Conjugation of 4-aminosalicylate with thiazolinones afforded non-cytotoxic potent in vitro and in vivo anti-inflammatory hybrids

Article abstract of DOI:10.1016/j.bioorg.2019.103378

Eicosanoids like leukotrienes and prostaglandins that produced within the arachidonic acid cascade are involved in the pathogenesis of pain, acute and chronic inflammatory diseases. A promising approach for an effective anti-inflammatory therapy is the de

Full text of DOI:10.1016/j.bioorg.2019.103378

Journal Pre-proofs
Conjugation of 4-aminosalicylate with thiazolinones afforded non-cytotoxic
potent in vitro and in vivo anti-inflammatory hybrids
Hajjaj H. M. Abdu-Allah, Alshaimaa A. B. Abdelmeoaz, Hamadeh Tarazi,
Abdel-Nasser A. El-Shorbagi, Raafat El-Awady
PII:
S0045-2068(19)31397-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103378
YBIOO 103378
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Bioorganic Chemistry
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26 August 2019
18 October 2019
19 October 2019
Please cite this article as: H. H. M. Abdu-Allah, A. A. B. Abdelmeoaz, H. Tarazi, A.A. El-Shorbagi, R. El-
Awady, Conjugation of 4-aminosalicylate with thiazolinones afforded non-cytotoxic potent in vitro and in vivo
anti-inflammatory hybrids, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103378
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Conjugation of 4-aminosalicylate with thiazolinones afforded non-cytotoxic
potent in vitro and in vivo anti-inflammatory hybrids
Hajjaj H. M. Abdu-Allah a *, Alshaimaa A. B. Abdelmeoaz a, Hamadeh Tarazi b, Abdel-Nasser A.
El-Shorbagi b, Raafat El-Awady b
a Department Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University, Assiut
71526, Egypt.
b Sharjah Institute for Medical Research and College of Pharmacy, University of Sharjah, Sharjah
27272, UAE
* Dr. Hajjaj Abdu-Allah
Work: (+2088)240-1521
Mobile: +201010211566 hajjaj@aun.edu.eg
ORCID: 0000-0002-4474-9086
1

Abstract
Eicosanoids like leukotrienes and prostaglandins that produced within the arachidonic acid
cascade are involved in the pathogenesis of pain, acute and chronic inflammatory diseases. A
promising approach for an effective anti-inflammatory therapy is the development of inhibitors
targeting more than one enzyme of this cascade. Aiming to develop balanced COX/LOX
inhibitors; 4-aminosalicylate based thiazolinones having different substituents at the 5th position
of the 4-thiazolinone ring (2-22) were designed, synthesized, characterized and evaluated in vitro
and in vivo for their anti-inflammatory activity. Most of the investigated compounds showed
high COX-2 inhibitory potencies (IC50 39-200 nM) with selectivity indexes (30-84). Two
compounds, 19 and 21, (IC50 = 41 and 44 nM), are equipotent to celecoxib (IC50 = 49 nM), while
compound 22 (IC50 = 39 nM) was the most potent. For 15-LOX, compounds 5, 11, 19, 21 and 22
revealed higher potency (IC50 1.5-2.2 µM) than zileuton (IC50 15 µM). Thus, compounds 5, 11,
19, 21 and 22 are potent dual inhibitors of COX-2 and 15-LOX. In vivo anti-inflammatory
testing of these compounds revealed that, compounds 5 and 21 had an anti-inflammatory activity
similar to indomethacin and celecoxib (% inhibition of oedema = 60 9) and higher than
diclofenac potassium (% inhibition = 52 29), while compound 22 (% inhibition = 63 5) was
more active than the reference drugs. The results showed that the activity is controlled by the
bulkiness and lipophilicity of the substituent at the 5th position. The cytotoxicity results revealed
that all compounds are not cytotoxic, additionally, in an experimental model of ulcerogenic
effect, the most active compounds 21 and 22 showed better safety profile than indomethacin.
Further, at the active sites of the COX-1, COX-2 and 15-LOX co-crystal, 19, 21, and 22 showed
high binding forces in free binding energy study, which is consistent with in vitro and in vivo
results. In conclusion, these compounds are good candidates for further biological investigation
as potential anti-inflammatory drugs with dual balanced inhibition of COX and 15-LOX and
good safety profile.
Keywords: 4-aminosalicylic; 4-thiazolinone; COX-2 and COX-1 inhibitors; LOX; anti-
inflammatory; docking
2

1. Introduction
Inflammation is a protective mechanism employed by tissues against endogenous or exogenous
antigens [1]. Prostaglandins (PGs) from cyclooxygenases (COX) pathway and leukotrienes (LTs)
from lipoxygenases (LOX) pathway are both mediators of the inflammatory process generated
from the same arachidonic acid cascade. Inflammation has been implicated in the initiation
and/or progression of a wide number of diseases including rheumatoid arthritis, neurological
disorders, diabetes, cardiovascular diseases, and cancer [2]. Non-steroidal anti-inflammatory
drugs (NSAIDs) such as salicylates are used widely for the treatment of inflammatory conditions
through inhibition of COXs [3-5]. COX enzymes exist in three isoforms; COX-1, COX-2, and
COX-3 [6,7]. COX-1 is a constitutive enzyme found in most cells and has important roles in the
protection of gastric mucosa, platelet aggregation, and renal blood flow. COX-2 is an inducible
isozyme; significantly expressed during inflammation, pain, and oncogenesis [7]. Most NSAIDs
interact with both (COX-1 and COX-2) and therefore their long-term administration often causes
gastrointestinal, renal, and hepatic side effects [8-10]. These findings have led to the hypothesis
that selective COX-2 inhibitors might provide better anti-inflammatory effect with fewer side
effects. Therefore, many selective COX-2 inhibitors were synthesized [11] and marketed as new
NSAIDs generation [12,13]. However, cardiovascular adverse effects associated with coxibs,
have been reported [14,15]. On the other hand, LOXs are involved in arachidonic acid metabolic
process giving Leukotrienes (LTs) in a compensatory mechanism for COX pathway [16].
Therefore, the use of cyclooxygenase inhibitors (NSAIDs) shunts the metabolism of arachidonic
acid toward 15-LOX [17]. LTs are, also, mediators of inflammatory and allergic diseases and are
believed to be involved in cancer, cardiovascular diseases [16] and the gastrointestinal reactions
caused by NSAIDs [18]. Thus, the mammalian 5-LOX and 15-LOX have become drug targets
since their inhibitors may be used for the treatment of pathological conditions such as allergy,
chronic inflammation, certain cancers and cardiovascular diseases [19,20]. Therefore, COX/LOX
co-inhibition can be a valid strategy to avoid gastrointestinal tract and cardiovascular side effects
of NSAIDs [21,22]. 4-thiazolidinone scaffold, and its derivatives have attracted considerable
attention of medicinal chemists as potential lead compounds for novel anti-inflammatory agents
[23-25]. The anti-inflammatory activity of 4-thiazolidinones is associated, primarily, with their
ability to inhibit isoforms of cyclooxygenase (COX) and lipoxygenase (LOX) [24]. Another
interesting core is salicylic acid. The broad spectrum of biological applications of salicylic acid
3

and its esters have fueled up the researchers in synthesizing hundreds of analogues [26-32].
Particularly, a series of 4- and 5-aryl/heteroarylsalicylic acid derivatives were synthesized as
anti-inflammatory agents [31,32]. Among the salicylate derivatives, aminosalicylate is a
privileged structure in various drug classes that exhibit a broad range of biological activities such
as anti-inflammatory, anticancer, antioxidant, antibacterial, and neuroprotective [33,34]. 4-
Aminosalicylic acid (4-ASA) and its positional isomer 5-aminosalicylic acid (5-ASA) are well
known aminosalicylates with distinctive chemical and pharmacological properties [35]. 5-ASA
has no antitubercular activity and is unstable and degrades rapidly. However, 4-ASA is a
comparatively safe antitubercular agent in multidrug resistant tuberculosis. The Anti-
inflammatory activity of 4-aminosalicylic acid (4-ASA) was first described by Lover in 1984
[35]. It exerts its anti-inflammatory effect through COX inhibition, and it is more stable and
inexpensive alternative to 5-ASA [35]. To this end and in continuation of our interest in
aminosalicylates [36-40] and in developing anti-inflammatory thiazolidinone hybrids with dual
inhibition of COX and 15-LOX [41], we postulated that combining the pharmacophores 4-
aminosalicylate and 4-thiazolidinone in one molecular frame could afford effective anti-
inflammatory agents with balanced potent dual inhibition of COX and 15-LOX with better safety
profile. To test this hypothesis, we have synthesized some 4-aminosalicylate-based 4-
thiazolinones having variable substituents at the 5th position of 4-thiazolinone to confer different
electronic and lipophilic properties (Fig. 1). All target compounds (2-22) were evaluated in vitro
for their inhibitory potency against COX-1, COX-2 and 15-LOX. The most potent compounds 5,
19, 21 and 22 were evaluated for their in vivo anti-inflammatory activity. Further, the target
compounds were evaluated for cytotoxicity and the most active compounds, 21 and 22, were
tested for their gastric ulcer liability. In addition, we also conducted free binding energies on
compounds 19, 21 and 22 to explore feasible interactions with the enzymes.
Fig. 1. The design strategy for 4-aminosalicylate-based thiazolinones.
4

2. Results and discussion
2.1. Chemistry
The target compounds were synthesized as shown in Scheme 1. Methyl ester of 4-ASA [42] was
chloroacetylated by heating with chloroacetyl chloride in dry benzene giving methyl-4-
(chloroacetylamino)salicylate (1). Heterocyclization of 1 in the presence of ammonium
thiocyanate in refluxing ethanol efficiently produced methyl 2-hydroxy-4-[(4-oxo-4,5-dihydro-
1,3-thiazol-2-yl)-amino]benzoate (2). The two active methylene protons appear as separate
doublets at d = 4.00 and 3.96 ppm. The final compounds (3-22) were obtained by refluxing 2
with commercially available aromatic aldehydes using a Knoevenagel condensation procedure in
the presence of sodium acetate in glacial acetic acid. The purity of the synthesized compounds
has been checked by TLC and their structures were confirmed by spectral data (IR, 1H, 13C
NMR) and elemental analyses. Thiazolinone structure was considered based on the 1HNMR
pattern and similar to the structure of their closely related analogues [38]. Some compounds in
this series are poorly soluble even in DMSO and it wasn’t possible to measure 13CNMR in
reasonable time.
5

2.2. Biological evaluation
2.2.1. In vitro cyclooxygenase (COX) and lipooxygenase (15-LOX) inhibition assay
We used in vitro biological activity tests such as in vitro COX-inhibition assay to study the
ability of the synthesized compounds to inhibit ovine COX-1 and human recombinant COX-2 by
use of an enzyme immunoassay (EIA) kit. On the other hand, Cayman’s Lipoxygenase Inhibitor
Screening Assay Kit was used to study the ability of the synthesized compounds to inhibit 15-
LOX. As shown in Table 1, all the tested compounds 2-22 exhibited moderate COX-1 isozyme
inhibitory activities (IC50 = 2.67-9.23 µM) except compound 2 (IC50 = 12.3 µM) with the un-
substituted 4-thiazolinone ring and compound 12 (IC50 = 11.21 µM) both had weak activities.
Also, the results showed that all compounds exhibited high COX-2 isozyme inhibitory activity
(IC50 < 1µM) with selectivity index (S.I. = 25.47-83.77) lower than celecoxib (S.I. = 308.16) but
still higher than diclofenac sodium and indomethacin (S.I. = 4.52 and 0.08, respectively). Hence,
the tested compounds are expected to show lower side effects on both gastric and cardiac system.
To investigate the influence of arylidene in activity, compounds (3-13) having different
substituents at phenyl ring were synthesized (Scheme 1) and their in vitro anti-inflammatory
activities were listed in Table 1. The 4-bromo (5) was more active than 3-bromo (6) against both
COX-1 and COX-2 isozymes. It is the most potent as it exhibited moderate inhibitory activity
against COX-1 and high activity against COX-2 isozymes (IC50 = 3.11 µM and 0.046 µM,
respectively). Replacing the phenyl group with a small heterocyclic ring as in compounds 15
(pyridyl) and 16 (thienyl) did not improve the inhibitory activity to a remarkable extent.
However, as illustrated in Table 1, increasing the bulkiness of the moiety at this position
enhanced both COX-1 and COX-2 inhibitory activities, as for compounds 17-22 with isatin
moiety (IC50 = 2.67-8.67 µM and 0.039-0.29 µM, respectively). Within this series (17-22), the
chloro (19) and methoxy (21) analogs were more active as COX-2 inhibitors than the
unsubstituted (17), flouro (18) and methyl (20) analogs. Both compounds 19 and 21 showed
COX-2 inhibitory potencies (IC50 = 41 nM and 44 nM, respectively) comparable to clecoxib
(IC50 = 49 nM). Furthermore, alkylation of isatin nitrogen atom with a bulky p-chlorobenzyl
group as in compound 22 afforded a higher potency (IC50 = 39 nM) than celecoxib.
On the other hand, the inhibitory potencies of the synthesized compounds 2-22 against 15-LOX,
were evaluated using zileuton (an orally active inhibitor of LOX) as a reference drug. Results
(Table 1) revealed that all compounds showed a significantly higher potency against 15-LOX
6

(IC50 = 1.5-8.4 µM) than zileuton (IC50 = 13.8 µM). Within the phenyl series, compounds 3-13,
the 4-bromo (5), 4-OMe (8), and 2,4-dichloro (13) derivatives exhibited moderate potency (IC50
= 2.21, 2.34, and 1.98 µM, respectively). For isatin series (17-22); unsubstituted- (17), 5-fluoro
(18) and 5-Me (20) isatin derivatives showed fair potency (IC50 = 5.64, 4.12, and 2.98 µM,
respectively). While compounds 19 (5-chloro), 21 (5-OMe), and 22 (N-4-chlorobenzyl) exhibited
moderate potency against 15-LOX (IC50 = 1.87, 1.98, and 1.47 µM, respectively). Accordingly,
compounds 19, 21 and 22 are the most potent dual inhibitors of COX and 15-LOX. These results
could be attributed to the bulkiness of substituent at the 5th position of 4-thiazolinone ring.
Table 1.
In vitro COX-1, COX-2, and 15-LOX inhibitory activities of 4-ASA-thiazolinone derivatives (2-
22) as well as reference anti-inflammatory drugs.
IC50 (µM)a
15-LOX
Compd. No.
S.I.b
IC50
(µM)
COX-1
COX-2
Celecoxib
15.1
3.8
0.049
0.84
0.51
-
308.16
4.52
-
diclofenac sodium
2.11
-
Indomethacin
0.041
-
0.08
Zileuton
13.84
8.14
5.28
4.65
2.21
6.52
2.34
3.54
3.41
5.22
1.98
2
3
12.3
8.11
6.75
3.11
7.65
4.32
5.74
4.12
6.98
4.33
0.33
0.26
0.12
0.046
0.11
0.095
0.19
0.084
0.10
0.064
37.27
31.19
56.25
67.61
69.54
45.47
30.21
49.05
69.8
4
5
6
7
8
9
10
11
67.65
7

12
13
14
15
16
17
18
19
20
21
22
11.21
5.41
9.11
9.23
10.21
8.67
5.11
2.98
3.51
2.94
2.67
0.44
0.092
0.29
25.47
58.80
31.41
51.28
30.03
29.89
83.77
72.68
51.62
66.82
68.46
6.54
4.51
3.41
5.29
8.41
5.64
4.12
1.87
2.98
1.98
1.47
0.18
0.34
0.29
0.061
0.041
0.068
0.044
0.039
a IC50 value represents the concentration of the compound that produce 50% inhibition of COX-
1, COX-2, or 15-LOX which is the mean value of two determinations where the deviation from
the mean is < 10% of the mean value. b Selectivity index (COX-1 IC50 /COX-2 IC50).
2.2.2. In vivo anti-inflammatory activity
We investigated the most potent compounds from the in vitro studies on carrageenan-induced
paw-edema in rats [43], a widely used in vivo model of acute inflammation that encompass all
stages of inflammation [44]. The anti-inflammatory activities exhibited by compounds 5, 19, 21
and 22 using equivalent doses to 10 mg/kg of indomethacin were listed in Table 2 and are shown
in Fig. 2. The curves show the effect of tested compounds and reference drugs on decreasing
edema size with time, where most of the tested compounds showed their maximum anti-
inflammatory activity after 3 h time interval (Fig. 2). Most of the tested compounds showed a
significant reduction of edema size versus control after 3 h time interval and comparable to the
reference drugs (Fig. 2B). As shown in Table 2, compounds 5 and 21, which showed high in
vitro COX-2 inhibitory activity (IC50 = 0.046 and 0.044 µM, respectively), gave an anti-
inflammatory activity (57.9% and 55.2% reduction in inflammation after 3 h, respectively)
higher than diclofenac potassium and close to indomethacin and celecoxib (51.6%, 60.5% and
60.5% reduction in inflammation after 3 h, respectively). We noticed that introduction of a bulky
electron withdrawing group such as 4-chlorobenzyl group influenced the activity as for
8

compound 22 with the highest COX-2 inhibitory activity (IC50 = 39 nM) and being the most
active one with 63.15% reduction in inflammation in vivo and it was higher than the reference
drugs. On the other hand, compound 19 with 5-chloroisatin showed moderate in vivo anti-
inflammatory activity.
Table 2.
Anti-inflammatory activity at different time intervals of the tested compounds 5, 19, 21, 22 and
reference drugs in equivalent doses to 10 mg/kg of indomethacin and by use of carrageenan-
induced paw edema method in rats.a
Compd. No.
% Inhibition of Edema
1 h
2 h
3 h
4 h
Indomethacin
55 10
52.5 9.57
63.15 6.07 60.52 10.08
45 5.77
35 5.77
5
57.89 8.59
57.89 0
19
45 8.66
39.47 4.55
38.15 7.78
22.5 4.33
21
22
40 7.07
50 7.7
42.1 5.26
47.36 7.44
55.26 13.67 37.5 4.33
63.15 5.26 42.5 8.29
Diclofenac potassium
47.5 14.79 63.15 5.26 51.62 29.08 47.5 8.29
Celecoxib
55 11.18 57.89 7.44 60.52 8.73 40 7.07
a
Data represent mean values for two separate experiments performed in duplicate where the
deviation from the mean is < 10% of the mean value.
Fig. 2. A) Effect of 5, 19, 21 and 22 and reference drugs on rat-paw thickness (edema size) at
different time intervals. Notice that most of the tested compounds reached their maximum
inhibitory activity after 3 h. B) In vivo anti-inflammatory activity of tested compounds and
9

reference drugs at 3 h time interval (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus
control, Two-tailed Student’s t-test).
2.2.3. Ulcerogenic liability
The most potent compounds 21 and 22 were subjected to further in vivo toxicological evaluation
in order to examine their ulcerogenic potential [45]. The ulcerogenic effect was compared to
indomethacin, a well-known anti-inflammatory drug with ulcerogenic effect, at a dose equivalent
to 10 mg/kg (Fig. 3). We found a significant edema, leukocyte migration and loss of gastric
mucosa integrity in rats treated with indomethacin (Fig. 3B). On the other hand, compounds 21
and 22 showed better safety profile on gastric mucosa than indomethacin as their ulcerogenic
liability was nearly negligible. When the dose of compound 22 was doubled, it did not cause any
gastric ulceration (Fig. 3C,3D). These promising results supported the preliminary COX-1/COX-
2 in vitro inhibitory activities where both compounds showed higher selectivity toward COX-2
but their selectivity remained five times lower than celecoxib. Hence, after administration of
compounds 21 and 22, no gastric injuries occurred. This supports our design to develop effective
anti-inflammatory agents with better safety profile.
10

Fig. 3. Effect of oral indomethacin and compound 22 on mucous membrane of rat stomach.
Histological sections (A–D) in the stomach of rat treated with: (A) control showing normal intact
mucosa (intact arrow), (B) indomethacin showing massive necrotic destruction of epithelium
with loss of the crypts and alteration in muscular layer (interrupted-arrow) with signs of edema
and recruitment of leukocytes (intact arrow). (C) and (D) compound 22 (10, 20 mg/Kg,
respectively) showing normal intact epithelium (intact arrow) and normal blood vessels as
noticed in D (small arrow with triangle head). Sections were stained by use of haematoxylin and
eosin stain. Magnification X10; scale bar: 100µm.
2.2.4. In vitro antitumor activity
In a previous study from our group; 5-aminosalicylate based thiazolinones showed high
antitumor potency against a panel of cancer cell lines; MCF-7, HCT-116, Hela, and A549 [44].
Thus, we tested the antitumor activity of this series (2-22) on two cancer cell lines (MCF7 and
A549). The results showed that these compounds had no significant effect on survival of these
cell lines, and they are not cytotoxic (supplementary data; Tables S1 and S2).
2.3. Molecular docking
For the purpose of investigating the most probable binding modes and type of interactions
secured by our compounds against COX-1, COX-2 and 15-LOX, a molecular docking study was
carried-out. The ovine COX-1 crystal structure (PDB-ID: 3KK6) was selected as template for
this study since the human COX-1 has not been resolved yet. Both structures showed a high
similarity with sequence identity of (~95%) in which the residues of the active site are fully
conserved [46]. On the other hand, the rabbit 15- LOX-1 (PDB-ID:1LOX) was chosen for the
same reason and justified by the high sequence similarity (87%) between the r15-LOX and the
h15-LOX, with the amino acid residue differences located at the N-terminal domain and not in
the catalytic domain [47]. The results of binding affinities for all compounds of this series (2-22)
against the studied enzymes are summarized in Table S3 (Supplementary data), while the results
of the most potent compounds as well as some reference compounds against the studied enzymes
are summarized in Table 3.
Results from binding mode analysis with respect to COX-1 enzyme revealed a consistent binding
pattern (Fig. 4A), in which the indolinone moiety projected deep inside the enzyme sub-pockets
and the salicylate exposed outward. Our most potent derivatives (19 and 22) showed ability to
11

form H-bond interaction with the corresponding Arg-120 residue via their thiazolinone ring
nitrogen (Fig. 4B, 4C). The compound 22 appeared to be superior due to its enhanced
hydrophobic interaction environment with residues of the active site (Supplementary Fig. S1).
Table 3.
Binding affinities
vina scoring
estimated using
algorithm.
Free binding energy
(Kcal/mol)
Compound
COX-1
COX-2
15-LOX-1
Celecoxib
-9.8
-8.1
-7.9
-
-11.3
-7.5
-7.0
-
-
diclofenac sodium
-7.9
-
Indomethacin
Zileuton
19
-7.5
-8.1
-7.9
-8.2
-8.2
-8.1
-8.5
-8.3
-8.4
-8.6
21
22
(A)
(B)
(C)
Fig. 4. Binding mode overview of selected derivatives within the COX-1 active site (PDB-ID:
3KK6). (A) Overlay of the best-docked poses of compounds 19, 21 and 22. (B) Best-docked
12

pose of compound 19. (C) Best-docked pose of compound 22. Ligands and important amino
acids residues are presented in stick rendering. Hydrogen bond interactions are shown as red
dashed lines.
As shown in Fig. 5, results of the best docked poses of our compounds against the COX-2 active
site showed similarity in their binding conformations (Fig. 5A). Compared to COX-1, our
derivatives appeared to be buried deeper inside the enzyme inner-sub-pockets with ability to
secure two H-bond interactions via their salicylate hydroxyl group with the corresponding Arg-
120 amino acid residue (Fig. 5B, 5C).This could be explained by the fact that COX-2 active site
possesses an additional secondary pocket which is not observed in case of COX-1 controlled by
the amino acid residues; His-90, Gln-192 and Tyr-355 [48, 49].
(A)
(B)
(C)
Fig. 5. Binding mode overview of selected derivatives within the COX-2 active site (PDB-ID:
5F19). (A) Overlay of the best-docked poses of compounds 19, 21 and 22. (B) Best-docked pose
of compound 19. (C) Best-docked pose of compound 22. Ligands and important amino acids
residues are presented in stick rendering. Hydrogen bond interactions are shown as red dashed
lines.
Interestingly, the binding mode of our derivatives within the 15-LOX-1 active site showed a
different pattern compared to COX-1 and COX-2, in which the salicylate appeared to be
projected deep inside the enzyme sub-pocket and the indolinone moiety outward (Fig. 6A).
Compound 19 was able to form two H-bond interactions with the corresponding amino acid
residues His-361 and Ile-663 (Fig. 6B), while compound 22 was involved in H-bond interaction
with the residue His-361 (Fig. 6C). The compound 22 appeared to be shifted from complete
13

alignment with other derivative due to its larger molecular structure and to accommodate the 4-
chlorobenzyl extension (Fig. 6A).
(A)
(B)
(C)
Fig. 6. Binding mode overview of selected derivatives within the 15-LOX-1 active site (PDB-ID:
15LOX). (A) Overlay of the best-docked poses of compounds 19, 21 and 22. (B) Best-docked
pose of compound 19. (C) Best-docked pose of compound 22. Ligands and important amino
acids residues are presented in stick rendering. Hydrogen bond interactions are shown as red
dashed lines.
3. Conclusion
Novel 4-aminosalicylate-based 4-thiazolinones (2-22) were designed, synthesized and examined
for their COX-1/COX-2 and 15-LOX inhibitory activity in vitro and anti-inflammatory activity
in vivo. Compound 2 without substituent at the 5th position of the 4-thiazolinone ring showed low
potency suggesting the necessity of additional moieties for receptor binding and hence
confirming that insertion of substituents at that position seems to be essential. Among the phenyl
subset (3-13), compound 5 with 4-bromo was the most potent. Replacing the phenyl group with
isosteric thiophene or pyridine led to less active compounds. Within the isatin subset (17-22),
alkylation of indolinone nitrogen with a bulky benzyl group augmented the activity (22). These
results refer that the activity is controlled mainly by the bulkiness and lipophilicity of the
substituent at the 5th position. All investigated compounds showed high in vitro COX-2
inhibitory potency (IC50 < 1µM). Compounds 19 and 21 are equipotent to celecoxib, while 22 is
more potent. More importantly, all tested compounds were significantly more potent than
zileuton against 15-LOX. Particularly, compounds 5, 11, 13, 19, 21 and 22 with IC50 1.5-2.0 µM.
14

These results are very interesting, since 4-aminosalicylate is not LOX inhibitor [40]. Therefore,
compounds 5, 19, 21 and 22 are highly potent dual inhibitors of COX and 15-LOX. These
compounds were evaluated for their anti-inflammatory activity, in vivo. Two of them
(compounds 5 and 21) had an anti-inflammatory activity close to indomethacin and celecoxib
and higher than diclofenac potassium, while compound 22 revealed promising activity than the
reference drugs. In an experimental model of ulcerogenic effect, compounds 21 and 22 showed
better safety profile than indomethacin. Further, there was no significant cytotoxic activity. In
addition, docking studies have explained and supported the in vitro and in vivo results. In
conclusion, this study led to the discovery of two promising compounds with high potency
against COX and 15-LOX and high in vivo anti-inflammatory activity making them suitable
candidates for safe and effective anti-inflammatory drugs.
4. Experimental
4.1. Chemistry
All reagents and solvents were obtained from commercial suppliers and used without further
purification. Melting points are uncorrected and were determined on an electrothermal melting
point apparatus (Stuart Scientific, model SM.P.3, England, UK), and were uncorrected.
Precoated silica gel plates (kieselgel 0.25 mm, 60G F254, Merck, Germany) were used for TLC
monitoring of reactions. UV light was used for detection. IR spectra (KBr discs) were recorded
on a Shimadzu IR-470 spectrometer (Shimadzu, Kyoto, Japan), at Faculty of Science, Assiut
University, Egypt. Wave numbers in the IR spectra are given in cm-1. 1H-NMR (400 MHz) and
13C-NMR (100 MHz) Spectra were scanned on AVANCE-III High Performance FT-NMR
spectrometer, (Brucker Biospin International AG, Swtizerland) at Faculty of Pharmacy, Ain
Shams University, Egypt or on AVANCE 500 spectrometer (Bruker Biospin K.K.; Yokohama,
Japan) at Tokai University, Japan. Chemical shifts are expressed in δ-values (ppm) relative to
TMS as an internal standard. Elemental microanalyses were performed on elemental analyzer
model flash 2000 thermo fisher at the Regional Center for Mycology and Biotechnology
(RCMB), Faculty of Science, Al-Azhar University, Nasr city, Cairo, Egypt. Elemental CHN are
within 0.45%. Doubling of signals in NMR was described by superscript star (*).
4.1.1. Methyl 4-chloroacetamidosalicylate (1)
Chloroacetyl chloride (2.26 g, 1.6 ml, 0.02 mole) in dry benzene (5 ml) was added dropwise to a
15

cooled suspension of methyl 5-aminosalicylate (3.43 g, 0.02 mole) in dry benzene (50 ml)
while stirring. The mixture was refluxed for 5 h. The reaction mixture was evaporated under
reduced pressure, where a solid residue was obtained; the residue was filtered, washed with
cooled water, hydrochloric acid (2%), again with cooled water, dried and crystallized from
aqueous ethanol). (2.13 g, 88% yield); mp = 184-185 oC; 1H NMR (60 MHz, CDCl3, δ = ppm ) δ
10.90 (s, 1H, NH), 9.90 (s, 1H, OH), 7.75 (d, J = 9 Hz, 1H, ArH-6), 7.40-7.20 ( m, 1H, ArH-5),
7.15, 7.00 (dd, J = 9.0, 3.5 Hz, 1H, ArH-3), 4.20 (s, 2H, CH2), 3.90 (s, 3H, OCH3).
4.1.2. Synthesis of methyl 2-hydroxy-4-[(4,5-dihydro-4-oxo-1,3-thiazol-2-yl)-amino]-
benzoate (2)
A solution of methyl 4-chloroacetamidosalicylate (2.3 g, 9.5 mmol) and ammonium thiocyanate
(1.5 g, 19.7 mmol) in 40 ml ethanol was refluxed for 2 h and allowed to stand overnight. The
mixture was evaporated, and the residue was washed with water and then recrystallized from
ethanol/water to give the target compound (2.13 g, 80% yield); mp = 243.5-245 oC; IR (KBr, ύ
cm-1): 3264, 3195, 3099, 3160, 3000, 2755, 1710, 1674, 1637, 1600, 1479, 1429, 1329, 1207.
3269, 3206, 3031, 2915, 1684, 1664, 1627, 1566, 1493, 1348, 1305, 1238, 788, 760. 1H NMR
(60 MHz, DMSO-d6, δ = ppm ) δ 11.50 (s, 1H; NH), 10.50 (s, 1H; OH), 7.65 (d, J = 9 Hz, 1H;
ArH-6), 7.40-7.20 (d, m, 1H; ArH-5), 7.30, 6.70 (dd, J = 9.0, 3.5 Hz, 1H; ArH-3), 4.25, 3.98
(2*d, J = 15.8 Hz, 2H; CH2), 3.90, 3.88 (2*s, 3H; OCH3). Anal. Calcd. for C11H10N2O4S
(266.07): C, 49.62, H, 3.79, N, 10.52, S, 12.04; Found: C, 49.74 H, 3.80, N, 10.69, S, 12.04.
4.1.3. General Procedure for Synthesis of methyl 2-hydroxy-4-[(5-benzylidene-4-oxo-4,5-
dihydro-1,3-thiazol-2-yl)amino]benzoate (3–22).
To a well-stirred solution of compound 2 (0.3 g, 1.13 mmol) in acetic acid (10 ml) buffered with
sodium acetate (0.18 g, 2.2 mmol), the appropriate arylaldehyde (1.6 mmol) was added. The
solution was refluxed overnight till the completion of the reaction as monitoring by TLC, and
then poured into ice-cold water. The precipitate was filtered, washed with water, and the
resulting crude product was purified by recrystallization from dioxane.
4.1.3.1. Methyl 2-hydroxy-4-[(5Z-benzylidene-4 (H)-oxo-1,3-thiazol-2-yl)-amino]benzoate
(3). Yield 82%, mp 293-294 oC; IR (KBr, ύ cm-1): 3264, 3195, 3099, 3160, 3000, 2755, 1710,
1674, 1637, 1600, 1479, 1429, 1329, 1207. 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ 10.69
(br. s, 1H; NH and OH), 7.83-7.81 (d, J = 8 Hz, 1H; ArH-6), 7.52-7.46 (m, 8H, ArH-5, CH=C,
ArH-3, Ph), 3.89 (s, 3H; OCH3). Anal. Calcd. for C18H14N2O4S (354.07): C, 61.01, H, 3.98, N,
16

7.90, S, 9.05; Found: C, 61.18, H, 3.96, N, 8.07, S, 9.06.
4.1.3.2. Methyl 2-hydroxy-5-[(5-(4-fluorobenzylidene)-4 (H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (4) Yield 80%, mp > 300 oC; 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ
12.37 (br. s, 1H; NH), 10.67 (s, 1H; OH), 7.79 (d, J = 8 Hz, 1H; ArH-6), 7.67-7.63 (m, 3H),
7.35-7.33 (m, 3H), H), 6.51 (s, 1H), 3.89 (s, 3H; OCH3). Anal. Calcd. for C18H13FN2O4S
(372.06): C, 58.06, H, 3.52, N, 7.52, S, 8.61. Found: C, 58.34, H, 3.58, N, 7.64, S, 8.69.
4.1.3.3. Methyl 2-hydroxy-4-[(5Z-(4-bromobenzylidene)-4 (H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (5) Yield 85%, mp > 300 oC, 1H NMR (400 MHz, DMSO-d6) δ= 12.58 (br. s,
1H; NH), 10.68 (s, 1H; OH), 7.83-7.48 (m, 6H; CH=C, Ar-H), 6.60 (br. s., 1H), 3.90 (s, 3H;
OCH3). 13C NMR (100 MHz, DMSO-d6) δ= 168.4, 161.6, 159.0, 139.5, 137.6, 132.3, 131.1,
128.6, 128.4, 125.7, 120.7, 120.1, 116.5, 113.6, 109.8, 109.5, 52.6. Anal. Calcd. for
C18H13BrN2O4S (431.98): C, 49.90, H, 3.02, N, 6.47, S, 7.40; Found: C, 50.05, H, 3.08, N, 6.40,
S, 7.50.
4.1.3.4. Methyl 2-hydroxy-4-[(5Z-(3-bromobenzylidene)-4 (H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (6) Yield 84%, mp 300-301 oC; IR (KBr, ύ cm-1): 3263, 3197, 3099, 3124,
3000, 2992, 2954, 2850, 1669, 1628, 1565, 1497, 1484, 1438, 1272, 885, 774, 720; 1H NMR
(500 MHz, DMSO-d6, δ = ppm ) δ 12.66, 12.53 (2*s, 1H; NH), 10.71 (s, 1H; OH), 7.82-7.34 (m,
CH=C, ArH, m-BrPh), 6.61 (br. s, 1H; ArH-3) 3.89 (br. s, 3H; OCH3). Anal. Calcd. for
C18H13BrN2O4S (431.98): C, 49.90, H, 3.02, N, 6.47, S, 7.40; Found: C, 50.05, H, 3.08, N, 6.40,
S, 7.50
4.1.3.5. Methyl 2-hydroxy-4-[(5Z-(4-dimethylaminobenzylidene)-4 (H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (7) Yield 65%, mp 304-305.5 oC; IR (KBr, ύ cm-1): 3194, 3091, 2951, 2853,
1651, 1624, 1577, 1506, 1484, 1443, 1249, 1211, 1187, 846; 1H NMR (500 MHz, DMSO-d6, δ
= ppm) δ 12.20 (br. s, 1H; NH), 10.69 (s, 1H; OH), 7.81 (d, d, J = 8.0 Hz 1H), 7.64-7.37 (m,
ArH-4), 6.81-6.61 (m, 3H; ArH-3), 3.89 (s, 3H; OCH3), 2.99 (br. s, 6H, NMe2). Anal. Calcd.
for C20H19N3O4S (397.11): C, 60.44, H, 4.82, N, 10.57, S, 8.07 Found: C, 60.72, H, 4.78, N,
10.74, S, 8.09.
4.1.3.6. Methyl 2-hydroxy-4-[(5Z-(4-methoxylbenzylidene)-4 (H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (8) Yield 78%, mp 266-267 oC; 1H NMR (400 MHz, DMSO-d6) δ= 12.36 (br.
s, NH), 10.70 (s, 1H; OH), 7.82 (d, J = 8 Hz, 1H; ArH-6), 7.63-7.49 (m, 3H, =CH, ArH), 7.40-
7.07 (m, 2H; ArH), 6.61 (br. S., 2H, ArH), 3.89 (s, 3H; OCH3), 3.80 (s, 3H; OMe), Anal. Calcd.
17

for C19H16N2O5S (384.08): C, 59.37, H, 4.20, N, 7.29, S, 8.34; Found: C, 59.48, H, 4.23, N, 7.54,
S, 8.80.
4.1.3.7. Methyl 2-hydroxy-4-[(5Z-(Benzo[d][1,3]dioxol-5-ylmethylene -4 (H)-oxo-1,3-thiazol-
2-yl)-amino]benzoate (9) Yield 75%, mp 283-285 oC; 1H NMR (400 MHz, DMSO-d6) δ= 12.46
(br. s, 0.5H; NH), 11.72 (br. s, 0.5H; NH), 10.70 (s, 1H; OH), 7.81 (d, J = 8 Hz, 1H; ArH-6),
7.70-7.60 (m, 2H, =CH, ArH), 7.23-7.05 (m, 3H; ArH), 6.60 (br. S., 1H, ArH), 6.10 (s, 2H,
OCH2O), 3.89 (s, 3H; OCH3); Anal. Calcd. for C19H14N2O6S (398.06): C, 57.28, H, 3.54, N,
7.03, S, 8.05; Found: C, 57.45, H, 3.58, N, 7.19, S, 8.13.
4.1.3.8. Methyl 2-hydroxy-4-[(5Z-(3, 4-dimethoxybenzylidene)-4-(H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (10) Yield 80%, mp 280-281.5 oC; 1H NMR (400 MHz, DMSO-d6) δ= 12.28
(br. s, 1H; NH), 10.70 (s, 1H; OH), 8.29 (s, 1H, =CH), 7.80 (d, J = 8 Hz, 1H; ArH-6), 7.66 (br.
S., 1H, ArH), 7.20-7.09 (m, 3H, ArH), 6.58 (br. s., 1H, ArH), 3.89 (s, 3H, OCH2O), 3.82 (s, 6H;
2OCH3); Anal. Calcd. for C20H18N2O6S (414.09): C, 57.96, H, 4.38, N, 6.76, S, 7.74; Found: C,
58.15, H, 4.42, N, 6.85, S, 7.82.
4.1.3.9. Methyl 2-hydroxy-4-[(5Z-(4-propybenzylidene)-4- (H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (11) Yield 82%; mp 271.5-273 oC; IR (KBr, ύ cm-1): 3198, 2955, 2930, 2870,
1665, 1627, 1565, 1498, 1438, 1244, 1213, 865, 779. 1H NMR (500 MHz, DMSO-d6, δ = ppm)
δ 12.12 (br. s, 1H; NH), 10.69 (s, 1H; OH), 7.80 (d, J= 8 Hz, 1H; ArH), 7.64 (br. s., 1H; =CH),
7.47 (br. s, 2H; ArH), 7.33 (br. s, 2H, ArH), 6.62 (br. s., 1H, ArH-3), 3.89 (s, 3H, OCH3), 2.58
(t, J= 10, 5 Hz, 2H, CH2), 1.60-1.56 (m, 2H, CH2), 0.88 (t, J= 10, 5 Hz, 3H, CH3) . 13C NMR
(125 MHz, DMSO-d6) δ= 172.5, 169.4, 161.6, 144.8, 131.7, 130.1, 129.7, 113.5, 109.3, 52.8,
37.5, 24.3, 21.5, 14.1; Anal. Calcd. for C21H20N2O4S (396.11): C, 63.62, H, 5.08, N, 7.07, S,
8.09; Found: C, 63.75, H, 5.18, N, 7.27, S, 8.19.
4.1.3.10. Methyl 2-hydroxy-4-[(5Z-(4-isopropybenzylidene)-4 (H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (12) Yield 80%; mp 281-282.5 oC; IR (KBr, ύ cm-1): 3200, 3084, 2958, 2871,
1664, 1640, 1603, 1828, 1565, 1509, 1496, 1437, 1348, 1297, 1245, 1213, 822, 781; 1H NMR
(500 MHz, DMSO-d6, δ = ppm) δ 12.52 (br. s, 0.5H; NH), 11.77 (br. s, 0.5H; NH), 10.70 (s, 1H;
OH), 7.82-7.25 (m, 7H, CH=C, ArH), 6.66 (br. s, 2H, ArH), 3.89 (s, 3H, OCH3), 2.89 (m, 1H,
CH), 1.19 (d, J = 15 Hz, 6H, 2CH3). 13C NMR (125 MHz, DMSO-d6) δ= 169.4, 161.6, 151.3,
132.0, 131.4, 130.4, 127.8, 113.6, 109.9, 109.6, 52.8, 33.8, 24.0; Anal. Calcd. for C21H20N2O4S
(396.11): C, 63.62, H, 5.08, N, 7.07, S, 8.09; Found: C, 63.56, H, 5.12, N, 7.02, S, 8.17.
18

4.1.3.11. Methyl 2-hydroxy-4-[(5Z-(2,4-dichlorobenzylidene)-4 (H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (13) Yield 85%; mp 292-294 oC; 1H NMR (500 MHz, DMSO-d6, δ = ppm) δ
12.71 (br. s, 0.5H; NH), 11.89 (br. s, 0.5H; NH), 10.68 (s, 1H; OH), 7.86-7.25 ( m, 6H, =CH and
ArH), 6.60 (br. s., 1H, ArH), 3.89 (s, 3H; OCH3). Anal. Calcd. for C24H16ClN3O2S: C, 64.64,
H, 3.62, N, 9.42, S, 7.19. Found: C, 64.92, H, 3.57, N, 9.68, S, 7.11. Anal. Calcd. for
C18H12Cl2N2O4S (421.99): C, 51.08, H, 2.86, N, 6.62, S, 7.58; Found: C, 51.23, H, 2.90, N, 6.74,
S, 7.65.
4.1.3.12. Methyl 2-hydroxy-4-[(5Z-(3-phenylprop-2-en-1-ylidene)-4 (H)-oxo-1,3-thiazol-2-
yl)-amino]benzoate (14) Yield 84%; mp 286.5-288 oC; IR (KBr, ύ cm-1): 3255, 3193, 3050,
2950, 2847, 1663, 1634, 1582, 1556, 1489, 1438, 1409, 1299, 1242, 1195, 1166, 1146, 1099,
955, 873, 741, 691; 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ 12.35 (br. s, 0.5H; NH), 11.70
(br. s, 0.5H; NH), 10.69 (s, 1H; OH), 7.81 (d, J= 4.8 Hz, 1H; ArH-6), 7.64 (br. s, 2H, ArH),
7.35-6.95 (m, 7H, CH=C, ArH), 6.60 (br. s., 1H, ArH-3), 3.89 (s, 3H, OCH3). 13C NMR (100
MHz, DMSO-d6) δ= 169.4, 161.6, 151.3, 136.2, 131.9, 129.9, 129.3, 128.2, 124.0, 109.7, 109.6,
52.8. Anal. Calcd. for C20H16N2O4S (380.08): C, 63.14, H, 4.24, N, 7.36, S, 8.43; Found: C,
63.17, H, 4.19, N, 7.29, S, 8.50.
4.1.3.13. Methyl 2-hydroxy-4-[5Z-(3-pyridinylmethylidene -4 (H)-oxo-1,3-thiazol-2-yl)-
o
amino]benzoate (15) Yield 73%; mp > 300 C; IR (KBr, ύ cm-1): 3264, 3205, 3084, 2953,
1682, 1662, 1628, 1566, 1498, 1483, 1432, 1347, 1299, 1242, 1214, 872, 730; 1H NMR (500
MHz, DMSO-d6, δ = ppm) δ 12.62 (br. s, 1H; NH), 10.70 (s, 1H; OH), 8.87-8.58 (m, 2H, ArH-
6, =CH), 7.99-7.27 (m, 5H, ArH), 6.62 (br. s., 1H, ArH-3), 3.89 (s, 3H, OCH3). Anal. Calcd. for
C17H13N3O4S (355.06): C, 57.46, H, 3.69, N, 11.82, S, 9.02; Found: C, 57.59, H, 3.72, N, 11.98,
S, 9.06.
4.1.3.14. Methyl 2-hydroxy-4-[(5Z-(3-thienylmethylidene -4 (H)-oxo-1,3-thiazol-2-yl)-
amino]benzoate (16) Yield 68%; mp 252-253 oC; IR (KBr, ύ cm-1): 3454, 3198, 2950, 1663,
1626, 1559, 1500, 1438, 1349, 1296, 1221, 1169, 1146, 1097, 780, 695; 1H NMR (500 MHz,
DMSO-d6, δ = ppm) δ 12.50 (br. s, 0.5H; NH), 11.76 (br. s, 0.5H; NH), 10.70 (s, 1H; OH), 8.01-
7.90 (m, 3H, ArH-6, =CH, ArH), 7.60 (br. s., 1.5H, ArH), 7.25 (br. s., 1.5H, ArH-3), 6.62 (br. s.,
1H, ArH), 3.89 (s, 3H, OCH3). Anal. Calcd. for C16H12N2O4S2 (360.02): C, 53.32, H, 3.36, N,
7.77, S, 17.79; Found: C, 53.49, H, 3.34, N, 7.89, S, 17.84.
4.1.3.15. Methyl 2-hydroxy-4-[(5Z-(2-oxoindolin-3-ylidene)--4 (H)-oxo-1,3-thiazol-2-yl)-
19

amino]benzoate (17) Yield 85%; mp > 300 oC; IR (KBr, ύ cm-1): 3168, 3053, 2950, 2820, 1709,
1684, 1663, 1619, 1440, 1338, 1211, 1142, 955, 858, 785; 1H NMR (500 MHz, DMSO-d6, δ =
ppm) δ 12.77 (br. s, 1H, NH), 11.82 (br. s, 1H, NH), 11.16 (s, 1H, OH), 10.71 (s, 1H, OH), 8.90
(d, J= 8 Hz, 1H, ArH-6), 8.82 (d, J= 8 Hz, 1H, ArH-6), 7.84, 7.64 (br. s*, 1H, =CH), 7.39-7.37
(m, 2H, ArH-6,), 7.09-7.07 (br. s, 1H, ArH), 6.95 (br. s, 1H; ArH), 6.62 (br. s, 1H; ArH), 3.89 (s,
3H, OCH3). Anal. Calcd. for C19H13N3O5S (395.06): C, 57.72, H, 3.31, N, 10.63, S, 8.11; Found:
C, 57.89, H, 3.34, N, 10.89, S, 8.16.
4.1.3.16. Methyl 2-hydroxy-4-[(5Z-(5-fluoro-2-oxoindolin-3-ylidene)-4-(H)-oxo-1,3-thiazol-
2-yl)-amino]benzoate (18) Yield 80%; mp 278-280 oC; 1H NMR (500 MHz, DMSO-d6, δ =
ppm) δ 12.85 (br. s, 1H, NH), 11.94 (br. s, 1H, NH), 11.18 (s, 1H, OH), 10.71 (s, 1H, OH), 8.77,
8.63* (d, J= 8 Hz, 0.5H, ArH-6), 7.63 (br. s, 1H; =CH), 7.30-7.23 (m, 2H, ArH), 6.94 (br. s, 1H,
ArH), 6.62 (br. s, 1H, ArH), 3.89 (s, 3H, OCH3). Anal. Calcd. for C19H12FN3O5S (413.05): C,
55.20, H, 2.93, N, 10.17, S, 7.76; Found: C, 55.48, H, 2.97, N, 10.44, S, 7.84.
4.1.3.17. Methyl 2-hydroxy-4-[(5Z-(5-chloro-2-oxoindolin-3-ylidene)-4-(H)-oxo-1,3-thiazol-
2-yl)-amino]benzoate (19) Yield 78%; mp > 300 oC; IR (KBr, ύ cm-1): 3351, 3247, 3155, 3051,
2949, 2825, 1706, 1671, 1615, 1570, 1454, 1440, 1342, 1211, 785; 1H NMR (400 MHz, DMSO-
d6, δ = ppm) δ 12.87 (br. s, 1H; NH), 11.91 (br. s, 1H; NH), 11.27 (d, J= 8 Hz, 1H), 10.70 (d, J=
8 Hz, 1H), 8.97 (s, 0.5H), 8.85 (s, 0.5H), 7.90 (d, J= 8 Hz, 1H; ArH-6), 8.63 (d, J= 8 Hz, 0.5H;
ArH-6), 7.80 (br. s. 1H, ArH), 7.82 (d, J= 8 Hz, 1H), 7.60 (br. s., 1H, =CH), 7.43-7.38 (m, 1H;
ArH), 7.25 (br. s, 0.5H; ), 6.98-6.92 (m, 1H; ArH), 6.61 (br. s, 1H; ArH), 3.93, 3.88 (s, 3H,
OCH3). Anal. Calcd. for C19H12ClN3O5S (429.02): C, 53.09, H, 2.81, N, 9.78, S, 7.46; Found: C,
53.24, H, 2.80, N, 9.86, S, 7.52.
4.1.3.18. Methyl 2-hydroxy-4-[(5Z-(5-methyl-2-oxoindolin-3-ylidene)-4-(H)-oxo-1,3-thiazol-
2-yl)-amino]benzoate (20) Yield 80%; mp > 300 oC; 1H NMR (400 MHz, DMSO-d6, δ = ppm)
δ 12.63 (br. s, 1H; NH), 11.67 (br. s, 1H; NH), 11.03 (s, 1H, OH), 10.71 (s, 1H, OH), 8.79 (s,
0.5H, NH), 8.66 (s,0.5H, NH), 7.83 (d, J = 8 Hz, 1H; ArH-6), 8.63 (br. s., 0.5H; =CH), 7.27-7.17
(m, 1.5H, ArH), 6.82 (br. s, 1.5H, ArH), 6.61 (br. s, 1H; ArH), 3.89 (s, 3H, OCH3), 2.31 (s, 3H,
H3). Anal. Calcd. for C20H15N3O5S (409.07): C, 58.67, H, 3.69, N, 10.26, S, 7.83; Found: C,
58.91, H, 3.72, N, 10.48, S, 7.90.
4.1.3.19. Methyl 2-hydroxy-4-[(5Z-(5-methoxy-2-oxoindolin-3-ylidene)--4 (H)-oxo-1,3-
thiazol-2-yl)-amino]benzoate (21) Yield 78%; mp 289-290 oC; IR (KBr, ύ cm-1): 3173, 3057,
20

2954, 2834, 1698, 1658, 1611, 1488, 1438, 1335, 1217, 1156, 1141, 950, 873, 785; 1H NMR
(500 MHz, DMSO-d6, δ = ppm) δ 12.79 (br. s, 1H, NH), 11.83 (br. s, 1H, NH), 11.03 (s, 1H,
OH), 10.96 (s, 1H, OH), 8.67 (s, 0.5H, NH), 8.53 (s, 0.5H, NH), 7.83 (d, J = 8 Hz, 1H, ArH-6),
7.64 (br. s., 0.5H, =CH), 7.28 (br. s, 0.5H, ArH), 6.98-6.82 (m, 3H, ArH), 6.62 (br. s, 1H, ArH),
3.89 (s, 3H, OCH3), 3.77 (s, 3H, OCH3). Anal. Calcd. for C20H15N3O6S (425.07): C, 56.47, H,
3.55, N, 9.88, S, 7.54; Found: C, 56.62, H, 3.59, N, 10.02, S, 7.61.
4.1.3.20. Methyl 4-[(5Z-(4-chlorobenzyl)-2-oxoindolin-3-ylidene)-4 (H)-oxo-1,3-thiazol-2-yl)
amino]-2-hydroxybenzoate (22) Yield 85%; mp 153-154 oC; IR (KBr, ύ cm-1): 3460, 3260,
2951, 2830, 1687, 1608, 1465, 1439, 1356, 1255, 1183, 1107, 878; 1H NMR (500 MHz, DMSO-
d6, δ = ppm) δ 12.00 (br. s, 1H, NH), 10.72 (s, 1H, OH), 10.68 (s, 1H, OH), 9.03, 7.83 (2*d, J=
5 Hz, 1H, ArH), 8.81, 7.93 (2*d, J = 10 Hz, 1H, ArH), 7.16-7.11 (m, 5H, ArH), 7.08-7.02 (m,
4H, ArH), , 1H, ArH-3), 5.05, 5.00 (2*d, J = 15 Hz, 2H, CH2), 3.93, 3.89 (2*s, 3H; OCH3). 13C
NMR (125 MHz, DMSO-d6, δ = ppm) δ = 172.5, 169.4, 168.6, 168.1, 167.6, 164.9, 160.2,
144.0, 143.1, 138.6, 135.6, 135.5, 135.3, 135.2, 133.2, 132.8, 132.6, 132.2, 131.6, 131.3, 129.7,
129.6, 129.6, 129.2, 129.2, 128.4, 128.3, 126.2, 123.5, 123.2, 123.0, 120.5, 119.8, 119.4, 117.6,
114.9, 110.4, 110.0, 53.1, 52.9; Anal. Calcd. for C26H18ClN3O5S (519.07): C, 60.06, H, 3.49, N,
8.08, S, 6.17; Found: 60.06, H, 3.49, N, 8.08, S, 6.17.
4.2. Biological evaluation
4.2.1. In vitro cyclooxygenase and lipooxygenase inhibition assay
The inhibitory activity of the tested compounds against both COX-1 and COX-2 isozymes was
measured by use of colorimetric COX-Inhibitor Screening Assay Kit (ACETM EIA Kit catalog
number 560131, Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s
instructions. Briefly, different concentrations of reference drugs or tested compounds were
incubated with the enzyme for a period of 5 min at 25 °C, followed by the addition of
arachidonic acid and a color developing substrate. Afterwards the absorbance was measured at
590 nm using plate reader. For evaluating the activity of the tested compounds against 15-LOX
enzyme, colorimetric Lipoxygenase Inhibitor Screening Assay Kit (LOX ISA Kit catalog
number 760700, Cayman Chemical, Ann Arbor, MI, USA) was used according to the
manufacturer’s instructions. Briefly, different concentrations of reference drugs or tested
compounds were mixed with hydrogen peroxide, and then incubated with substrate (either
arachidonic acid or linolenic acid) for 5 minutes at 25 °C. After the addition of a color
21

developing substrate the absorbance was measured at 500 nm using plate reader and soybean 15-
LOX as positive control.
4.2.2. In vivo anti-inflammatory activity
Healthy male albino wistar rats (120-150 g) were obtained from the animal house, Faculty of
Medicine, Assiut University, Egypt. Rats were kept in sterilized cages and were maintained
under repeated cycles of light and dark at 24 1 °C and relative humidity of 55 15%. They were
provided with standard rat chow and water ad libitum. Ethical allowance was obtained from the
university animal ethics committee for handling the experimental animals. The carrageenan-
induced rat model was used to test the anti-inflammatory activity of the most active compounds
from in vitro assay and MOE study (compounds 5, 19, 21 and 22), and according to a specified
protocol [43]. Generally, 1% of carrageenan was injected in one hind footpad, half an hour
before they were treated with the test compounds while the other footpad is injected with saline
as a control. Each rat was injected i.p. with 28 µmol/Kg rat, where indomethacin, diclofenac
potassium, and celecoxib were used as reference drugs. The assessment of paw volume/thickness
was performed at specified time points (0, 0.5 h, 1 h, 2 h, 3 h, and 4 h) following compounds
injection. The average paw volume was measured by Vernier calliper and compared with control
and reference drugs. Reduction in the paw volume in test compounds compared with control
animals was considered as anti-inflammatory response. The percentage of paw edema inhibition
was calculated by using the following formula [43,44].
Inhibition of Paw edema (%) = Oc-Ot / Oc X 100.
Where Oc is edema volume (the average difference in thickness between the two hind paws)
of control group and Ot is edema volume of treated group.
4.2.3. Ulcerogenic liability
The ulcerogenic effect of compounds 21, 22, and indomethacin was evaluated according to a
reported method [45]. In this study twenty adult male albino rats were used, divided into five
groups and had been fasted for 18 h before the administration of drugs. One group received 1%
aqueous solution of CMC (w/v) as a control group, then the other three groups received a single
dose of the tested compounds (21 and 22) equivalent to 10 mg indomethacin per kg rat and
indomethacin (as a drug causing gastric ulcer), respectively. Each dose was first dissolved in 1%
22

aqueous solution of CMC (w/v). Furthermore, the dose of compound 22 was doubled and given
to the last group to ensure its safety at higher doses. 6 h later, stomachs were collected from all
the experimental rats after sacrificing them through cervical dislocation. Each stomach was
opened along the greater curvature; rinsed with saline and examined for any morphological
changes. Presence of lesions in gastrointestinal mucosa was taken as an indication for the
ulcerogenic effect. Then, stomach specimens were fixed in 1% formalin, dehydrated with ethanol
and embedded in paraffin wax. Serial sections were taken and stained with hematoxylin and
eosin according to a specified protocol [45]. The stained sections were observed under
microscope (Labomed Lx400 microscope, Labomed, NY microscope Company INC., USA).
Images were recorded using iVu 3100 3.0 Mega Pixel CMOS Digital Camera and Labomed
PixelProTM software (Labomed, NY microscope Company INC., USA). Image processing was
carried out with the ImageJ software.
2.2.4. In vitro antitumor activity
The synthesized compounds have been tested for their cytotoxicity at one high concentration (10
µM) using doxorubicin as internal positive standard as previously reported [38]. The fraction of
cells remaining survival is given in the Tables S1 and S2. The results given are the average of 6
different readings.
4.3. In-silico docking
All the docking experiments were carried-out using the program Autodock Vina [50]. The X-ray
crystal structures of COX-1 (PDB-ID: 3KK6), COX-2 (PDB-ID: 5F19) and 15LOX-1 (PDB-ID:
1LOX) were obtained from the RSCB protein data bank (http://www.rcsb.org). In the aspirin-
acetylated human COX-2 structure (PDB-ID: 5F19), the acetylated Ser-530 was mutated into the
corresponding Ser-530 employing rotamers library [51,52]. The complexed inhibitors were
extracted from the initial X-ray structures followed by removal of water molecules. Polar
hydrogens and Gas tieger charges were added and the corresponding charge files were generated
using the MGL Tools. The compounds under study were drawn using the software ChemDraw
Ultra 8.0 (Cambridge Soft Corporation, USA) and were optimized for energy and geometry
using MMFF94 force field. All the compounds were treated employing the preparation
procedure mentioned earlier. Later, a grid boxes were established and centered to cover the
active sites in each macromolecule (Table S4, supplementary data), with a spacing of 1.0 Å
23

between the grid points. The exhaustiveness and the number of poses were set to 12 and 10
respectively. The 3D-best docked poses were visualized using PyMOL molecular viewer
(Schrödinger Inc., New York, NY, USA).
Acknowledgements
This work was supported by Faculty of pharmacy, Assiut University.
Declaration of Competing Interest
The authors have declared no conflict of interest
Appendix A. Supplementary data
Supplementary data to this article can be found online at ……….
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