Design, Synthesis, and Anti-Inflammatory Evaluation of Novel Diphenylthiazole-Thiazolidinone Hybrids

Article abstract of DOI:10.1002/ardp.201500104

A series of diphenylthiazole-thiazolidinone hybrids was synthesized and evaluated in vitro and in vivo as anti-inflammatory/analgesic agents. The inhibition of cyclooxygenase (COX) enzymes was suggested as a molecular mechanism for the hybrids to exert their anti-inflammatory action. Of these compounds, 13b, 14, and 15b showed the most potent COX inhibitory activity with IC₅₀ values between 2.03 and 12.27 μM, but with different selectivity profiles. All compounds were further evaluated in vivo for their anti-inflammatory/analgesic activities using three animal models. Interestingly, the results of the COX assay were in agreement with those of in vivo assays where the most potent COX inhibitors, 13b, 14, and 15b, exhibited the highest anti-inflammatory/analgesic activities compared to diclofenac. On the contrary, compounds 11 and 12 were the least potent ligands in vitro and in vivo as well.

Full text of DOI:10.1002/ardp.201500104

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Full Paper
Design, Synthesis, and Anti-Inflammatory Evaluation of Novel
Diphenylthiazole–Thiazolidinone Hybrids
1
,2
3,4
5
Ahmed H. Abdelazeem , Samir A. Salama , and Ibrahim A. Maghrabi
1
Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
Department of Pharmaceutical Chemistry, Taif University, Taif, Saudi Arabia
Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
Department of Pharmacology, Division of Biochemistry and GTMR Unit, Taif University, Taif, Saudi
Arabia
2
3
4
5
Department of Clinical Pharmacy, College of Pharmacy, Taif University, Taif, Saudi Arabia
A series of diphenylthiazole–thiazolidinone hybrids was synthesized and evaluated in vitro and in vivo
as anti-inflammatory/analgesic agents. The inhibition of cyclooxygenase (COX) enzymes was
suggested as a molecular mechanism for the hybrids to exert their anti-inflammatory action. Of these
compounds, 13b, 14, and 15b showed the most potent COX inhibitory activity with IC50 values
between 2.03 and 12.27 mM, but with different selectivity profiles. All compounds were further
evaluated in vivo for their anti-inflammatory/analgesic activities using three animal models.
Interestingly, the results of the COX assay were in agreement with those of in vivo assays where the
most potent COX inhibitors, 13b, 14, and 15b, exhibited the highest anti-inflammatory/analgesic
activities compared to diclofenac. On the contrary, compounds 11 and 12 were the least potent
ligands in vitro and in vivo as well.
Keywords: Anti-inflammatory/analgesic / COX / Diphenylthiazole–thiazolidinone hybrids / NSAIDs
Received: March 18, 2015; Revised: April 16, 2015; Accepted: April 17, 2015
DOI 10.1002/ardp.201500104
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
found to be the key enzymes that catalyze the rate-limiting
step in the biotransformation of arachidonic acid into
prostaglandins, biologically active materials that are respon-
sible for various physiological processes and pathological
conditions (such as inflammation) as well [4, 5]. Currently,
COX enzymes exist in three different isoforms: mainly COX-1
and COX-2 isoforms and recently, COX-3 [6]. COX-1 is a
constitutive isoform and is basically involved in the biosyn-
thesis of cytoprotective prostaglandins in GIT and thrombox-
ane which stimulates platelet aggregation. On the contrary,
COX-2 is an inducible isoform which is responsible for the
biosynthesis of prostaglandins that cause peripheral inflam-
mation [7]. During the past few years, the long-term use of
NSAIDs has been severely hampered by the emerging of
several serious effects such as gastrointestinal ulcers, hepato-
toxicity, renal dysfunction, and cardiotoxicity, in particular
with COX-2 selective inhibitors [8]. Therefore, the develop-
ment of novel anti-inflammatory agents with better thera-
peutic profile and devoid of these classical and severe
The classical non-steroidal anti-inflammatory drugs (NSAIDs)
remain the most commonly marketed anti-inflammatory and
analgesic medications [1]. NSAIDs are considered as the drugs
of choice for the treatment of several inflammatory diseases
such as rheumatoid arthritis, osteoarthritis, and acute and
chronic pain [2]. They exert their biological effects via
inhibition of the major anti-inflammatory targets; cyclooxy-
genase (COX) enzymes which were recognized in the
inflammatory cascades [3]. Cyclooxygenases (COXs) were
Correspondence: Dr. Ahmed H. Abdelazeem, Department of
Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University,
Beni-Suef 62514, Egypt.
E-mail: ahmed.abdelazeem@pharm.bsu.edu.eg,
ahmed.pharm@yahoo.com
Fax: þ20 82 2317958
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drawbacks remains an important issue in the field of
medicinal chemistry.
molecules would certainly open up new doors for the
treatment of inflammation with fewer side effects.
One of the most common and frequently used templates in
the design of novel anti-inflammatory motifs, especially COX
inhibitors, is the diphenylheterocylic privileged structure [9].
Celecoxib, rofecoxib, and valedoxib are the most familiar
examples of NSAIDs incorporating different diphenylheter-
ocyles as main building blocks in their structures [6].
Meanwhile, a series of diarylthiazole derivatives (1) was
reported as anti-inflammatory agents that showed high
affinity for COX enzymes [10], preferentially for COX-2
isoform (Fig. 1).
On the other hand, numerous reports have appeared in the
literature describing thiazolidinone as a core structure in a
wide variety of pharmacologically active compounds that
possess high activity and affinity for various biotargets [11]. A
number of 4-thiazolidinones-containing compounds provided
a broad spectrum of biological activities such as anti-
inflammatory [12, 13], complex COX-2/5-LOX inhibitors [14],
or phospholipase A2 (PLA2) [15] possessing anti-inflammatory
action, anti-diabetic [16], anti-microbial [17], and anticancer
activities [18]. A panel of 4-thiazolidinone derivatives (2–4)
was reported to be associated with better degree of anti-
inflammatory activity compared to indomethacin [13, 19, 20]
and showed high levels of carrageenan-induced paw edema
inhibition (Fig. 2).
Recently, we have reported a series of thiazolidinone-based
diphenylthiazoles with good anticancer activity. These com-
pounds were found to exert their anti-tumor effect, at least in
part, through inhibition of COX enzymes [18]. These findings
gave us a great impetus to speculate for new thiazolidinone
derivatives and evaluate their anti-inflammatory and analge-
sic potential. One of the most powerful and interesting
strategies in modern medicinal chemistry to explore novel and
highly active therapeutic molecules is the combination of two
pharmacophores into a single hybrid molecule. In view of the
above data, we describe herein the synthesis of a series of
Results and discussion
Chemistry
The preparation of the target compounds (11–15) was
achieved as outlined in Scheme 1. The starting material 9,
4,5-diphenylthiazol-2-amine, was synthesized in a high yield
from the readily condensation of desyl chloride, obtained
from the reaction of benzoin 8 with thionyl chloride in
pyridine, with thiourea in absolute ethanol using the
previously reported method [21]. The intermediate 11 was
prepared using two methods; first, the starting material 9 was
reacted with chloroacetyl chloride in methylene chloride and
triethylamine at 0°C followed by heterocyclization using
ammonium isothiocyanate in refluxing ethanol to efficiently
furnish 2-((4,5-diphenylthiazol-2-yl)imino)thiazolidin-4-one
11 [18]. The second method was accomplished through the
initial preparation of the starting material 2-carboethoxyme-
thylthio-2-thiazoli-4-one 7 from the reaction of 2-thioxo-4-
oxothiazolidine triethylammonium salt 6 with ethyl chlor-
oacetate in acetone. Subsequently, the intermediate salt 7
was coupled with the amino compound 9 in ethanol to
provide the thiazolidinone derivative 11 in a good yield [13].
The mechanism that has been proposed for the formation of
thiazolidine-4-one derivative 11 is well-documented and
previously mentioned in many reports [18, 22]. The availability
of active methylene group in position 9 of 2-oxyarylamino-2-
thiazolin-4one 11 in addition to the previous reports on the
pharmacological significance of the substitution at that
particular position encouraged us to synthesize the final 5-
arylidene derivatives (12–15) utilizing Knoevenagel reaction
conditions. Accordingly, the intermediate 11 was condensed
with various aldehydes in toluene in the presence of
ammonium acetate to afford the final targeted compounds
1
“hybrid COX inhibitors” wherein the diphenylthiazole skele-
(12–15). It is worth noting that the H NMR revealed the
ton is coupled to thiazolidinone moiety (Fig. 3). To validate
our design, in vitro COX inhibitory activity and in vivo anti-
inflammatory screening were assessed. Considering the
aforementioned reports on the pharmacological activities
of both diphenylthiazole derivatives and thiazolidinone
moiety, it was envisioned that the newly designed hybrid
appearance of the methine proton downfield as a singlet
peak with chemical shift equal to or slightly more than
7.00 ppm due to the reported deshielding effect attributed to
the adjacent carbonyl group. This observation is a good proof
for the presence of the Knoevenagel reaction products as only
Z isomers [23]. All physical data of the newly synthesized
Figure 1. Some diphenylheterocycle-based COX enzymes inhibitors.
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Figure 2. Some thiazolidinone-based anti-inflammatory compounds.
compounds are presented in Table 1 and all compounds were
anticipated mechanism of the newly synthesized hybrids (11–
15) as anti-inflammatory and analgesic agents, COX inhibitory
assay was accomplished at concentrations of 2, 10, and 50 mM
for both COX-1 and COX-2, using diclofenac and celecoxib as
positive controls. The results were expressed in terms of IC50
values and presented in Table 2. The overall results revealed
that most compounds, except 11 and 12, showed moderate to
high inhibitory activities against COX subtypes in comparing
with diclofenac and celecoxib with different selectivity
profiles. Obviously, compound 11 was the least potent COX
inhibitor and this could be attributed to the absence of the
substitution at 4-thiazolidinone core. Thus, a reverse trend of
relatively increased inhibitory activities was noticed upon
addition of substituted 5-benzylidine moiety on the rest of
compounds. However, compound 12 with thiophene moiety
showed weak activity indicating the significant effect of
the substituents’ size on COX inhibition. Interestingly, the
replacement of the five-membered thiophene ring in 12 with
pyridine in 13a or phenyl ring in 15a resulted in a marked
increase in the inhibitory activity and selectivity for COX-1. It
was found that compounds 13b and 15b were the most potent
and preferentially COX-1 selective inhibitors with IC50 values
of 3.51 and 2.03 mM, respectively, while potent compound 14
confirmed by elemental analysis as well as by other spectral
1
13
data ( H NMR, C NMR, and HRmass).
Pharmacology
The newly synthesized diphenylthiazole–thiazolidinone
hybrids were assessed as potential anti-inflammatory and
analgesic agents using both in vitro and in vivo bioassays. All
hybrid molecules initially underwent in vitro screening assay
to evaluate their inhibitory activities on COX enzymes and the
results were expressed in terms of IC50 values (the concentra-
tion that caused a 50% inhibition) as shown in Table 2.
Consequently, all compounds were examined for their in vivo
efficacy utilizing three in vivo measures; carrageenan-induced
paw edema assay was used for assessment of the anti-
inflammatory activity while the carrageenan-induced
paw hyperalgesia and hot-plate assays were used for
testing the anti-hyperalgesia and anti-nociception potential,
respectively.
Inhibition of COX subtypes activity
It is well documented that COX enzymes are robust and valid
targets for treatment of inflammation. In order to explore the
Figure 3. The newly designed anti-inflammatory scaffold.
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Scheme 1. Synthesis of the target compounds 11–15. Reaction reagents and conditions: a) TEA, EtOH, reflux, 1 h; b) ClCH
acetone, reflux, 2 h; c) SOCl , pyridine, 1 h; d) thiourea, EtOH, reflux, 2 h; e) EtOH, reflux; f) ClCH COCl, TEA, CH Cl , rt, 1 h; g) NH
acetone; h) appropriate aldehyde, CH COONH , toluene, reflux, 2 h.
2
COOC
2 5
H ,
2
2
2
2
4
SCN,
3
4
bearing the bulky naphthyl moiety was COX-2 selective ligand
with IC50 values; COX-1 ¼ 6.22 mM and COX-2 ¼ 3.84 mM. The
increased COX-2 selectivity of compound 14 might be
attributed to the larger size of COX-2 active site over COX-
affect the COX inhibitory activities but it could alter their
selectivity profiles.
Carrageenan-induced mice paw edema assay
1
which allows accommodation of bulky compounds [24].
The in vivo anti-inflammatory activity of the new compounds
(11–15) was performed using carrageenan-induced mice paw
edema model. Diclofenac was used as a reference drug. Each
drug was immediately given (10 mg/kg orally) to the
corresponding animal group after induction of inflammation
by carrageenan s.c. injection. Mean changes in paw edema
thickness were recorded at 1, 3, and 5 h after induction of
Moreover, compound 15e bearing electron donating methyl
group exhibited good activity with increased COX-2 selectivity
index. On the contrary, compounds 15c and 15d with electron
withdrawing groups were moderate COX inhibitors with
slight selectivity to COX-1 subtype. Clearly from the results, we
can conclude that the electronic effect did not significantly
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Table 1. List of the newly synthesized compounds (11–15) with molecular weights, melting points, and yields.
Compounds
R’
Molecular
formula
Mol. wt.
m.p. (°C)
Yield (%)
1
1
2
H
C
C
18
H
13
N
3
3
OS
OS
2
3
351.45
445.58
240–242
268–270
67
78
1
H
23 15
N
1
1
3a
3b
C
C
24
H
16
N
4
4
OS
OS
2
2
440.54
440.54
270–271
275–277
72
74
H
24 16
N
1
4
C
C
29
H
19
N
3
3
OS
OS
2
2
489.61
241–244
73
1
1
5a
5b
H
25 17
N
439.55
484.55
253–254
261–262
74
72
25 16 4 3 2
C H N O S
1
5c
25
C H16ClN
3
OS
2
474.00
224–225
78
1
1
5d
5e
C
25
H
17
N
3
O
2
S
2
455.55
453.58
285–287
263–265
75
78
26 19 3 2
C H N OS
inflammation and displayed in Table 3. The anti-inflammatory
activity of these compounds could be attributed to their
ability to inhibit COX enzymes activities which in turn inhibit
the synthesis of prostaglandins. Interestingly, the results were
in agreement to good extent with the in vitro results of COX
inhibitory assay where compounds 13b, 14, 15b, and 15e
exhibited the most potent anti-inflammatory activities
compared to diclofenac. It was noticed that compound 13b
was more active than diclofenac with edema inhibition
percentage of 67%. On the other hand, compounds 13a, 15a,
owing to their weak to moderate activities on both COX
enzymes. However, the least potent compounds were 11 and
12 which demonstrated the least in vitro inhibitory activity
against COXs. Consistently, all compounds displayed the
highest edema inhibition percentages at 5 h after their
administration, proposing that they are not rapidly metabo-
lized (Fig. 4). These results emphasize the importance of 5-
benzylidene moiety at the 5-position of the 4-thiazolidinone
for the biological activities, including anti-inflammatory
activity, based on the well-established and reported postu-
late [25, 26]. Moreover, we can conceptualize that the
1
5c, and 15d showed moderate anti-inflammatory activities
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Table 2. The results of in vitro COX enzymes inhibitory assay expressed in IC50 and selectivity index.
a)
COX inhibition (IC50mM)
b)
Compounds
COX-1
COX-2
SI
1
1
1
1
1
1
1
1
1
1
1
2
22.45
19.16
13.34
3.51
6.22
12.25
2.03
10.39
14.21
9.36
2.60
16.2
>30
>30
17.62
12.27
3.84
16.98
6.74
12.50
15.64
6.41
14.11
0.34
0.74
0.64
0.76
0.28
1.62
0.72
0.30
0.83
0.91
1.46
0.18
47.64
3a
3b
4
5a
5b
5c
5d
5e
Diclofenac
Celecoxib
a)
b)
IC50 is the concentration of the test compound that gives 50% inhibition of enzyme activity. Duplicates of each concentration
were used for calculation of IC50
SI ¼ selectivity index (COX-1 IC50/COX-2 IC50).
.
electronic effect of the various substitutions did not
significantly influence the activity.
percentages of 79.26, 87.63, and 86.42% respectively.
Meanwhile, compounds 13a, 14, 15a, and 15d showed
moderate analgesic activities. However, compounds 11 and 12
exhibited the least withdrawal latency percentages of 21.48
and 37.04%, respectively. It was noteworthy from the
comparison of these results with that of the carrageenan-
induced paw edema assay, both assays results are consistent.
Carrageenan-induced thermal hyperalgesia assay
Subsequently, all prepared hybrids (11–15) were subjected to
in vivo evaluation for their analgesic activity using carrageen-
an-induced thermal hyperalgesia assay. Figure 5 shows the
withdrawal latency percentages of the synthesized probes at
5
h post-drug administration. Almost all compounds, except
Hot-plate test
1
1 and 12, exhibited appreciable withdrawal latency com-
All the newly synthesized compounds (11–15) were further
assessed for their analgesic activity using hot-plate assay in
mice. In this assay, all of the test compounds along with the
positive control diclofenac were given orally in a dose of
pared with diclofenac indicating a good analgesic activity.
Compounds 13b, 15b, and 15e showed excellent analgesic
activities compared to diclofenac with withdrawal latency
Table 3. Anti-inflammatory activity of test compounds (11–15) using carrageenan-induced paw edema.
Edema thickness in mm mean ꢀ SD (% edema inhibition)
Compounds
Basal
1 h
3 h
5 h
NC
Dic.
2.41 ꢀ 0.23
2.39 ꢀ 0.27
2.42 ꢀ 0.20
2.41 ꢀ 0.26
2.39 ꢀ 0.28
2.41 ꢀ 0.22
2.41 ꢀ 0.22
2.39 ꢀ 0.23
2.41 ꢀ 0.19
2.42 ꢀ 0.15
2.42 ꢀ 0.16
2.41 ꢀ 0.23
3.57 ꢀ 0.34
4.12 ꢀ 0.24
4.261 ꢀ 0.28
3.48 ꢀ 0.33 (5.95)
3.38 ꢀ 0.18 (16.99)
3.52 ꢀ 0.24 (4.66)
3.45 ꢀ 0.47 (7.84)
3.40 ꢀ 0.08 (14.48)
3.43 ꢀ 0.32 (3.36)
3.52 ꢀ 0.25 (2.24)
3.54 ꢀ 0.51 (2.59)
3.56 ꢀ 0.37 (2.07)
3.49 ꢀ 0.18 (7.32)
3.58 ꢀ 0.36 (7.76)
3.26 ꢀ 0.23 (49.03)
3.75 ꢀ 0.22 (22.23)
3.75 ꢀ 0.19 (21.53)
3.53 ꢀ 0.36 (33.06)
3.31 ꢀ 0.38 (47.67)
3.55 ꢀ 0.41 (33.59)
3.43 ꢀ 0.15 (38.91)
3.46 ꢀ 0.25 (38.85)
3.43 ꢀ 0.12 (65.30)
3.32 ꢀ 0.39 (47.51)
3.48 ꢀ 0.25 (37.86)
3.04 ꢀ 0.30 (65.10)
3.91 ꢀ 0.44 (19.43)
3.70 ꢀ 0.27 (30.09)
3.43 ꢀ 0.26 (43.67)
3.02 ꢀ 0.34 (67.37)
3.21 ꢀ 0.37 (56.66)
3.52 ꢀ 0.28 (38.80)
3.12 ꢀ 0.39 (61.90)
3.53 ꢀ 0.17 (40.26)
3.43 ꢀ 0.30 (45.18)
3.12 ꢀ 0.09 (61.90)
1
1
1
1
1
1
1
1
1
1
1
2
3a
3b
4
5a
5b
5c
5d
5e
Mice in different groups were subjected to carrageenan-induced paw edema test to evaluate the anti-inflammatory activity of
the test compounds. The dorso-ventral thickness of edema in the hind paw was measured in mm at zero time (Basal), 1, 3, and 5 h
post-drug treatment. Negative control, NC (given vehicle) and positive control, Dic (given diclofenac, 10 mg/kg) were included
for comparison. The results are presented as mean ꢀ SD, (n ¼ 6) and percent inhibition.
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Figure 4. The inhibition percentage of edema at
5
h post-drug administration. Negative control,
NC (given vehicle) and positive control, Dic
given diclofenac, 10 mg/kg) were included for
(
comparison.
1
0 mg/kg b.w. It was found that almost all prepared
of the amino acid residue, Ile523 in COX-1 with the smaller
Val523 residue in COX-2 creates an additional side pocket and
enlarges the size of the COX-2 active site that enables the
more bulky compounds to fit nicely and well accommodated
[24]. Accordingly, it was noticed from the docking of
compounds 11, 13b, and 15b within the COX-1 binding site
that these compounds were pushed to the bottom of the
active site and forced to adopt a longitudinal disposition due
to presence of Ile523 that closed the side additional pocket.
Figure 7 shows the aromatic hydrophobic stacking between
phenyl groups in compounds (11, 13b, and 15b) and Trp387
and Tyr385 aromatic rings. Moreover, these compounds
formed an essential H-bond with Arg120 residue which is
important for COX-1 affinity and selectivity, (Fig. 7). Obviously,
this orientation of compound 11 could explain its selectivity
toward COX-1 enzymes.
On the other hand, compound 14, exhibiting in vitro COX-2
selectivity, was docked into the COX-2 active site. The results
revealed that compound 14 assumed a similar binding pattern
to that of the cocrystallized ligand 1-phenylsulfonamide-3-
trifluoromethyl-5-parabromophenylpyrazole (S58). Interest-
ingly, the side additional corridor of COX-2 could accommo-
date the bulky naphthyl moiety as well as the 4-thiazolidinone
heterocycle forming a network of H-bonding with Ile517 and
compounds significantly delayed the latency time compared
with the basal values, indicating reduced nociception. Of
these compounds, 13a, 13b, 14, 15c, and 15e exhibited the
most potent analgesic activity compared to the used standard
analgesic drug, diclofenac, with latency change percentage of
more than 100%. However, compounds 15a and 15d showed
intermediate activity owing to their intermediate to weak
inhibitory activities on COX enzymes. On the other hand,
compound 12 incorporation thiophene moiety was the
weakest drug probe with inhibition percentage of 43.84%.
These results are displayed in Table 4 and graphically
presented in Fig. 6.
Molecular docking study
In an attempt to understand the proper binding modes and to
explain the difference in selectivity profiles of the newly
synthesized hybrids toward COX subtypes, some of the active
and selective compounds were docked into the active sites of
COX-1 and COX-2, respectively, using LIGANDFIT embedded in
Discovery Studio software [27]. The 3D crystal structures of
COX-1 and COX-2 protein complexed with their cocrystallized
ligands (PDB codes: 1PGF and 1CX2, respectively) were used
for this approach. It is well-documented that the substitution
Figure 5. Withdrawal latency change percent-
age at 5 h post-drug administration.
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Table 4. Analgesic effect of test compounds on thermal pain induced by hot-plate.
Response latency in seconds (change %)
Compounds
Basal
1 h
2 h
NC
Dic.
13.33 ꢀ 2.4
15.00 ꢀ 1.6
14.00 ꢀ 2.2
14.60 ꢀ 3.2
13.00 ꢀ 2.2
14.00 ꢀ 2.4
12.33 ꢀ 3.8
15.50 ꢀ 1.7
14.80 ꢀ 3.7
13.67 ꢀ 2.5
15.00 ꢀ 1.5
13.83 ꢀ 2.7
14.40 ꢀ 3.1
14.33 ꢀ 2.8
*
*
25.50 ꢀ 4.1 (70.00)
31.00 ꢀ 5.1 (106.67)
*
1
1
1
1
1
1
1
1
1
1
1
2
15.10 ꢀ 2.7 (7.86)
19.20 ꢀ 3.5 (31.51)
22.80 ꢀ 4.0 (62.86)
*
21.00 ꢀ 5.1 (43.84)
*
*
*
*
3a
3b
4
5a
5b
5c
5d
5e
19.21 ꢀ 5.7 (47.69)
27.40 ꢀ 3.6 (110.77)
34.83 ꢀ 4.9 (148.81)
28.00 ꢀ 4.0 (127.03)
*
28.00 ꢀ 2.1 (100.00)
*
25.50 ꢀ 3.6 (106.76)
*
15.90 ꢀ 3.8 (2.58)
25.40 ꢀ 3.6 (63.87)
*
*
22.20 ꢀ 3.4 (50.00)
28.00 ꢀ 5.1 (91.22)
*
*
*
22.33 ꢀ 3.9 (63.41)
30.83 ꢀ 4.9 (125.60)
*
*
23.30 ꢀ 4.1 (55.33)
25.00 ꢀ 3.8 (66.67)
*
20.33 ꢀ 3.2 (46.99)
33.5 ꢀ 5.3 (142.17)
Mice in different groups were subjected to hot-plate test to evaluate the analgesic effect. The latency time in seconds at zero
time (Basal), 1 and 2 h post-drug treatment is presented as mean ꢀ SD and percent change (change %). Negative control NC
(
given vehicle) and positive control Dic (given diclofenac) were included for comparison.
Statistically significant difference from the corresponding Basal, p < 0.05 (n ¼ 6).
*
Figure 6. Percentage change in latency time at
2
h post-drug treatment.
Figure 7. A) Docking and binding mode of compound 11 within COX-1 active site (PDB code: 1PGF), B) docking and binding mode of
compound 13b into the same COX-1 binding pocket, C) docking and binding mode of compound 15b into the same COX-1 binding
pocket. Hydrogen bonds are represented by dashed green lines. All hydrogens are removed for the purpose of clarity.
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525

Arch. Pharm. Chem. Life Sci. 2015, 348, 518–530
A. H. Abdelazeem et al.
ARC HH PHARM
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Figure 8. A) Docking and binding mode of compound 14 within COX-2 active site (PDB code: 1CX2), B) docking and binding mode of the
co-crystallized ligand, 1-phenylsulfonamide-3-trifluoromethyl-5-parabromophenylpyrazole (S58) into the same COX-2 binding pocket.
Hydrogen bonds are represented by dashed green lines. All hydrogens are removed for the purposes of clarity.
Leu352 in addition to an electrostatic interaction with
Arg513. Furthermore, positioning the diphenyl rings within
the hydrophobic pouch, consisted of the side chains of Tyr348,
Val349, and Trp387, was well-correlated with the position of
the bromophenyl fragment of S58 into the same pouch
through p-stacking interactions (Fig. 8).
compound 14. It is worthwhile that the in vitro COX inhibitory
assay results were consistent with that of in vivo bioassays.
The docking study of some selected hybrids into both COX
subtypes active sites supported the in vitro results and
illustrated the variation in affinity and selectivity profiles
of the newly synthesized ligands. Taken together, these
promising findings would be helpful to explore novel
therapeutics for potential treatment of inflammatory
diseases.
Conclusion
In this study, a series of diphenylthiazole–thiazolidinone
hybrids was designed and synthesized. All the newly
synthesized compounds were evaluated for their anti-
inflammatory and analgesic activity in vitro against COX
enzymes and in vivo using three common animal models.
Compounds 13b, 14, 15b, and 15e were the most active COX
inhibitors with IC50 values between 2.03 and 12.27 mM.
However, compounds 13a, 15a, 15c, and 15d showed
intermediate COX inhibitory activity. On the contrary com-
pounds 11 and 12 exhibited the weakest activity confirming
that the 5-benzylidine moiety is essential for activity. It was
observed that the electronic effect of the various substitutions
has not significantly influenced the activity but it affects the
selectivity profiles of both COX subtypes. On the other hand,
the results of the in vivo studies revealed that compounds 13b,
Experimental
Chemistry
Chemical reagents and solvents were obtained from
commercial sources. Solvents are dried by standard methods
when necessary. Melting points (m.p.) were uncorrected and
were carried out by open capillary tube method using IA
9100MK-Digital Melting Point Apparatus. Microanalyses
were carried out at the micro-analytical Center, Faculty
of Science, Cairo University using Vario EL III elemental
analyzer (Vario, Germany). Infrared spectra were made on
Bruker Vector 22 (Japan) infrared spectrophotometers and
ꢁ
1
were expressed in wavenumber (cm ) using potassium
bromide disc. The proton magnetic resonance H NMR
1
1
4, 15b, and 15e showed the highest edema inhibition
spectra were recorded on a Varian Mercury VX-300 NMR
spectrometer at 400 MHz and a Bruker APX400 spectrometer
at 400 MHz in the specified solvent, chemical shifts were
reported on the d scale and were related to that of the
solvent and J-values are given in Hz. C NMR spectra were
obtained on a Bruker APX400 at 100 MHz at the Faculty of
Pharmacy, Beni-Suef University. Mass spectra were recorded
on a Fennigan MAT, SSQ 7000 mass spectrometer, at 70 eV
(EI) at the Microanalytical Center, Faculty of Science, Cairo
University. The high resolution mass spectra (HRMS) were
percentages of 67.37, 56.66, 61.90, and 61.90%, respectively
compared with 65.1% for diclofenac. Similarly, these four
compounds were the most potent analgesic agents in hot-
plate test. Moreover, compounds 13b, 15b, and 15e showed
excellent analgesic activities in carrageenan-induced thermal
hyperalgesia assay with withdrawal latency percentages of
1
3
7
9.26, 87.63, and 86.42% respectively. On contrast, it was
found that compounds 11 and 12 were the weakest drug
probes in vitro against COX enzymes as well as in vivo in the
three used animal bioassays. From the study of structure
activity, it could be concluded that the substitution at position
five of thiazolidinone heterocycle is important for activity. In
addition, compounds bearing bulky moieties were found
to be more effective and COX-2 selective inhibitors as in
recorded on
a Waters Micromass Q-Tof Micro mass
spectrometer with a lock of spray source. Thin layer
chromatography was done using Macherey–Nagel Alugram
Sil G/UV254 silica gel plates and methylene chloride/
methanol (9.5:0.5) as the eluting system.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 518–530
Anti-Inflammatory Diphenylthiazole–Thiazolidinone Hybrids
ARC HH PHARM
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2
-Thioxo-4-oxothiazolidine triethylammonium (6) [13]
(1.4 g, 18.2 mmol) was refluxed for 5 h in acetone (40 mL).
After the reaction was completed, the mixture was cooled.
The precipitate was collected by filtration, washed with
water, and recrystallized from ethanol/acetone mixture to
afford compound 11 as a light brown solid.
Triethylamine (3.8 g, 37.5 mmol) was added slowly to a
mixture of rhodanine 5 (5 g, 37.5 mmol) in isopropanol
(
20 mL). The mixture was gently heated until obtaining a
clear solution. Thereafter, the mixture was cooled and the
solid formed was filtered off, washed with isopropanol, and
then recrystallized from toluene to afford the salt 6 in a high
yield (91%), m.p. 80–81°C.
Method B [13]: A mixture of 2-carboethoxymethylthio-2-
thiazoli-4-one, 7 (3 g, 13.7 mmol) and 4,5-diphenylthiazol-2-
amine (3.5 g, 13.7 mmol) was heated under reflux in
isopropanol (20 mL) for 3 h. After the reaction was completed,
the mixture was cooled and the obtained precipitate was
filtered off, washed with water, and recrystallized from
2
-Carboethoxymethylthio-2-thiazoli-4-one (7) [13]
A mixture of ethyl chloroacetate (3.12 g, 25.5 mmol) and
1
2
-thioxo-4-oxothiazolidine
triethylammonium
6
(5 g,
ethanol to furnish compound 11 as a brown solid. H NMR
2
1.2 mmol) was refluxed in acetone (20 mL) for 3 h. The
6 2
(400 MHz, DMSO-d ): d 4.03 (s, 2H, CH ), 7.29–7.49 (m, 10H),
12.17 (s, 1H, NH). C NMR (101 MHz, DMSO): d 174.57 (C–O),
167.25, 163.87, 146.08, 134.76, 132.01, 129.82, 129.48, 128.86,
1
3
separated solid was filtered off and the solution was
evaporated under vacuum. The residue obtained was then
recrystallized from isopropanol to give the intermediate
compound 7, yield (65%), m.p. 78–79°C.
128.79, 128.74, 128.36, 35.53 (CH
2
). HRMS calcd. for
þ
C
H N
18 13 3
OS
2
[MþH] 351.0500, found 352.0615. Anal. calcd.
for: C18H N
13 3
OS
2
: C, 61.52; H, 3.73; N, 11.96. Found: C, 61.15;
2
-Chloro-1,2-diphenylethanone (8) [21]
H, 3.48; N, 12.03.
A mixture of benzoin (10 g, 47mmol) and pyridine (5.7 mL) was
heated until a solution wasobtained, then cooled in an ice bath
until solid. The mass obtained was coarsely ground and thionyl
chloride (7.5 g, 63 mmol) was added slowly with vigorous
stirring and cooling in an ice bath. After about an hour, water
was added and the solid was coarsely ground, filtered, and
washed several times with water. The crude product was dried
by suction and left overnight over calcium chloride until full
dryness. The white powder obtained was recrystallized from
ethanol to give colorless crystals of 2-chloro-1,2-diphenyletha-
General procedure for the synthesis of compounds 12,
13a,b, 14, and 15a–e
A mixture of compound 11 (1.57 mmol) and appropriate
aldehyde (1.57 mmol) was refluxed with ammonium acetate
(0.25 g, 3.20 mmol) in anhydrous toluene (20 mL). The reaction
mixture was refluxed for 3 h and then the solvent was
evaporated. The residue was dissolved in water acidified with
conc. HCl. The product was extracted into ethyl acetate, dried
over sodium sulfate, and evaporated under vacuum to obtain
the purified final target compounds.
þ
none 8 (79%). MS (EI) m/z 231.15 (M þ 1).
4
A
4
,5-Diphenylthiazol-2-amine (9) [21]
solution of 2-chloro-1,2-diphenylethanone
1.5 mmol) in ethanol (40 mL) and thiourea (3.5 g, 45 mmol)
2-((4,5-Diphenylthiazol-2-yl)imino)-5-(thiophen-2-
8
(10 g,
ylmethylene)thiazolidin-4-one (12)
1
Dark yellow solid. H NMR (400 MHz, DMSO-d
6
) d 7.31–7.41
were refluxed for 2 h. The solvent was removed under vacuum
and the solid product was soaked in 100 mL of 30% NaOH at
(m, 8H), 7.50 (t, J ¼ 8.27 Hz, 1H), 7.61 (d, J ¼ 7.39 Hz, 2H), 7.70
(t, J ¼ 4.19 Hz, 1H), 7.98–8.01 (m, 1H), 8.09 (d, J ¼ 4.73 Hz, 1H),
1
3
5
0°C for 5 h. The crude product was filtered and washed with
12.66 (s, 1H, NH). C NMR (101 MHz, DMSO) d 168.33 (C –– O),
167.77, 158.86, 158.82, 151.33, 149.02, 147.54, 135.74,
133.00, 130.99, 130.63, 130.11, 130.08, 129.87, 129.61,
water to neutrality and then was recrystallized from 95%
ethanol to obtain a yellow solid (75%) of compound 10a. MS
þ
(
EI) m/z 252.00 (M ).
123.21, 120.15, 120.04, 115.21. HRMS calcd. for C23
H
15
N
3
OS
3
þ
[
MꢁH]
445.0377, found 444.0246. Anal. calcd. for:
OS : C, 62.00; H, 3.39; N, 9.43. Found: C, 62.32;
2
-Chloro-N-(4,5-diphenylthiazol-2-yl)acetamide (10) [18]
To a solution of compound 9 (5 g, 19.8 mmol) andtriethylamine
6 g, 59.5 mmol) in CH Cl at 0°C, chloroacetyl chloride (2.9 g,
5.7 mmol) was added. The reaction mixture was stirred for 1 h
C
23
H
15
N
3
3
H, 3.51; N, 9.38.
(
2
2
2
2-((4,5-Diphenylthiazol-2-yl)imino)-5-(pyridin-2-
at 0°C then for 5 h at room temperature. The organic layer was
then separated and evaporated in vacuo. The residue obtained
ylmethylene)thiazolidin-4-one (13a)
Greenish yellow solid. H NMR (400 MHz, DMSO-d
1
6
) d 7.29–
was purified by flash column chromatography (SiO
dichloromethane/methanol (9.5:0.5) as eluent to give brown
2
) using
7.49 (m, 9H), 7.63 (d, J ¼ 7.59 Hz, 2H), 7.78 (s, 1H), 7.86 (d,
J ¼ 8.17 Hz, 1H), 7.95 (t, J ¼ 8.04 Hz, 1H), 8.77 (d, J ¼ 4.23 Hz,
1
3
solid (62%) of 2-chloro-N-(4,5-diphenylthiazol-2-yl)acetamide
1H), 12.61 (s, 1H, NH). C NMR (101 MHz, DMSO) d 167.63
(C–O), 166.84, 161.68, 152.32, 149.62, 146.33, 137.94, 134.67,
1
1
(
1a. H NMR (400 MHz, DMSO-d
6
): d 4.45 (s, 2H, CH
2
), 7.29–7.42
þ
m, 10H), 10.48 (s, 1H, NH). MS (EI) m/z 328.05 (M ).
131.94, 129.90, 129.81, 129.62, 129.54, 129.48, 129.02, 128.87,
1
28.78, 128.51, 128.09, 124.33. HRMS calcd. for C24
H
16
N
4
OS
2
þ
2
-((4,5-Diphenylthiazol-2-yl)imino)thiazolidin-4-one (11)
Method A [18]: A mixture of 2-chloro-N-(4,5-diphenylthiazol-
-yl)acetamide 10 (3 g, 9.1 mmol) and ammonium thiocyanate
[MþH]
440.0766, found 441.0947. Anal. calcd. for:
OS : C, 65.43; H, 3.66; N, 12.72. Found: C, 65.57; H,
C
H N
24 16 4
2
2
3.46; N, 12.60.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 518–530
A. H. Abdelazeem et al.
ARC HH PHARM
Archiv der Pharmazie
2
-((4,5-Diphenylthiazol-2-yl)imino)-5-(pyridin-3-
2-((4,5-Diphenylthiazol-2-yl)imino)-5-(2-
hydroxybenzylidene)thiazolidin-4-one (15d)
Dark yellow solid. H NMR (400 MHz, DMSO-d
ylmethylene)thiazolidin-4-one (13b)
Yellow solid. H NMR (400 MHz, DMSO-d
1
1
6
) d 7.36–7.41 (m, 8H),
6
) d 4.02 (s, 1H,
7
.54 (t, J ¼ 6.24, 2H), 7.62 (s, 1H), 7.79 (s, 1H), 8.05 (d,
OH), 6.95–7.02 (m, 3H), 7.15–7.24 (m, 2H), 7.33–7.40 (m, 5H),
7.49–7.55 (m, 4H), 7.99 (s, 1H), 12.70 (s, 1H, NH). C NMR
(101 MHz, DMSO) d 167.62 (C–O), 166.59, 157.86, 157.20,
1
3
J ¼ 8.17 Hz, 1H), 8.65 (d, J ¼ 4.53 Hz, 1H), 8.91 (s, 1H), 12.90 (s,
13
1
1
1
1
4
6
H, NH). C NMR (101 MHz, DMSO) d 167.28 (C–O), 166.33,
56.09, 151.59, 150.83, 146.49, 136.77, 134.59, 131.74, 130.15,
29.87, 129.86, 129.53, 129.14, 129.06, 128.92, 128.77, 128.61,
27.66, 124.59. HRMS calcd. for
40.0766, found 441.0809. Anal. calcd. for: C24
5.43; H, 3.66; N, 12.72. Found: C, 65.09; H, 3.73; N, 12.63.
146.32, 136.84, 134.65, 132.63, 129.87, 129.50, 129.35, 128.85,
128.75, 128.65, 127.84, 125.76, 123.65, 122.76, 120.91, 119.95,
þ
þ
24
C H
16
N
4
OS
2
[MþH]
116.73. HRMS calcd. for C25
found 456.0769. Anal. calcd. for: C25
H
17
N
3
O
H
2
S
2
[MþH] 455.0762,
H
16
N
4
OS
2
: C,
17
N
3
O
2
S
2
: C, 56.91; H,
3.76; N, 9.22. Found: C, 57.13; H, 3.86; N, 9.51.
2
-((4,5-Diphenylthiazol-2-yl)imino)-5-(naphthalen-1-
2-((4,5-Diphenylthiazol-2-yl)imino)-5-(4-
ylmethylene)thiazolidin-4-one (14) [18]
Dark orange solid. H NMR (400 MHz, DMSO-d
methylbenzylidene)thiazolidin-4-one (15e) [18]
1
1
6
) d 7.23–7.49
Shiny yellow solid. H NMR (400 MHz, DMSO-d
6
) d 1.55 (s, 3H,
(
m, 10H), 7.60–7.83 (m, 4H), 7.98–8.13 (m, 3H), 8.35 (d,
CH
3
), 7.14 (d, J ¼ 12.42 Hz, 2H), 7.34–7.66 (m, 12H), 7.71 (s, 1H),
13
13
J ¼ 19.74 Hz, 1H), 12.77 (s, 1H, NH). C NMR (101 MHz, DMSO)
d 168.14 (C¼O), 165.45, 151.03, 150.06, 138.35, 133.80, 132.00,
12.70 (s, 1H, NH). C NMR (101 MHz, DMSO) d 174.41 (C–O),
167.02, 163.71, 150.62, 145.85, 145.08, 136.58, 134.53, 131.80,
129.61, 129.26, 129.14, 128.64, 128.58, 128.54, 128.44, 128.14,
123.02, 119.65, 25.31. HRMS calcd. for C26
453.0970, found 454.1008. Anal. calcd. for: C26
68.85; H, 4.22, N, 9.26. Found: C, 68.71; H, 3.99 N, 9.67.
1
1
1
4
7
29.86, 129.84, 129.52, 129.49, 129.34, 129.05, 128.88, 128.87,
þ
28.83, 128.80, 128.74, 128.69, 128.47, 128.37, 127.30, 125.88,
19
H N
3
OS
2
[MþH]
þ
22.85, 100.00. HRMS calcd. for
89.0970, found 490.1122. Anal. calcd. for: C29
C
29
H
19
N
3
OS
2
[MþH]
H N
19 3
OS
2
: C,
H
19
N
3
OS
2
: C,
1.14; H, 3.91; N, 8.58. Found: C, 71.39; H, 3.78; N, 8.76.
Pharmacology
COX in vitro inhibition assay
5
-Benzylidene-2-((4,5-diphenylthiazol-2-yl)imino)-
thiazolidin-4-one (15a) [18]
Yellow solid. H NMR (400 MHz, DMSO-d
In vitro inhibition of COX-1 and COX-2 was evaluated
colorimetrically using COX (ovine) Colorimetric Inhibitor
Screening Assay Kit (Cayman Chemical Ann Arbor, MI, USA)
in accordance with the manufacturer instructions [28]. Briefly,
COX-1/COX-2 enzymes were incubated with different con-
centrations of our test compounds, diclofenac or vehicle for
5 min at 25°C. Colorimetric substrate and arachidonic acid
were then added. The COX peroxidase inhibition activity of
the test compounds was determined by monitoring the
production of the oxidized N,N,N ,N -tetramethyl-p-phenyl-
enediamine (TMPD) at 590 nm. The COX-inhibiting activity of
the tested compounds (11–15) was calculated using the
following equation:
1
6
) d 7.34–7.41 (m, 8H),
7
1
1
1
1
4
9
.51–7.61 (m, 5H), 7.71 (d, J ¼ 7.58 Hz, 2H), 7.76 (s, 1H), 12.80 (s,
13
H, NH). C NMR (101 MHz, DMSO) d 170.03 (C–O), 166.49,
57.68, 156.80, 154.12, 146.36, 143.80, 134.59, 134.02, 132.54,
30.79, 130.59, 129.88, 129.75, 129.55, 128.92, 128.74, 128.60,
þ
25.44. HRMS calcd. for C25
40.0977. Anal. calcd. for: C25
.56. Found: C, 68.11; H, 3.59; N, 9.46.
H
17
N
3
OS
2
[MþH] 439.0813, found
17 3 2
H N OS : C, 68.31; H, 3.90; N,
0
0
2
-((4,5-Diphenylthiazol-2-yl)imino)-5-(3-nitrobenzylidene)-
thiazolidin-4-one (15b) [18]
1
6
Orange solid. H NMR (400 MHz, DMSO-d ) d 7.34–7.60 (m, 9H),
7
8
1
1
1
4
6
.85–7.93 (m, 3H), 8.15–8.34 (m, 2H), 8.53 (d, J¼ 12.07 Hz, 1H),
13
.96(s,1H,NH). CNMR(101 MHz,DMSO)d167.22(C –– O),148.71,
ð% inhibitionÞ ¼ ½1 ꢁ ðA2 ꢁ A0Þ=ðA1 ꢁ A0Þꢂ ꢃ 100
48.42, 147.68, 135.56, 134.63, 134.63, 131.83, 131.32, 131.07,
29.91, 129.86, 129.58, 129.12, 128.96, 128.75, 124.87, 123.45,
where A
0
is the absorbance of the blank, A
1
is the absorbance
þ
22.62, 118.49, 113.04. HRMS calcd. for C25
84.0664, found 485.0715. Anal. calcd. for: C25
1.97; H, 3.33; N, 11.56. Found: C, 62.15; H, 3.61; N, 11.29.
H
16
N
4
O
3
S
N
2
[MþH]
of the vehicle control, and A
2
is the absorbance of the test.
H
16
4
O
3
S
2
: C,
In vivo assays
5
-(2-Chlorobenzylidene)-2-((4,5-diphenylthiazol-2-yl)-
Animals
imino)thiazolidin-4-one (15c)
Brown solid. H NMR (400 MHz, DMSO-d
Swiss albino mice weighing 20–25 g were obtained from the
animal house of King Fahd Medical Research Center, King
Abdulaziz University, Jeddah, Saudi Arabia. Animals were
housed in polypropylene cages (three mice/cage) at controlled
environment conditions (temperature 23 ꢀ 2°C, humidity
60 ꢀ 10%, and a 12 h light/dark cycle) and were acclimatized
for 1 week before starting the experiments. Standard
commercial chow and water were allowed ad libitum. All
procedures relating to animal care and treatments were
1
6
) d 7.11–7.87
13
(
m, 15H), 10.35 (s, 1H, NH). C NMR (101 MHz, DMSO)
d 190.31 (C–O), 164.19, 162.21, 136.23, 135.38, 133.08, 132.76,
1
1
31.99, 131.50, 131.22, 130.78, 130.51, 130.21, 129.61, 129.23,
28.83, 128.37, 128.26, 128.05, 127.96, 126.85. HRMS calcd. for
þ
C
25
H
16CIN
for: C25 16CIN
H, 3.58; N, 8.73.
3
OS
2
[MꢁH] 473.0423, found 472.0405. Anal. calcd.
H
3 2
OS : C, 63.35; H, 3.40; N, 8.87. Found: C, 63.19;
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528

Arch. Pharm. Chem. Life Sci. 2015, 348, 518–530
Anti-Inflammatory Diphenylthiazole–Thiazolidinone Hybrids
ARC HH PHARM
Archiv der Pharmazie
conducted in compliance with the guidelines of Taif
University Research Ethical Committee.
measured at zero (Basal, just before drug administration), 1
and 2 h post-drug administration. The level of analgesia was
expressed as percentage change in latency time after 1 and 2 h
according to the following equation:
Carrageenan-induced rat paw edema assay
Mice in different groups were injected subcutaneously on the
plantar surface of the left hind paw each with 50 mL of vehicle
or carrageenan (1% w/v in saline) [29, 30]. Mice were then
immediately treated orally with vehicle, test compounds (11–
ð% changeÞ ¼ ½ðLt ꢁ L0Þ=L0ꢂ ꢃ 100
t 0
where L is the latency time after 1 or 2 h and L is the latency
1
5) (10 mg/kg) or standard (diclofenac, 10 mg/kg). The paw
time at zero time (Basal).
edema was evaluated by measuring the paw dorso-ventral
diameter at 1, 3, and 5 h after drug administration. The dorso-
ventral diameter of paw was measured using a high precision
caliper. Results are expressed as percent inhibition that was
calculated based on the following equation:
Statistical analysis
Statistical significance among groups was analyzed by one
way analysis of variance (ANOVA) followed by Dunnett’s post-
test. Data were presented as mean ꢀ standard deviation (SD).
Differences were considered significant at p < 0.05 (n ¼ 6).
Statistical analysis was done using SigmaPlot 12 statistics
software (Systat Software, Inc., San Jose, CA).
ð% inhibitionÞ ¼ f½ðTt ꢁ T0Þ ꢁ ðNCt ꢁ NC0Þꢂ=ðNCt ꢁ NC0Þg
ꢃ 100
where T
is the paw thickness for the test group at zero time, NC
the paw thickness for the negative control group (NC) at 1, 3,
or 5 h, and NC is the paw thickness for the negative control
t
is the paw thickness for the test group at 1, 3, or 5 h,
The authors deeply thank Prof. Dr. Mutasem Taha, The
University of Jordan, for his help in performing the docking
study and his valuable discussion. Also, the authors thank
Mohamed Al-Zahrany, Abdelaziz Al-Kahtany, and Fahd Al-
Otaiby for their excellent technical help in carrying out the in
vivo three bioassays.
T
0
t
is
0
group at zero time.
Carrageenan-induced thermal hyperalgesia
The authors have declared no conflict of interest.
Mice were injected each with 50 mL of vehicle or carrageenan
(
1% w/v in saline) subcutaneously on the plantar surface of
the left hind paw [29]. Animals were then immediately
treated with vehicle, test compounds (11–15) (10 mg/kg, per
oral) or standard (diclofenac, 10 mg/kg, per oral). Animals in
all groups were subjected to the hot-plate assay and the
latency time was recorded at 0, 1, and 2 h as described above.
A cutoff latency time of 60 s was used to avoid tissue damage
caused by prolonged exposure to the hot-plate. The level of
hyperalgesia was expressed as percentage change in latency
time after one and two hours according to the following
equation:
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