Novel 1,2,4-triazine-quinoline hybrids: The privileged scaffolds as potent multi-target inhibitors of LPS-induced inflammatory response via dual COX-2 and 15-LOX inhibition

Article abstract of DOI:10.1016/j.ejmech.2021.113457

Based on the observed pharmacophoric structural features for the reported dual COX/15-LOX inhibitors and inspired by the abundance of COX/LOX inhibitory activities reported for the 1,2,4-triazine and quinoline scaffolds, we designed and synthesized novel 1,2,4-triazine-quinoline hybrids (8a-n). The synthesized hybrids were evaluated in vitro as dual COXs/15-LOX inhibitors. The new triazine-quinoline hybrids (8a-n) exhibited potent COX-2 inhibitory profiles (IC₅₀ = 0.047–0.32 μM, SI ～ 20.6–265.9) compared to celecoxib (IC₅₀ = 0.045 μM, SI ～ 326). Moreover, they revealed potent inhibitory activities against 15-LOX enzyme compared to reference quercetin (IC₅₀ = 1.81–3.60 vs. 3.34 μM). Hybrid 8e was the most potent and selective dual COX-2/15-LOX inhibitor (COX-2 IC₅₀ = 0.047 μM, SI = 265.9, 15-LOX IC₅₀ = 1.81 μM). These hybrids were further challenged by their ability to inhibit NO, ROS, TNF-α, IL-6 inflammatory mediators, and 15-LOX product, 15-HETE, production in LPS-activated RAW 264.7 macrophages cells. Compound 8e was the most potent hybrid in reducing ROS and 15-HETE levels showing IC₅₀ values of 1.02 μM (11-fold more potent than that of celecoxib, IC₅₀ = 11.75 μM) and 0.17 μM (about 43 times more potent than celecoxib, IC₅₀ = 7.46 μM), respectively. Hybrid 8h exhibited an outstanding TNF-α inhibition with IC₅₀ value of 0.40 μM which was about 25 times more potent than that of celecoxib and diclofenac (IC₅₀ = 10.69 and 10.27 μM, respectively). Docking study of the synthesized hybrids into the active sites of COX-2 and 15-LOX enzymes ensures their favored binding affinity. To our knowledge, herein we reported the first 1,2,4-triazine-quinoline hybrids as dual COX/15-LOX inhibitors.

Full text of DOI:10.1016/j.ejmech.2021.113457

European Journal of Medicinal Chemistry 219 (2021) 113457
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Novel 1,2,4-triazine-quinoline hybrids: The privileged scaffolds as
potent multi-target inhibitors of LPS-induced inflammatory response
via dual COX-2 and 15-LOX inhibition
,
c
,
d,
e
,
,
, d, e, f
Amany M. Ghanim a, Samar Rezq b
f, Tarek S. Ibrahim a g, Damian G. Romero c
,
,
*
Hend Kothayer h
a Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, 44519, Egypt
b Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zagazig University, Egypt
c Departments of Cell and Molecular Biology, University of Mississippi Medical Center, Jackson, MS, USA
d Mississippi Center for Excellence in Perinatal Research, University of Mississippi Medical Center, Jackson, MS, USA
e Women’s Health Research Center, University of Mississippi Medical Center, Jackson, MS, USA
f Cardio Renal Research Center, University of Mississippi Medical Center, Jackson, MS, USA
g Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
h Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the observed pharmacophoric structural features for the reported dual COX/15-LOX inhibitors
and inspired by the abundance of COX/LOX inhibitory activities reported for the 1,2,4-triazine and
quinoline scaffolds, we designed and synthesized novel 1,2,4-triazine-quinoline hybrids (8a-n). The
synthesized hybrids were evaluated in vitro as dual COXs/15-LOX inhibitors. The new triazine-quinoline
Received 1 March 2021
Received in revised form
31 March 2021
Accepted 5 April 2021
Available online xxx
hybrids (8a-n) exhibited potent COX-2 inhibitory profiles (IC50 ¼ 0.047e0.32
compared to celecoxib (IC50 ¼ 0.045 M, SI ~ 326). Moreover, they revealed potent inhibitory activities
against 15-LOX enzyme compared to reference quercetin (IC50 ¼ 1.81e3.60 vs. 3.34 M). Hybrid 8e was
the most potent and selective dual COX-2/15-LOX inhibitor (COX-2 IC50 ¼ 0.047 M, SI ¼ 265.9, 15-LOX
M). These hybrids were further challenged by their ability to inhibit NO, ROS, TNF- , IL-6
mM, SI ~ 20.6e265.9)
m
m
Keywords:
Dual COX-2/15-LOX inhibitors
1,2,4-Triazine
Quinoline
Invitro anti-inflammatory
Docking
m
IC50 ¼ 1.81
m
a
inflammatory mediators, and 15-LOX product, 15-HETE, production in LPS-activated RAW 264.7 mac-
rophages cells. Compound 8e was the most potent hybrid in reducing ROS and 15-HETE levels showing
IC50 values of 1.02
times more potent than celecoxib, IC50 ¼ 7.46
inhibition with IC50 value of 0.40 M which was about 25 times more potent than that of celecoxib and
diclofenac (IC50 ¼ 10.69 and 10.27 M, respectively). Docking study of the synthesized hybrids into the
m
M (11-fold more potent than that of celecoxib, IC50 ¼ 11.75
mM) and 0.17 mM (about 43
M), respectively. Hybrid 8h exhibited an outstanding TNF-
m
a
m
m
active sites of COX-2 and 15-LOX enzymes ensures their favored binding affinity. To our knowledge,
herein we reported the first 1,2,4-triazine-quinoline hybrids as dual COX/15-LOX inhibitors.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
sclerosis, neurodegenerative disorders such as Alzheimer’s, osteo-
arthritis, psoriasis, diabetes mellitus and even cancer [2].
Inflammation is a multifactorial body response to different
intrinsic and extrinsic stimuli [1]. Control of inflammation is now
taking priority as it is involved in the initiation and/or progression
of numerous diseases like asthma, rheumatoid arthritis, cardio-
vascular disorders, diabetes mellitus, Crohn’s disease, multiple
During inflammation huge amounts of inflammatory mediators
are released through various biological pathways [2]. Among
which, the arachidonic acid (AA) cascade plays a major role as the
precursor for eicosanoids including prostaglandins (PGs) from
cyclooxygenase (COX) pathway and leukotrienes (LTs), hydrox-
yeicosatetraenoic acids (HETEs), hydroperoxyeicosatetraenoic acids
(HPETEs), and hydroxyoctadecadienoic acids (HODEs) from lip-
oxygenase (LOX) pathway [1,3,4].
* Corresponding author.Department of Medicinal Chemistry, Faculty of Phar-
macy, Zagazig University, 44511, Zagazig, Egypt.
E-mail addresses: hendo1311@hotmail.com, hkelhamalawy@pharmacy.zu.edu.
eg (H. Kothayer).
There are three COX enzymes isoforms: COX-1, COX-2, and COX-
3. COX-1 is a constitutive enzyme that synthesizes prostaglandins
https://doi.org/10.1016/j.ejmech.2021.113457
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
responsible for the protection of gastric mucosa, regulation of
platelet aggregation, and renal blood flow. COX-2 is induced during
inflammation, pain, and oncogenesis. Most classical non-steroidal
anti-inflammatory drugs (NSAIDs) that are widely used for the
treatment of inflammatory conditions inhibit both COX isoforms,
therefore their prolonged administration causes gastrointestinal
ulceration, renal damage, and hepatic side effects [5,6].
Those concerns triggered the efforts towards designing selective
COX-2 inhibitors such as coxibs to overcome the classical NSAIDs
related side effects. Although coxibs showed good anti-
inflammatory activity with good gastric and kidney safety pro-
files, cardiovascular adverse effects have been reported leading to
the withdrawal of rofecoxib and valdecoxib from the market [6e8].
On the other hand, the other arachidonate pathway comprises
lipoxygenases (LOXs) which exist as 3 different isozymes; 5-LOX,
12-LOX, and 15-LOX and they catalyze the production of inflam-
matory mediators from arachidonic acid (AA) like leukotrienes,
eoxins and lipoxins [9,10].
Since arachidonic acid is the same substrate for both COXs and
LOXs enzymes, COXs inhibition (either by NSAIDs or coxibs) leads
to increased substrate availability and hence increased LOXs in-
flammatory mediators production. Leukotrienes have been re-
ported to be involved in cancer, cardiovascular diseases and the
gastrointestinal reactions caused by NSAIDs. Thus, 5-LOX and 15-
LOX represent promising drug targets and development of COX-1/
COX-2 inhibitors with LOX inhibitory properties can be a valid
strategy to avoid the gastrointestinal and cardiovascular side effects
of NSAIDs [6,7,10e12].
Recently, 15-LOX has been reported as a target for reduction of
the biosynthesis of eoxines, pro-inflammatory mediator and cancer
promotor [13]. Also, 15-LOX is hypothesized to promote athero-
sclerosis through low-density lipoprotein (LDL) oxidation. More-
over, 15-LOX is one of the key mediators in neurodegenerative
diseases such Alzheimer’s disease [13]. It is documented that 15-
LOX-inhibitors (such as PD146176 among others) could serve as
effective therapeutics for various types of cancer, airway inflam-
mation, diabetes, Alzheimer’s disease, hypertension, osteoarthritis,
obesity, atherosclerosis and chronic adipose tissue inflammation,
and multiple sclerosis [11,13,14]. Recent evidence showed that 15-
LOX plays fundamental roles in the molecular mechanisms
related to the pathophysiology of these diseases [11,14].
showed an additional 15-LOX inhibitory activity (Fig. 1). Quinolines
V-VII [18e20] represent promising COXs inhibitors. Furthermore,
quinoline VIII [21] exhibited an extra 5-LOX inhibitory activity
(Fig. 1).
Recently, there have been reports of successful trials to develop
dual inhibitors of COX-2 and 15-LOX. Most of these studies applied
molecular hybridization strategy to combine a COX active moiety to
another one that exhibits LOX inhibitory activity [7,8,10,22e24].
Analyzing these reported dual COX-2/15-LOX inhibitors, we
found common pharmacophoric structural features. These features
include three regions: a nitrogen containing bicyclic ring, a central
five or six membered nitrogen containing heterocycle (azole or
azine) and a hydrophobic tail usually contains aromatic ring
(compounds IX-XI) [8,22,23] (Fig. 2). Sometimes, the nitrogen
containing bicyclic ring is replaced by nitrogen containing hetero-
cycle bearing phenyl substituent as in compounds XII and XIII
[7,10]. Replacement of the central azole or azine by open ring
analogue is also reported as in compound XIV [24] (Fig. 2).
In the current work, The plethora of established COX/LOX
inhibitory activities associated with the 1,2,4-triazine and quinoline
entities inspired us to combine both pharmacologically active
scaffolds and design novel 1,2,4-triazine-quinoline hybrids
mimicking the common pharmacophoric structural features
observed for the previously reported dual COX-2/15-LOX inhibitors
(Fig. 2).
Our hydrophobic tail moiety was sometimes chosen to be an
aryl bearing electron-rich substituent (tri-methoxyphenyl or
vanillyl) being reported as criteria for LOX inhibitors [25,26].
In addition, our design provides better selectivity towards
COX-2 over COX-1 enzyme as the bulkiness of the designed hy-
brids prevents them from fitting into the COX-1 active site which
is 20% smaller than that of COX-2 due to the replacement of the
smaller Val 434 and Val 523 in COX-2 with the larger Ile 434 and
Ile 523 in COX-1. This amino acid difference opens an additional
side pocket in the COX-2 binding site accessible for drug binding
[2,7,12,27,28].
2. Results and discussion
2.1. Chemisrty
Accordingly, the use of multitarget directed ligands (MTDLs) to
make dual inhibition for both 15-LOX and COX enzymes is a
promising strategy in drug therapy with maximum efficiency and
minimal side effects [3,11,14].
Herein, we designed and synthesized novel 1,2,4-triazine -
quinoline hybrids (8a-n) as dual COX-2 and 15-LOX inhibitors and
they were evaluated for their COX-1/COX-2 selectivity and 15-LOX
inhibitory activity using in vitro assays. Moreover, the anti-
inflammatory properties of these newly synthesized hybrids were
investigated based on their ability to block the generation of
intracellular reactive oxygen species (ROS) generation. Their ability
to suppress the expression of pro-inflammatory factors, including
The synthesis of our target 1,2,4-triazine e quinoline hybrids
(8a-n) was accomplished by coupling 2-chlorosubstituted quino-
line-3-carbaldehydes (3a-h) with 4-amino-6-substituted-3-thioxo-
3,4-dihydro-1,2,4-triazin-5(2H)-ones (7a-d).
The two previously reported steps (Scheme 1) were utilized to
synthesize the required quinoline moieties (3a-h) starting with
acetylation of the appropriate anilines (1a-h) by acetic anhydride in
glacial acetic acid at 0e5 ꢀC to give the corresponding anilides (2a-
h) [29e35], which then underwent VilsmeiereHaack reaction to
produce the desired quinolines (3a-h) [30e37].
Cyclization of hippuric acid (4) with the appropriate aldehydes
(5a-d) in acetic anhydride containing sodium acetate yielded the
corresponding azlactones (6a-d). These azlactones (6a-d) were
subjected to alkaline hydrolysis followed by condensation with
thiocarbohydrazide to obtain the required 1,2,4-triazines (7a-d)
[38] (Scheme 2).
iNOS, TNF-a, and IL-6 as well as 15(S)-HETE, which is produced
from arachidonic acid by 15-LOX, was further evaluated in LPS-
stimulated murine RAW264.7 macrophages. Molecular modelling
was used to predict their binding modes with COX-2 and 15-LOX.
1.1. Rationale for the design of multi-target directed ligands
(MTDLs)
Finally, the target novel hybrids (8a-n) were synthesized by
condensation of 2-chlorosubstituted quinoline-3-carbaldehydes
(3a-h) with 4-amino-6-substituted-3-thioxo-3,4-dihydro-1,2,4-
triazin-5(2H)-ones (7a-d) in either acetic acid or benzene. Utiliz-
ing benzene as a solvent gave the corresponding chloro derivatives
(8g,h) while acetic acid produced the hydroxy analogues [39e42]
8a-f and 8i-n (Scheme 3).
1,2,4-Triazine and quinoline scaffolds have been reported to
possess significant inhibitory activity against different COXs and
LOXs isozymes. For example, Triazines I-III [15e17] (Fig. 1) showed
selective inhibition of COX-2 over COX-1. Moreover, triazine IV [5]
2

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
Fig. 1. Reported 1,2,4-triaizines and quinolines with significant inhibitory activity against different COXs and LOXs isozymes.
2.2. Biological activity
On the other hand, hybrid 8a (containing 6-benzyl triazine and
2-hydroxy-6-isopropoxyquinoline) was the least potent and se-
lective COX-2 inhibitor (COX-2 IC50 ¼ 0.32, SI ¼ 20.6 respectively).
Maintaining the chloro substituent at quinoline C2 (hybrids 8g and
h) did not show any improvement in either COX-2 potency or
selectivity.
2.2.1. In vitro COX-1 and COX-2 inhibitory assay
All synthesized compounds (8a-n) were tested by in vitro COX-
1/COX-2 inhibition assays using an ovine COX-1/human recombi-
nant COX-2 assay kit. Celecoxib and diclofenac sodium were used
for reference. The half-maximal inhibitory concentration (IC50
values were determined, and the COX-2 selectivity index (SI) values
were calculated as IC50 (COX-1)/IC50 (COX-2).
,
m
M)
It is notable that between the trimethoxyphenyl triazine-
quinoline hybrids (8g-k) 6-substituted quinolines (either 6-
methoxy or 6-methyl) 8i and k is favored to any other substitu-
tion position of quinolines (8g,h and j). While between the benzyl
triazine-quinoline hybrids 8a-e, substitution of quinoline arranged
as 6-benzyloxy > unsubstituted > 6-methoxy > 7-methoxy > 6-
methyl > 6-isopropoxy considering both COX-2 potency and
selectivity.
The new triazine - quinoline hybrids (8a-n) exhibited potent
COX-2 inhibitory profiles (IC50 ¼ 0.047e0.32
mM) that were com-
M), except for hybrids 8a,b, and
parable to celecoxib (IC50 ¼ 0.045
m
h that were less potent than celecoxib (p < 0.001). Notably, the new
hybrids are 2.6e17 folds superior to the activity of diclofenac so-
dium (IC50 ¼ 0.84
moderate COX-1 inhibitory activity (IC50 ¼ 5.9e12.5
parison to references; celecoxib and diclofenac sodium (IC50 ¼ 14.7
and 3.80 M, respectively, p < 0.001) (Table 1).
Concerning COX-2 selectivity, all tested hybrids showed mod-
erate to potent COX-2 inhibition selectivity. Amongst all, hybrid 8e
which contains 6-benzyl triazine and 6-benzyloxy 2-
hydroxyquinoline, was the most potent and selective COX-2 in-
hibitor (COX-2 IC50 ¼ 0.047, SI ¼ 265.9) about 59 times exceeding
that of diclofenac sodium (COX-2 SI ¼ 4.52, p < 0.01) and slightly
lower than celecoxib (COX-2 SI ~ 326, p ¼ 0.63), which could be
considered as an advantage to avoid the cardiovascular side effects
of highly selective COX-2 inhibitors (Table 1).
m
M, p < 0.001). On the other hand, they showed
m
M) in com-
2.2.2. In vitro 15-LOX inhibition assay
Evaluation of 15- LOX inhibitory activities of all the synthesized
hybrids 8a-n were carried out using soybean 15-LOX inhibitor
screening assay using quercetin as reference.
m
All the hybrids 8a-n revealed potent inhibitory activities against
15-LOX enzyme (IC50 ¼ 1.81e3.60
or comparable potency to that of the reference quercetin
(IC50 ¼ 3.34 M). Hybrids 8e was the most active 15-LOX inhibitor
with IC50 ¼ 1.81 m, which represent nearly double the inhibitory
activity of reference quercetin (IC50 ¼ 3.34 M, p < 0.01) (Table 1).
Benzyl triazine derivatives 8e and f (IC50 ¼ 1.81 and 1.97
respectively) were favored to their trimethoxyphenyl triazines
mM) that showed either greater
m
m
m
mm
3

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
Fig. 2. Some reported compounds as dual COX-2/15-LOX inhibitors and rationale for the design of multi-target directed ligands (MTDLs).
4

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
Scheme 1. Meth-Cohn synthesis of 2-chlorosubstituted quinoline-3-carbaldehydes (3a-h) using Vilsmeier’s reagent (DMF and POCl3). Reagents and conditions: (i) Ac2O, AcOH,
0e5 ꢀC,1h; (ii) DMF, POCl3, 70e90 ꢀC, 18 h.
Scheme 2. Synthesis of 1,2,4-triazines (7a-d). Reagents and conditions: (i) Ac2O, NaOAc, 100 ꢀC, 1h; (ii) 20% aq. NaOH, reflux, 2.5 h; (iii) Thiocarbohydrazide, AcOH, reflux 15 min.
Scheme 3. Synthesis of target 1,2,4-triazine e quinoline hybrids (8a-n). Reagents and conditions: (i) For compounds 8a-f and 8i-n, HOAc, reflux, 8e12 h; (ii) for compounds 8g, h,
benzene, reflux, 8e12 h.
counterparts 8g and k (IC50 ¼ 3.39 and 2.57
m
m, respectively).
the mechanism of action of new anti-inflammatory candidates. The
challenge of bacterial toxins LPS to RAW 264.7 cells triggers inflam-
mation and induces COX-2 expression [43]. Additionally, LPS upre-
gulates the inducible isoforms of nitric oxide synthase to induce nitric
oxide (NO) production, which is required to maintainprolonged COX-
2 expression [44]. Activated macrophages also generate ROS which in
turn augment the inflammatory response by inducing the production
of different inflammatory cytokines [45]. Numerous in vivo and
in vitro studies have shown that compounds with antioxidant po-
tential are effective as anti-inflammatory [45e47]. Compounds 8a-n
were evaluated for their ability to inhibit NO and ROS production in
LPS-activated RAW 264.7 macrophage cells.
Interestingly, 15-LOX inhibitory activities of the benzyl triazine-
quinoline hybrids 8a-f were consistent with their respective COX-
2 inhibitory activities regarding the preferred substitution of
quinoline with nearly the same arrangement 6-benzyloxy ˃ 6-
methoxy ˃ unsubstituted ˃ 7-methoxy ˃ 6-methyl ˃ 6-isopropoxy.
2.2.3. Effects on NO and ROS production in LPS-activated RAW
264.7 macrophages cells
Macrophages represent one of the main immune cells that are
activated under inflammatory conditions to produce various inflam-
matory mediators. We utilized lipopolysaccharides (LPS) the main
constituent of Gram-negative bacteria outer membrane to activate
RAW 264.7 macrophage cells, a widely used in vitro model to screen
Compounds 8b,c,e-i,m and n inhibited the production of the
inflammatory mediator NO with IC50 ¼ 4.78e36.81
mM. Notably,
5

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
Table 1
In vitro COX-1, COX-2, and 15-lipoxygenase enzyme inhibitory activities of triazine-quinoline hybrids and references, in addition to COX-2 selectivity index.
Compd. no.
aCOX-1 IC50
(m
M)
aCOX-2 IC50
(m
M)
bCOX-2 SI
a15-LOX
IC50(mM)
Celecoxib
Diclofenac sodium
Quercetin
(8a)
(8b)
(8c)
(8d)
(8e)
(8f)
(8g)
(8h)
(8i)
(8j)
(8k)
(8l)
(8 m)
(8n)
14.7 0.29
3.8 0.23*
e
0.045 0.01
0.84 0.05*
e
326.6
4.52*
e
e
e
3.34 0.27
3.49 0.25
2.66 0.23
2.32 0.35@
2.12 0.29@
1.81 0.23@
1.97 0.35@
3.39 0.23
1.86 0.29@
2.06 0.29@
2.18 0.46@
2.57 0.29
1.97 0.40@
2.38 0.22@
2.13 0.40@
6.6 0.07*#
0.32 0.06*#
0.28 0.12*#
0.073 0.01#
0.054 0.02#
0.047 0.01#
0.068 0.02#
0.12 0.01#
0.16 0.05*#
0.065 0.02#
0.097 0.01#
0.060 0.02#
0.085 0.02#
0.11 0.003#
0.079 0.02#
20.6*
21.07*
139.7*
209.3
265.9
154.4*
64.2*
51.87*
176.9
100*
5.9 0.06*#
10.2 0.03*#
11.3 0.15*#
12.5 0.12*#
10.5 0.15*#
7.7 0.84*#
8.3 0.07*#
11.5 0.03*#
9.7 0.07*#
11.8 0.23*#
10.3 0.35*#
9.0 0.35*#
11.1 0.09*#
196.6
121.2*
81.8*
140.5*
*p < 0.05 vs celecoxib, #p < 0.05 vs diclofenac, @p < 0.05 vs quercetin.
a
IC50 in
(
m
M) concentration as expressed as mean SEM, for three replicates.
b
Selectivity index ¼ (COX-1 IC50/COX-2 IC50). Values are expressed as mean SEM.
compounds 8c,e-i and n were significantly more potent compared
to diclofenac (IC50 ¼ 38.33 M, p < 0.001). Compounds 8 c,e,f,g,i
and n (IC50 ¼ 4.78e13.07 M) showed superior scores compared to
the reference drug celecoxib, (IC50 ¼ 19.51 M, p < 0.05). The best
hybrids 8f,g and i (IC50 ¼ 6.93, 6.76, and 6.34 M, respectively)
showed approximately 3-fold better IC50 values than that of cele-
coxib (IC50 ¼ 19.51 M) and 6-fold better IC50 values than that of
diclofenac (IC50 ¼ 38.33 M) (Table 2). It is notable that between the
levels was hybrid 8e that showed IC50 value of 1.02
mM (11 folds
m
more potent than that of celecoxib, 11.75 M, p < 0.001) (Fig. 3C).
m
m
Within the benzyl triazine-quinoline hybrids (8a-f) 6-bulky quin-
olines (either 6- isopropoxy 8a or 6- benzyloxy 8e) were favored.
While between the trimethoxyphenyl triazine-quinoline hybrids
(8g-k), 2-chloro,8- methyl (8h) was favored (Table 2). Notably, none
of the tested concentrations were toxic to RAW 264.7 macrophages
as tested by MTS cell bioavailability assay except for compounds 8c
and 8g, which showed decreased viability only at the highest
m
m
m
m
benzyl triazine-quinoline hybrids (8a-f), 6- methoxy quinolines
(8f) is favored to any other substitution position of quinolines then
6- benzyloxy quinoline (8e) is coming next (Fig. 3A and B). While
between the trimethoxyphenyl triazine-quinoline hybrids (8g-k),
7- benzyloxy (8g) and 6-methyl (8i) quinolines were favored. Un-
fortunately, compounds 8a,d,j,k,l did not show significant activities
concerning inhibition of NO production (Table 2).
Regarding ROS production, most of the tested compounds
exhibited potent inhibitory activity, indicating much improved
antioxidant activity comparing with that of the two references
celecoxib and diclofenac. The most potent hybrid in reducing ROS
concentration (50 mM).
2.2.4. Effects on TNF-
264.7 macrophages cells
LPS can also activate macrophages to secrete TNF-
a and IL-6 production in LPS-activated RAW
a
and IL-6. The
excessive production of these cytokines results in severe systemic
inflammatory response, tissue damage, and sepsis [48]. Our find-
ings show that hybrids 8c,h,j, and m inhibited the production of the
inflammatory mediator TNF-
pounds 8h,j, and m were significantly more potent than the two
references celecoxib and diclofenac (IC50 ¼ 10.69 and 10.27 M,
respectively, p < 0.01). Also, compound i had comparable IC50
values (11.49 M) while compounds 8e (Fig. 3D), and g showed
higher IC50 values (19.19 and 16.02 M, respectively, p < 0.05)
a
with IC50 ¼ 0.40e8.41
mM. Com-
m
Table 2
m
In vitro inhibitory activities of triazine-quinoline hybrids and references on LPS-
induced NO and ROS production in RAW 264.7 cells.
m
compared to both references’ IC50 values. Interestingly, hybrid 8h
(trimethoxyphenyltriazine- 2-chloro, 8-methylquinoline hybrid)
Compound
NO
IC50
ROS
IC50
(
m
M)
(mM)
exhibited an outstanding IC50 value (0.40
mM) which is about 25
8a
8b
8c
>50
m
M
1.91 0.56*#
7.31 0.10*#
3.78 0.04*#
times more potent than that of the two references, celecoxib and
33.49 1.17*
diclofenac (IC50 ¼ 10.69 and 10.27
mM, respectively, p < 0.0001)
16.06 1.45*#
(Table 3).
8d
>50
m
M
>50 mM
12.75 1.35*#
6.93 1.34*#
6.76 1.26*#
25.78 1.74*#
6.34 1.51*#
1.02 0.2*#
3.95 0.05*#
4.36 0.05*#
1.80 0.05*#
6.67 0.04*#
4.52 0.23*#
Concerning inhibitiong IL-6 production, hybrids 8c,e-h (Fig. 3E),
and j exhibited IC50 ¼ 3.99e7.7 M and were more effective than
the two references celecoxib and diclofenac (IC50 ¼ 11.17 and
12.91 M respectively, p < 0.05) by 1.5e3 folds. Also, compounds
8a, b, I, and m showed comparable IC50 values (11.14e13.54 M).
Unfortunately, compounds 8d,k,l, and n did not show any signifi-
8e
8f
8g
8h
8i
8j
8k
8l
m
m
m
>50
>50
>50
m
m
m
M
M
M
>50
>50
m
m
M
M
cant activities concerning inhibiting the production of either TNF-
or IL-6.
a
8m
8n
Celecoxib
Diclofenac
36.81 2.19*
9.37 1.27*#
19.58 1.10
36.28 1.42*
10.94 0.08#
>50 mM
11.73 1.10
25.51 1.06*
2.2.5. Effects on 15(S)-HETE production in LPS-activated RAW 264.7
macrophages cells
15(S)-HETE is the product of arachidonic acid metabolism by 15-
LOX. Incubating human macrophages with 15-HETE showed
IC50
(
m
M) concentration as expressed as mean
SEM, for three replicates.
in
*p < 0.05 vs celecoxib. #p < 0.05 vs diclofenac. NO (Nitric Oxide); ROS (Reactive
Oxygen Species).
6

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
Fig. 3. Effect of compound 8e on the production of NO (A, B): (A) Representative image showing reduction of NO production in RAW264.7 cells treated with 8e and celecoxib
(25 M). LPS induced NO production, which is indicated as green fluorescence by the fluorescent probe, DAF-FM Diacetate. (B) The fluorescence intensity was quantified by using a
fluorescence microplate reader. Effects of compound 8e on ROS (C), TNF- (D), IL-6 (E), and 15(s)-HETE (F) in LPS-induced RAW264.7 macrophages. The cells were pretreated with
M) for 2 h and then treated with LPS (1 g/mL) for 20 h. The results shown are representative of three independent
m
a
different concentrations of 8e (3.125, 6.25, 12.5, 25 or 50
m
m
experiments. *p < 0.05 vs untreated controls. #p < 0.05 vs LPS-treated cells.
significant increases in LPS-mediated inflammatory response,
indicating its potential role in LPS-induced inflammatory response
[49]. Therefore, its inhibition could be targeted for more efficient
anti-inflammatory activity. Our present findings showed that
compounds 8a,c,e,f-h inhibited the production of 15(S)-HETE with
It is notable that between the trimethoxyphenyl triazine-
quinoline hybrids (8g-k) 2-chloroquinolines (either 7-benzyloxy
or 8-methyl) 8g, and h were favored to any other quinolines (8i,-
k). While between the benzyl triazine-quinoline hybrids 8a-e,
substitution of quinoline arranged as 6-benzyloxy > 6-isopropoxy
> 7-methoxy > 6-methoxy > 6-methyl > unsubstituted.
IC50 ¼ 0.17e3.91
and diclofenac (IC50 ¼ 7.46 and 12.19
Moreover, compounds 8i and m (IC50 ¼ 8.86 and 7.13, respectively)
were superior to diclofenac (IC50 ¼ 12.19 M, p < 0.0001) and
comparable to celecoxib. Also, compounds 8b, and j (IC50 ¼ 21.26
and 18.55 M, respectively) showed lower potencies compared to
mM exceeding that of the two references celecoxib
mM, respectively, p < 0.0001).
2.3. Molecular modeling
m
The orientations of compounds 8a-n within the active binding
sites of the cyclooxygenase-2 enzyme (COX-2) co-crystallized with
SC-558 (PDB entry 1CX2) [50] and the homo sapiens 15-LOX co-
crystallized with substrate mimic (PDB:4NRE) [51] were investi-
gated using Molecular Operating Environment (MOE) version MOE
2019.0102 (Chemical Computing Group, Montreal, CA). The best
m
both reference drugs (p < 0.0001). Interestingly, again benzyl
triazine derivatives 8e (Fig. 3F) exhibited an outstanding IC50 value
(0.17
mM) which was about 43 times more potent than that of
celecoxib (IC50 ¼ 7.46
mM, p < 0.0001) (Table 3).
7

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
Table 3
coordination with the imidazole nitrogen of His 373, His378, and
His 553 [22,51].
In vitro inhibitory activities of triazine-quinoline hybrids and references on LPS-
induced TNF-
a, IL-6 and 15(s)-HETE production in RAW 264.7 cells.
The iron metal is well conserved in lipoxygenases and its
enzyme catalysis role is considered to be the elimination of the
hydrogen from the central carbon of the fatty acid [52,53]. Substi-
Compound
TNF-
a
IC50
(m
M)
IL-6 IC50
(
m
M)
15(s)-HETE IC50 (mM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
34.60 1.13*#
30.29 1.39*#
8.41 1.29
12.92 1.12
13.54 1.41
7.36 1.03*#
0.42 0.44*#
21.26 0.20*#
3.60 0.14*#
tution of this metal for other redox-active metals (e.g., Co2þ, Mn2þ
,
Ni2þ) makes the enzyme inactive [53].
>50
m
M
>50
m
M
>50 mM
All the new compounds 8a-n displayed a U-shaped binding
orientation within 15-LOX binding site in the same way as the co-
crystalized substrate mimic with favored docking scores ranging
from ꢁ9.09 to ꢁ5.25 kcal/mol (Supplementary Material Table 1).
Interestingly, compounds (8a-e, and i) can coordinate with the
iron atom via their 1,2,4-triazin-5(2H)-one carbonyl groups. This
finding could explain their capability to inhibit 15-LOX through
interference with the availability of the iron for use during enzy-
matic function.
19.19 1.17*#
7.70 1.05#
5.98 1.06*#
6.11 1.04*#
3.99 1.05*#
11.14 1.02
5.54 1.08*#
0.17 2.1*#
3.91 0.05*#
2.03 0.55*#
1.46 0.21*#
8.86 0.03#
18.55 0.25*#
>50 mM
16.02 1.31*#
0.40 1.06*#
11.49 1.06
2.61 1.11*#
>50
>50
m
m
M
M
>50
>50
11.80 1.03
>50
m
m
M
M
>50
>50
m
m
M
M
8 m
8n
Celecoxib
Diclofenac
3.40 1.58*#
7.13 0.69#
>50
mM
m
M
>50 mM
10.69 1.09
10.27 1.12
11.17 1.18
12.91 1.87
7.46 0.10
12.19 0.04*
Besides binding to Fe, compounds (8a-e, and i) were capable of
forming hydrogen bonds with Asn 413 via their thioxo-triazine
groups and hydrophobic interactions with Glu 369 through their
phenyl rings. Additional hydrophobic interactions with either
His378 or Leu420 are also observed with docking scores ranged
from ꢁ9.094 to ꢁ7.07 kcal/mol.
IC50
(
m
M) concentration as expressed as mean
*p < 0.05 vs celecoxib. #p < 0.05 vs diclofenac. TNF-
(interleukin-6); 15(s)HETE (hydroxyeicosatetraenoic acid).
SEM, for three replicates.
in
a
(Tumor necrosis factor- ); IL-6
a
docking poses were selected according to the scoring functions
along with binding interactions formed with the surrounding
amino acids, and the relative orientation of the docked compounds
compared to the co-crystallized ligand.
Notably, Compound 8e showed the best docking score ꢁ9.094
which match with the in vitro 15-LOX binding assay, as it also
showed the lowest IC50 value (1.81 mM) against 15-LOX (Fig. 5).
To validate our docking protocol settings, the co-crystallized
ligands SC-558 into the COX-2 active site and the substrate mimic
into the 15-LOX active site were redocked and their original con-
formations were reproduced (RMSD: 0.97 and 1.25 Score ꢁ9.35
and ꢁ8.77 respectively).
Moreover, compound 8e was the most potent among all tested
compounds to inhibit LPS-induced 15(S)-HETE production, the
direct arachidonate metabolite of 15-LOX, in RAW macrophages
(IC50 ¼ 0.17
mM).
Although compounds 8f-h, and j-n did not show any interaction
with iron, they all formed hydrophobic interaction with His378
(iron coordinate). Moreover, they completely filled the pocket
forming hydrogen/hydrophobic interactions with one or more of
surrounding amino acid residues (Ile 412, Ile 676, Gln 425, Leu420,
Phe 192, leu 419, and His 372). Compounds 8g,h, and j also formed
hydrogen bond interactions with Arg 429 at the entrance of the
pocket [54] and this could also be responsible for the good inhib-
itory activities shown by these compounds. (supplementary ma-
terial table).
2.3.1. Docking into COX-2 active site
The main difference between COX-1 and COX-2 binding sites is
the larger size of the COX-2 binding site due to the opening of an
additional side pocket. This side pocket is surrounded by Gln192,
Tyr355, His 90 and Arg513 (the latter is replaced by His 513 in COX-
1). Binding with Arg513 is responsible for the high selectivity of
COX-2 inhibitors (usually through the sulfone of sulfonamide
groups) [7,23,27,28].
The docking scores for the compounds 8a-n within COX-2 active
site ranged from ꢁ7.62 to ꢁ4.81. All the compounds 8a-n not only
were able to reach the additional side pocket but also formed ionic
or hydrogen bond interactions with Arg513 amino acid (responsible
for the high selectivity of COX-2 inhibitors) either via their 1,2,4-
triazin-5(2H)-one carbonyl or thioxo groups.
Beside Arg513, compounds 8a-d, and i-l were able to form hy-
drophobic interaction with Ser 353 (one of the key residues in the
binding mode of the ligand SC-558) via their triazine rings. Com-
pounds 8a-d, h,i, and k were able to make hydrogen bonds with
both Ala516 and Ph518. (supplementary material table).
Moreover, compounds 8e,-k, and m made additional hydrogen
bonds with one or more of surrounding amino acid residues (Gly
354, Ph518, Ala516, Gln192). Compounds 8e,-h,k, and m showed
hydrophobic interaction with at least one of the surrounding amino
acid residues (Supplementary Material Table 1).
3. Conclusion
In summary, a library of 14 triazine - quinoline hybrids (8a-n)
were synthesized and evaluated in vitro as dual COXs/15-LOX in-
hibitors. All of them exhibited potent COX-2 and 15-LOX inhibitory
profiles. Hybrid 8e was the most potent and selective dual COX-2/
15-LOX inhibitor (COX-2 IC50 ¼ 0.047
IC50 ¼ 1.81 M). Moreover, the hybrids were evaluated for their
suppression effect of LPS-stimulated NO, ROS, TNF- , IL-6, and 15-
m
M, SI ¼ 265.9, 15-LOX
m
a
HETE production in RAW 264.7 macrophages. The results showed
that compound 8e was the most potent hybrid in reducing ROS and
15-HETE levels showing 11 and 43 folds more potent than cele-
coxib, respectively. Hybrid 8h showed an outstanding TNF-
bition IC50 value (0.40 M) which is about 25 times more potent
than that of the two references celecoxib and diclofenac
M, respectively). Furthermore, docking
a inhi-
m
Intriguingly, compound 8e, that expressed the least COX-2 IC50
value (0.047 mm) in in vitro binding assay and also had the best SI,
(IC50 ¼ 10.69 and 10.27
m
study of 8e into active sites of COX-2 and 15-LOX enzymes revealed
was able to make 8 binding interactions with the surrounding
amino acid residues within COX-2 active site including hydrogen
bond with Arg513 with docking score ꢁ6.01 (Fig. 4).
its favored binding mode.
4. Materials and methods
2.3.2. Docking into 15-LOX active site
4.1. Chemistry
The 15-LOX active site is a hydrophobic channel that is U-
shaped to accommodate arachidonic acid. A catalytic iron (Fe)
located proximal to the base of the “U”, held in place by
Melting points (ꢀC) were measured with a Gallenkamp (London,
U.K.) melting point apparatus and are uncorrected. NMR spectra
8

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
Fig. 4. Two/Three-dimensional (2-D, 3-D) binding interaction pattern of 8e (green) in the binding site of COX-2 (PBD:1CX2).
were recorded on Bruker high performance Digital FT-NMR spec-
trometer AVANCE III operating at 400 MHz for proton and 100 MHz
for carbon nuclei 400 MHz using dimethyl sulfoxide (DMSO)-d6 or
chloroform (CDCl3)-d as solvent and tetramethylsilane (TMS) as an
visualized by UV-lamp at wavelength (l) 254 nm. Intermediates 2a-
h, 3a-h, 6a-d and 7a-d were prepared according to the reported
procedures [29e38].
internal standard (chemical shift in d, ppm). Mass spectra were
determent using a GC/MS Shimadzu Qp-2010 plus (Shimadzu
Corporation, Tokyo, Japan). Elemental analyses were determined
using the Vario MICRO cube (Elementar) CHNS analyzer.
4.1.1. 4-Amino-3-thioxo-6-(3,4,5-trimethoxybenzyl)-3,4-dihydro-
1,2,4-triazin-5(2H)-one (7b)
1H NMR (400 MHz, CDCl3)
d 10.51 (s, 1H, NH), 6.55 (s, 2H, C2eH
All reactions were monitored by thin layer chromatography
(TLC) using silica gel 60 GF245 (E-Merck, Germany) and were
& C6eH trimethoxybenzyl), 6.15 (s, 2H, NH2), 3.91 (s, 2H, CH2), 3.84
(s, 6H, 2 OCH3), 3.82 (s, 3H, OCH3).
9

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
Fig. 5. Two/Three-dimensional (2-D, 3-D) binding interaction pattern of 8e (green) in the binding site of 15-LOX (PBD:4NRE) displaying a U- shaped binding orientation in the same
way as the co-crystalized substrate mimic (yellow).
10

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
4.1.2. 4-Amino-6-(4-hydroxy-3-methoxybenzyl)-3-thioxo-3,4-
NMR (101 MHz, DMSO) d 171.0, 169.2, 162.8, 161.1, 150.0, 149.3,
dihydro-1,2,4-triazin-5(2H)-one (7c)
d 13.85 (s, 1H, NH), 8.83 (s, 1H, OH),
6.82 (s, 1H, C2eH vanillyl), 6.68e6.61 (m, 2H, C5eH & C6eH
141.0, 140.2, 136.7, 133.6, 130.6, 129.7, 128.8, 127.1, 123.2, 118.9, 115.9,
36.4. MS, m/z: 389.73 (Mþ). Analysis calcd. for C20H15N5O2S: C,
61.68; H, 3.88; N, 17.98. Found: C, 61.83; H, 4.09; N, 18.11.
1H NMR (400 MHz, DMSO)
vanillyl), 6.48 (s, 2H, NH2), 3.78 (s, 2H, CH2), 3.72 (s, 3H, OCH3).
4.1.9. 6-Benzyl-4-(((6-(benzyloxy)-2-hydroxyquinolin-3-yl)
methylene)amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one
(8e)
4.1.3. 4-Amino-6-(thiophen-2-ylmethyl)-3-thioxo-3,4-dihydro-
1,2,4-triazin-5(2H)-one (7d)
1H NMR (400 MHz, DMSO)
thiophene), 6.93e6.86 (m, 2H, C3eH & C4eH thiophene), 6.53 (s,
d
7.28 (dd, J ¼ 5.0, 1.3 Hz, 1H, C5eH
Yield (89%). M. p.: 272e274 ꢀC. 1H NMR (400 MHz, DMSO)
d 13.78 (s, 1H, NH), 12.25 (s, 1H, OH), 8.80 (s, 1H, C4eH quinoline),
2H, NH2), 4.06 (s, 2H, CH2).
8.77 (s,1H, CH]N), 7.82e6.90 (m,13H, C5,7,8-H quinoline & 10 ArH),
5.14 (s, 2H, CH2O), 3.93 (s, 2H, CH2). 13C NMR (100 MHz, DMSO)
4.1.4. General procedure for synthesis of 6-substituted-4-(((2-
hydroxy/chloro quinolin-3-yl)methylene)amino)-3-thioxo-3,4-
dihydro-1,2,4-triazin-5(2H)-one (8a-n)
To a solution of triazine derivative 7a-d (2 mmol) in acetic acid/
benzene (10 mL), the appropriate 2-chloro- substituted quinoline-
3-carbaldehyde 3a-h (2 mmol) was added and the reaction mixture
was refluxed for 8e12 h. The obtained precipitate was filtered off
and crystallized from ethanol to give the target compounds 8a-h.
d 171.0, 169.2, 160.6, 154.0, 150.0, 149.3, 139.6, 137.2, 136.8, 135.9,
129.7, 128.9, 128.8, 128.5, 127.1, 123.9, 123.5, 119.4, 117.3, 112.3, 70.2,
36.4. MS, m/z: 495.85 (Mþ). Analysis calcd. for C27H21N5O3S: C,
65.44; H, 4.27; N, 14.13. Found: C, 65.62; H, 4.35; N, 14.02.
4.1.10. 6-Benzyl-4-(((2-hydroxy-6-methoxyquinolin-3-yl)
methylene)amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one
(8f)
Yield (88%). M. p.: 281e283 ꢀC. 1H NMR (400 MHz, DMSO)
4.1.5. 6-Benzyl-4-(((2-hydroxy-6-isopropoxyquinolin-3 yl)
methylene)amino) -3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one
(8a)
d 13.74 s, 1H, NH), 12.18 (s, 1H, OH), 8.78 (s, 1H, C4eH quinoline),
8.77 (s, 1H, CH]N), 7.60e6.68 (m, 8H, C5,7,8-H quinoline & 5 ArH),
3.92 (s, 2H, CH2), 3.80 (s, 3H, OCH3). 13C NMR (101 MHz, DMF)
Yield (89%). M. p.: 271e272 ꢀC. 1H NMR (400 MHz, DMSO)
d 168.7, 160.1, 157.2, 154.6, 149.5, 148.8, 146.0, 139.1, 136.3, 135.3,
d
13.78 (s, 1H, NH), 12.21 (s, 1H, OH), 8.79 (s, 1H, C4eH quinoline),
129.3, 128.3, 126.6, 123.0, 110.9, 110.6, 95.1, 55.6, 35.9. MS, m/z:
419.13 (Mþ). Analysis calcd. for C21H17N5O3S: C, 60.13; H, 4.09; N,
16.70; Found: C, 60.33; H, 4.12; N, 16.50.
8.78 (s, 1H, CH]N), 7.54e7.18 (m, 8H, C5,7,8-H quinoline & 5 ArH),
4.69e4.58 (m, 1H, OCH), 3.92 (s, 2H, CH2), 1.29 (d, J ¼ 5.9 Hz, 6H,
2CH3). 13C NMR (100 MHz, DMSO)
d 171.0, 169.3, 160.7, 153.1, 150.0,
149.3, 139.7, 136.8, 135.6, 129.7, 128.8, 127.1, 124.7, 123.3, 119.6, 117.3,
4.1.11. 4-(((7-(Benzyloxy)-2-chloroquinolin-3-yl)methylene)
amino)-3-thioxo-6-(3,4,5-trimethoxybenzyl)-3,4-dihydro-1,2,4-
triazin-5(2H)-one (8g)
113.2, 70.3, 36.4, 22.2. MS, m/z: 447.58 (Mþ). Analysis calcd. for
C
23H21N5O3S: C, 61.73; H, 4.73; N, 15.65. Found: C, 61.91; H, 4.63; N,
15.44.
Yield (78%). M. p.: 228e229 ꢀC. 1H NMR (400 MHz, DMSO)
d
13.82 (s, 1H, NH), 9.16 (s, 1H, C4eH quinoline), 9.09 (s, 1H, CH]N),
4.1.6. 6-Benzyl-4-(((2-hydroxy-6-methylquinolin-3-yl)methylene)
amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (8b)
Yield (86%). M. p.: 282e283 ꢀC. 1H NMR (400 MHz, DMSO)
8.25 (d, J ¼ 9.2 Hz, 1H, C5eH quinoline), 7.63e7.28 (m, 7H, C6,8-H
quinoline-H & 5 ArH), 6.61 (s, 2H, C2eH & C6eH trimethoxybenzyl),
5.35 (s, 2H, CH2O), 3.88 (s, 2H, CH2), 3.74 (s, 6H, 2 OCH3), 3.62 (s, 3H,
d
13.74 (s, 1H, NH), 12.20 (s, 1H, OH), 8.76 (s, 1H, C4eH quinoline),
OCH3). 13C NMR (101 MHz, DMSO)
d 170.9, 170.0, 162.9, 153.2, 151.3,
8.71 (s, 1H, CH]N), 7.80e6.84 (m, 8H, C5,7,8-H quinoline-H & 5
150.0, 149.8, 149.3, 138.5, 136.7, 136.6, 132.4, 131.7, 129.1, 128.7,
128.6, 122.2, 121.9, 121.4, 108.2, 107.1, 70.5, 60.4, 56.3, 36.6. MS, m/z:
604.39 (Mþ). Analysis calcd. for C30H26ClN5O5S: C, 59.65; H, 4.34; N,
11.59. Found: C, 59.86; H, 4.75; N, 11.78.
ArH), 3.90 (s, 1H, CH2), 2.34 (s, 1H, CH3). 13C NMR (101 MHz, DMSO)
d
171.0, 169.2, 161.0, 150.0, 149.4, 139.8, 139.2, 136.8, 135.7, 135.0,
132.3, 129.7, 128.8, 127.1, 123.1, 118.8, 115.9, 36.4, 20.8. MS, m/z:
403.67 (Mþ). Analysis calcd. for C21H17N5O2S: C, 62.52; H, 4.25; N,
17.36. Found: C, 62.56; H, 4.53; N, 17.42.
4.1.12. 4-(((2-Chloro-8-methylquinolin-3-yl)methylene)amino)-3-
thioxo-6-(3,4,5-trimethoxybenzyl)-3,4-dihydro-1,2,4-triazin-5(2H)-
one (8h)
4.1.7. 6-Benzyl-4-(((2-hydroxy-7-methoxyquinolin-3-yl)
methylene)amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one
(8c)
Yield (90%). M. p.: 213e214 ꢀC. 1H NMR (400 MHz, DMSO)
d
13.82 (s, 1H, NH), 9.19 (s, 1H, C4eH quinoline), 9.16 (s, 1H, CH]N),
Yield (75%). M. p.: 263e264 ꢀC. 1H NMR (400 MHz, DMSO)
8.14 (d, J ¼ 8.1 Hz, 1H, C5eH quinoline), 7.84 (d, J ¼ 6.8 Hz, 1H, C7eH
quinoline), 7.65 (t, J ¼ 7.6 Hz, 1H, C6eH quinoline), 6.62 (s, 2H, C2eH
& C6eH trimethoxybenzyl), 3.89 (s, 2H, CH2), 3.75 (s, 6H, 2 OCH3),
3.63 (s, 3H, OCH3), 2.70 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3)
d
13.76 (s, 1H, NH), 12.15 (s, 1H, OH), 8.76 (s, 1H, C4eH quinoline),
8.73 (s, 1H, CH]N), 7.88 (d, J ¼ 8.8 Hz, 1H, C5eH quinoline),
7.38e7.16 (m, 5H, ArH), 7.05e6.78 (m, 2H, C6eH & C8eH quinoline),
3.92 (s, 2H, CH2), 3.87 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO)
d
171.2, 169.0, 153.4, 149.5, 148.9, 148.6, 148.5, 139.2, 137.2, 136.9,
d
171.1, 169.1, 163.8, 161.4, 150.1, 149.3, 143.3, 140.0, 136.8, 132.3,
133.3, 130.7, 127.9, 127.1, 126.7, 123.1, 106.4, 60.9, 56.2, 37.0, 17.9. MS,
m/z: 511.16 (Mþ), 513.17 (Mþ2). Analysis calcd. for C24H22ClN5O4S: C,
56.30; H, 4.33; N, 13.68. Found: C, 56.62; H, 4.54; N, 14.03.
129.7, 128.8, 127.1, 119.6, 113.3, 112.9, 98.2, 56.1, 36.4. MS, m/z:
419.68 (Mþ). Analysis calcd. for C21H17N5O3S: C, 60.13; H, 4.09; N,
16.70. Found: C, 60.26; H, 4.31; N, 16.86.
4.1.13. 4-(((2-Hydroxy-6-methylquinolin-3-yl)methylene)amino)-
3-thioxo-6-(3,4,5-trimethoxybenzyl)-3,4-dihydro-1,2,4-triazin-
5(2H)-one (8i)
4.1.8. 6-Benzyl-4-(((2-hydroxyquinolin-3-yl)methylene)amino)-3-
thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (8d)
Yield (80%). M. p.: 282e283 ꢀC. 1H NMR (400 MHz, DMSO)
Yield (89%). M. p.: 284e286 ꢀC. 1H NMR (400 MHz, DMSO)
d
13.78 (s, 1H, NH), 12.30 (s, 1H, OH), 8.82 (s, 1H, C4eH quinoline),
d 13.73 (s, 1H, NH), 12.20 (s, 1H, OH), 8.79 (s, 1H, C4eH quinoline),
8.80 (s, 1H, CH]N), 7.95 (d, J ¼ 7.8 Hz, 1H, C5eH quinoline), 7.65 (t,
8.72 (s, 1H, CH]N), 7.73 (s, 1H, C5eH quinoline), 7.49 (dd,
J ¼ 8.4,1.5 Hz, 1H, C7eH quinoline), 7.29 (d, J ¼ 8.4 Hz, 1H, C8eH
quinoline), 6.60 (s, 2H, C2eH & C6eH trimethoxybenzyl), 3.86 (s,
J ¼ 7.4 Hz, 1H, C7eH quinoline), 7.38 (d, J ¼ 7.4 Hz, C8eH quinoline),
7.32e7.22 (m, 6H, C6eH quinoline & 5 ArH), 3.93 (s, 2H, CH2). 13
C
11

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
2H, CH2), 3.75 (s, 6H, 2 OCH3), 3.63 (s, 3H, OCH3), 2.36 (s, 3H, CH3).
4.1.18. 4-(((2-Hydroxyquinolin-3-yl)methylene)amino)-6-
(thiophen-2-ylmethyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-
one (8n)
13C NMR (100 MHz, DMSO)
d 170.9, 170.0, 153.2, 149.9, 149.4, 148.3,
147.6, 138.7, 138.4, 136.7, 136.1, 132.3, 128.7, 128.0, 126.9, 124.0, 107.1,
60.4, 56.3, 36.6, 21.6. MS, m/z: 493.14 (Mþ). Analysis calcd. for
Yield (88%). M. p.: 273e275 ꢀC. 1H NMR (400 MHz, DMSO)
C
24H23N5O5S: C, 58.41; H, 4.70; N, 14.19 Found: C, 58.54; H, 4.89; N,
d 13.85 (s, 1H, NH), 12.31 (s, 1H, OH), 8.85 (s, 1H, C4eH quinoline),
14.12.
8.82 (s, 1H, CH]N), 7.97 (d, J ¼ 7.8 Hz, 1H, C5eH quinoline), 7.67 (t,
J ¼ 7.6 Hz, 1H, C7eH quinoline), 7.42e7.38 (m, 2H, C5eH thiophene
& C8eH quinoline), 7.30e7.27 (t, J ¼ 7.6 Hz 1H, C6eH quinoline),
4.1.14. 4-(((2-Hydroxy-7-methoxyquinolin-3-yl)methylene)
amino)-3-thioxo-6-(3,4,5-trimethoxybenzyl)-3,4-dihydro-1,2,4-
triazin-5(2H)-one (8j)
6.99e6.96 (m, 2H, C3eH & C4eH thiophene), 4.14 (s, 2H, CH2). 13
C
NMR (100 MHz, DMSO) d 171.1,169.2,161.1,149.8,148.4,141.0,140.3,
Yield (77%). M. p.: 267e269 ꢀC. 1H NMR (400 MHz, DMSO)
138.1, 133.6, 130.6, 127.4, 127.4, 125.8, 123.2, 123.2, 118.9, 116.0, 30.8.
MS, m/z: 395.07 (Mþ). Analysis calcd. for C18H13N5O2S2: C, 54.67; H,
3.31; N, 17.71. Found: C, 54.84; H, 3.43; N, 17.57.
d
13.73 (s, 1H, NH), 12.15 (s, 1H, OH), 8.76 (s, 1H, C4eH quinoline),
8.73 (s, 1H, CH]N), 7.89 (d, J ¼ 8.8 Hz, 1H, C5eH quinoline), 6.91
(dd, J ¼ 8.8, 2.2 Hz, 1H, C6eH quinoline), 6.86 (bs, 1H, C8eH quin-
oline), 6.59 (bs, 2H, C2eH & C6eH trimethoxybenzyl), 3.87 (s, 3H,
4.2. Biological activity
OCH3), 3.86 (s, 2H, CH2), 3.75 (s, 6H, 2 OCH3), 3.63 (s, 3H, OCH3). 13
C
4.2.1. In vitro COX-1 and COX-2 inhibition assay
NMR (100 MHz, DMSO)
d 171.1, 169.1, 163.8, 161.4, 153.2, 150.2,
Cayman colorimetric COX (ovine) inhibitor screening assay kit
(Catalog No. 560131) supplied by Cayman Chemicals (Ann Arbor,
MI, USA) was used to assess the COX-1 and COX-2 inhibitory ac-
tivities of the new hybrids according to the manufacturer’s in-
structions and in accordance to a previously reported method
[6,10,12].
149.2, 143.4, 140.0, 139.9, 136.6, 132.3, 119.6, 113.3, 113.0, 107.0, 98.2,
60.4, 56.3, 56.1, 36.7. MS, m/z: 509.45 (Mþ). Analysis calcd. for
C
24H23N5O6S: C, 56.57; H, 4.55; N, 13.74. Found: C, 56.43; H, 4.76; N,
13.71.
4.1.15. 4-(((2-Hydroxy-6-methoxyquinolin-3-yl)methylene)
amino)-3-thioxo-6-(3,4,5-trimethoxybenzyl)-3,4-dihydro-1,2,4-
triazin-5(2H)-one (8k)
4.2.2. In vitro 15-LOX inhibition assay
The 15-LOX inhibitory activities of tested compounds were
measured using Cayman lipoxygenase inhibitor screening assay kit
Catalog No. (760,700) supplied by Cayman Chemicals (Ann Arbor,
MI, USA) according to the manufacturer’s instructions and in
accordance to a previously reported method [6,10,12].
Yield (86%). M. p.: 291e293 ꢀC. 1H NMR (400 MHz, DMSO)
d
13.72 (s, 1H, NH), 12.18 (s, 1H, OH), 8.79 (s, 1H, C4eH quinoline)
8.77 (s, 1H, CH]N), 7.52 (s, 1H, C5eH quinoline), 7.34e7.31 (m, 2H,
C7eH & C8eH quinoline), 6.60 (s, 2H, C2eH & C6eH trimethox-
ybenzyl), 3.86 (s, 2H, CH2), 3.81 (s, 3H, OCH3), 3.75 (s, 6H, 2 OCH3),
3.63 (s, 3H, OCH3).13C NMR (100 MHz, DMSO)
d 170.5, 168.6, 160.2,
4.2.3. Cell culture studies
154.6,152.7, 149.6, 148.7, 139.1, 136.3,135.3, 131.9,123.0,119.0, 116.8,
110.6, 106.7, 59.9, 55.8, 55.6, 36.2. MS, m/z: 509.14 (Mþ). Analysis
calcd. for C24H23N5O6S: C, 56.57; H, 4.55; N, 13.74. Found: C, 56.33;
H, 4.28; N, 13.54.
RAW 264.7 macrophages were obtained from ATCC (Manassas,
VA) and cultured in Dulbecco’s minimal essential media (DMEM,
Invitrogen) containing 10% HI-FBS, sodium pyruvate (1 mM),
penicillin (100 mg/mL), and streptomycin (100 mg/mL) [55,56].
4.1.16. 6-(4-Hydroxy-3-methoxybenzyl)-4-(((2-hydroxy-6-
methylquinolin-3-yl)methylene)amino)-3-thioxo-3,4-dihydro-
1,2,4-triazin-5(2H)-one (8l)
4.2.3.1. NO production in LPS-activated RAW 264.7 macrophages.
NO
was
detected
using
4-amino-5-methylamino-2,7-
difluorofluorescein diacetate (DAF-FM Diacetate; Molecular
Probes). Briefly, RAW 264.7 cells were seeded in black 96 well
Yield (91%). M. p.: 290e291 ꢀC. 1H NMR (400 MHz, DMSO)
d
13.74 (s, 1H, NH), 12.23 (s, 1H, OH), 8.86 (s, 1H, OH), 8.78 (s, 1H,
plates (200,000 cells/mL, 100
cubation with the individual test compounds or the reference drugs
at different concentrations (1.56, 3.125, 6.25, 12.5, 25, and 50 M)
for 2 h at 37 ꢀC. LPS was then added at a final concentration of 1
g/
ml/well) for 24 h followed by the in-
C4eH quinoline), 8.73 (s, 1H, CH]N), 7.74 (s, 1H, C5eH quinoline),
7.50 (dd, J ¼ 8.4,1.5 Hz, 1H, C7eH quinoline), 7.30 (d, J ¼ 8.4 Hz, 1H,
C8eH quinoline), 6.84 (bs, 1H, C2eH vanillyl), 6.73e6.62 (m, 2H,
C5eH & C6eH vanillyl), 3.81 (s, 2H, CH2), 3.74 (s, 3H, OCH3), 2.37 (s,
m
m
mL for an additional 20 h [57]. The assay was performed by washing
the cells with phosphate buffered saline (PBS) and incubating with
3H, CH3). 13C NMR (100 MHz, DMSO)
d 171.0, 169.2, 165.8, 161.0,
150.0, 147.9, 145.8, 142.6, 139.8, 135.7, 130.5, 129.8, 127.3, 123.1,
122.0, 118.8, 115.9, 115.8, 113.9, 56.0, 36.0, 20.8. MS, m/z: 449.81
(Mþ). Analysis calcd. for C22H19N5O4S: C, 58.79; H, 4.26; N, 15.58.
Found: C, 58.89; H, 4.40; N, 15.41.
DAF-FM (2 mM) in serum free medium. The fluorescence intensity,
which is directly proportional to NO levels, was quantified, as
detailed in our previous study [58]. IC50 values for each compound
were calculated from the dose-response curves. For the fluores-
cence image acquisition, cells were cultured on cover slips in 6
well-plates and treated as detailed above. The images were
captured using an Olympus fully motorized BX63 microscope
(USA).
4.1.17. 4-(((2-Hydroxy-6-methylquinolin-3-yl)methylene)amino)-
6-(thiophen-2-ylmethyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-
one (8m)
Yield (89%). M. p.: 265e267 ꢀC. 1H NMR (400 MHz, DMSO)
d
13.80 (s, 1H, NH), 12.20 (s, 1H, OH), 8.78 (s, 1H, C4eH quinoline),
4.2.3.2. ROS production in LPS-activated RAW 264.7 macrophages.
To test the antioxidant potential of the test compounds following
the induction of inflammation in RAW 264.7 by LPS, the probe of
oxidative species 2,7-dichlorofluorescein diacetate (DCFH-DA)
(Molecular Probes), was used. Following the incubation of the cells
8.74 (s, 1H, CH]N), 7.73 (s, 1H, C5eH quinoline), 7.48 (d, J ¼ 8.4 Hz,
1H, C7eH quinoline), 7.40 (d, J ¼ 4.5 Hz, 1H, C5eH thiophene), 7.29
(d, J ¼ 8.4 Hz, 1H, C8eH quinoline), 6.99e6.96 (m, 2H, C3eH & C4eH
thiophene), 4.12 (s, 2H, CH2), 2.36 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO)
d
170.6, 168.8, 160.5, 149.3, 147.8, 139.4, 138.7, 137.6, 134.5,
with the different test compounds and activation with LPS (1
mL) as detailed above, the cells were incubated with DCFH-DA
(25 M). The fluorescence intensity, which is directly proportional
to intracellular ROS levels, was measured as detailed in our
mg/
131.8, 129.3, 126.9,126.9, 125.2, 122.6, 118.4, 115.4, 30.3, 20.4. MS, m/
z: 409.28 (Mþ). Analysis calcd. for C19H15N5O2S2: C, 55.73; H, 3.69;
N, 17.10. Found: C, 55.97; H, 3.87; N, 17.13.
m
12

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
previous report [58]. IC50 values were calculated from the dose-
response curves.
Funding
The biological work was supported by National Institutes of
Health National Institute of General Medical Sciences grant
P20GM121334 (D.G.R.). The content is solely the responsibility of
the authors and does not necessarily represent the official views of
the National Institutes of Health.
4.2.3.3. TNF-
264.7 macrophages cells. RAW 264.7 cells were seeded in 24-well
plates (200,000 cells/mL, 500 l/well) for 24 h followed by the in-
cubation with the test compounds or the reference drugs at
different concentrations (1.56, 3.125, 6.25, 12.5, 25, and 50 M) for
2 h at 37 ꢀC. The cells were incubated with LPS (1
g/mL) for an
additional 20 h, then the cell culture supernatants were collected.
Commercially available ELISA kits were used to measure TNF-
(catalogue No. DY410, R&D Systems, Inc., MN, USA), IL-6 (catalogue
No. DY406, R&D Systems, Inc., MN, USA), and 15(S)-HETE (cata-
logue No. 534721, Cayman Chemicals) according to the manufac-
turer’s instructions.
a, IL-6 and 15(S)-HETE production in LPS-activated RAW
m
m
References
m
[1] S. Manju, K. Ethiraj, G. Elias, Safer anti-inflammatory therapy through dual
COX-2/5-LOX inhibitors: a structure-based approach, Eur. J. Pharmaceut. Sci.
121 (2018) 356e381.
[2] M.T.-E. Maghraby, O.M. Abou-Ghadir, S.G. Abdel-Moty, A.Y. Ali, O.I. Salem,
Novel class of benzimidazole-thiazole hybrids: the privileged scaffolds of
potent anti-inflammatory activity with dual inhibition of cyclooxygenase and
15-lipoxygenase enzymes, Biorg. Med. Chem. (2020) 115403.
a
[3] P.A. Elzahhar, R. Alaaeddine, T.M. Ibrahim, R. Nassra, A. Ismail, B.S. Chua,
R.L. Frkic, J.B. Bruning, N. Wallner, T. Knape, Shooting three inflammatory
targets with a single bullet: novel multi-targeting anti-inflammatory glita-
zones, Eur. J. Med. Chem. 167 (2019) 562e582.
4.3. Molecular modeling
€
€
[4] T.K. Uski, E.D. Hogestatt, Effects of various cyclooxygenase and lipoxygenase
metabolites on Guinea-pig cerebral arteries, Gen. Pharmacol. 23 (1992)
109e113.
The molecular modelling studies of the compounds 2d, and 3d
were carried out using Molecular Operating Environment (MOE)
version MOE 2019.0102 (Chemical Computing Group, Montreal, CA).
The X-ray crystallographic complex structure of Cyclooxygenase-2
enzyme (COX-2) with ligand SC-558 (PDB entry 1CX2) [50], and
15-LOX (PDB code 4NRE) [51] with substrate mimic. Both were
downloaded from protein data bank website (http://www.rcsb.org).
The protein structures were prepared after downloading using MOE
quick preparation tool. We deleted H2O molecules, repeated chains,
and unwanted surfactants except the metal ion (Fe) in 15-LOX. Each
compound data was prepared by minimizing energy, adding
hydrogen atoms, calculating partial charges and potential energy.
Validation was achieved by restoring the original orientation upon
redocking SC-558 and substrate mimic within their corresponding
enzymes binding sites.
[5] M. Khoshneviszadeh, O. Shahraki, M. Khoshneviszadeh, A. Foroumadi,
O. Firuzi, N. Edraki, H. Nadri, A. Moradi, A. Shafiee, R. Miri, Structure-based
design, synthesis, molecular docking study and biological evaluation of 1, 2, 4-
triazine derivatives acting as COX/15-LOX inhibitors with anti-oxidant activ-
ities, J. Enzym. Inhib. Med. Chem. 31 (2016) 1602e1611.
[6] H.H. Abdu-Allah, A.A. Abdelmoez, H. Tarazi, A.-N.A. El-Shorbagi, R. El-Awady,
Conjugation of 4-aminosalicylate with thiazolinones afforded non-cytotoxic
potent in vitro and in vivo anti-inflammatory hybrids, Bioorg. Chem. 94
(2020) 103378.
[7] Y.M. Omar, H.H. Abdu-Allah, S.G. Abdel-Moty, Synthesis, biological evaluation
and docking study of 1, 3, 4-thiadiazole-thiazolidinone hybrids as anti-
inflammatory agents with dual inhibition of COX-2 and 15-LOX, Bioorg.
Chem. 80 (2018) 461e471.
[8] G. Moussa, R. Alaaeddine, L.M. Alaeddine, R. Nassra, A.S. Belal, A. Ismail, A.F. El-
Yazbi, Y.S. Abdel-Ghany, A. Hazzaa, Novel click modifiable thioquinazolinones
as anti-inflammatory agents: design, synthesis, biological evaluation and
docking study, Eur. J. Med. Chem. 144 (2018) 635e650.
[9] O.S. Afifi, O.G. Shaaban, H.A. Abd El Razik, S.E.-D.A.S. El, F.A. Ashour, A.A. El-
Tombary, M.M. Abu-Serie, Synthesis and biological evaluation of purine-
pyrazole hybrids incorporating thiazole, thiazolidinone or rhodanine moiety
as 15-LOX inhibitors endowed with anticancer and antioxidant potential,
Bioorg. Chem. 87 (2019) 821e837.
4.4. Statistical analysis
[10] P.A. Elzahhar, S.M. Abd El Wahab, M. Elagawany, H. Daabees, A.S. Belal, A.F. El-
Yazbi, A.H. Eid, R. Alaaeddine, R.R. Hegazy, R.M. Allam, Expanding the anti-
cancer potential of 1, 2, 3-triazoles via simultaneously targeting
Cyclooxygenase-2, 15-lipoxygenase and tumor-associated carbonic anhy-
drases, Eur. J. Med. Chem. 200 (2020) 112439.
[11] Y.M. Omar, S.G. Abdel-Moty, H.H. Abdu-Allah, Further insight into the dual
COX-2 and 15-LOX anti-inflammatory activity of 1, 3, 4-thiadiazole-thiazoli-
dinone hybrids: the contribution of the substituents at 5th positions is size
dependent, Bioorg. Chem. 97 (2020) 103657.
The data were analyzed using GraphPad Prism 8 software
(GraphPad Software, San Diego, California). One-way analysis of
variance (ANOVA) or repeated-measures analysis of variance (RM-
ANOVA), followed by Tukey’s post hoc test, were used to state
significance between groups. Data are presented as the
mean SEM. Differences were considered significant at p < 0.05.
[12] B.G. Youssif, M.F. Mohamed, M.M. Al-Sanea, A.H. Moustafa, A.A. Abdelhamid,
H.A. Gomaa, Novel aryl carboximidamide and 3-aryl-1, 2, 4-oxadiazole ana-
logues of naproxen as dual selective COX-2/15-LOX inhibitors: design, syn-
thesis and docking studies, Bioorg. Chem. 85 (2019) 577e584.
[13] S.A. Ali, S.M. Awad, A.M. Said, S. Mahgoub, H. Taha, N.M. Ahmed, Design,
synthesis, molecular modelling and biological evaluation of novel 3-(2-
naphthyl)-1-phenyl-1H-pyrazole derivatives as potent antioxidants and 15-
Lipoxygenase inhibitors, J. Enzym. Inhib. Med. Chem. 35 (2020) 847e863.
[14] R. van der Vlag, H. Guo, U. Hapko, N. Eleftheriadis, L. Monjas, F.J. Dekker,
A.K. Hirsch, A combinatorial approach for the discovery of drug-like inhibitors
of 15-lipoxygenase-1, Eur. J. Med. Chem. 174 (2019) 45e55.
Declaration of competing interest
The authors declare that they have no financial, conflict of in-
terest, or personal relationships that may appear to hinder or affect
this work.
Acknowledgements
[15] A.G. Banerjee, N. Das, S.A. Shengule, R.S. Srivastava, S.K. Shrivastava, Synthesis,
characterization, evaluation and molecular dynamics studies of 5,
6ediphenyle1, 2, 4etriazine3 (2H)eone derivatives bearing 5esubstituted 1,
3, 4eoxadiazole as potential antieinflammatory and analgesic agents, Eur. J.
Med. Chem. 101 (2015) 81e95.
[16] A.G. Banerjee, N. Das, S.A. Shengule, P.A. Sharma, R.S. Srivastava,
S.K. Shrivastava, Design, synthesis, evaluation and molecular modelling
studies of some novel 5, 6-diphenyl-1, 2, 4-triazin-3 (2H)-ones bearing five-
member heterocyclic moieties as potential COX-2 inhibitors: a hybrid phar-
macophore approach, Bioorg. Chem. 69 (2016) 102e120.
We are thankful to the efforts of Faculty of Pharmacy at Zagazig
University for the purchase of MOE license version 2019 (Chemical
Computing Group, Montreal, CA). Our appreciation of the efforts of
Dr. Waleed Ali, Biochemistry lab, Cairo General Hospital for his
collaboration in in vitro COXs/15-LOX enzyme assays. Graphical
abstract created with BioRender.com.
[17] L.W. Mohamed, O.M. El-Badry, A.K. El-Ansary, A. Ismael, Design & synthesis of
novel oxazolone & triazinone derivatives and their biological evaluation as
COX-2 inhibitors, Bioorg. Chem. 72 (2017) 308e314.
Appendix A. Supplementary data
[18] S.A. El-Feky, Z.K. Abd El-Samii, N.A. Osman, J. Lashine, M.A. Kamel, H.K. Thabet,
Synthesis, molecular docking and anti-inflammatory screening of novel
quinoline incorporated pyrazole derivatives using the Pfitzinger reaction II,
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113457.
13

A.M. Ghanim, S. Rezq, T.S. Ibrahim et al.
European Journal of Medicinal Chemistry 219 (2021) 113457
Bioorg. Chem. 58 (2015) 104e116.
[38] A.M. Ghanim, D.W. Knight, N.A. Osman, H.A. Abdel-Fattah, A.M. Kadry, New
methods for the selective alkylation of 3-thioxo-1, 2, 4-triazin-5-ones, Tet-
rahedron Lett. 57 (2016) 2215e2218.
[39] A. Mazumder, M. Shaharyar, Synthesis, characterization, and in vitro anti-
cancer evaluation of novel 2, 5-disubstituted 1, 3, 4-oxadiazole analogue, in:
BioMed Research International, 2014, 2014.
[40] M.K. Singh, A. Chandra, B. Singh, R.M. Singh, Synthesis of diastereomeric 2, 4-
disubstituted pyrano [2, 3-b] quinolines from 3-formyl-2-quinolones through
OeC bond formation via intramolecular electrophilic cyclization, Tetrahedron
Lett. 48 (2007) 5987e5990.
[41] Z.-C. Liu, B.-D. Wang, B. Li, Q. Wang, Z.-Y. Yang, T.-R. Li, Y. Li, Crystal structures,
DNA-binding and cytotoxic activities studies of Cu (II) complexes with 2-oxo-
quinoline-3-carbaldehyde Schiff-bases, Eur. J. Med. Chem. 45 (2010)
5353e5361.
[19] C.S. Manohar, A. Manikandan, P. Sridhar, A. Sivakumar, B.S. Kumar, S.R. Reddy,
Drug repurposing of novel quinoline acetohydrazide derivatives as potent
COX-2 inhibitors and anti-cancer agents, J. Mol. Struct. 1154 (2018) 437e444.
[20] A.M. Mohassab, H.A. Hassan, D. Abdelhamid, M. Abdel-Aziz, K.N. Dalby,
T.S. Kaoud, Novel quinoline incorporating 1, 2, 4-triazole/oxime hybrids:
synthesis, molecular docking, anti-inflammatory, COX inhibition, ulcer-
oginicity and histopathological investigations, Bioorg. Chem. 75 (2017)
242e259.
[21] I. Chaaban, O.H. Rizk, T.M. Ibrahim, S.S. Henen, E.-S.M. El-Khawass, A.E. Bayad,
I.M. El-Ashmawy, H.A. Nematalla, Synthesis, anti-inflammatory screening,
molecular docking, and COX-1, 2/-5-LOX inhibition profile of some novel
quinoline derivatives, Bioorg. Chem. 78 (2018) 220e235.
[22] A.N. Boshra, H.H. Abdu-Allah, A.F. Mohammed, A.M. Hayallah, Click chemistry
synthesis, biological evaluation and docking study of some novel 20-hydrox-
ychalcone-triazole hybrids as potent anti-inflammatory agents, Bioorg. Chem.
95 (2020) 103505.
[42] S. Bondock, H. Gieman, Synthesis, antibacterial and anticancer evaluation of
some new 2-chloro-3-hetarylquinolines, Res. Chem. Intermed. 41 (2015)
8381e8403.
[23] M.H. Abdelrahman, B.G. Youssif, A.H. Abdelazeem, H.M. Ibrahim, A.M. Abd El
Ghany, L. Treamblu, S.N.A. Bukhari, Synthesis, biological evaluation, docking
study and ulcerogenicity profiling of some novel quinoline-2-carboxamides as
dual COXs/LOX inhibitors endowed with anti-inflammatory activity, Eur. J.
Med. Chem. 127 (2017) 972e985.
[24] E.D. AlFadly, P.A. Elzahhar, A. Tramarin, S. Elkazaz, H. Shaltout, M.M. Abu-
Serie, J. Janockova, O. Soukup, D.A. Ghareeb, A.F. El-Yazbi, Tackling neuro-
inflammation and cholinergic deficit in Alzheimer’s disease: multi-target in-
hibitors of cholinesterases, cyclooxygenase-2 and 15-lipoxygenase, Eur. J.
Med. Chem. 167 (2019) 161e186.
[43] S.-Y. Lee, H.-J. Kim, J.-S. Han, Anti-inflammatory effect of oyster shell extract in
LPS-stimulated Raw 264.7 cells, Preventive nutrition and food science 18
(2013) 23.
[44] D.J. Perkins, D.A. Kniss, Blockade of nitric oxide formation down-regulates
cyclooxygenase-2 and decreases PGE2 biosynthesis in macrophages, J. Leukoc.
Biol. 65 (1999) 792e799.
[45] S. Qi, Z. Feng, Q. Li, Z. Qi, Y. Zhang, Myricitrin modulates NADPH oxidase-
dependent ROS production to inhibit endotoxin-mediated inflammation by
blocking the JAK/STAT1 and NOX2/p47phox pathways, Oxid. Med. Cell. Lon-
gev. (2017) 2017.
[25] E.K. Abdelall, P.F. Lamie, W.A. Ali, Cyclooxygenase-2 and 15-lipoxygenase
inhibition, synthesis, anti-inflammatory activity and ulcer liability of new
celecoxib analogues: determination of region-specific pyrazole ring formation
by noesy, Bioorg. Med. Chem. Lett 26 (2016) 2893e2899.
[26] S. Muthuraman, S. Sinha, C. Vasavi, K.M. Waidha, B. Basu, P. Munussami,
M. Balamurali, M. Doble, R.S. Kumar, Design, synthesis and identification of
novel coumaperine derivatives for inhibition of human 5-LOX: antioxidant,
pseudoperoxidase and docking studies, Biorg. Med. Chem. 27 (2019)
604e619.
[27] E.M. Gedawy, A.E. Kassab, A.M. El Kerdawy, Design, synthesis and biological
evaluation of novel pyrazole sulfonamide derivatives as dual COX-2/5-LOX
inhibitors, Eur. J. Med. Chem. 189 (2020) 112066.
[28] A. Sakr, H. Kothayer, S.M. Ibrahim, M.M. Baraka, S. Rezq, 1, 4-
dihydroquinazolin-3 (2h)-yl benzamide derivatives as anti-inflammatory
and analgesic agents with an improved gastric profile: design, synthesis,
cox-1/2 inhibitory activity and molecular docking study, Bioorg. Chem. 84
(2019) 76e86.
[46] M. Zafarullah, W. Li, J. Sylvester, M. Ahmad, Molecular mechanisms of N-
acetylcysteine actions, Cellular and Molecular Life Sciences CMLS 60 (2003)
6e20.
[47] M. Fujita, V. Somasundaram, D. Basudhar, R.Y. Cheng, L.A. Ridnour, H. Higuchi,
K. Imadome, J.H. No, G. Bharadwaj, D.A. Wink, Role of nitric oxide in
pancreatic cancer cells exhibiting the invasive phenotype, Redox biology 22
(2019) 101158.
[48] L.W. Soromou, Z. Zhang, R. Li, N. Chen, W. Guo, M. Huo, S. Guan, J. Lu, X. Deng,
Regulation of inflammatory cytokines in lipopolysaccharide-stimulated RAW
264.7 murine macrophage by 7-O-methyl-naringenin, Molecules 17 (2012)
3574e3585.
[49] D. Namgaladze, R.G. Snodgrass, C. Angioni, N. Grossmann, N. Dehne,
G. Geisslinger, B. Brüne, AMP-activated protein kinase suppresses arach-
idonate 15-lipoxygenase expression in interleukin 4-polarized human mac-
rophages, J. Biol. Chem. 290 (2015) 24484e24494.
[50] R.G. Kurumbail, A.M. Stevens, J.K. Gierse, J.J. McDonald, R.A. Stegeman, J.Y. Pak,
D. Gildehaus, T.D. Penning, K. Seibert, P.C. Isakson, Structural basis for selec-
tive inhibition of cyclooxygenase-2 by anti-inflammatory agents, Nature 384
(1996) 644e648.
[29] Y. Peng, H. Liu, M. Tang, L. Cai, V. Pike, Highly efficient N-monomethylation of
primary aryl amines, Chin. J. Chem. 27 (2009) 1339e1344.
[30] R. Schneider, A. Gohla, J.R. Simard, D.B. Yadav, Z. Fang, W.A. van Otterlo,
D. Rauh, Overcoming compound fluorescence in the FLiK screening assay with
red-shifted fluorophores, J. Am. Chem. Soc. 135 (2013) 8400e8408.
[31] I. Sato, T. Nakao, R. Sugie, T. Kawasaki, K. Soai, Enantioselective Synthesis of
Substituted 3-quinolyl Alkanols and Their Application to Asymmetric Auto-
catalysis, Synthesis, 2004, pp. 1419e1428, 2004.
[32] D. Bigg, O. Lavergne, F.P. Rodas, J. Pommier, G. Ulibarri, J. Harnett, A. Rolland,
A.-M. Liberatore, C. Lanco, J.-B. Cazaux, Comptothecin analogues, preparation
methods therefor, use thereof as drugs, and pharmaceutical compositions
containing said analogues, in: Google Patents, 2002.
[33] M. Abdel-Aziz, H.M. Abdel-Rahman, Synthesis and anti-mycobacterial evalu-
ation of some pyrazine-2-carboxylic acid hydrazide derivatives, Eur. J. Med.
Chem. 45 (2010) 3384e3388.
[34] P. Rajeev, S. Rajendran, Facile Synthesis of 1-Hydroxy-5-methoxy-Benzo [f][2,
7] naphthyridines, Synth. Commun. 40 (2010) 2837e2843.
[35] T.S. Ibrahim, M.M. Hawwas, E.S. Taher, N.A. Alhakamy, M.A. Alfaleh,
M. Elagawany, B. Elgendy, G.M. Zayed, M.F. Mohamed, Z.K. Abdel-Samii,
Design and synthesis of novel pyrazolo [3, 4-d] pyrimidin-4-one bearing
quinoline scaffold as potent dual PDE5 inhibitors and apoptotic inducers for
cancer therapy, Bioorg. Chem. 105 (2020) 104352.
[36] D.K. Kumar, R. Shankar, S.P. Rajendran, Cannizzaro reaction of 2-chloro-3-
formylquinolines and its synthetic utility for 2-acetylfuro [2, 3-b] quino-
lines: the alkaloid analogues, J. Chem. Sci. 124 (2012) 1071e1076.
[37] J. Guillemont, C. Meyer, A. Poncelet, X. Bourdrez, K. Andries, Diarylquinolines,
synthesis pathways and quantitative structureeactivity relationship studies
leading to the discovery of TMC207, Future Med. Chem. 3 (2011) 1345e1360.
[51] M.J. Kobe, D.B. Neau, C.E. Mitchell, S.G. Bartlett, M.E. Newcomer, The structure
of human 15-lipoxygenase-2 with a substrate mimic, J. Biol. Chem. 289 (2014)
8562e8569.
[52] T. Ogunwa, Insights into interaction profile and inhibitory potential of
amentoflavone with
a-glucosidase, tyrosinase and 15-lipoxygenase as vali-
dated therapeutic targets, J Syst Biol Proteome Res 2 (2018) 10e20.
[53] A.R. Offenbacher, T.R. Holman, Fatty acid allosteric regulation of CH activation
in plant and animal lipoxygenases, Molecules 25 (2020) 3374.
ꢀ
[54] C. Theoduloz, J. Alzate-Morales, F. Jimenez-Aspee, M.I. Isla, M.R. Alberto,
M.W. Pertino, G. Schmeda-Hirschmann, Inhibition of key enzymes in the in-
flammatory pathway by hybrid molecules of terpenes and synthetic drugs:
in vitro and in silico studies, Chem. Biol. Drug Des. 93 (2019) 290e299.
[55] E. Soliman, R. Van Dross, Anandamide-induced endoplasmic reticulum stress
and apoptosis are mediated by oxidative stress in non-melanoma skin cancer:
receptor-independent endocannabinoid signaling, Mol. Carcinog. 55 (2016)
1807e1821.
[56] M. Talactac, J.S. Lee, H. Moon, M.Y. Chowdhury, C.J. Kim, The antiviral effect of
high-molecular weight poly-gamma-glutamate against newcastle disease vi-
rus on murine macrophage cells, Adv. Virol. (2014) 301386, 2014.
[57] S.Y. Lee, H.J. Kim, J.S. Han, Anti-inflammatory effect of oyster shell extract in
LPS-stimulated raw 264.7 cells, Preventive nutrition and food science 18
(2013) 23e29.
[58] S. Rezq, A.A. Abdel-Rahman, Central GPR109A activation mediates glutamate-
dependent pressor response in conscious rats, J. Pharmacol. Exp. Therapeut.
356 (2016) 456e465.
14