Synthesis, evaluation and docking studies of some 4-thiazolone derivatives as effective lipoxygenase inhibitors

Article abstract of DOI:10.1007/s11696-018-0520-9

Some promising 4-thiazolone derivatives as lipoxygenase inhibitors were designed, synthesized, characterized and evaluated for anti-inflammatory activity and respective ulcerogenic liabilities. Compounds (1b, 1e, 3b, and 3e) exhibited considerable in vivo

Full text of DOI:10.1007/s11696-018-0520-9

Chemical Papers
https://doi.org/10.1007/s11696-018-0520-9
ORIGINAL PAPER
Synthesis, evaluation and docking studies of some 4‑thiazolone
derivatives as efective lipoxygenase inhibitors
Sushant Kumar Shrivastava1 · Brijesh K. Patel1 · Prabhash Nath Tripathi1 · Pavan Srivastava1 · Piyoosh Sharma1 ·
Avanish Tripathi1 · Ankit Seth1 · Manish Kumar Tripathi1
Received: 9 August 2017 / Accepted: 26 May 2018
© Institute of Chemistry, Slovak Academy of Sciences 2018
Abstract
Some promising 4-thiazolone derivatives as lipoxygenase inhibitors were designed, synthesized, characterized and evaluated
for anti-infammatory activity and respective ulcerogenic liabilities. Compounds (1b, 1e, 3b, and 3e) exhibited considerable
in vivo anti-infammatory activity (57.61, 79.35, 75.00, and 79.35%) against carrageenan-induced rat paw edema model,
whereas compounds (1e, 3b, and 3e) were found active against the arachidonic acid-induced paw edema model (55.38, 55.38,
and 58.46%). The most potent compound (3e) exhibited lesser ulcerogenic liability compared to the standard diclofenac and
zileuton. Further, the promising compounds (1e and 3e) were evaluated for in vitro lipoxygenase (LOX; IC50 =12.98 µM
and IC50 =12.67 µM) and cyclooxygenase (COX) inhibition assay (COX-1; IC50 >50 µM and, COX-2; IC50 >50 µM). The
enzyme kinetics of compound 3e was evaluated against LOX enzyme and supported by in silico molecular docking and
molecular dynamics simulations studies. Overall, the results substantiated that 5-benzylidene-2-phenyl-4-thiazolones are
promising pharmacophore for anti-infammatory activity.
Keywords 4-Thiazolone · One-pot multicomponent reaction · Anti-infammatory · Lipoxygenase
Introduction
infammatory leukotrienes that regulate infammation, pain,
and fever, as well as some diverse physiological reactions
such as blood fow, thrombosis, renal and gastrointestinal
functions (Allaj et al. 2013). The LOX enzyme is known as
non-haem iron-containing dioxygenase having an elongated
active cavity without access to the bulk solvent. The enzyme
is lined with both invariant amino acids (Leu368, Leu373,
Leu414, Leu607 and Ile406) and LOX-specifc amino acids
(Ala603, Ala606, His600, and Thr364) (Gilbert et al. 2011).
The reported LOX inhibitors show the interactions with
amino acids through respective hydrogen bonding (His372),
hydrophobic (Phe359, Leu414, Leu607, and Ala603) and
electrostatic interactions. However, exploratory research is
required to develop a potent molecule as a drug to clear the
phase trials (Shrivastava et al. 2017).
Lipoxygenase (LOX) enzyme produces leukotrienes (LTs)
and lipoxins (LXs) from arachidonic acid (AA) LT–LX
metabolism. These are potent inflammatory mediators,
responsible for allergic infammation and gastric ulceration
(Martel-Pelletier et al. 2003; Shrivastava et al. 2017). The
LOX enzymes exist in human beings in three functional
isoforms 5-, 12-, and 15-LOXs, whereas isoforms 9- and
13-LOX exist in plants (Reddy et al. 2015). The LOX
enzyme generally produce 5-hydroperoxyeicosatetraenoic
acid metabolites (HPETEs) by the addition of molecu-
lar oxygen into the substrate (Carter et al. 1997; Murphy
and Gijón 2007; Srivastava et al. 2016), and generates
Zileuton is the most common LOX inhibitor, frequently
prescribed by physicians to treat asthma (Drazen et al. 1999),
infammation of the upper airway, and chronic obstructive
pulmonary disease (COPD) (Berger et al. 2007). However,
its long-term use is restricted due to side efects such as
dyspepsia (Wenzel and Kamada 1996), and hepatotoxic-
ity (Rossi et al. 2010). Therefore, to discover and develop
the safe and potent LOX inhibitors devoid of toxicities is
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-018-0520-9) contains
supplementary material, which is available to authorized users.
* Sushant Kumar Shrivastava
skshrivastava.phe@itbhu.ac.in
1
Department of Pharmaceutical Engineering and Technology,
Indian Institute of Technology (Banaras Hindu University),
Varanasi 221 005, India
Vol.:(0123456789)
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a challenge for the drug discovery and medicinal chemist
in the feld of pulmonary therapeutics (Zaitsu et al. 2003).
In the modern age of drug development and therapeu-
tics, pharmacophore-based methods are successfully utilized
in drug discovery over the years. Literature revealed that
4-thiazolone nucleus (Barzen et al. 2012; Hofmann et al.
2011) coupled with diferent aromatic moieties is proven
to be efective against infammation (Ali et al. 2007; Bekhit
et al. 2010; Deep et al. 2010; Ottana et al. 2005). Taking
a cue from those studies, we have explored several 4-thia-
zolone derivatives reported as the potential LOX inhibitors
(Franke et al. 2009; Pergola and Werz 2010; Schneider et al.
2009; Scholz et al. 2009). In the design of the LOX inhibi-
tors, the pharmacophore “5-benzylidene-2-phenyl-4-thia-
zolones” was equipped with two hydrogen bond acceptors,
two hydrogen bond donors, and two hydrophobic groups
attached to the aromatic ring (Aparoy et al. 2012; Barreiro
et al. 2002) (Fig. 1).
Experimental
Chemicals were purchased from Sigma-Aldrich (India)
and used without further purifcation. The reaction was
monitored by thin-layer chromatography on Merck silica
gel 60 F254 aluminum sheets (Merck, Germany) using
ethyl acetate (EtOAc):hexane (3:2) as the mobile phase.
Melting points were determined using open capillary
tubes on a Stuart melting point apparatus (SMP10) and are
uncorrected. FT-IR spectra were recorded on Shimadzu
8400S FTIR (Shimadzu Corporation, Japan) using KBr
1
pellets. H-NMR (500 MHz) and 13C-NMR (125 MHz)
spectra were recorded on a Bruker Avance FT-NMR spec-
trophotometer at room temperature using TMS as an inter-
nal standard. Elemental analysis was performed on Exeter
CE-440 analyzer.
Literature also revealed that 4-thiazolone has a poor aque-
ous solubility that leads to the reduced bioavailability in the
central compartment. (Franke et al. 2009; Hofmann et al.
2012). Therefore, our specifc aim was to design more hydro-
philic as well as potent LOX inhibitors devoid of ulcerogenic
liabilities. In the present study, the designed and synthesized
compounds were evaluated for anti-infammatory activity
against carrageenan and arachidonic acid-induced rat paw
edema models, and the active compounds were further
screened through in vitro LOX, COX-1, and COX-2 enzyme
inhibition assay. The enzyme kinetics was determined and
collectively supported by computational molecular docking
and dynamics simulations studies that were thrown light on
the type of enzyme inhibition.
Synthesis of (Z)‑benzylidene‑2‑(4‑chlorophenyl)
thiazol‑4(5H)‑one derivative (1a–1e)
The substituted aromatic aldehydes (5.34 mmol) were
added to the mixture of thioglycolic acid (7.89 mmol),
4-chlorobenzonitrile (5.78 mmol) and methanolic trieth-
ylamine (6.58 mmol). The reaction mixture was stirred
at room temperature for 30 min and refuxed for 16–24 h.
On the completion of the reaction, the solvent was evapo-
rated under reduced pressure, and the crude product was
recrystallized from methanol and washed with acetone to
get the pure solid compounds 1a–1e (Scheme 1) (Zayed
et al. 1985).
Fig. 1 Pharmacophoric consid-
eration of designed molecule
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Chemical Papers
R1
R1
Cl
Cl
HO
SH
TEA, CH3OH
16-24h, reflux
+
+
S
O
R1=
Thioglycolic acid
N
CHO
1a = H,
1b = 4-Cl,
CN
Substituted
O
4-chlorobenzonitrile
Benzaldehyde
1a-1e
1c = 4-CH3,
1d = 4-F,
1e = 4-OH
Scheme 1 Synthesis of target compounds 1a–1e
Hindu University, Varanasi, India. The animals were housed
at room temperature of 25 2 °C, 45–55% relative humid-
ity under 12 h light/dark cycles. Animals had free access to
water ad libitum and fed with semi-synthetic balanced diet.
The in vivo experimental protocol was duly approved by the
Central Animal Ethical Committee of BHU, Varanasi, India
(Protocol No. Dean/12-13/CAEC/18].
Synthesis of 5‑cyano‑2‑hydroxybenzoic acid (2)
Compound (2) was synthesized from the 5-aminosalicylic
acid by the reported procedure (Qiu et al. 1999; Sandmeyer
1884).
Synthesis of (Z)‑5‑(5‑benzylidene‑4‑oxo‑4,5‑dihy‑
drothiazol‑2‑yl)‑2‑hydroxybenzoic acid (3a–3e)
Acute oral toxicity study
The substituted aromatic aldehydes (5.34 mmol) were added
to the mixture of thioglycolic acid (9.19 mmol), compound
2 (6.13 mmol), and methanolic triethylamine (7.36 mmol).
The reaction mixture was stirred at room temperature for
30 min and then refuxed for 20–30 h. The reaction mixture
was evaporated under reduced pressure, and the crude prod-
uct was recrystallized from methanol. Further, the obtained
product was washed with acetone to aford the pure crystals
of compounds 3a–3e (Scheme 2) (Zayed et al. 1985).
The acute toxicity of synthesized compounds was deter-
mined as per the OECD 423 guidelines (OECD 423-2002) on
healthy nulliparous female rats. Tested compounds (1a–1e
and 3a–3e) were well tolerated up to a dose 500 mg kg−1.
Also, no mortality was observed till 14 days post-administra-
tion of the test compounds. The result showed the signifcant
safety margin of all the evaluated compounds.
Carrageenan‑induced rat paw edema (acute infammatory
model)
Physicochemical characterization
The partition coefcient (log P) of synthesized compounds
(1a–1e and 3a–3e) was determined using n-octanol and
bufer (pH 7.4) by the shake fask method. The log P was
calculated by correlating the absorbance with the concentra-
tion using a standard plot.
Carrageenan-induced rat paw edema model was used as
standard screening experiment to observe the acute infam-
mation. The paw edema was induced by sub-plantar admin-
istration of 0.1 ml of a 1% w/v lambda-carrageenan in nor-
mal saline on the left hind paw of the rats. The animals were
divided into various groups along with pre-treated standard
diclofenac (10 mg kg−1, p.o.) and equimolar dose of test
compounds (1a–1e and 3a–3e) in 0.3% CMC. The vehicle
was administered orally to the control group. The paw size
was measured in volume (mL) using plethysmometer after 2,
4, and 6 h of the intoxication of carrageenan injection. The
animals were pre-treated with the test compounds (1a–1e
Pharmacology
Animals
In vivo studies were performed on healthy Wistar rats
weighing around 200–250 gm procured from Central Ani-
mal Breeding House, Institute of Medical Sciences, Banaras
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Scheme 2 Synthesis of target compounds 3a–3e
and 3a–3e) and standard drug 1 h before the administration
forfeited with cervical dislocation. The incision was done
on the peritoneal cavity; stomach was removed, washed
with distilled water, rinsed with normal saline and
inspected through the 3X magnifying lens for any evidence
of ulcer. Lesions were counted, and ulcer index was calcu-
lated (Banerjee et al. 2015; Szelenyi and Thiemer 1978).
Further, histological studies were carried out on embedded
parafn blocks, fxed in 10% v/v formalin and stained with
hematoxylin and eosin.
of carrageenan (Winter et al. 1962).
Arachidonic acid‑induced rat paw edema
AA-induced rat paw edema model was used to assess anti-
infammatory activity specifc for LOX enzyme. The paw
edema was induced by sub-plantar administration of 0.1 ml
of 0.5% w/v AA in 0.2 M carbonate bufer (pH 8.2) on the
right hind paw of rats after 30 min post-drug treatment. The
0.3% CMC was used as a control, and the test compounds
were given at an equimolar dose relative to standard zileuton
10 mg kg−1 p.o. A digital vernier caliper measured changes
in thickness (mm) after 1 h of the AA injection (DiMartino
et al. 1987).
In‑vitro COX inhibition assay
The COX-1 and COX-2 inhibitory potential of active com-
pounds were evaluated using colorimetric COX (ovine)
Inhibitor Screening Assay Kit (Cayman Chemicals; Item
No. 560131). Diferent dilutions of celecoxib, diclofenac,
and compounds (1e and 3e) were incubated with the enzyme
for 5 min at 25 °C. After the incubation, the substrate and
AA were added, and the absorbance was recorded at 412 nm
using multimode microplate reader (Pradelles et al. 1985).
Acute ulcerogenic studies
Ulcerogenic liability of the most active compound (3e)
and standard diclofenac was evaluated on the 21st day of
the AA-induced rat paw edema bioassay. The rats were
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Chemical Papers
In‑vitro LOX inhibition assay
ligand-binding site was defned using Glide Grid Generation
module. The ligands were built-up using LigPrep module
application through the OPLS2005 force feld. OPLS stands
for optimized potential liquid simulations that give the cor-
responding minimum energy conformers of the ligands. The
default settings were used for all other parameters to perform
the docking simulations.
Soybean lipoxygenase (linoleate 13S-lipoxygenase) is fre-
quently used as a dependable screen for LOX inhibition. It
has been studied that AA-binding sites in soybean lipoxy-
genase contribute to almost the similar resemblance with
animal LOX enzyme (Tsurumi et al. 1986). The assay was
performed according to continuous spectrophotometric
rate determination method. The stock solution of lipoxy-
genase was prepared by dissolving 5000–10,000 units/ml
of lipoxidase in 200 mM borate bufer at pH 9.0. Further,
inhibitors (1e and 3e) in six diferent concentrations range
of 0.025–50 µM were selected and used for determination
of the enzyme inhibition activity. Increasing concentrations
of inhibitors were added to the enzyme solution and kept
for 5 min followed by the substrate addition. The result-
ing solution was incubated at room temperature for 20 min
with continuous shaking. The enzyme–substrate reaction
was considered maximum in the vehicle due to the forma-
tion of enzyme–substrate (ES) complex. Blank contains all
the substances except the enzyme solution to account for
non-enzymatic reactions. The concentration of each test
compound was recorded in triplicates at 234 nm using mul-
timode microplate reader, and their IC50 values were deter-
mined graphically from absorption v/s concentration curve
(Banerjee et al. 2016).
Molecular dynamics simulations
Molecular dynamics simulations confrmed the binding
mode and conformational stability of compound (3e) on
LOX enzyme. Desmond package incorporated in Schro-
dinger Maestro 10.5.014 program using OPLS-AA force
feld was used to predict the thermodynamic stability of
the protein–ligand system. Simulations were performed by
placing the docked complex (3e+LOX enzyme) in prede-
fned TIP3 water and orthorhombic periodic boundary con-
dition. Initially, the volume of periodic box condition was
calculated as 750,210 Å3 and was minimized to 716,092 Å3.
The solvated system was neutralized with the addition of
18 Na+ counterions. The system was reduced to 2000 steps
and relaxed using the default protocols. Molecular dynamics
simulations were carried out at normal volume, and tem-
perature and pressure were kept at 300 K and 1 atmospheric
pressure. Finally, the system was allowed to run for 50 ns
time scale and trajectories were recorded at 5 ps interval.
Enzyme kinetic studies
In‑silico ADME studies
Continuous spectrophotometric rate determination method
was used to identify the type of enzyme inhibition by the
compound (3e). The diferent concentrations of substrate
(AA) were prepared in Tween-20 in 200 mM borate bufer
at pH 9.0. The fxed concentration of LOX enzyme (10,000
units/ml) was prepared in 200 mM borate bufer at pH 9.0.
The enzyme kinetics was analyzed at the diferent concen-
trations of the substrate either in the absence or presence
of compound (3e). The enzyme kinetics was studied by
Lineweaver and Burk method (Lineweaver and Burk 1934).
The QikProp module of Schrodinger Maestro 10.5.014
Release 2016-1 was used to predict the ADME properties
of the active compound 3e and standards (zileuton, C06, and
ST1098). It predicts the pharmaceutically relevant properties
of the compounds along with several descriptors. Ligands
were initially neutralized and used for the QikProp analysis.
The program was processed in a normal mode to observe the
physiological properties including the total solvent accessi-
ble surface area (SASA), Van der Waals surface area of polar
nitrogen and oxygen atoms (PSA), hydrogen bond donor,
hydrogen bond acceptor, predicted hexadecane/gas partition
coefcient (QPlogPC16), Predicted octanol/gas partition
coefcient (QPlogPoct), and predicted octanol/water parti-
tion coefcient (QPlogPo/w).
Computational studies
Molecular docking study
The synthesized compounds (1e and 3e) were computa-
tionally docked into the LOX enzyme pockets (PDB Code:
3V99) using Glide XP module (Schrodinger, LLC, USA,
2016-1 Maestro 10.5.014) to gain structural insights of bind-
ing of a ligand to the enzyme. The crystal structure of the
enzyme–substrate complex was retrieved from the protein
data bank and used for molecular docking. The enzyme
was optimized with the “protein preparation wizard” work-
flow. The co-crystallized ligand was extracted, and the
Results and discussion
Chemistry and spectral characterization
The 4-thiazolone derivatives were designed by substituting
more polar groups such as hydroxyl on 5-benzylidene-4-thi-
azolone moieties for improving hydrophilic characteristics.
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Table 1 Results of spectral characterization
Compound Spectral data
1a
1b
1c
1d
1e
FT-IR, cm−1: 1664 (>C=O), 1564 (>C=N)
1H-NMR (DMSO-d6, 500 MHz), δ: 8.20 (d, J=8.0 Hz, 2H, Cl-Ph-3,5H), 8.01 (s, 1H, –CH=), 7.64 (d, J=8.5 Hz, 2H, Cl-Ph-2,6H),
7.55–7.24 (m, 5H, benzylidene).
13C-NMR (DMSO-d6, 125 MHz), δ: 185.4 (thia-4C), 183.1 (thia-2C), 140.0 (thia-5C), 138.2 (Cl-Ph-2,6C), 138.1 (Cl-Ph-1C), 132.3
(benzylidene-4C), 131.2 (–CH=), 131.0 (Cl-Ph-3,5C), 129.5 (Cl-Ph-4C), 128.0 (benzylidene-2,6C), 127.3 (benzylidene-1C),
126.2 (benzylidene-3,5C).
FT-IR, cm−1: 1681 (>C=O), 1593 (>C=N)
1H-NMR (DMSO-d6, 500 MHz), δ: 8.23 (d, J=8.5 Hz, 2H, Cl-Ph-3,5H), 8.07 (s, 1H, –CH=), 7.84 (d, J=7.5 Hz, 2H, Cl-ben-
zylidene-2,6H), 7.77 (d, J=8.0 Hz, 2H, Cl-benzylidene-3,5H), 7.67 (d, J=8.0 Hz, 2H, Cl-Ph-2,6H).
13C-NMR (DMSO-d6, 125 MHz), δ: 184.1 (thia-4C), 182.1 (thia-2C), 140.3 (thia-5C), 139.8 (Cl-Ph-2,6C), 138.1 (Cl-Ph-1C), 135.3
(Cl-benzylidene-1C), 132.4 (–CH=), 130.7 (Cl-benzylidene-2,6C), 130.3 (Cl-benzylidene-4C), 130.2 (Cl-Ph-3,5C), 129.5 (Cl-Ph-
4C), 128.5 (Cl-benzylidene-3,5C).
FT-IR, cm−1: 1675 (>C=O), 1591 (C=N)
1H-NMR (DMSO-d6, 500 MHz), δ: 7.93 (s, 1H, –CH=), 7.78 (d, J=8.0 Hz, 2H, Cl-Ph-3,5H), 7.65 (d, J=8.0 Hz, 2H, Cl-Ph-2,6H),
7.45 (d, J=8.2 Hz, 2H, CH3-benzylidine-3,5), 7.12 (d, J=8.0 Hz, 2H, CH3-benzylidene-2,6H), 2.65 (s, 3H, –CH3-Ph).
13C-NMR (DMSO-d6, 125 MHz), δ: 185.1 (thia-4C), 182.2 (thia-2C), 141.1 (thia-5C), 139.1 (Cl-Ph-1C), 137.2
(CH3-benzylidene-1C), 131.1 (–CH=), 130.2 (Cl-Ph-3,5C), 130.1 (CH3-benzylidene-3,5C), 129.8 (Cl-Ph-4C), 129.7 (Cl-Ph-
2,6C), 129.1 (CH3-benzylidene-4C), 131.0 (CH3-benzylidene-2,6C), 22.1 (–CH3).
FT-IR, cm−1: 1664 (>C=O), 1592 (>C=N)
1H-NMR (DMSO-d6, 500 MHz), δ: 8.23-7.98 (m, 3H, F-benzyledine-3,5H and –CH=), 7.78 (d, J=8.5 Hz, 2H, Cl-Ph-3,5H), 7.68
(d, J=8.0 Hz, 2H, Cl-Ph-2,6H), 7.30 (d, JH,H=8.4 Hz, JH,F=8.9 Hz, 2H, F-benzyledine-2,6H).
13C-NMR (DMSO-d6, 125 MHz), δ: 184.7 (thia-4C), 182.3 (thia-2C), 167.1 (JC,F =249.0 Hz, F-benzylidene-1C), 139.0 (thia-5C),
139.4 (Cl-Ph-1C), 131.1 (–CH=), 131.2 ((JC,F =8.6 Hz, F-benzylidene-3,5C), 130.2 (Cl-Ph-3,5C), 129.7 (Cl-Ph-2,6C), 129.8 (Cl-
Ph-4C), 127.8 (JC,F =3.5 Hz, F-benzylidene-4C), 116.7 (JC,F =23.2 Hz, F-benzylidene-2,6C).
FT-IR, cm−1: 3163 (-OH), 1664 (>C=O), 1597 (>C=N)
1H-NMR (DMSO-d6, 500 MHz), δ: 10.53 (s, 1H, –OH), 8.19 (d, J=8.0 Hz, 2H, HO-benzylidene-3,5H), 7.97 (s, 1H, –CH=), 7.75
(d, J=8.5 Hz, 2H, Cl-Ph-3,5H), 7.69 (d, J=8.0 Hz, 2H, Cl-Ph-2,6H), 6.96 (d, J=8.5 Hz, 2H, HO-benzylidene-2,6H).
13C-NMR (DMSO-d6, 125 MHz), δ: 184.4 (thia-4C), 182.1 (thia-2C), 161.1 (HO- benzylidene-1C), 140.0 (thia-5C), 139.0 (Cl-
Ph-1C), 133.4 (–CH=), 130.1 (Cl-Ph-3,5C), 130.0 (Cl-Ph-2,6C), 129.8 (Cl-Ph-4C), 124.3 (HO-benzylidene-4C), 121.8 (HO-
benzylidene-3,5C), 116.6 (HO-benzylidene-2,6C).
2
FT-IR, cm−1: 3458 (–OH), 2235 (–CN), 1685 (>C=O, –COOH)
1H-NMR (DMSO-d6, 500 MHz), δ: 8.14 (s, 1H, Ph-6H), 7.85 (d, J=8.5 Hz 1H, Ph-4H), 7.06 (d, J=9.0 Hz 1H, Ph-3H).
13C NMR (DMSO-d6, 125 MHz), δ: 169.9 (>C=O, –COOH), 164.8 (Ph-2C), 137.9 (Ph-4C), 135.1 (Ph-6C), 118.7 (Ph-3C), 118.6
(Ph-1C), 115.6 (–CN), 100.8 (Ph-5C).
3a
FT-IR, cm−1: 3365 (–COOH), 3028 (Ph-H), 1684, 1657 (>C=O), 1591 (–C=N)
1H-NMR (DMSO-d6, 500 MHz), δ: 10.40 (s, 1H, –COOH), 8.35 (d, J=2.5 Hz, 1H, COOH-Ph-6H), 7.87 (s, 1H, –CH=), 7.71 (d,
J=3.0 Hz, 1H, COOH-Ph-4H), 7.54 (d, J=7.5 Hz, 2H, benzylidene-3,5H), 7.21–7.45 (m, 3H, benzylidene-1,2,6H), 6.89 (d,
J=8.5 Hz,1H,COOH-Ar-3H),
13C-NMR (DMSO-d6, 125 MHz), δ: 185.4 (thia-4C), 182.0 (thia-2C), 169.0 (–COOH), 162.4 (HO-Ph-2C), 140.0 (thia-5C), 135.1
(HO-Ph-4C), 128.2 (benzylidene-1C), 135.4 (HO-Ph-6C), 131.8 (–CH=), 130.2 (benzylidene-4C), 127.5 (benzylidene-2,6C),
126.5 (benzylidene-3,5C), 125.2 (HO-Ph-5C), 118.3 (HO-Ph-3C), 116.0 (HO-Ph-1C).
3b
3c
FT-IR, cm−1: 3365 (–COOH), 3039 (Ph-H), 1681, 1668 (>C=O), 1555 (–C=N)
1H-NMR (DMSO-d6, 500 MHz), δ: 10.39 (s, 1H, –COOH), 8.35 (d, J=2.5 Hz, 1H, COOH-Ph-6H), 7.89 (s, 1H, –CH=), 7.78 (d,
J=8.5 Hz, 2H, Cl-benzylidene-3,5H), 7.70 (d, J=2.5 Hz, 1H, COOH-Ph-4H), 7.58 (d, J=8.5 Hz, 2H, Cl-benzylidene-2,6H),
6.80 (d, J=8.5 Hz, 1H, COOH-Ph-3H).
13C-NMR (DMSO-d6, 125 MHz), δ: 185.2 (thia-4C), 183.1 (thia-2C), 168.9 (–COOH), 162.1 (HO-Ph-2C), 139.2 (thia-5C), 136.4
(HO-Ph-4C), 136.1 (Cl-benzylidene-1C), 135.1 (HO-Ph-6C), 132.0 (–CH=), 130.2 (Cl-benzylidene-4C), 130.6 (Cl-benzylidene-
2,6C), 129.0 (Cl-benzylidene-3,5C), 124.2 (HO-Ph-5C), 118.4 (HO-Ph-3C), 117.0 (HO-Ph-1C).
FT-IR, cm−1: 3440 (–COOH), 3039 (Ar–H), 1685, 1664 (>C=O), 1561 (–C=N).
1H-NMR (DMSO-d6, 500 MHz), δ: 10.39 (s, 1H, –COOH), 8.35 (d, J=3.0 Hz, 1H, COOH-Ph-6H), 7.74 (s, 1H, –CH=), 7.71 (d,
J=2.5 Hz, 1H, COOH-Ph-4H), 7.33 (d, J=8.0 Hz, 2H, CH3-benzylidene-3,5H), 7.15 (d, J=8.5 Hz, 2H, CH3-benzylidene-2,6H),
6.85 (d, J=8.5 Hz, 1H, COOH-Ar-3H), 2.74 (s, 3H, –CH3-Ar).
13C-NMR (DMSO-d6, 125 MHz), δ: 184.8 (thia-4C), 182.2 (thia-2C), 168.2 (–COOH), 162.0 (HO-Ph-2C), 141.2 (thia-5C),
136.9 (CH3-benzylidene-1C), 135.8 (HO-Ph-4C), 135.0 (HO-Ph-6C), 131.2 (–CH=), 130.8 (CH3-benzylidene-2,6C), 130.2
(CH3-benzylidene-3,5C), 128.9 (CH3-benzylidene-4C), 124.2 (HO-Ph-5C), 118.7 (HO-Ph-1C), 117.5 (HO-Ph-3C), 22.9
(CH3-Ph).
1 3

Chemical Papers
Table 1 (continued)
Compound Spectral data
3d
FT-IR, cm−1: 3441 (–COOH), 3041 (Ph-H), 1677, 1664 (>C=O), 1561.41 (–C=N)
1H-NMR (DMSO-d6, 500 MHz), δ: 10.34 (s, 1H, –COOH), 8.48 (d, J=2.5 Hz, 1H, COOH-Ph-6H), 7.99 (d, J=2.5 Hz, 1H,
COOH-Ph-4H), 7.79 (s, 1H, –CH=), 7.73 (d, JH,H=8.5 Hz, JH,F=5.8 Hz, 2H, F-benzylidene-3,5H), 7.35 (d, JH,H=8.0 Hz,
JH,F=9.1 Hz, 2H, F-benzylidene-2,6H), 6.77 (d, J=8.5 Hz, 1H, COOH-Ar-3H).
13C-NMR (DMSO-d6, 125 MHz), δ: 184.8 (thia-4C), 182.6 (thia-2C), 169.7 (–COOH), 167.2 (JC,F =246.2 Hz, F-benzylidene-1C),
161.7 (HO-Ph-2C), 138.1 (thia-5C), 136.5 (HO-Ph-4C), 134.8 (HO-Ph-6C), 131.0 (–CH=), 128.0 (JC,F =3.5 Hz, F-benzylidene-
4C), 125.2 (HO-Ph-5C), 130.7 (JC,F =7.7 Hz, F-benzylidene-3,5C), 118.2 (HO-Ph-3C), 118.4 (HO-Ph-1C), 116.6 (JC,F =22.8 Hz,
F benzylidene-2,6 C).
3e
FT-IR, cm−1: 3416, 3363 (–OH), 3066 (Ph-H), 1629 (>C=O), 1566 (–C=N).
1H-NMR (DMSO-d6, 500 MHz), δ: 10.35 (s, 1H, –COOH), 8.48 (d, J=2.5 Hz, 1H, COOH-Ph-6H), 8.01 (d, J=2.5 Hz, 1H,
COOH-Ph-4H), 7.77 (s, 1H, –CH=), 7.65 (d, J=8.5 Hz, 2H, HO-benzylidene-3,5H), 6.95 (d, J=8.5 Hz, 2H, HO-benzylidene-
2,6H), 6.74 (d, J=9.0 Hz, 1H, COOH-Ph-3H).
13C-NMR (DMSO-d6, 125 MHz), δ: 185.0 (thia-4C), 182.6 (thia-2C), 169.7 (–COOH), 161.7 (HO-Ph-2C), 161.2 (HO-benzylidene-
1C), 139.9 (thia-5C), 136.5 (HO-Ph-4C), 134.8 (HO-Ph-6C), 132.9 (–CH=), 125.0 (HO-Ph-5C), 124.4 (HO-benzylidene-4C),
122.2 (HO-benzylidene-3,5 C), 118.3 (HO-Ph-3 C), 118.2 (HO-Ph-1C), 116.4 (HO-benzylidene-2,6 C).
C06
1H-NMR (DMSO-d6, 500 MHz), δ: 8.12 (d, J=8.0 Hz, 2H, CH3-Ph-3,5H), 8.02 (s, 1H, –CH=), 7.66 (d, J=8.5 Hz, 2H,
CH3O-benzyledine-3,5),7.37 (d, J=8.0 Hz, 2H, CH3-Ph-2,6H), 7.03 (d, J=8.5 Hz, 2H, CH3O-benzylidene-2,6H), 3.91 (s, 3H,
-OCH3-Ph), 2.49 (s, 3H, –CH3-Ph).
13C-NMR (DMSO-d6, 125 MHz), δ: 186.4 (thia-4C), 183.6 (thia-2C), 161.9 (CH3O-benzylidene-1C), 146.3 (thia-5C), 138.1
(CH3-Ph-1C), 132.7 (CH3-Ph-3,5C), 129.9 (CH3-Ph-2,6C), 129.3 (–CH=), 128.8 (CH3O-benzylidene-3,5C), 126.3 (CH3-Ph-4C),
123.7 (CH3O-benzylidene-4C), 114.8 (CH3O-benzylidene-2,6C), 55.5 (–OCH3), 21.9 (–CH3).
ST1098
1H-NMR (DMSO-d6, 500 MHz), δ: 8.16 (d, J=8.5 Hz, 2H, Cl-Ph-3,5H), 8.05 (s, 1H, –CH=), 7.66 (d, J=9.0 Hz, 2H,
CH3O-benzyledine-3,5), 7.55 (d, J=8.5 Hz, 2H, Cl-Ph-2,6H), 7.04 (d, J=8.5 Hz, 2H, CH3O-benzylidene-2,6H), 3.91 (s, 3H, –
OCH3-Ph).
13C-NMR (DMSO-d6, 125 MHz), δ: 185.1 (thia-4C), 183.2 (thia-2C), 162.2 (CH3O-benzylidene-1C), 141.3 (thia-5C), 139.1
(Cl-Ph-1C), 132.9 (Cl-Ph-3,5C), 130.4 (–CH=), 129.8 (Cl-Ph-2,6C), 129.3 (CH3O-benzylidene-3,5C), 126.3 (Cl-Ph-4C), 123.9
(CH3O-benzylidene-4C), 114.9 (CH3O-benzylidene-2,6C), 55.5 (–OCH3).
Apart from the substituted hydroxyl group at 4th position,
some lipophilic compounds have been developed by substi-
tuting methyl, chloro, and fuoro groups at the same position
on 5-benzylidene. In the same way, more hydrophilic com-
pounds were also developed by placing O-hydroxybenzoic
acid at 2nd position of 4-thiazolones.
p-hydroxybenzylidene (HO-benzylidene-1C) displayed a
peak at 161.1 ppm.
Further, the nitrosation of 5-aminosalicylic acid with
nitrous acid (in situ from sodium nitrite and hydrochloric
acid) leads to diazonium salts, and subsequent substitution
of a cyanide nucleophile (–CN) under copper (I) cyanide
catalysis generated the 5-cyano-2-hydroxy benzoic acid (2)
(Sandmeyer reaction) with the removal of nitrogen gas. The
FT-IR spectrum confrmed its structure, eliciting an original
strong stretching band of nitrile group (–CN) at 2235 cm−1,
and (>C=O) of carboxylic acid at 1685 cm−1. The 13C-NMR
exhibited a unique up-feld value of phenyl-1C containing
carboxylic acid, cyano (–CN) and cyano-attached carbon
(phenyl-5C) at 118.6, 115.6, 100.8 ppm, respectively.
The 4-thiazolone derivatives (3a–3e) were synthesized
by reacting thioglycolic acid, 5-cyano-2-hydroxybenzoic
acid (2) and substituted aromatic benzaldehyde to generate
the 4-thiazolone compounds (Scheme 2). The FT-IR spec-
tra of derivatives (3a–3e) exhibited characteristic stretch-
ing bands for carbonyl (>C=O) groups of carboxylic acid
between 1684 and 1670 cm−1, (–OH) of carboxylic acid at
3365–3340 cm−1 and imine (–C=N) appeared in the range
of 1591–1555 cm−1. The 1H-NMR of compounds (3a–3e)
showed one proton peak at ~ 10.4 ppm for a carboxylic
acid (–OH). The 13C-NMR peak of carbonyl (>C=O)
of carboxylic acid was observed at 169.7–168.2 ppm,
The 4-thiazolone derivatives (1a–1e and 3a–3e) were
synthesized by reacting of thioglycolic acid with 4-chlo-
robenzonitrile and substituted benzaldehyde through one-pot
multicomponent reaction (Zayed et al. 1985). The thiogly-
colic acid and 4-chlorobenzonitrile were reacted in etha-
nol with triethylamine to get the 4-thiazolone core which
was then condensed in situ with substituted benzaldehyde
under the refuxed condition to generate the 4-thiazolone
derivatives (1a–1e) (Scheme 1). The FT-IR spectra of com-
pounds (1a–1e) showed distinctive stretching bands for
carbonyl group >C=O in the range of 1682–1664 cm−1
,
(–C=CH–) of a benzylidene at 1478–1425 cm−1 and (–C=N)
of a 4-thiazolone nucleus at 1597–1555 cm−1. Compound
(1e) displayed a characteristic hydroxyl (–OH) stretching
1
band at 3163 cm−1. The H-NMR of (1e) showed broad
singlet proton peak at 10.53 ppm of a phenolic hydroxyl
group and (–C=CH–) proton of a benzylidene at around
of 8.07 ppm. The 13C-NMR peak of the carbonyl group
(>C=O) was observed between 185.4 and 184.1 ppm, and
imine (>C=N) peak in the range of 182.6–182.0 ppm. The
1 3

Chemical Papers
Table 2 Results of physical
characterization and elemental
analysis
Compound
Formula (Mr)
wi(calc.)/%
Rfa
Yield (%)
M.P.b
Log Pc
wi(found)/%
C
H
N
1a#
1b
C16H10ClNOS
299.02
64.11
64.35
3.36
3.35
4.67
4.68
0.55
0.35
0.42
0.45
0.32
–
45
38
34
43
37
65
29
40
24
31
21
62
66
273–275
279–281
315–317
253–255
345–347
–
3.93
4.21
4.32
4.40
4.07
–
C16H9Cl2NOS
57.50
2.71
4.19
334.22
57.30
2.72
4.20
1c
C17H12ClNOS
313.80
65.07
65.30
3.85
3.84
4.46
4.44
1d
C16H9ClFNOS
60.48
2.85
4.41
317.77
60.47
2.86
4.40
1e*
2
C16H10ClNO2S
315.77
60.86
61.10
3.19
3.53
4.44
4.91
C8H5NO3
58.90
3.09
8.59
163.13
59.14
3.07
8.56
3a
C17H11NO4S
325.34
62.76
62.01
3.41
3.42
4.31
4.30
0.45
0.18
0.51
0.58
0.53
0.44
0.41
286–288
272–274
303–305
242–244
357–359
222–224
186–188
3.11
3.36
3.59
2.98
3.72
4.40
4.52
3b
C17H10ClNO4S
56.75
2.80
3.89
359.78
56.61
2.79
3.88
3c
C18H13NO4S
339.37
63.71
63.90
3.86
3.87
4.13
4.14
3d
C17H10FNO4S
59.47
2.94
4.08
343.03
59.48
2.95
4.10
3e
C17H11NO5S
341.34
59.82
59.98
3.25
3.56
4.10
4.24
C06
ST1098
C18H15NO2S
69.88
4.89
4.53
309.38
69.81
4.90
4.49
C17H12ClNO2S
329.80
61.91
61.95
3.67
3.59
4.25
4.29
#Liang et al. Liang et al. 2016
*Hofmann et al. Hofmann et al. 2008
aSolvent system: EtOAc:hexane (3:2)
bMelting points were determined using open capillary tubes on a Stuart melting point apparatus (SMP10)
and were uncorrected
cLog P values were determined using shake fask method
whereas the carbonyl (>C=O) of the 4-thiazolone nucleus
at around 185.4–184.8 ppm, and imine (–C=N) in range
182.6–182.0 ppm. These spectral data confrmed the pres-
ence of carbonyl groups, a hydroxyl group, benzylidene
group and carboxylic acid in 4-thiazolone derivatives. Spec-
tral characterization of all derivatives by FT-IR, 1H-NMR,
and 13C-NMR is summarized in Table 1. The results of the
elemental analyses were within 0.4% of theoretical values.
These thiazolones were further subjected to physiochemical
characterization (Table 2).
ulcerogenic liability remain unexplored till this date. This
was also the fact that compound C06 could not achieve the
desired plasma and tissue concentration due to poor aqueous
solubility (Hofmann et al. 2012). In the present work, we
have explored the anti-infammatory activity of synthesized
compound (1a–1e and 3a–3e) against the carrageenan and
AA-induced rat paw edema model for assessing the contri-
bution of mediators involved in vascular changes associated
with acute infammation. The ulcerogenic liability was also
observed to establish the gastrointestinal toxicity profle.
Pharmacology
Carrageenan‑induced rat paw edema
In the literature, 4-thiazolone compound (Z)-5-(4-
methoxybenzylidene)-2-(p-tolyl)thiazol-4(5H)-one (C06)
was reported as potential LOX inhibitor on the cellu-
lar assay, but its in vivo anti-infammatory activity and
Carrageenan-induced rat paw edema model is frequently
used for evaluation of the acute infammatory response gen-
erated by cyclooxygenase (COX) enzyme. The progression
of the carrageenan-induced hind paw edema by injection
1 3

Chemical Papers
Table 3 Paw volume and % inhibition as calculated by carrageenan-
Table 4 % Mean protection
by AA-induced rat paw edema
model
Compound
Mean
induced rat paw edema model
protectiona
(%)
Compound
aMean protection (% inhibition)
Control
Zileuton
1a
–
2 h
4 h
6 h
66.15
36.92
41.53
40.00
43.07
55.38
47.69
55.38
50.76
35.38
58.46
Control
Diclofenac
0.63 0.03
0.86 0.04
0.92 0.06
0.19 0.01 (69.84) 0.14 0.01 (83.72) 0.12 0.01
1b
(86.96)
1c
1a
1b
1c
1d
1e
3a
3b
3c
3d
3e
0.43 0.03 (31.75) 0.46 0.03 (46.51) 0.49 0.03
(46.74)
0.32 0.02 (49.21) 0.30 0.02 (65.12) 0.39 0.02
1d
1e
(57.61)
3a
0.39 0.03 (38.10) 0.38 0.02 (55.81) 0.37 0.02
(59.78)
0.41 0.03 (34.92) 0.41 0.03 (52.32) 0.42 0.03
3b
3c
3d
(54.35)
3e
0.24 0.02 (61.90) 0.22 0.01 (74.42) 0.19 0.02
(79.35)
0.39 0.03 (38.10) 0.35 0.02 (59.30) 0.33 0.02
All other compounds were
administered at an equimolar
oral dose relative to 10 mg kg−1
standard
(64.13)
0.27 0.02 (57.14) 0.24 0.02 (72.09) 0.23 0.02
(75.00)
0.36 0.03 (42.86) 0.31 0.02 (63.95) 0.39 0.03
aStandard zileuton (10 mg kg−1
p.o) in 0.3% CMC solution
(57.61)
Protection (%) was expressed as
% edema inhibition of the tested
compounds relative to % edema
inhibition of standard
0.44 0.04 (30.16) 0.42 0.03 (51.16) 0.43 0.01
(53.26)
0.22 0.02 (65.08) 0.20 0.02 (76.74) 0.19 0.02
(79.35)
inhibitory activity (54.35, and 53.26%). The results thus
reafrm the anti-infammatory potential of compounds (1e
and 3e).
All other compounds were administered at an equimolar oral dose
relative to 10 mg kg−1 diclofenac
Control 0.3% carboxymethylcellulose sodium (CMC) solution in dis-
tilled water (10 ml/kg/p.o.), Standard diclofenac (10 mg kg−1 p.o) in
0.3% CMC solution
aMean protection was expressed as (%) edema inhibition of the tested
compounds relative to (%) edema inhibition of standard
Arachidonic acid‑induced rat paw edema
AA-induced rat paw edema is used extensively to evalu-
ate the anti-infammatory activity through LOX inhibition
pathway. This model is more sensitive to observe the LOX
than COX enzyme inhibition (DiMartino et al. 1987). The
LOX inhibitors appreciably reduce the paw edema induced
by AA. The results (Table 4) showed that synthesized com-
pounds (1a–1e and 3a–3e) exhibited anti-inflammatory
activity against AA-induced rat paw edema. Compound
3e and the standard zileuton considerably inhibit the AA-
induced infammation with the mean protection percent-
age of 58.46 and 66.15%, respectively. The test compounds
of lambda-carrageenan in the rat is a biphasic event. The
early phase (1–2 h) releases pro-infammatory agents such
as histamine and serotonin, whereas the later stage (post-3-h
treatment) release prostaglandins (Winter et al. 1962).
The anti-inflammatory activity of the 4-thiazolone
compounds (1a–1e and 3a–3e) was evaluated against car-
rageenan-induced hind paw edema model and reported
in Table 3. Amongst all ten evaluated compounds, three
compounds (1e, 3e, and 3b) showed considerable anti-
infammatory activity 79.35, 79.35, and 75.00%, respec-
tively. Compounds 1e and 3e comprising of strong elec-
tron-donating hydroxyl (-OH) substituent on benzylidene
elicited the maximal anti-infammatory activity amongst
the synthesized derivatives. Compounds (3a, 1c, and 3c)
comprising of an electron-donating phenyl (3a) and methyl
(-CH3) substituent on benzylidene (1c and 3c) exhibited
an average reduction of 64.13, 59.78, and 57.61% of
infammation. Compounds (1d and 3d) comprising of
strong electron withdrawing substituents exhibited weak
Table 5 The ulcerogenic
Compounds
Ulcer
activity of compound 3e
index range
(mean SEM)
3e
10.2 1.4
46.0 4.1
52.0 4.9
Diclofenac
Zileuton
1 3

Chemical Papers
Fig. 2 Photomicrographs (10x magnifcation) of
a
control;
b
detachment of surface epithelium resulting in the formation of gastric
lesions. Black arrow shows the normal stomach structure with less or
no ulcer formation
diclofenac; c Zileuton and d compound 3e-treated groups in rat stom-
ach tissues (hematoxylin and eosin staining). Green arrow shows
Table 6 IC50 values of
Compounds
COX-1 IC50
(µM) SEM
COX-2IC50
LOX IC50 (µM) SEM
Type of inhibition
LOX
compounds 1e and 3e
(µM) SEM
Ki (µM)
1e
>50
>50
>50
>50
12.98 0.17
12.67 0.24
nda
nda
nda
3e
Non-competitive
0.73
Celecoxib
Diclofenac
Zileuton
C06b
6.70 0.03
0.15 0.01
nda
0.87 0.04
0.05 0.01
nda
nda
nda
nda
nda
nda
3.43 0.04
9.86 0.02
8.20 0.02
Non-competitive
Non-competitive
Non-competitive
0.49
0.58
0.56
nda
nda
ST1098c
nda
nda
aNot determined
bHofmann et al. 2012
cBarzen et al. 2012
(1a–1e and 3a–3e) inhibited the formation of edema up to
35.38–58.46%. Based on the outcome of the study, mono-
substituted electronegative (-OH) group on benzylidene of
4-thiazolone can be considered as a key marker in the deter-
mination of anti-infammatory activity of p-substituted ben-
zylidene 4-thiazolone derivatives. Our fndings revealed that
5-benzylidene-4-thiazolones with substituted electronegative
(-OH) groups are efective inhibitors of LOX enzyme.
Acute ulcerogenic studies
The most active compound (3e) and standard drugs
(diclofenac and zileuton) were subjected to their ulcero-
genic liabilities in rats. The results of ulcer index (UI) of
the test compound and standard drugs are calculated and
mentioned in Table 5. The study demonstrated that com-
pound (3e) has minimal gastric insult compared to standard
1 3

Chemical Papers
drugs diclofenac and zileuton. Gastric mucosa lesion liabil-
ity was evaluated and supported by histological evaluation
(Fig. 2). These outcomes revealed that the test compound
(3e) elicited less damage to the gastric mucosa compared
to standard drugs.
showed to occupy the active binding pocket of the enzyme
and are involved in the interactions with the active site
amino acid residues (Fig. 4a, b).
Molecular dynamics simulations
Molecular dynamics simulations of compound 3e with LOX
enzyme (PDB Code: 3V99) was performed up to 50 ns time
scale to predict the binding stability of the docked confor-
mation. The results were analyzed using root mean square
deviation (RMSD) and root mean square fuctuation (RMSF)
protocols. The protein–ligand contacts during the entire sim-
ulation time scale were also analyzed.
In‑vitro COX inhibition assay
In-vitro COX inhibition of compound (1e and 3e) was per-
formed on ovine COX-1 and human COX-2 enzymes using
colorimetric enzyme immunoassay (EIA). Along with
the compounds (1e and 3e), the standard diclofenac and
celecoxib were tested and results are reported in Table 6. The
results suggested that compounds (1e and 3e) have signif-
cantly lower inhibitory potential against COX-1 and COX-2
(COX-1; IC50 >50 µM, COX-2; IC50 >50 µM) compared to
standard drugs diclofenac (COX-1; IC50 =0.15 0.01 µM,
COX-2; IC50 = 0.05 0.01 µM) and celecoxib (COX-1;
IC50 =6.70 0.03 µM, COX-2; IC50 =0.87 0.04 µM).
The RMSD value revealed that initially up to 25 ns time
scale, trajectories were unstable and showed several fuctua-
tions. Stable trajectories were observed after 25 ns timescale
with average fuctuation less than 1 Å (Fig. 5).
Further, root mean square fuctuations (RMSF) of active
amino acid residues of the enzyme with compound 3e exhib-
ited stable movements (Supplementary Fig. S17).
The interactions of the ligand with protein play a sig-
nificant role in the stability of the protein–ligand com-
plex. The interaction pattern of compound 3e with LOX
enzyme was also evaluated (Supplementary Fig. S18).
The results showed that amino acid residues Phe177,
Thr364, Lys409, Ala410, Glu412, His432, and Leu607
contribute to strong hydrogen bonding interaction
throughout the simulation timescale. The interactions
observed in docking were also retained throughout the
simulation run. The percentage of interaction by the inter-
acting residue is analyzed and represented as a histogram
in Fig. 6.
In‑vitro LOX inhibition assay
The active compounds (1e and 3e) were evaluated against
in vitro LOX enzyme to determine the respective IC50
values to ascertain their specifcity. The results showed
that compounds 1e (IC50 = 12.98 0.17 µM) and 3e
(IC50 = 12.67 0.24 µM) have considerable lipoxygenase
(LOX) enzyme inhibitory activity with standard zileuton
(IC50=3.43 0.04 µM), ST1098 (IC50=8.20 0.02 µM)
and C06 (IC50=9.86 0.02 µM), respectively (Table 6).
Enzyme kinetic studies
The Lineweaver–Burk plot was used to determine the type of
enzyme inhibition. The active compound (3e) was evaluated
for substrate-dependent enzyme kinetics (Fig. 3). In the pres-
ence or absence of inhibitor, diferent 1/Vmax (y-intercept)
values were obtained, and inhibition constant (Ki) was cal-
culated (Table 6). The Lineweaver–Burk plot analysis sug-
gested that compound (3e) is the non-competitive inhibitor.
1200
1000
800
Computational studies
600
3e
400
Molecular docking study
Substrate
200
The compounds (1e and 3e) with efective in vitro LOX
inhibitory activity were evaluated for in silico docking stud-
ies to measure the binding interactions at the active binding
pocket of LOX enzyme (PDB Code: 3V99). The docking
simulations were performed using the Glide-XP protocol
in the Schrödinger software suite (Schrödinger, LLC, USA,
2016-1 Maestro 10.5.014). The compounds (1e and 3e)
0
-0.15
-0.1
-0.05
-200
0
0.05
0.1
0.15
1/[S] X 10-6 M
Fig. 3 In-vitro LOX enzyme kinetics showing the non-competitive
type of inhibition by compound 3e
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Chemical Papers
Fig. 4 a Binding mode of 1e docked into the ‘active site’ of LOX enzyme. b Binding mode of 3e docked into the ‘active site’ of LOX enzyme
In‑silico ADME studies
Conclusions
The compound 3e was also evaluated for drug likeliness
characteristics using QikProp module of Schrödinger Suite.
The results suggested that compound 3e has more polar sur-
face area and lower predicted partition coefcient values,
compared to standards C06 and ST1098 (Table 7).
The synthesized compounds (1e and 3e) exhibited poten-
tial anti-infammatory activities against carrageenan and
AA-induced rat paw edema models with a lesser ulcero-
genic insult compared with standard drugs diclofenac and
zileuton. The kinetic study of (3e) revealed a non-com-
petitive type of inhibition. The strong electron-donating
1 3

Chemical Papers
Fig. 5 RMSD graph of the compound (3e) and LOX enzyme complex for the period of 50 ns simulation
hydroxyl group on benzylidene of compound (3e) exhib-
ited high mean protection against infammation compared
to compounds (1a–1d and 3a–3d). The docking studies
illustrated that compound (3e) has an excellent ft into
the pocket of LOX enzyme with minimum energy com-
pared with compound (1e), which might be attributed
to better hydrogen bonding interaction of p-hydroxy
benzylidene at 5th position and o-hydroxybenzoic acid
at 2nd position of 4-thiazolone. Further, molecular
dynamics studies confrmed that active site interaction
of compound (3e) was stable throughout simulation time-
scale. With these fndings, it can be postulated that the
Fig. 6 Protein–ligand contacts through 50 ns simulation of compound 3e–LOX enzyme
1 3

Chemical Papers
Table 7 Comparative
QikProp analysis properties of
compounds 3e, zileuton, C06,
and ST1098
Code
SASAa
PSAb
DonorHBc
AcceptHBd
QPlogPC16e
QPlogPoctf
QPlogPo/wg
3e
Zileuton
C06
581.1
447.7
581.5
574.11
132.7
73.5
49.9
49.9
2
3
0
0
6
11.8
8.4
10.1
10.5
17.8
14.6
14.1
14.1
1.9
0.9
3.6
3.8
3.7
4.3
4.3
ST1098
aSASA Total solvent accessible surface area in square angstroms using a probe with a 1.4 Å radius (range
300–1000 Å)
bPSA Van der Waals surface area of polar nitrogen and oxygen atoms (range 7–200)
cDonorHB H-bond donors (range 0–6)
dAcceptHB H-bond acceptors (range 2–20)
eQPlogPC16 predicted log of hexadecane/gas partition coefcient (range 4–18)
fQPlogPoct predicted log of octanol/gas partition coefcient (range 8–43)
gQPlogPo/w predicted log of octanol/water partition coefcient (range 2–6)
fármacos analgésicos, antiinfamatórios e anti-trombóticos. Quim
Nova 25:129–148. https://doi.org/10.1590/S0100-4042200200
0100022
5-benzylidene-2-phenyl-4-thiazolones moiety is an essen-
tial pharmacophore necessary to elicit the inhibitory activ-
ity against LOX enzyme along with the lesser ulcerogenic
liability.
Barzen S, Rödl CB, Lill A, Steinhilber D, Stark H, Hofmann B (2012)
Synthesis and biological evaluation of a class of 5-benzylidene-
2-phenyl-thiazolinones as potent 5-lipoxygenase inhibitors.
Bioorg Med Chem 20:3575–3583. https://doi.org/10.1016/j.
bmc.2012.04.003
Acknowledgements The authors gratefully acknowledge Indian Insti-
tute of Technology (BHU) for providing fnancial assistance through
Institute Research Project (IRP) scheme and Design Innovation Center
(DIC) scheme. Grant No. IIT (BHU)/R&D/IRP/2015-16/3471/L and
Grant No. DIC-IIT(BHU)/L-11, respectively. The authors are also
thankful to Department of Health Research, Ministry of Health and
Family Welfare for providing the Young Scientist Project in newer
areas of Drugs Chemistry (DHR/HRD/YS-15-2015-16).
Bekhit AA, Fahmy HT, Rostom SA, Bekhit AE-DA (2010) Synthesis
and biological evaluation of some thiazolylpyrazole derivatives
as dual anti-infammatory antimicrobial agents. Eur J Med Chem
45:6027–6038. https://doi.org/10.1016/j.ejmech.2010.10.001
Berger W, De Chandt M, Cairns C (2007) Zileuton: clinical impli-
cations of 5-lipoxygenase inhibition in severe airway dis-
ease. Int J Clin Pract 61:663–676. https://doi.org/10.111
1/j.1742-1241.2007.01320.x
Carter BZ, Wiseman AL, Orkiszewski R, Ballard KD, Ou C-N, Lieber-
man MW (1997) Metabolism of leukotriene C4 in γ-glutamyl
transpeptidase-defcient mice. J Biol Chem 272:12305–12310.
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