatory activity results showed excellent inhibition of rat paw edema. The methoxybenzene/nitro-
furyl derivative of title compounds showed 83% inhibition of infammation during 2–6 h after carrageenan injection. All
compounds showed anti-tuberculosis activity at MIC of 50 µg/cm3. The molecular docking studies revealed that the oxa-
diazole and nitrofuran groups played a signifcant role in the inhibiting site of the enzymes COX1, COX2, 5-LOs, and InhA
by forming hydrogen bonding with Tyr 385, Ser 530, Tyr 467, and Tyr 158 amino acid residues, respectively. The novel
compounds are active antibacterial agents with potential inhibition on E. coli bacteria. The toxicity results showed good
percentage viability of human kidney cell lines with IC50 value greater than 100 µg/cm3 concentration. The Hirshfeld surface
analysis and electrostatic potential map of compound showed good intermolecular contacts and hydrogen bonding donor
and acceptor potential.
Graphic abstract
Keywords Michael addition · Alkynes · Carbonyl compounds · Protein · Hydrothiolation · Toxicity
Electronic supplementary material The online version of this
Introduction
article (https://doi.org/10.1007/s00706-019-02507-2) contains
supplementary material, which is available to authorized users.
Non-steroidal drugs are widely used for the treatment of
pain, fever, and infammation. The prominent side efect
of non-steroidal drug is formation of gastric or intestine
ulcers and hemorrhage, in addition to the suppression of
prostaglandin biosynthesis from arachidonic acid by inhibit-
ing the enzyme cyclooxygenases (COX). The adverse side
efects may be controlled by an inhibitor of 5-lipoxygenase
(5-LOs) [1–3]. The 1,3,4-oxadiazoles are found to be the
* Balakrishna Kalluraya
bkalluraya@gmail.com
1
Department of Chemistry, Mangalore University,
Mangalagangothri 574199, India
2
Department of Materials Science, Mangalore University,
Mangalagangothri 574199, India
3
Department of Chemical Sciences, IISER, Mohali 140306,
India
Vol.:(0123456789)
1 3

V. Turukarabettu et al.
best remedies against infammation due to their dual mecha-
nism for inhibition of both COX and LOs [4–12]. In chronic
tuberculosis, exacerbated infammation leads to severe lung
tissue necrosis and the promotion of mycobacterial dissemi-
nation and transmission [13]. Anti-infammatory drugs are
currently used for therapy in most severe life-threatening
forms of tuberculosis such as meningitis and pericarditis,
while antibiotics are used to kill the bacteria [13, 14]. The
sulfur linked 1,3,4-oxadiazole-propenone derivatives also
showed promising potency against M. tuberculosis [15].
Pyrazinamide, isoniazid, ethambutol, and rifampicin are
few antitubercular drugs which are known to be frst-line
antituberculosis drugs. The increasing resistance to these
frst-line drugs, leading to multidrug-resistant tuberculo-
sis, has resulted in the need for second-line drugs. These
second-line drugs are less potent and more toxic compared
to the frst-line drugs [16]. Nitrofurantion (I), nifuroxazide
(II), and furamizole (III) are nitrofuran-containing drugs
which possess antibacterial properties. The 5-Nitrofuroyl
moiety attached to several other chemical motifs IV–VI was
found to be active against multidrug-resistant M. tubercu-
losis and non-toxic [17] (Fig. 1). Therefore, a combination
of nitrofuryl moiety and sulfur-linked 1,3,4-oxadiazole-
propenone derivative may result in a dual role with anti-
infammatory and antimycobacterial activity.
Considering the above findings, and in continuation
of our study on bioactive heterocycles [18–21], we have
designed a series of 1,3,4-oxadiazole-propenone deriva-
tives with nitrofuryl/nitrothienyl moiety as promising dual
inhibitors of infammation and Mycobacterium tuberculo-
sis. The signifcance of this study is sulfur cross-linking of
5-aryl-1,3,4-oxadiazole and nitrofuran/nitrothiophene-pro-
penone pharmacophores, which are known for their wide
range of biological activity [4–12]. This cross linking was
achieved through the Michael-type addition of 5-aryl-1,3,4-
oxadizole-2-thiols 6a, 6b to acetylenic ketones 5a–5h con-
taining nitrofuran/nitrothiophene in benzene under refux
condition (Schemes 1, 2).
The in vitro antitubercular activity was carried out on all
synthesized compounds. The in vivo anti-infammatory anal-
ysis was performed on compounds 7a, 7b, 7c, 7h, and 7i,
which showed active ligand–protein interactions in the dock-
ing study. The antibacterial activity was studied by using
agar well difusion method. The toxicity study on human
Fig. 1 Antibacterial drugs con-
O
NO2
O
taining a nitrofuryl moiety
N N
O
N
NH
N
O
HN N
O
O
O
HO
O2N
O
H2N
I
II
III
O
NO2
O
N
O
Cl
OH
O
O2N
O
O
NH
MeO
N
O2N
NO2
O
V
IV
VI
Scheme 1
1 3

Design and synthesis of sulfur cross-linked…
Scheme 2
kidney cell line was carried out for active anti-infammatory
Table 1 Series of (Z)-3-[5-nitro(furan/thiophen)-2-yl]-1-aryl-3-(5-
aryl-1,3,4-oxadiazol-2-ylthio)prop-2-en-1-one derivatives 7a–7j
compounds to establish the IC50 level by in vitro MTT assay.
Compd.
R1
R2
X
Yield/%
M.p./°C
7a
7b
7c
7d
7e
7f
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
o-HO-C6H4
o-HO-C6H4
o-HO-C6H4
H
O
O
O
O
S
S
S
S
O
S
66
64
60
65
60
57
62
67
56
58
198–202
214–216
200–202
229
198–200
179–181
174
182–184
171–173
191–193
Results and discussion
CH3
OCH3
Cl
CH3
OCH3
F
H
CH3
OCH3
Chemistry
The synthetic route for acetylenic ketones 5a–5h is given
in Scheme 1. The acid-catalyzed condensation of 5-nitro-
2-(furyl/thienyl)methyl diacetate 1 and para substituted
acetophenone gave corresponding propenones 3 in acetic
acid. Bromination of propenones in acetic acid and their
subsequent dehydrobromination in dry benzene employing
triethylamine as base gave acetylenic ketones 5a–5h.
7g
7h
7i
7j
Further, the hydrothiolation of acetylenic ketones 5a–5h
with compounds 6a, 6b is shown in Scheme 2. The reaction
proceeded via Michael-type addition to give only Z regioiso-
mer (as confrmed by single crystal XRD) of the propenone
rather than a mixture of E/Z isomers, as seen in the litera-
ture protocol for hydrothiolation of internal alkynes [22, 23].
and no alkenyl isomeric protons were observed. Elemental
analyses (C, H, N) results were found to be in good agree-
ment ( 0.3%) with the calculated values. The m/z values of
‘calculated exact mass’ of compounds 7a–7j are ([M+1]+):
420.06, 434.08, 450.08, 454.03, 450.06, 466.05, 454.03,
452.04, 450.08, 482.05; and the ‘experimental accurate
mass’ data are: 419.50, 434.00, 449.50, 453.40, 449.50,
465.55, 454.20, 451.45, 449.55, 481.45. The m/z data is a
result of single measurements, and diferences in exact and
accurate masses are due to instrument errors [24]. Detailed
spectral and analytical data are given in the experimental
section.
1
The H NMR spectrum of crude compound was recorded
which showed resonance signals corresponding to the single
isomer. The presence of electron-withdrawing nitro group
on acetylenic ketone causes the negative inductive efect on
alkyne carbon and facilitates the nucleophilic attack of sulfur
at the β carbon of alkyne. Thus, the sp3 hybridized sulfur
atom bridges the propenone and 1,3,4-oxadiazole moieties.
The newly synthesized 3-[5-nitro(furan/thiophen)-2-yl]-1-
phenyl-3-(5-aryl-1,3,4-oxadiazol-2-ylthio)prop-2-en-1-ones
Single crystal XRD data
1
7a–7j were characterized by FT-IR, H NMR, 13C NMR,
mass spectra, and elemental analysis. The yields and melting
point data are given in Table 1.
The single crystal data were collected by X-ray difraction
technique for 3-(5-nitrofuran-2-yl)-1-phenyl-3-[5-(p-tolyl)-
1,3,4-oxadiazol-2-ylthio]prop-2- en-1-one (7b) (CCDC No.
1499538). The ORTEP diagram of compound 7b drawn
with thermal displacement ellipsoids at 50% probability
level is given in Fig. 2. The single crystal was character-
ized as follows: crystal system: monoclinic; space group:
P21/C; a = 18.724(1) Å, b = 6.982(1) Å, c = 16.667(16)
Å; α = 90°, β = 113.84°, γ = 90°; V = 1993.0(4) Å3; Z = 4;
μ = 0.204 mm−1; ρcalc - = 1.444 Mg m−3; F(000) = 896.0;
2.378≤θ≤26.429; R=0.0412; wR=0.1296.
The FT-IR spectra of compounds 7a–7j were recorded in
KBr. The characteristic absorption bands at 1585–1600 cm−1
are due to C=C stretching vibrations. The C=O stretching
was observed at 1624–1635 cm−1 as a sharp absorption band.
The asymmetric and symmetric stretching of the nitro group
was observed around 1460 and 1375 cm−1, respectively. In
the 1H NMR spectra of compounds 7a–7j, the alkene pro-
ton at α carbon resonated as a singlet at δ=7.57–8.27 ppm.
The 13C NMR spectra of compounds 7a–7j showed a peak
at δ=186–188 ppm, corresponding to the carbonyl carbon.
A portion of the molecule, comprising the nitrofuran
ring, benzoyl ring, and the central segment (O1/C7-C9)
1
The HMBC spectra showed H–13C signal correlations
1 3

V. Turukarabettu et al.
Fig. 2 The ORTEP diagram of
the compound 7b viewed along
the c-axis
Table 2 The intermolecular hydrogen bonding interactions in 7b
crystal
–D–H··A–/Å
D –H/Å
H··A/Å
D··A/Å
D-H··A/°
C12–H12···N2
C20–H20···O4
C5–H5···N3
1.083
1.083
1.083
2.450
2.606
2.446
3.533 (6)
3.689 (2)
3.529 (2)
159.83
175.72
134.03
to the nearest nucleus and de is the closest external contacts
from the surface. The higher percentages of inter-contacts
are arrived for C–H (19.8%), H–H (27.9%), N–H (10.1%),
and O–H (20%) interactions along with other short contacts
which are shown below the respective plots in Fig. 4.
The electrostatic potential of compounds 7b (Fig. 5)
was mapped using wave function STO-3G basis set with
Hartree–Fock theory over the range of 0.025 au [28]. The
positive electrostatic potential (blue) region represents the
hydrogen donor potential and the negative electrostatic
potential (red) region represents the hydrogen bond accep-
tors. The volume of electrostatic potential for compound 7b
was found to be 489.65 Å3.
Fig. 3 The crystal packing structure of compound 7b viewed along
the a-axis
connecting the two rings, was almost planar as indicated
by the dihedral angles, 2.25(8)° (between nitrofuran and
benzoyl rings), 12.08(10)° (benzoyl rings and the central
segment), and 13.94(10)° (nitrofuran ring and the central
segment). The oxadiazole ring (C15/N2–N3/C16/O5) made
dihedral angles of 12.41(10)°, 58.39(9)°, and 59.32(8)° with
phenyl (C17–C22), nitrofuran (C11–C14/O2), and benzoyl
(C1–C6) rings, respectively. The molecule was twisted at
sulfur atom (S1) with torsion angle C8–C9–S1–C15 being
equal to 154.46(5)°. The torsion angle of the alkene seg-
ment H8–C8–C9–S1 was found to be 178.84(6)°. The crystal
packing structure was drawn using CrystalExplorer [25, 26]
and viewed along the a-axis of unit cell as shown in Fig. 3.
The crystal structure was stabilized by C–H···O type of inter-
molecular contacts and their intermolecular hydrogen bond
geometries are given in Table 2.
The Hirshfeld analysis results indicated that the com-
pounds possess hydrogen bond donors and acceptors, which
would extend the possible interactions with biomolecules for
good biological activity.
In vivo anti‑infammatory activity and molecular
docking studies
The in vivo anti-infammatory activity study was con-
ducted by carrageenan-induced rat right hind paw edema
method [29]. The test results are presented in Table 3, as
mean paw volume SEM, n = 6. Compounds 7a, 7b, 7c,
and 7i, which contain nitrofuryl moiety showed moder-
ate to good anti-infammatory activity than compound
7h, which contains nitrothiophene moiety, as indicated
by the control in the paw edema after 2 h of carrageenan
injection. The compounds 7b, 7c, and 7i showed 66.65%,
85.71%, and 80.94% of inhibition during the 2nd hour
of study, respectively. Interestingly, among the tested
Hirshfeld surface analysis
The short contacts in single crystal of compound 7b were
identifed by Hirshfeld surface analysis [27, 28]. The Hir-
shfeld surfaces and their corresponding 2D-fnger plots are
shown in Fig. 4. In the 2D fngerprint plot, di is the closest
internal distance from a given point on the Hirshfeld surface
1 3

Design and synthesis of sulfur cross-linked…
Fig. 4 Hirshfeld surfaces mapped with dnorm ranging from − 0.24 to 1.61 Å and its 2D fnger plots visualizing the intercontacts of the compound
7b
Fig. 5 The electrostatic
potential mapped on Hirshfeld
surfaces (front and rear view)
over a range of 0.025 au for
compound 7b
1 3

V. Turukarabettu et al.
Table 3 Anti-infammatory
activity results of
Compound
Time/h
0
1,3,4-oxadiazole derivatives as
mean increase in paw volume in
cm3 SEM values
1
2
4
6
Control
Diclofenac
0.466 0.06
0.450 0.02
0.650 0.04
0.583 0.03
0.500 0.04
0.533 0.02
0.400 0.04
0.716 0.06
0.816 0.040
0.750 0.05
0.95 0.04***
0.700 0.05
0.550 0.04**
0.600*
1.067 0.07
1.167 0.06
0.550 0.22
0.800 0.03***
0.883 0.07**
0.700 0.02
0.883 0.09
0.826 0.06
0.746 0.08*
0.716 0.03
0.65 0.10
0.600 0.02
1.133 0.01
0.90 0.10
7a
7b
7c
7i
0.60 0.02** 0.616 0.04***
0.933 0.06
1.067 0.09
0.783 0.04***
0.750 0.09***
7h
0.733 0.07
Levels of signifcance: *P=0.05, **P<0.01, ***P< 0.001
compounds, 7c was found to be the most active (83.3%)
for the inhibition of paw edema (Table S1 of ESI).
The in silico molecular docking results of compounds
7b, 7c, and 7i docked with COX1 and COX2 protein mole-
cule showed hydrogen bonding network in the same pocket
as that of reference drug diclofenac. The compounds are
shown as ball and stick models. The protein is shown in
cartoons for best docked conformations and as lines in
the enlarged pictures. The diclofenac is shown in magenta
color and labeled as DIF 701. The best conformation of
7b with COX1 and their hydrogen bonding interactions
are shown in Fig. 6.
Tyr 385. The docking scores for 7c and 7i were − 44.35 kJ/
mol and − 46.86 kJ/mol, respectively.
The active site of COX2 protein for the ligands 7b, 7c,
7i, and diclofenac was identifed as Ser 530, Tyr 355, Tyr
385, Tyr 115, His 90, Leu 352, Leu 531, Phe 518, Gly 526,
and Val 349. The compound 7b exhibited the highest gliding
score of − 53.56 kJ/mol. The hydrogen bonding interactions
were found between His 90 and the nitro group, and Tyr
355 and 1,3,4-oxadiazole (Fig. 7). The compound 7c showed
hydrogen bond with Tyr 355 at nitrogen of the 1,3,4-oxadia-
zole ring with docking score − 53.17 kJ/mol. Similarly, the
compound 7i formed hydrogen bonding interactions with
Tyr 355 at the nitro group and oxygen of the furan ring (Fig.
S2 of ESI) with a gliding score of − 46.44 kJ/mol.
The ligands showed their stable conformation in the
active site of COX1 with amino acid residues Tyr 385, Tyr
355, Ser 530, Gly 526, Val 349, Phe 381, Trp 381, Leu 384,
and Val 349, which is a key requirement for anti-infamma-
tory activity as seen in several studies [4]. The compound 7b
exhibited the highest gliding score of − 49.79 kJ/mol. The
1,3,4-oxadiazole nitrogen formed a hydrogen bonding with
Tyr 385 and the nitro group formed polar interactions with
Ser 530. The compounds 7c and 7i showed the same active
pocket for stable binding conformation as that of 7b (Fig. S1
of ESI) and hydrogen bonding interactions with Ser 530 and
The docking experiments of 5-LOs with compounds 7b,
7c, and 7i showed moderate ligand–protein interactions,
as indicated by their low binding energy compared to the
COX studies. The active binding pocket for compound 7b
in the protein 5-LOs was identifed as Ser 444, Tyr 467, Gln
546, Thr 363, Ala 450, and Arg 367, with polar interactions
(Fig. 8). The gliding score value was found to be − 19.25 kJ/
mol. Similarly, the compounds 7c and 7i showed gliding
score of − 23.43 kJ/mol and − 30.96 kJ/mol, respectively,
Fig. 6 a The docked view of
compound 7b with diclofenac in
the active site of COX1 (3N8Y)
and b hydrogen bonding inter-
action
1 3

Design and synthesis of sulfur cross-linked…
Fig. 7 a The docking pocket of
compound 7b with re-docked
diclofenac in COX2 (1PXX)
and b the hydrogen bonding
interaction
Fig. 8 a The docked view of
compound 7b in 5-LOs (3O8Y)
and b their hydrogen bonding
interactions
in the active binding pocket of protein as shown in Fig. S3
of ESI.
with Tyr 158 seem to be a conserved feature among all the
InhA-inhibitor compounds synthesized so far [31]. In addi-
tion to this, the nitro group and carbonyl group formed three
polar co-ordination bonds with NAD+.
Antimycobacterial activity
Microplate Alamar Blue assay (MABA) methodology was
used for antitubercular study [30]. The in vitro antimyco-
bacterial results showed that all compounds 7a–7j are active
against multidrug-resistant M. tuberculosis (ATCC 35835) at
a minimum inhibitory concentration of 50 μg/cm3 compared
to the standard drug streptomycin which showed an MIC
value of 6.25 μg/cm3. The docking analysis of compound 7c
showed that the compound has a stable conformation in the
InhA active site of protein (Fig. 9). The Tyr 158 and oxidized
form of nicotinamide adenine dinucleotide (NAD+, shown
in yellow) are active sites for the antimycobacterial activity
as referred to in the literature [16]. The compound formed
hydrogen bonding network and gliding score of −43.93 kJ/
mol in the vicinity of amino acids: Tyr 158, Phe 149, Phe
97, Met 98, Met 103, Met 161, Met 199, Ser 200, Ala 198,
His 265, and Asp 150. This active site and hydrogen bonding
Antibacterial activity
The antibacterial activity of synthesized compounds was
determined by agar well diffusion method [32] and the
results were represented as diameter of zone of inhibition
of bacterial growth in millimeters. The following test micro-
organisms were used: Gram-positive bacteria Bacillus subti-
lis ATCC 33712, Staphylococcus aureus ATCC 25923, and
Gram-negative bacteria Escherichia coli ATCC 25922 and
Klebsiella pneumoniae ATCC 700603. The antibacterial
activity results of test compounds are given in Table 4.
All compounds showed moderate to good antibacterial
activity compared to the standard drug ampicillin for the
tested bacterial strains. The compounds showed very good
activity against Gram-negative bacteria E. coli, and among
the tested compounds 7f was found to be the most efective.
1 3

V. Turukarabettu et al.
Fig. 9 a The docked view of
compound 7c in the active
site of MTb (4TZK) and b the
hydrogen bonding interaction
The compound 7j was found to be most active against B.
subtilis, and 7a and 7b were active against S. aureus. In
general, compounds carrying alkyl and alkoxy substituents
have shown good antibacterial activity.
Table 4 Antibacterial activity data of (Z)-3-[5-nitro(furan/thiophen)-
2-yl]-1-aryl-3-(5-aryl-1,3,4-oxadiazol-2-ylthio)prop-2-en-1-ones
7a–7j
Test compounds Antibacterial activity at 10 mg/cm3 (Zone of
inhibition/mm)
Toxicity studies
B. subtilis S. aureus E. coli
K. pneumoniae
Control
Ampicillin
–
–
–
12
–
–
The compounds 7b, 7c, and 7i which showed good anti-
infammatory and antituberculosis activity were tested for
their toxicity by in vitro MTT assay [33]. The human kidney
cell line HEK293 was used for the study. The test results are
documented in Table 4 as the percentage viability of kidney
cell lines against various concentrations of compounds and
the standard drug mitomycin C at 500 µg/cm3 concentration
(Fig. 10).
21 0.1
10
12 0.6
15 0.2
10 0.3
11 0.2
20
15 0.4
10 0.6
15 0.1
20 0.1
26 0.1
20 0.2
21 0.3
12 0.1
14 0.1
12
11 0.3
9
8
13
11
29 0.2
12 0.3
12 0.1
7a
7b
7c
7d
7e
7f
10
10 0.2 12 0.2
15 0.1 10
9
23 0.1
15 0.2
10 0.5
15
0.1
–
8
–
8
0.1
7g
7h
7i
0.3
0.2
The toxicity study revealed that the test compounds
7b, 7c, and 7i showed good IC50 value of 133.65 µg/cm3,
243.02 µg/cm3, and 274.03 µg/cm3, respectively. Interest-
ingly, the compounds 7c and 7i showed better percentage
0.4
18 0.2
13 0.3
7j
–
Fig. 10 The percentage viability
of HEK293 cell lines
1 3

Design and synthesis of sulfur cross-linked…
viability at 500 µg/cm3 concentration (14.89% and 24.12%,
respectively) to that of the standard drug (10.46%).
were added. The reaction mixture was stirred for 1 h and
kept aside at room temperature. The propenone crystals 3
formed were collected by fltration and washed with etha-
nol. The crude product 3 (5 mmol) was dissolved in 20 cm3
glacial acetic acid by heating. 30% v/v bromine solution was
added drop by drop until bromination was complete. The
reaction mixture was stirred for 2 h and kept aside overnight.
The α,β-dibromochalcones 6 formed were fltered, washed
with ethanol, and recrystallized from glacial acetic acid. The
dibromochalcone 4 (5 mmol) was taken in a round-bottomed
fask and 25 cm3 of dry benzene was added. To this, tri-
ethylamine (6 mmol) was added and the fask was closed
with a lid. The mixture was stirred for 4 h and the separated
triethylammonium hydrobromide fltered of. The fltrate
was roto-evaporated and the solid separated was collected
by fltration and further purifed by recrystallization from
ethanol. The compounds were characterized by reference to
their melting point [34, 35] and the data are given in ESI.
Conclusion
In summary, a series of 1,3,4-oxadiazole-propenone deriva-
tives were prepared and evaluated for their biological activ-
ity. The in vivo anti-infammatory study indicated that the
nitrofuran derivatives possess good inhibition of infamma-
tion and the highest inhibition was observed between the 2nd
to 6th hour for 7c. The docking studies showed that the target
compounds are both COX1 and COX2 active site binders as
well as moderate inhibitors of 5-LOs. All compounds exhib-
ited antituberculosis activity and were found to be active
InhA blockers. The antibacterial activity study showed that
the compounds are moderate to good antibacterial agents.
Compounds 7c and 7i were found to be least toxic to the
human kidney cell lines, with IC50 value of greater than
240 µg/cm3, in comparison with the standard drug mitomy-
cin 500. The Hirshfeld surface analysis showed that the com-
pounds possess hydrogen bond donors and acceptors which
would extend the possible interactions with biomolecules for
good biological activity. These fndings indicated that syn-
thesized nitrofuran/nitrothiophene containing 1,3,4-oxadia-
zole-propenone derivatives would exhibit anti-infammatory
as well as antimicrobial activity. Further study in the struc-
ture–activity relationship of these lead compounds would
beneft in the development of drugs for treatment of severe
tuberculosis associated with infammation.
General procedure for synthesis of compounds
7a–7j
The acetylenic ketone 5a–5h (5 mmol) and 5-aryl-1,3,4-
oxadiazole-2-thiol 6a, 6b (5 mmol) were taken in a round-
bottomed fask containing 50 cm3 of dry benzene. A water-
cooled condenser was attached to the round-bottomed
fask and the reaction mixture was refuxed on an oil bath
at 100 °C under atmospheric pressure conditions. The pro-
gress of the reaction was monitored by TLC (ethylacetate
and petroleum ether 1:3). The solution was then rota-evap-
orated to remove the excess solvent. The solid obtained was
recrystallized from DMF/ethanol mixture.
Experimental
All chemicals were procured commercially, and further puri-
fcation was done wherever needed. The melting point of
newly synthesized compounds was determined using Inno-
vative DTC-967A instrument. IR spectra were recorded on
(Z)‑3‑(5‑Nitrofuran‑2‑yl)‑1‑phenyl‑3‑(5‑phenyl‑1,3,4‑oxa‑
diazol‑2‑ylthio)prop‑2‑en‑1‑one (7a, C21H13N3O5S) Yellow
crystalline; IR (KBr): v̄ =1629 (C=O), 1595 (C=C), 1541
(C=N), 1469 (NO2 assym. str.), 1377 (NO2 sym. str.), 970
(C–S) cm−1; 1H NMR (CDCl3): δ=8.0 (d, 2H, J=8.8 Hz),
7.97 (s, 1H, = C–H), 7.46–8.95 (m, 8H), 7.23 (d, 1H,
J=3.6 Hz, NF-4H), 6.87 (d, 1H, J=3.6 Hz, NF-3H) ppm;
13C NMR (CDCl3): δ=188.6 (C=O), 151.3, 136.4, 134.1,
132.9, 129.1, 129.0, 128.7, 128.2, 126.9, 122.9, 115.1, 115,
112.6 ppm; MS: m/z=419.50 ([M+1]+).
a Shimadzu FTIR 157 spectrophotometer. The 1H and 13
C
NMR spectra were recorded on Agilent VNMRS-400 MHz
NMR spectrometer using TMS as internal standard. Mass
spectra were recorded using Shimadzu 8030-LCMS/MS
mass spectrometer. Elemental analysis was carried out on
Vario-EI elementar III model elemental analyzer. The single
crystal data were collected on Bruker D8 Venture Photon
100 CMOS X-ray difractometer at 293 K.
3‑(5‑Nitrofuran‑2‑yl)‑1‑phenyl‑3‑[5‑(p‑tolyl)‑1,3,4‑oxadi‑
azol‑2‑ylthio]prop‑2‑en‑1‑one (7b, C22H15N3O5S) Yellow
crystalline; IR (KBr): v̄ =1627 (C=O), 1521 (C=N) cm−1;
1H NMR (DMSO-d6): δ = 8.20 (s, 1H, = C–H), 7.88 (d,
2H, J = 8.4 Hz), 7.76 (d, 1H, J = 4 Hz, NF-4H), 7.58 (d,
1H, J = 5.6 Hz, NF-3H), 7.56–8.08 (m, 5H), 7.42 (d, 2H,
J=8.4 Hz), 2.49 (s, 3H, CH3) ppm; 13C NMR (DMSO-d6):
δ=188.9 (C=O), 152.7, 144.6, 137.2, 135.5, 134.7, 130.5,
General procedure for preparation of acetylenic
ketones 5a–5h
To a well-stirred solution of 5-nitro-2-(furyl/thienyl)methyl
diacetate 1 (5 mmol) in 20 cm3 glacial acetic acid, substi-
tuted acetophenone 2 (5 mmol) and 0.5 cm3 of conc. H2SO4
1 3

V. Turukarabettu et al.
130.2, 129, 128.3, 127.8, 126.1, 117.3, 114.1, 111.8, 21.2
(DMSO-d6): δ=8.14 (s, 1H,=C–H), 8.04 (d, 1H, J=4.4 Hz,
NT-4H), 7.54 (d, 1H, J= 4.4 Hz, NT-3H), 7.42–7.84 (m,
9H) ppm; 13C NMR (DMSO-d6): δ = 187.7 (C=O), 166.7
(1JC-F =230 Hz, Ar C-F), 164.8, 160.2, 151.9, 146.8, 139.5,
133.2 (3JC-F, 12.4 Hz, Ar C-F), 133, 132.5 (2JC-F =39 Hz, Ar
C-F), 130.2, 129.7, 128.8, 126.9, 122.8, 116.7, 116.5 ppm;
MS: m/z=454.2 ([M+1]+).
(CH3) ppm; MS: m/z=434 ([M+1]+).
1‑(4‑Methoxyphenyl)‑3‑(5‑nitrofuran‑2‑yl)‑3‑(5‑phenyl‑1,3,4‑
oxadiazol‑2‑ylthio)prop‑2‑en‑1‑one (7c, C22H15N3O6S) Yel-
low crystalline; IR (KBr): v̄ = 1637 (C=O), 1597 (C=C),
1
1516 (C=N), 966 (C-S) cm−1; H NMR (CDCl3): δ=7.93
(s, 1H,=C-H), 7.02 (d, 1H, J=4 Hz, NF-4H), 6.99–8.07 (m,
9H), 6.83 (d, 1H, J=3.6 Hz, NF-3H), 3.90 (s, 3H, OCH3)
ppm; 13C NMR (DMSO-d6): δ=188.7 (C=O), 166.7, 160.7,
151.7, 145.4, 135.6, 134.0, 132.8, 130.1, 129.9, 129.5, 129.4,
127.0, 123.1, 114.6, 52.4 (OCH3) ppm; MS: m/z=449.50
([M+1]+).
3‑[5‑(2‑Hydroxyphenyl)‑1,3,4‑oxadiazol‑2‑ylthio]‑3‑
(5‑nitrothiophen‑2‑yl)‑1‑phenylprop‑2‑en‑1‑one (7h,
C21H13N3O5S2) Yellow crystalline; IR (KBr): v̄ = 1633
1
(C=O), 1589 (C=C), 1543 (C=N), 900 (C-S) cm−1; H
NMR (CDCl3): δ=9.69 (s, 1H, OH), 7.98 (s, 1H, =C-H),
7.95 (d, 2H, J=8 Hz), 7.25 (d, 1H, J=4 Hz, NT-4H), 6.94–
7.60 (m, 9H), 6.86 (d, 1H, J=4 Hz, NT-3H), 2.44 (s, 3H,
CH3) ppm; 13C NMR (CDCl3): δ=186.2 (C=O), 166, 158.7,
157.2, 150.5, 147.1, 135.7, 133.4, 132.3, 131.4, 130.8,
129.8, 129.5, 127.5, 120.1, 117.5, 117.2, 114.2, 110.2 ppm;
MS: m/z=451.45 ([M+1]+).
1‑(4‑Chlorophenyl)‑3‑(5‑nitrofuran‑2‑yl)‑3‑(5‑phenyl‑1,3,4‑
oxadiazol‑2‑ylthio)prop‑2‑en‑1‑one (7d, C21H12ClN3O5S) Yel-
low crystalline; IR (KBr): v̄ = 1627 (C=O), 1585 (C=C),
1
1537, 1523 (C=N), 970 (C–S) cm−1; H NMR (DMSO-
d6): δ=8.27 (s, 1H,=C-H), 7.61 (d, 1H, J=4 Hz, NF-4H),
7.46–8.26 (m, 9H), 7.43 (d, 1H, J=5.6 Hz, NF-3H) ppm;
13C NMR (DMSO-d6): δ=189 (C=O), 170.5, 165.7, 155.8,
155.5, 135.3, 135.2, 132.5, 130.7, 131.9, 130.7, 129.3, 127,
125, 120.5, 115.2 ppm; MS: m/z=453.40 ([M+1]+), 455.40
([M+3]+) (37Cl), with peak intensity ratio of 3:1.
3‑[5‑(2‑Hydroxyphenyl)‑1,3,4‑oxadiazol‑2‑ylthio]‑3‑(5‑nitro‑
furan‑2‑yl)‑1‑(p‑tolyl)prop‑2‑en‑1‑one (7i, C22H15N3O6S) Yel-
low crystalline; IR (KBr): v̄ = 3309 (O–H), 1624 (C=O),
1
1591 (C=C), 1543 (C=N), 894 (C–S) cm−1; H NMR
(CDCl3): δ=9.69 (s, 1H, OH), 7.95 (d, 2H, J=8 Hz, ArH),
7.98 (s, 1H, = C-H), 7.58 (d, 1H, J = 8 Hz, ArH), 7.43 (t,
1H, J=7.2 Hz, 6.8 Hz, salicyl-4H), 7.32 (d, 2H, J=8.4 Hz,
ArH), 7.25 (d, 1H, J=4 Hz, NF-4H), 7.06 (d, 1H, J=8.4 Hz,
ArH), 6.93 (t, 1H, J = 7.6 Hz, 8 Hz, salicyl-5H), 6.86 (d,
1H, J = 4 Hz, NF-3H), 2.44 (s, 3H, CH3), ppm; 13C NMR
(CDCl3): δ=188.5 (C=O), 167, 157.5, 159.9, 151.3, 145.5,
135.2, 134.5, 132.1, 130.2, 129.5, 129, 126.5, 120.1, 118.5,
115.2, 113.2, 107.2, 22 (CH3) ppm; MS: m/z = 449.55
([M+1]+).
3‑(5‑Nitrothiophen‑2‑yl)‑1‑phenyl‑3‑[5‑(p‑tolyl)‑1,3,4‑oxa‑
diazol‑2‑ylthio]prop‑2‑en‑1‑one (7e, C22H15N3O4S2) Yellow
crystalline; IR (KBr): v̄ =1637 (C=O), 1589 (C=C), 1548
(C=N), 950 (C–S) cm−1; 1H NMR (DMSO-d6): δ=8.09 (s,
1H,=C-H), 8.04 (d, 2H, J=8.4 Hz), 7.99 (d, 1H, J=4.4 Hz,
NT-4H), 7.79 (d, 2H, J=7.6 Hz), 7.53–7.60 (m, 3H), 7.50
(d, 1H, J=4.4 Hz, NT-3H), 7.37 (d, 2H, J=8 Hz), 2.38 (s,
3H, CH3) ppm; 13C NMR (DMSO-d6): δ = 188.7 (C=O),
166.7, 160.3, 151.8, 146.9, 145.4, 138.7, 134, 132.9, 130.2,
130, 129.9, 129.5, 129.3, 126.9, 122.8, 21.7 ppm; MS:
m/z=449.50 ([M+1]+).
3‑[5‑(2‑Hydroxyphenyl)‑1,3,4‑oxadiazol‑2‑ylthio]‑1‑(4‑me‑
thoxyphenyl)‑3‑(5‑nitrothiophen‑2‑yl)prop‑2‑en‑1‑one‑
(7j, C22H15N3O6S2) Yellow crystalline; IR (KBr): v̄ = 3327
(O–H), 1624 (C=O), 1589 (C=C), 1544 (C=N), 898 (C–S)
cm−1; 1H NMR (CDCl3): δ=9.66 (s, 1H, OH), 8.01 (d, 2H,
J = 7.2 Hz, ArH), 7.69 (d, 1H, J = 2 Hz, NT-4H), 7.60 (s,
1H, = C–H), 7.17 (d, 1H, J = 2 Hz, NT-3H), 7.06 (d, 1H,
J = 8.8 Hz, salicyl-3H), 6.93–7.52 (m, 8H), 6.93 (t, 1H,
J=7.2 Hz, 6.8 Hz, salicyl-4H), 3.90 (s, 3H, OCH3) ppm; 13C
NMR (CDCl3): δ=186.7 (C=O), 166.4, 164.5, 159.6, 157.5,
146.2, 134.3, 131.1, 129.3, 128.3, 128, 127.8, 126.3, 120.1,
117.8, 114.3, 107.2, 55.6 (OCH3) ppm; MS: m/z = 481.45
(M+).
1‑(4‑Methoxyphenyl)‑3‑(5‑nitrothiophen‑2‑yl)‑3‑
(5‑phenyl‑1,3,4‑oxadiazol‑2‑ylthio)prop‑2‑en‑1‑one (7f,
C22H15N3O5S2) Yellow crystalline; IR (KBr): v̄ = 1637
(C=O), 1595 (C=C), 1546, 1520 (C=N), 900 (C-S) cm−1;
1H NMR (CDCl3): δ = 8.01 (d, 2H, J = 8 Hz), 7.85 (d,
2H, J = 8.4 Hz), 7.65 (d, 1H, J = 4.4 Hz, NT-4H), 7.57
(s, 1H, = C–H), 7.43–7.57 (m, 3H), 7.15 (d, 1H, J= 4 Hz,
NT-3H), 6.99 (d, 2H, J=8.4 Hz, ArH), 3.90 (s, 3H, OCH3)
ppm; 13C NMR (CDCl3): δ = 186.7 (C=O), 164.4, 146.3,
132.3, 131, 129.4, 129.1, 128.3, 127.3, 126.7, 122.8, 114.3,
55.6 (OCH3) ppm; MS: m/z=465.55 ([M+1]+).
1 ‑ ( 4 ‑ Fl u o ro p h e ny l ) ‑ 3 ‑ ( 5 ‑ n i t ro t h i o p h e n ‑ 2 ‑ y l ) ‑ 3 ‑
(5‑phenyl‑1,3,4‑oxadiazol‑2‑ylthio)prop‑2‑en‑1‑one (7g,
In vivo anti‑infammatory activity
C
21H12FN3O4S2) Brown needles; IR (KBr): v̄ =1629 (C=O),
Male Wistar rats were taken, fasted for 12 h, and given free
access to water. Since the synthesized compounds were less
1
1600 (C=C), 1541 (C=N), 902 (C–S) cm−1; H NMR
1 3

Design and synthesis of sulfur cross-linked…
soluble in 0.1% Tween 80–saline solution than in diclofenac
sodium, the compounds were administered at higher dosage,
orally at 100 mg/kg body weight by gavage to each group of
six animals. Diclofenac sodium at a dose of 10 mg/kg body
weight was used as a standard anti-infammatory agent. The
negative control group received 0.1% Tween 80 in saline
solution. Acute infammation was produced by sub-plantar
injection of 0.1 cm3 of 1% suspension of carrageenan in
normal saline in the right hind paw just below the tibiotar-
sal junction of the rats 1 h after the oral administration of
the test compounds. The paw volume was measured by a
plethysmometer at 0 h, 1 h, 2 h, 4 h, and 6 h after the carra-
geenan injection. The anti-infammatory activity of the test
compounds and the standard reference drug was determined
using the formula:
Molecular docking
The molecular docking studies were carried out using Auto-
docksuite-4.2.5.1 molecular docking software [36]. The
aspirin acetylated COX1 (3N8Y with X-ray resolution 2.6
Å), diclofenac bound to COX2 (1PXX with X-ray resolution
2.9 Å), human 5-LOs (3O8Y with X-ray resolution 2.38 Å),
and 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-
3-carboxamide (CDOC) bound MTb (4TZK with X-ray
resolution 1.62 Å) were the protein molecules obtained from
the protein data bank [31, 37–39]. The structure of protein
molecules was simplified by removing HETATOMS, co-
crystallized molecules, and water molecules. The force feld
parameters and topology fles for all ligands were generated
using PRODRUG server [40]. Flexible torsions were defned
using AutoTors provided with AutoDock suite. The reference
drug molecule (diclofenac for COX1 and COX2; CDOC for
MTb) was redocked together with the docked compounds in
the inhibitor binding site. PyRx 0.8 interface was used with
grid box size 86×85×80, spacing 0.361 and assigning three
degrees of freedom. The Lamarckian genetic algorithm was
employed with ten conformations for each ligand and the best
pose with the highest binding energy was considered as the
stable docking site and visualized using PyMOL Molecular
Graphics System [41].
(
)
Vt
Percentage of anti-inflammatory activity = 1 −
× 100,
Vc
where Vt is the mean increase in paw volume in rats treated
with test compounds and Vc represents the mean increase in
paw volume in the control group of rats.
In vitro antimycobacterial activity
Sterile deionized water (200 mm3) was added to all outer
perimeter wells of sterile 96-well plates to minimize evapo-
ration of medium in the test wells during incubation. The
96-well plate received 100 mm3 of the Middlebrooks 7H9
broth and serial dilution of compounds were made directly
on plate. The fnal drug concentrations tested were from 0.2
to 100 µg/cm3. Plates were covered and sealed with paraflm
and incubated at 37 °C for 5 days. 25 mm3 of freshly pre-
pared 1:1 mixture of Alamar Blue reagent and 10% Tween
80 was added to the plate and incubated again for 24 h. A
blue color in the well was interpreted as no bacterial growth,
and pink color was scored as growth. The MIC was defned
as lowest drug concentration which prevented the color
change from blue to pink.
Toxicity study
The cells were taken in a confuent cell line fask and trypsi-
nized. Phosphate bufer saline wash was given twice and
centrifuged. The cells were resuspended in 10% fetal bovine
serum medium and counted using a hemocytometer. These
cells (3000–10,000) were plated to 96-well plates and incu-
bated at 37 °C in a CO2 incubator for 24 h. Then the medium
was discarded from 96-well plates. The drug solution was
prepared in a series of concentrations from 1 to 500 µg/cm3
in serum and added to diferent test groups. The test plates
were incubated again for 48 h. 20 mm3 of MTT dye (5 mg/
mm3) was added to all wells and incubated for 4 h. 100 mm3
of DMSO was added to all the wells and shaken well. The
absorbance of all the test samples was recorded at 540 nm.
Antibacterial activity
Acknowledgements The authors are grateful to NGSM Institute of
Pharmaceutical Sciences Nitte (Deemed to be University) for carrying
out animal studies and USIC Mangalore University for spectral data.
The overnight-grown culture was prepared by inoculating a
loopful of culture to broth media. The test samples were pre-
pared at a concentration of 10 mg/cm3 by dissolving in DMSO
followed by sonication. Five millimeter diameter wells on agar
plates were made using a sterile cork-borer. 20 mm3 of test
sample/standard at the concentration of 10 mg/cm3 was loaded
on to the wells. All bacterial cultures were incubated at 37 °C
and observed for the zone of inhibition. The diameter of the
zone of inhibition was measured.
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