Design, synthesis and analgesic/anti-inflammatory evaluation of novel diarylthiazole and diarylimidazole derivatives towards selective COX-1 inhibitors with better gastric profile

Article abstract of DOI:10.1016/j.bmc.2016.11.037

The inhibition of gastric cyclooxygenase 1 (COX-1) enzyme was believed to be the major cause of non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastric ulcer. Recent studies disproved this belief and showed that the gastric tissues vulnerability is not solely connected to COX-1 inhibition. This work aimed at exploring and rationalizing the differential analgesic and anti-inflammatory activities of novel selective COX-1 inhibitors with improved gastric profile. Two novel series of 4,5-diarylthiazole and diarylimidazole were designed, synthesized in analogy to selective COX-1 inhibitors (mofezolac and FR122047) which lack gastric damaging effects. The new compounds were evaluated in vitro for their COXs inhibitory activity and in vivo for their anti-inflammatory and analgesic potentials. Four compounds; diphenylthiazole glycine derivatives (15a, 15b) and diphenylimidazolo acetic acid derivatives (19a, 19b), which possess carboxylic acid group exhibited significant activity and selectivity against COX-1 over COX-2. Of these compounds, (4,5-bis(4-methoxyphenyl)thiazol-2-yl)glycine 15b was the most potent compound against COX-1 with an inhibitory half maximal concentration (IC₅₀) of 0.32 μM and a selectivity index (COX-2 IC₅₀/COX-1 IC₅₀) of 28.84. Furthermore, an ulcerogenicity study was performed where the tested compounds demonstrated a significant gastric tolerance. Interestingly, the most selective COX-1 inhibitor showed higher analgesic activity in vivo as expected compared to their moderate anti-inflammatory activity. This study underscores the need for further design and development of novel analgesic agents with low tendency to cause gastric damage based on improving their COX-1 affinity and selectivity profile.

Full text of DOI:10.1016/j.bmc.2016.11.037

Accepted Manuscript
Design, Synthesis and Analgesic/Anti-inflammatory Evaluation of Novel Dia-
rylthiazole and Diarylimidazole Derivatives Towards Selective COX-1 Inhibi-
tors with Better Gastric Profile
Ahmed H. Abdelazeem, Mohammed T. El-Saadi, Asmaa G. Safi El-Din, Hany
A. Omar, Samir M. El-Moghazy
PII:
S0968-0896(16)31070-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.037
BMC 13404
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 October 2016
16 November 2016
21 November 2016
Please cite this article as: Abdelazeem, A.H., El-Saadi, M.T., Safi El-Din, A.G., Omar, H.A., El-Moghazy, S.M.,
Design, Synthesis and Analgesic/Anti-inflammatory Evaluation of Novel Diarylthiazole and Diarylimidazole
Derivatives Towards Selective COX-1 Inhibitors with Better Gastric Profile, Bioorganic & Medicinal Chemistry
(2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.037
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Design, Synthesis and Analgesic/Anti-inflammatory Evaluation of
Novel Diarylthiazole and Diarylimidazole Derivatives Towards
Selective COX-1 Inhibitors with Better Gastric Profile
Ahmed H. Abdelazeema,b,*, Mohammed T. El-Saadia, Asmaa G. Safi El-Dina, Hany A.
Omarc,d and Samir M. El-Moghazye,*
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514,
Egypt.
b Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Taif 21974, Saudi
Arabia.
c
Sharjah Institute for Medical Research and College of Pharmacy, University of Sharjah, Sharjah
27272, United Arab Emirates.
d Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt.
e
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo 11562,
Egypt.
*To whom correspondence should be addressed:
Samir M. El-Moghazy, Ph.D. Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Cairo University, Kasr-El-Eini Street 11562 Cairo, Egypt.
Tel.: 002-01223662720‏
E-mail address: samirelmoghazy@gmail.com
Ahmed H. Abdelazeem, Ph.D. Department of Medicinal Chemistry, Faculty of Pharmacy,
Beni-Suef University, Beni-Suef 62514, Egypt.
Tel.: (002)-0100-2877405
Fax: (002)-082-2317958
E-mail address: ahmed.abdelazeem@pharm.bsu.edu.eg, ahmed.pharm@yahoo.com
1

Abstract
The inhibition of gastric cyclooxygenase 1 (COX-1) enzyme was believed to be the major cause
of non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastric ulcer. Recent studies
disproved this belief and showed that the gastric tissues vulnerability is not solely connected to
COX-1 inhibition. This work aimed at exploring and rationalizing the differential analgesic and
anti-inflammatory activities of novel selective COX-1 inhibitors with improved gastric profile.
Two novel series of 4,5-diarylthiazole and diarylimidazole were designed, synthesized in
analogy to selective COX-1 inhibitors (mofezolac and FR122047) which lack gastric damaging
effects. The new compounds were evaluated in vitro for their COXs inhibitory activity and in
vivo for their anti-inflammatory and analgesic potentials. Four compounds; diphenylthiazole
glycine derivatives (15a, 15b) and diphenylimidazolo acetic acid derivatives (19a, 19b), which
possess carboxylic acid group exhibited significant activity and selectivity against COX-1 over
COX-2. Of these compounds, (4,5-bis(4-methoxyphenyl)thiazol-2-yl)glycine 15b was the most
potent compound against COX-1 with an inhibitory half maximal concentration (IC50) of 0.32
μM and a selectivity index (COX-2 IC50/COX-1 IC50) of 28.84. Furthermore, an ulcerogenicity
study was performed where the tested compounds demonstrated a significant gastric tolerance.
Interestingly, the most selective COX-1 inhibitor showed higher analgesic activity in vivo as
expected compared to their moderate anti-inflammatory activity. This study underscores the
need for further design and development of novel analgesic agents with low tendency to cause
gastric damage based on improving their COX-1 affinity and selectivity profile.
Keywords: Selective COX-1; mofezolac, diarylthiazole; diarylimidazole; analgesic; anti-
inflammatory; ulcerogenicity
Graphical abstract
2

1. Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are the most prevalent drugs, either prescribed
or non-prescribed, for alleviating pain, inflammation, fever and rheumatic disorders.1 However,
these drugs suffer from several associated drawbacks, including gastric ulceration, kidney injury
and cardiotoxicity.2,3 It’s well-documented that the traditional NSAIDs exert their
pharmacological effects through the inhibition of cyclooxygenases (COXs)-dependent
prostaglandins biosynthesis. The COXs are a class of bifunctional enzymes which are
responsible for bis-oxygenation followed by reduction of arachidonic acid to generate
prostaglandin H2 (PGH2).4 To date, three COX isoforms, COX-1, COX-2 and COX-3 have been
identified. COX-1 enzyme is expressed constitutively generating PGE2 and TXA2 which are
responsible for gastrointestinal protection and platelet aggregation, respectively.5 On the other
hand, COX-2 is an inducible isoform which is upregulated in response to inflammatory or any
immunological stimuli.6 Therefore, COX-2 attracted great attention to be a proper target for the
development of powerful anti-inflammatory and analgesic agents with no tendency to cause
gastric damage. Despite the widespread belief that the inhibition of COX-1 enzyme is the main
cause of gastric ulcer, recent studies confirmed that the gastric tissues vulnerability is not
connected solely to COX-1 inhibition.7-9 Indeed, the pharmacological role of COX-1 in human
was cloudy, except for platelet activation and gastrointestinal protection, until Langenbach and
coworkers enlightened the horizons by confirmation that the inhibition of COX-1 alone is not
sufficient to induce gastric ulceration.7,8 Later, Wallace et al. disproved this accusation and
ensured that COX-1 selective inhibition does not cause any gastric damage.10 Moreover, studies
by Tanaka et al. strengthened these findings by showing that the inhibition of COX-1 causes up-
regulation of COX-2 expression which, in turn, increases the production of PGE2 to a level
necessary for mucosal integrity.11 Since then, COX-1 has represented a potential therapeutic
target for the design of potent analgesic agents with improved gastric safety profile. However,
only few selective COX-1 inhibitors have been discovered and introduced as potential analgesic
and anti-platelets agents such as 1 (SC-560), 2 (Mofezolac), 3 (FR122047), 4 (P6), 5 (TFAP),
Fig. 1.9,12-15 Of these compounds, mofezolac, a selective COX-1 inhibitor, was developed and
marketed in Japan as a powerful pain killer.12,16
3

Fig. 1. Structures of some known COX-1 selective inhibitors (1-5) and valdecoxib 6
It was conceptualized from comparing the structures of mofezolac 2 and its COX-2 selective
analog valdecoxib 6 that there are some important keys for their converse COXs activities.
Firstly, the replacement of the sulfonamide group in valdecoxib 6 with two methoxy groups in
mofezolac 2 resulted in the reversal of COX-2 selectivity in favor of COX-1. Secondly, the
presence of an acetic acid moiety attached to the five-membered ring in mofezolac 2 was found
to be influential primarily on the COX-1 selectivity. However, the absence of acetic acid moiety
in FR122047 3 did not affect its COX-1 selectivity. Moreover, it seems that the 4-
methylpiperazino moiety linked to the thiazolo heterocyclic nucleus in compound 3 with a
carbonyl spacer has a great impact on COX-1 activity and selectivity as acetic acid moiety.
Interestingly, the diaryl heterocyclic system as a known and frequently used scaffold in COX-2
selective inhibitors was likewise common among the majority of COX-1 selective compounds
but with different substitutions.
In the light of these observations, two novel sets of compounds were designed and synthesized
in analogy to mofezolac 2 and FR122047 3 with preserving the structural features required for
COX-1 selectivity that have been previously conceptualized including the two methoxy groups
and acetic acid moiety or methylpiperazine one. Furthermore, an iterative structure activity
relationship was conducted through varying the five-membered ring (thiazole and imidazole),
using different linkers and replacement of methylpiprazine by various cyclic amines, Fig. 2. The
novel compounds were evaluated in vitro against COX-1 and COX-2 and in vivo for their
analgesic and anti-inflammatory potential. Finally, our main focus was to examine the effect of
these new compounds on the gastric mucosa using acute ulcerogenicity studies.
4

Fig. 2. The design strategy of our novel compounds based on Mofezolac and FR122047 structures
2. Result and discussion
2.1. Chemistry
The synthetic pathways used for preparing the novel compounds were outlined in schemes 1 and
2. The substituted benzoin 8a or 8b were synthesized as starting materials from substituted
benzaldehyde in presence of NaCN as a catalyst using the reported method.17 The intermediates
8a and 8b were subjected to a chlorination process using thionyl chloride in pyridine forming
desyl chloride derivatives 9a and 9b, respectively which in turn were cyclized upon
condensation reaction with thiourea in ethanol to afford 4,5-diphenylthiazol-2-amine derivatives
10a and 10b, respectively in a good yield according to Hantez-thiazole synthesis protocol.18-20 In
order to prepare the intermediates 13a and 13b, we firstly tried the direct N-alkylation reaction
with ethyl chloroacetate using NaH as a base and it surprisingly resulted in the cyclized product
11a or 11b due to the incessant attack of the thiazole’s nitrogen on the acetate group. Thus,
efficient N-formylation of the amino group followed by reaction with ethyl chloroacetate, as
reported,21 was used to prevent the second attack and it successfully afforded the targeted
intermediates 13a and 13b as shown in Scheme 1. 1H NMR spectrum of the cyclized derivatives
11a and 11b revealed a singlet signal at 4.42 and 4.40 ppm, respectively attributed to the
methylene group. Also, the mass spectrum of 11a endorsed that interpretation via the appearance
of its molecular ion peak at m/z 294. On the other hand, 1H NMR spectra of compounds 13a and
13b revealed the presence of the characteristic triplet and quartet patterns of the ethyl moiety at
5

1.32 and 4.25 ppm, respectively. The final targeted compounds 14a-h were obtained in excellent
yield from the condensation reaction of 13a and 13b with various secondary amines. Finally, the
alkaline hydrolysis of 13a and 13b afforded the acetic acid containing compounds 15a and 15b,
respectively. The IR spectrum showed a broad absorption band at 3300 cm-1 assigned to the
1
carboxylic OH group. Moreover, H NMR spectrum displayed a singlet peak at 12.89 ppm that
disappeared upon deuteration attributed to carboxylic acid proton.13C NMR revealed a peak at
171 ppm assigned to the carbonyl group of the carboxylic acid.
Scheme 1. Reagents and reaction conditions: (a) NaCN, ethanol, reflux, 1 h; (b) SOCl2, pyridine, rt, 1
h; (c) thiourea, ethanol, reflux, 3 h; (d) ethyl chloroacetate, NaH, DMF, rt, overnight; (e) formic acid,
acetic anhydride, diethyl ether, rt, overnight; (f) appropriate secondary amine, reflux, 4 h; (g) KOH,
MeOH, reflux, 12 h.
A similar approach was used for the synthesis of 4,5-diarylimidazole derivatives 18a-h as shown
in Scheme 2.22,23 First, the substituted benzoins 8a and 8b were cyclized using formamide to
form the imidazole intermediates 16a and 16b which were subsequently reacted with ethyl
chloroacetate followed by condensation with various amines to afford the final targeted
compounds 18a-h. On the other hand, the final acetic acid containing compounds 19a and 19b
6

were obtained upon alkaline hydrolysis of the acetate intermediates 17a and 17b using
potassium hydroxide in methanol.
Scheme 2. Reagents and reaction conditions: (a) Formamide, reflux, 3 h; (b) ethyl chloroacetate, NaH,
DMF, rt, overnight; (c) appropriate secondary amine, reflux, 4 h; (d) KOH, MeOH, reflux, 12 h.
2.2. Biological activity
Considering the previous reports indicating COX-1 inhibition as a molecular mechanism of the
analgesic activity of mofezolac 2,16 our novel diarylthiazole and diarylimidazole derivatives as
mofezolac analogs were assessed in vitro for their COXs inhibitory activity and in vivo for their
analgesic and anti-inflammatory potential in addition to an acute ulcerogenicity study. The
results of COXs inhibitory assay were expressed in terms of IC50 values, Table 1. Two animal
models were utilized in the in vivo studies; the acetic acid-induced writhing test was employed
to assess the anti-nociception activity and the carrageenan-induced paw edema assay was used
for evaluating the anti-inflammatory potential.
2.2.1. In vitro COXs Inhibitory Assay
The COXs inhibitory activity of the newly synthesized compounds was examined as reported
before.24,25 The activity in terms of IC50 values in addition to the selectivity index were
summarized in table 1. The results revealed that compounds possessing acetic acid moiety,
either thiazole or imidiazole derivatives, were the most potent and selective COX-1 ligands as
presented in compounds 15a,b and 19a,b with IC50 values between 0.32-0.67 μM. However, the
presence of the two methoxy groups improved the COX-1 activity to some extent where
compounds 15b and 19b were more potent than 15a and 19a, respectively. In general, the
7

diarylthiazole derivatives showed more activity and selectivity than for COX-1 over COX-2
subtype in both scaffolds A and B. On the other hand, compounds 14a-f and 18a-f having
scaffold B as FR122047 analogs exerted lower activity than compounds bearing free carboxylic
groups (15a,b and 19a,b) in scaffold A. It was worth noting that compounds possessing
methylpiperazine moiety 14e,f and 18e,f were the most active derivatives with IC50 values
between 2.72 and 4.17 μM. Upon replacement of methylpiperazine with morpholine or
piperidine moieties, the COX-1 activities were remarkably decreased. Comparing the activity of
these compounds with that of the parent compound, FR122047, it was suggested that the change
of the length of the spacer between the heterocyclic nucleus and the secondary amine has a great
effect on decreasing COX-1 activity and selectivity. However, the piperidine containing
derivatives 14a,b and 18a,b showed the least activity between these FR122047 analogs with
scaffold B. On the contrary, almost all the tested compounds showed weak activity with no
selectivity against COX-2 subtype owing to the absence of sulfonamide or methylsulfone groups
as essential structural basis for COX-2 selectivity where they form H-bonding with Arg513
residue in the side pocket of COX-2 active site as reported.19,26 Taken together, it could be
conceptualized from these findings that there are some structural requirements that are optimal
for COX-1 activity and selectivity. First, the free carboxylic acid attached to the five-membered
heterocyclic ring is pivotal for COX-1 inhibitory activity and selectivity as represented in
compounds 15a,b and 19a,b. Secondly, the isosteric replacement of isoxazole ring in mofezolac
2 with thiazole ring preserved the COX-1 activity and selectively while the replacement with
imidazole resulted in a slight decrease in activity as demonstrated in compounds 19a and 19b.
Furthermore, the removal of the two methoxy groups from both scaffolds A and B did not
abolish the COX-1 activity however, their presence enhanced the potency. Finally, the spacer
length in the second series with scaffold B was crucial for COX-1 activity and selectivity where
the carbonyl group as a linker in FR122047 was optimal and largely contributed to their
significant COX-1 selectivity. Interestingly, compound 15b containing all these structural
features showed the most COX-1 affinity and selectivity with IC50 of 0.32 μM and the
selectivity index (COX-2 IC50/COX-1 IC50) of 28.84.
Table 1. The results of in vitro COXs inhibition assay in terms of IC50 values and selectivity index (SI)
COXs inhibition (IC50 µM)a
Selectivity
Compound
Index (SI)b
COX-1
COX-2
5.76 ± 1.34
4.01± 1.56
8.28 ± 2.06
6.89 ± 1.42
1.44
1.72
14a
14b
8

7.99 ± 2.31
6.01 ± 1.70
3.04 ± 1.98
2.72 ± 1.02
0.42 ± 1.03
0.32 ± 1.12
6.32 ± 1.76
5.09 ± 1.04
8.51 ± 1.04
7.34 ± 1.04
4.17 ± 1.74
3.06 ± 1.51
0.67 ± 1.56
0.54 ± 1.77
16.20 ± 1.21
0.71 ± 0.03
15.12 ± 1.74
16.12 ± 2.21
11.02 ± 2.04
7.95 ± 2.10
10.71 ± 1.08
9.23 ± 1.12
11.12 ± 1.75
9.12 ± 1.03
13.12 ± 1.15
14.12 ± 2.23
10.23 ± 1.94
8.32 ± 1.43
13.45 ± 1.97
12.57 ± 1.43
0.34 ± 0.02
11.07 ± 2.0
1.89
2.68
3.63
2.92
25.50
28.84
1.76
1.79
1.54
1.92
2.45
2.72
20.07
23.28
0.02
15.59
14c
14d
14e
14f
15a
15b
18a
18b
18c
18d
18e
18f
19a
19b
Celecoxib
Indomethacin
aValues are expressed as mean ± SEM (n = 3)
bSelectivity index (SI) = COX-2 IC50/COX-1 IC50
2.2.2. Acetic acid-induced writhing test
In order to measure the analgesic activity of the newly synthesized compounds, the acetic acid-
induced writhing test was employed using diclofenac as a positive control according to the
reported method.27 The activity of each compound was determined based on the reduction in the
number of acetic acid-induced writhing. At a glance, the results were in concordance with that of
the in vitro COXs inhibitory assay where the most active and COX-1 selective compounds 15a,b
and 19a,b bearing the free carboxylic group showed the in vivo highest potency with number of
writhes between 10.67-14.40 compared to the nonselective COXs diclofenac (reference drug)
with number of writhes 24. Moreover, almost all compounds possessing thiazole ring were more
active than that derivatives having an imidazole ring. However, the compounds (FR122047
analogs) with scaffold B, either thiazole or imidazole derivatives, exhibited low to moderate
analgesic activity. Compounds 14a,b and 18a,b bearing morpholine revealed lower activity
compared to N-methylpiperazine containing compounds 14e,f and 18e,f, Fig. 3. These results
affirmed the important role of the free carboxylic group, the two methoxy groups and the spacer
length in the in vitro and in vivo activities of our compounds as well.
9

Fig. 3. The results of acetic acid-induced writhing assay. Statistical analysis was performed using one-
way ANOVA followed by Dunnett Multiple Comparisons Test, Data are mean ± S.D., *Significant
different from control at p<0.05, #Significant different from diclofenac at p<0.05, N=5.
2.2.3. Carrageenan-induced rat paw edema assay
The anti-inflammatory activity of these new compounds was evaluated using the reported
carrageenan-induced rat paw edema assay where diclofenac sodium was a positive reference
drug.28,29 The tested compounds were suspended in DMSO and administered orally to the
experimental animals. The mean sizes of the resulted edema thickness of rats pretreated with the
tested compounds were observed and measured at 0, 1, 3 and 7 h from the induction of
inflammation. The percentage of inhibition in thickness of edema was calculated in comparison
to diclofenac sodium. Conversely to the analgesic assay results, all tested compounds exhibited
modest anti-inflammatory activity with average edema inhibition percentages between 50.30-
61.91% compared to the reference drug with average edema inhibition percentage of 84.52% as
shown in Table 2. It was clear from the results that compounds 14a, 14b, 14f, 18e and 18f
showed slightly higher activity than the rest of other derivatives. Compound 14f bearing the
methylpiperazine moiety was the most potent compounds with average edema inhibition
percentage of 61.91%. Interestingly enough, the most potent and COX-1 selective compound
15b possessing an acetic acid moiety exerted the least in vivo anti-inflammatory activity with
average edema inhibition percentage of 50.30%. These results reflected the great influence of
the significant COX-1 affinity and selectivity on improving the analgesic activity at expense of
anti-inflammatory activity.
10

Table 2. The results of carrageenan-induced rat paw edema assay of the tested compounds compared to
diclofenac as a reference drug.
Average
edema
inhibition%
Mean edema thickness (mm) ± SEM
1 h 3 h
Compound
0 h
7 h
0.171 ± 0.017 0.394 ± 0.220 0.401 ± 0.019 0.464 ± 0.023
0.148 ± 0.007 0.148 ± 0.067 0.167 ± 0.008 0.192 ± 0.012
0.170 ± 0.016 0.170 ± 0.164 0.190 ± 0.017 0.213 ± 0.018
0.173 ± 0.016 0.173 ± 0.160 0.197 ± 0.017 0.205 ± 0.010
0.144 ± 0.007 0.144 ± 0.066 0.163 ± 0.004 0.172 ± 0.009
0.147 ± 0.006 0.166 ± 0.008 0.185 ± 0.008 0.231 ± 0.009
0.155 ± 0.007 0.165 ± 0.008 0.213 ± 0.009 0.251 ± 0.006
0.155 ± 0.008 0.166 ± 0.009 0.186 ± 0.010 0.218 ± 0.016
0.142 ± 0.008 0.146 ± 0.010 0.159 ± 0.008 0.190 ± 0.012
0.147 ± 0.006 0.147 ± 0.006 0.168 ± 0.007 0.182 ± 0.006
0.147 ± 0.006 0.170 ± 0.012 0.191 ± 0.010 0.152 ± 0.008
0.148 ± 0.007 0.161 ± 0.005 0.189 ± 0.008 0.235 ± 0.009
0.149 ± 0.008 0.149 ± 0.008 0.180 ± 0.011 0.215 ± 0.009
0.162 ± 0.008 0.090 ± 0.008 0.060 ± 0.004 0.040 ± 0.003
---
Control
14a
59.80
54.52
54.26
61.91
53.99
50.30
54.55
60.78
60.52
58.82
53.79
56.98
84.52
14b
14e
14f
15a
15b
18a
18b
18e
18f
19a
19b
Diclofenac
All test compounds were given orally in a dose of 100 mg/kg while diclofenac sodium in a dose of 60
mg/kg. Treatments began 1 h before induction of inflammation by the injection of 1% carrageenan-
sodium gel into the sub-planter region of the right hind paw. The mean size of the induced paw edema
thickness of rats pretreated with the tested compounds were observed and measured at 0, 1, 3 and 7 h
from the induction of inflammation. The percentage of inhibition in thickness of edema was calculated in
comparison to diclofenac sodium. (N=5).
2.2.4. Acute ulcerogenicity study
Based on the in vitro COXs assay results, the most active and COX-1 selective compounds
15a,b and 19a,b were selected to be examined for their gastric-ulcerogenic potential using
indomethacin as a reference drug.30 Observation of the gastric mucosa for the presence of
lesions following oral administration of 20 mmol/kg of the tested compounds or the reference
drug was used as an indication for the ulcerogenic effects. The results revealed that the four
compounds have superior safety profile compared to indomethacin as shown in Table 3 and Fig.
4. Obviously, compound 15b demonstrated a remarkable improvement in ulcer index (UI = 8.5)
in comparing with indomethacin (UI =19.5).
11

Table 3. Ulcerogenic effects of synthesized compounds and indomethacin as a reference drug
Average number
Compound
Ulcer indexa
of ulcers
2.25
9.39
8.5
15a
15b
1.5
4
13.3
9.75
Nil
19a
2.5
--
19b
Control
Indomethacin
11
19.5
aThe ulcer index is the sum of % incidence, average severity and average number of ulcers following oral
administration of 20 mmol/kg of the tested compounds as well as the reference drug. (N=5).
Fig. 4. Representative pictures of the stomachs of rats treated with tested compounds 15a, 15b, 19a and
19b compared to indomethacin indicating the ulcer lesions. All test compounds and indomethacin were
used orally (20 mmol/kg).
2.3. Molecular Docking studies
In order to explore some clues on the most significant structural features governing COX-1
affinity and selectivity, a docking simulation was conducted using LIGANDFIT embedded in
Discovery Studio software.31 The results of this in silico study would correlate the in vitro COXs
inhibitory activity and the difference in selectivity profiles of the newly synthesized with their
chemical structures based on their orientation and binding patterns inside the COXs active sites.
In doing so, two representative compounds 15b and 14f were docked into the active site of
COX-1 where the 3D crystal structure complex (PDB codes: 1PGF) was employed for this
study. In addition, mofezolac 2 and FR122047 3 were used for comparison as two well-known
12

COX-1 selective agents. It was found that compound 15b with the highest COX-1 activity and
selectivity exhibited a binding pattern and interactions similar to that of mofezolac 2, Fig. 5.
Clearly from Fig. 5(A), the diphenyl rings bearing the two methoxy groups were oriented
towards the hydrophobic region lined by Tyr348, Phe381, Tyr385 and Trp387 while the acetic
acid moiety was leaned towards Arg120 with establishing an important H-bond interaction
likewise the free carboxylic group of mofezolac, Fig. 5(B). In addition, one of the two methoxy
groups formed H-bond with the backbone of Ser530 residue. The superimposition of the docked
poses 15b and Mofezolac 2 within COX-1 active site as shown in Fig. 5(C,D) pointed out the
high similarity between both compounds and explained the significant activity and selectivity of
15b against COX-1.
Fig. 5. (A) Docking and binding pattern of compound 15b into COX-1 active site (PDB code: 1PGF);
(B) Docking and binding pattern of Mofezolac 2 into the same COX-1 binding pocket; (C) The
superimposition of the docked pose 15b (violet) and Mofezolac 2 (cyan) within active site of COX-1; (D)
The superimposition of the docked pose 15b (violet) and Mofezolac 2 (cyan) without active site residues.
The poses were rendered as ball and stick models. Hydrogen bonds were represented as dashed green
lines. All hydrogens were removed for the purposes of clarity.
On the other hand, the docking results of compound 14f possessing methylpiperazine moiety
into COX-1 active site indicated that this compound was forced to adopt a longitudinal binding
pattern and it was pushed to the bottom of the active site, Fig. 6. However, the disposition of the
diphenyl bearing the two methoxy groups was in the same hydrophobic room formed of Tyr348,
13

Phe381, Tyr385 and Trp387 residues, the methylpiperazine moiety seems to extremely protrude
outside the COX-1 active site, compared to FR122047 due to the variation in the spacer length
between methylpiperazine and the thiazole heterocycle, Fig. 6(A-C). This observation could be
the reason behind the inferior affinity of 14f against COX-1 subtype, which reflected its low in
vivo analgesic potential.
Fig. 6. (A) Docking and binding pattern of compound 14f into COX-1 active site (PDB code: 1PGF); (B)
Docking and binding pattern of FR122047 3 into the same COX-1 binding pocket; (C) The
superimposition of the docked pose 14f (red) and Mofezolac 2 (blue) within active site of COX-1. The
poses were rendered as ball and stick models. Hydrogen bonds were represented as dashed green lines.
All hydrogens were removed for the purposes of clarity.
3. Conclusion
In the present investigation, a series of diarythiazole and diarylimidazole have been designed
and synthesized as mofezolac and FR122047 analogs. The newly synthesized compounds were
evaluated in vitro against COXs subtypes and in vivo for their analgesic, anti-inflammatory and
ulcerogenicity potential. The in vitro assays results revealed that compounds 15a,b and 19a,b
possessing acetic acid moiety were the most potent and COX-1 selective inhibitors with IC50
values of 0.42, 0.32, 0.67 and 0.54 µM, respectively. From the structure activity relationship
study, it could be concluded that the presence of free carboxylic or methylpiperazine moiety in
addition to the two methoxy groups are optimal for COX-1 affinity and selectivity. However, the
spacer length was crucial for COX-1 activity and selectivity especially in the FR122047 analogs.
Conversely, the tested compounds showed weak activity with no selectivity against COX-2
subtype due to the absence of sulfonamide or methylsulfone groups which highly contribute to
COX-2 activity and selectivity. On the other hand, the in vivo study results were consistent with
that of the in vitro COXs inhibitory assay where the most potent and COX-1 selective
compounds, especially 15b, exhibited the highest analgesic activity and least anti-inflammatory
potential. Meanwhile, compound 15b revealed a much better gastric tolerance profile (UI = 8.5)
than indomethacin (UI = 19.5). In the docking simulation, compound 15b adopted binding
pattern and H-bonding interactions inside COX-1 active site similar to that of mofezolac
14

explaining its remarkable activity and selectivity. This study indicates the potential medicinal
value of these compounds as analgesic agents with better safety margin less gastric damage.
4. Experimental protocols
4.1. Chemistry
Generally, reagents and solvents were of commercial quality and were used without further
purification. Melting points 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. Infrared spectra were done on
Bruker FT-IR spectrophotometer Vector 22 and expressed in wave number (cm-1) using KBr
discs at the micro-analytical center, Faculty of Science, Cairo University. The proton magnetic
13
resonance 1H NMR and C NMR spectra were recorded on BRUKER APX400 spectrometer at
400 MHz and 100 MHz, respectively in the specified solvent at the Faculty of Pharmacy, Beni-
Suef University. The chemical shifts were reported on the d scale and were related to that of the
solvent and J values were given in Hz. Mass spectra were recorded on Fennigan MAT, SSQ
7000, Mass spectrometer, at 70 eV (EI) at the micro-analytical center, Faculty of Science, Cairo
University. Thin layer chromatography (TLC) was done using Macherey–Nagel Alugram Sil
G/UV254 silica gel plates and Hexane-Ethyl acetate (8:2) as the eluting system.
2-Hydroxy-1,2-diphenylethanone (8a).18 A mixture of benzaldehyde (50 g, 4.7 mol) and
sodium cyanide (5 g) were heated for one-half hour in a mixture of absolute ethanol (62.5 mL)
and water (50 mL). The solution was cooled and the precipitate was filtered and washed with
water. The crude solid was recrystallized from ethanol to afford 9a as a white solid (80%)
m.p.129 oC.
2-Hydroxy-1,2-bis(4-methoxyphenyl)ethanone (8b).17 Yield 35%, bright yellow solid, m.p.
113-114 oC.
2-Chloro-1,2-diphenylethanone (9a).19 Compound 2a (10 g, 0.047 mol) was heated in pyridine
(5.7 mL) until a solution was obtained, then cooled till solidification has been occurred. The
mass was coarsely ground and thionyl chloride (7.5 g, 0.063 mol) was added slowly with
vigorous stirring in cold water bath for 1 h and at room temperature for another 1 h. Water was
added and the solid was filtered and recrystallized from ethanol to obtain compound 3a as
colorless crystals (74%), m.p. 66-67 oC.
2-Chloro-1, 2-bis(4-methoxyphenyl)ethanone (9b).32 Yield 36% as yellow solid, m.p. 80-81
oC.
15

4,5-Diphenylthiazol-2-amine (10a).19 A mixture of compound 3a (2 g, 8.3 mmol) and thiourea
(0.7 g, 9 mmol) in ethanol was refluxed for 2 h. After cooling, the resulting precipitate was
collected by filtration. The solid was filtered, washed with water and then recrystallized from
ethanol to furnish compound 4a as a grey, needle-shaped solid (72%). m.p. 188-189 oC.
4,5-Bis(4-methoxyphenyl)thiazol-2-amine (10b).20 Yield 89% as yellow solid, m.p. 178-180
oC.
2,3-Diphenylimidazo[2,1-b]thiazol-5(6H)-one (11a). To a stirred suspension of sodium
hydride 60% (0.2 g, 5 mmol) in DMF (5 mL), compound 10a (1.4 g, 5 mmol) was added
portionwise. The stirring was continued for 30 minutes, and then a solution of ethyl
chloroacetate (0.92 g, 5.5 mmol) was added. The reaction mixture was stirred at rt for 4 h and
subsequently was poured onto ice-water mixture. The precipitate was filtered off and
o
recrystallized from ethanol to afford compound 11a as brown solid (65%), m.p. 125-126 C. IR
(cm-1) 3050 (CH aromatic), 2945 (CH aliphatic), 1688 (amide C=O), 1607, 1511 (C=C, C=N).
1H NMR (400 MHz, DMSO-d6) δ 4.42 (s, 2H, CH2), 7.16-7.49 (m, 10H, aromatic). 13C NMR
(101 MHz, DMSO-d6) δ 22.9, 125.6, 128.1, 128.3, 128.7, 128.8, 129.4, 129.7, 132.3, 135.1,
144.1, 156.3, 169.1. MS (EI) m/z 294 (M++1). Anal. Calcd. For C17H12N2OS: C, 69.84; H, 4.14;
N, 9.58. Found: C, 69.47; H, 4.84; N, 9.68.
2,3-Bis(4-methoxyphenyl)imidazo[2,1-b]thiazol-5(6H)-one (11b). Yield 76% as brown solid,
m.p. 177-180 oC. IR (cm-1): 3040 (CH aromatic), 2956 (CH aliphatic), 1662 (amide C=O), 1607,
1511 (C=C, C=N). 1H NMR (400 MHz, DMSO-d6) δ 3.7 (s, 3H, OCH3), 3.8 (s, 3H, OCH3), 4.40
(s, 2H, CH2), 6.85-7.38 (m, 8H, aromatic). 13C NMR (101 MHz, DMSO-d6) δ 22.8, 55.4, 55.5,
114.13, 114.8, 124.1, 124.5, 127.7, 130.0, 131.0, 143.2, 155.6, 159.0, 159.3, 169.0. MS (EI) m/z
354 (M++1). Anal. Calcd. For C19H16N2O3S: C, 64.76; H, 4.58; N, 7.95. Found: C, 64.53; H,
5.19; N, 8.11.
N-(4,5-diphenylthiazol-2-yl)formamide (12a). To a mixture of the formic acetic anhydride (4.4
g, 0.05 mol) prepared by heating a mixture of acetic anhydride and 98 % formic acid at 60 oC for
2 h, 10a (6.3 g, 0.025 mol) in ether (12 mL) was added. The mixture was stirred at rt for 12 h
where the formed precipitate was filtered off, washed with ether and recrystallized from ethanol
o
to obtain compound 12a as white solid (67%), m.p. 110 C. IR (cm-1): 3400 (NH), 3100 (CH
1
aromatic), 2964 (CH aliphatic), 1674 (amide C=O), 1572, 1512 (C=C, C=N). H NMR (400
MHz, DMSO-d6) δ 7.15-8.16 (m, 10H, aromatic), 8.56 (s, 1H, CH), 12.49 (S, 1H, NH
13
exchangeable with D2O). C NMR (100 MHz, DMSO) δ 126.3, 128.3, 128.5, 128.7, 128.94,
16

129.4, 129.7, 132.0, 134.9, 144.4, 154.5, 160.3. MS (EI) m/z 280 (M++1). Anal. Calcd. For
C16H12N2OS: C, 68.55; H, 4.31; N, 9.99. Found: C, 68.79; H, 4.37; N, 10.12.
N-(4,5-bis(4-methoxyphenyl)thiazol-2-yl)formamide (12b). Yield 69% as light yellow solid,
m.p. 125-126 oC. IR (cm-1): 3392 (NH), 3100 (CH aromatic), 2993 (CH aliphatic), 1692 (amide
C=O), 1609, 1511 (C=C, C=N). 1H NMR (400 MHz, CDCl3) δ 3.83 (s, 3H, OCH3), 3.86 (s, 3H,
OCH3), 6.85-7.42 (m, 8H, aromatic), 7.58 (s, 1H, CH), 12.84 (s, 1H, NH, exchangeable with
13
D2O). C NMR (100 MHz, DMSO-d6) δ 55.2, 55.3, 114.1, 114.2, 123.7, 126.4, 126.9, 130.3,
130.5, 143.0, 155.4, 158.9, 159.3, 159.6. MS (EI) m/z 340 (M++1). Anal. Calcd. For
C18H16N2O3S: C, 63.51; H, 4.74; N, 8.23. Found: C, 63.64; H, 4.81; N, 8.41.
Ethyl 2-((4,5-diphenylthiazol-2-yl)amino)acetate (13a). To a stirred suspension of 60%
sodium hydride (0.2 g, 5 mmol) in DMF (5 mL), N-(4,5-diphenylthiazol-2-yl)formamide 12a
(1.4 g, 5 mmol) was added. The stirring was continued for 1 h and then a solution of ethyl
chloroacetate (0.92 g, 5.5 mmol) in DMF (2 mL) was added dropwise. The reaction mixture was
left to stirred overnight at rt. The mixture was poured onto ice-water and the precipitate was
filtered off and recrystallized from ethanol to give compound 13a as brown solid (66%), m.p.
o
98-99 C. IR (cm-1): 3372 (NH), 3056 (CH aromatic), 2980 (CH aliphatic), 1744 (amide C=O),
1
1597, 1495 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 1.31 (t, J = 7.1 Hz, 3H, CH3), 4.11 (s,
1H, NH exchangeable with D2O), 4.26 (q, J = 7.1 Hz, 2H, CH2), 4.40 (s, 2H, CH2), 7.57-7.18
(m, 10H, aromatic). 13C NMR (100 MHz, CDCl3) δ 14.3, 46.5, 61.6, 126.3, 126.7, 128.6, 128.8,
129.4, 129.5, 130.2, 131.0, 134.9, 137.1, 138.9, 168.6. MS (EI) m/z 338 (M++1). Anal. Calcd.
For C19H18N2O2S: C, 67.43; H, 5.36; N, 8.28. Found: C, 67.52; H, 5.41; N, 8.37.
Ethyl-2-((4,5-bis(4-methoxyphenyl)thiazol-2-yl)amino)acetate (13b). Yield 65% as yellow
solid, m.p. 102-103 oC. IR (cm-1): 3432 (NH), 3100 (CH aromatic), 2959 (CH aliphatic), 1745
1
(ester C=O), 1608, 1511 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 1.31 (t, J = 6.9 Hz, 3H,
CH3), 3.79 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 4.25 (q, J = 6.8 Hz, 2H, CH2), 4.37 (s, 2H, CH2),
13
6.80-7.44 (m, 8H, aromatic), 9.1 (1H, NH, exchangeable with D2O). C NMR (100 MHz,
CDCl3) δ 14.0, 46.2, 55.1, 55.3, 61.9, 113.5, 114.5, 121.9, 127.1, 127.4, 127.6, 132.2, 137.1,
137.7, 158.2, 160.0, 167.8. MS (EI) m/z 398 (M++1). Anal. Calcd. For C21H22N2O4S: C, 63.30;
H, 5.56; N, 7.03. Found: C, 63.45; H, 5.64; N, 7.11.
2-((4,5-diphenylthiazol-2-yl)amino)-1-morpholinoethanone (14a). A mixture of ethyl-2-
((4,5-diphenylthiazol-2-yl)amino)acetate 13a (1.6 g, 0.005 mol) and morpholine (0.86 g, 0.01
mol) was refluxed for 4 h. The mixture was cooled to rt and then poured onto ice cold water.
The formed precipitate was filtered and recrystallized from aqueous ethanol to give compound
17

14a as off-white solid (70%), m.p. 165-166 oC. IR (cm-1): 3431 NH), 3056 (CH aromatic), 2968
1
(CH aliphatic), 1648 (amide C=O), 1558, 1492 (C=C, C=N). H NMR (400 MHz, DMSO-d6) δ
3.44 (s, 1H, NH exchangeable with D2O), 3.56 (t, J = 4, 2 H, CH2), 3.65 (t, J=4; 2 H, CH2),
13
3.71-3.80 (m, 4H, 2*CH2), 4.42 (s, 2H, CH2), 7.18-7.47 (m, 10H, aromatic). C NMR (100
MHz, DMSO-d6) δ 42.2, 45.5, 51.5, 66.8, 121.5, 127.1, 127.5, 128.0, 128.5, 129.0, 129.4, 132.7,
135.4, 146.2, 167.0, 167.3. MS (EI) m/z 379 (M+-1). Anal. Calcd. for C21H21N3O2S: C, 66.47; H,
5.58; N, 11.07. Found: C, 66.59; H, 5.64; N, 11.25.
2-((4,5-bis(4-methoxyphenyl)thiazol-2-yl)amino)-1-morpholinoethanone (14b). Yield 71%
brown solid, m.p. 140-142 oC. IR (cm-1): 3435 (NH), 3071 (CH aromatic), 2959 (CH aliphatic),
1
1650 (amide C=O), 1606, 1511 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 3.49 (s, 1H, NH
exchangeable with D2O), 3.58 (t, J = 4 Hz, 2H, CH2), 3.65 (t, J = 4 Hz, 2H, CH2), 3.70-3.75 (m,
4H, 2* CH2), 3.80 (s, 3H, OCH3), 3.81 (s, 3H, OCH3) 4.51 (s, 2H, CH2), 6.77-7.41 (m, 8H,
13
aromatic). C NMR (100 MHz, CDCl3) δ 42.25, 45.52, 51.45, 55.20, 55.25, 66.85, 113.39,
113.97, 120.14, 125.17, 128.21, 130.10, 130.76, 145.39, 158.74, 158.82, 166.76, 167.13. MS
(EI) m/z 439 (M++1). Anal. Calcd. for C23H25N3O4S: C, 62.85; H, 5.73; N, 9.56. Found: C,
63.01; H, 5.791; N, 9.67.
2-((4,5-diphenylthiazol-2-yl)amino)-1-(piperidin-1-yl)ethanone (14c). Yield 73% brown
o
solid, m.p. 133-135 C. IR (cm-1): 3423 (NH), 3053 (CH aromatic), 2930 (CH aliphatic), 1645
1
(amide C=O), 1551, 1532 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 1.59-1.63 (m, 6H,
3*CH2), 3.38 (s, 1H, NH exchangeable with D2O), 3.45 (t, J = 4 Hz, 2H, CH2), 3.58 (t, J = 4 Hz,
13
2H, CH2), 4.54 (s, 2H, CH2), 7.18-7.50 (m, 10H, aromatic). C NMR (100 MHz, CDCl3) δ
24.45, 25.59, 26.38, 43.17, 46.04, 51.81, 121.14, 126.99, 127.30, 127.92, 128.45, 129.10,
129.48, 133.02, 135.70, 146.26, 166.57, 167.72. MS (EI) m/z 377 (M++1). Anal. Calcd. for
C22H23N3OS: C, 70.00; H, 6.14; N, 11.13. Found: C, 70.17; H, 6.22; N, 11.29.
2-((4,5-bis(4-methoxyphenyl)thiazol-2-yl)amino)-1-(piperidin-1-yl)ethanone (14d). Yield
o
72%, brown solid, m.p. 144-145 C. IR (cm-1): 3432 (NH), 3052 (CH aromatic), 2994 (CH
1
aliphatic), 1646 (amide C=O), 1623, 1511 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 1.59-
1.68 (m, 6H, 3*CH2), 1.68 (s, 1H, NH exchangeable with D2O), 3.47 (t, J = 2 Hz, 2H, CH2),
3.59 (t, J = 4 Hz, 2H, CH2), 3.80 (s, 3H, OCH3), 3.81(s, 3H, OCH3), 4.53 (s, 2H, CH2), 6.64-
7.46 (m, 8H, aromatic). 13C NMR (100 MHz, CDCl3) δ 24.48, 25.59, 26.40, 43.15, 46.06, 51.68,
55.19, 55.24, 113.28, 113.89, 119.64, 125.55, 128.52, 130.18, 130.82, 145.38, 158.59, 158.67,
166.70, 167.15. MS (EI) m/z 437 (M++1). Anal. Calcd. for C24H27N3O3S: C, 65.88; H, 6.22; N,
9.60. Found: C, 66.03; H, 6.31; N, 9.78.
18

2-((4,5-diphenylthiazol-2-yl)amino)-1-(4-methylpiperazin-1-yl)ethanone (14e). Yield 82%,
o
off-white solid, m.p. 143-145 C. IR (cm-1): 3383 (NH), 3053 (CH aromatic), 2934 (CH
aliphatic), 1645 (amide C=O), 1533, 1440 (C=C, C=N). 1H NMR (400 MHz, CDCl3) δ 2.21 (t, J
= 4 Hz, 2H, CH2) 2.27 (s, 3H, CH3), 2.32 (t, J = 4 Hz, 2H, CH2), 3.21 (t, J = 4 Hz, 2H, CH2)
3.60 (t, J = 4 Hz, 2H, CH2), 4.53 (s, 2H, CH2), 7.14-7.47 (m, 10H, aromatic), 7.69 (s, 1H, NH
13
exchangeable with D2O). C NMR (100 MHz, CDCl3): δ 42.3, 44.8, 45.6, 45.9, 54.4, 54.6,
126.3, 126.6, 128.0, 128.3, 128.9, 129.1, 130.4, 131.0, 134.5, 137.9, 138.0, 164.8. MS (EI) m/z
392 (M++1). Anal. Calcd. for C22H24N4OS: C, 67.32; H, 6.16; N, 14.27. Found: C, 67.59; H,
6.28; N, 14.49.
2-((4,5-bis(4-methoxyphenyl)thiazol-2-yl)amino)-1-(4-methylpiperazin-1-yl)ethanone (14f).
Yield 70%, brown solid, m.p. 170-171 oC. IR (cm-1): 3384 (NH), 3000 (CH aromatic), 2934 (CH
1
aliphatic), 1647 (amide C=O), 1608, 1510 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 2.21-
2.23 (t, J = 4 Hz, 2H, CH2), 2.27 (s, 3H, CH3), 2.33-2.35 (t, J = 4 Hz, 2H, CH2), 3.24-3.26 (t, J =
4 Hz, 2H, CH2), 3.59-3.61 (t, J = 4 Hz, 2H, CH2 ), 3.76 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.51
13
(s, 2H, CH2), 6.76-7.43 (m, 8H, aromatic), 7.68 (s, 1H, NH exchangeable with D2O). C NMR
(100 MHz, CDCl3) δ 42.2, 44.8, 45.6, 45.9, 54.4, 54.6, 55.1, 55.3, 113.5, 114.5, 122.3, 127.2,
127.7, 130.9, 132.3, 137.5, 137.6, 158.1, 159.9, 165.0. MS (EI) m/z 452 (M++1). Anal. Calcd.
for C24H28N4O3S: C, 63.69; H, 6.24; N, 12.38. Found: C, 63.84; H, 6.31; N, 12.53.
(4,5-diphenylthiazol-2-yl)glycine (15a). A solution of potassium hydroxide (0.112 g, 2 mmol)
in methanol (18 mL) was added to a mixture of ethyl2-((4,5-diphenylthiazol-2-yl)amino)acetate
derivatives 13a (0.33 g, 1 mmol) in methanol (25 mL). The resulting mixture was heated under
reflux overnight. The mixture was allowed to cool to room temperature and then poured onto
water and acidified with 1N HCl. The precipitate was filtered, washed with water and
recrystallization occurred from aqueous ethanol to afford compound 15a as yellow crystals
(77%), m.p. 98-99 oC. IR (cm-1): 3406 (NH), 3243 (acidic OH), 3054 (CH aromatic), 2924 (CH
1
aliphatic), 1727 (acidic C=O), 1624, 1500 (C=C, C=N). H NMR (400 MHz, DMSO-d6) δ 4.05
(s, 1H, NH exchangeable with D2O), 4.27 (s, 2H, CH2), 7.13-7.52 (m, 10H, aromatic), 12.89 (s,
1H, COOH exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6) δ 46.43, 126.38, 126.67,
128.59, 128.90, 129.39, 129.57, 130.40, 131.03, 134.95, 136.88, 138.92, 170.10. MS (EI) m/z
311 (M++1). Anal. Calcd. For C17H14N2O2S: C, 65.79; H, 4.55; N, 9.03. Found: C, 65.94; H,
4.52; N, 9.17.
(4,5-bis(4-methoxyphenyl)thiazol-2-yl)glycine (15b). Yield 70% yellow solid, m.p. 100-101
oC. IR (cm-1): 3404 (NH), 3200 (acidic OH), 3100 (CH aromatic), 2934 (CH aliphatic), 1685
19

1
(amide C=O), 1608, 1509 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 3.73 (s, 3H, OCH3) 3.83
(s, 3H, OCH3), 3.87 (s, 1H, NH exchangeable with D2O), 4.2 (s, 2H, CH2), 6.91-7.30 (m, 8H,
13
aromatic), 9.30 (s, 1H, COOH exchangeable with D2O). C NMR (100 MHz, CDCl3) δ 47.3,
55.6, 55.7, 113.5, 114.5, 122.3, 127.1, 127.3, 127.7, 132.2, 137.5, 137.6, 158.1, 159.9, 165.2.
MS (EI) m/z 370 (M+). Anal. Calcd. For C19H18N2O4S: C, 61.61; H, 4.90; N, 7.56. Found: C,
61.75; H, 4.98; N, 7.69.
4,5-diphenyl-1H-imidazole (16a).22 A mixture of compound 8a (4.8 g, 0.023 mol) and
formamide (30 mL) was heated to reflux for 3 h. The reaction mixture was poured onto water
and stirred vigorously to dissolve the gummy product. The resulting solid was filtered, washed
o
with water and suspended in 5% HCl (200 mL). The solution was heated to 80-90 C and
filtered while hot. The filtrate was treated with excess NH4OH where a white precipitate which
was formed, filtered and washed with water to afford compound 16a as white solid (80%), m.p.
230-234 oC.
4,5-bis(4-methoxyphenyl)-1H-imidazole (16b).23 Yield 67% a white solid, m.p. 178-180 oC.
Ethyl-2-(4,5-diphenyl-1H-imidazol-1-yl)acetate (17a). Yield 71% as yellowish green solid.
m.p. 163-164 oC. IR (cm-1): 3100 (CH aromatic), 2970 (CH aliphatic), 1747 (C=O), 1667, 1600
1
(C=C, C=N). H NMR (400 MHz, DMSO-d6) δ 1.06 (t, J = 7.0 Hz, 3H, CH3), 4.02 (q, J = 7.1
Hz, 2H, CH2), 4.75 (s, 2H, CH2), 7.13-7.49 (m, 10H, aromatic), 7.85 (s, 1H, CH). 13C NMR (100
MHz, DMSO-d6) δ 14.3, 46.5, 61.6, 126.3, 126.7, 128.6, 128.8, 129.4, 129.5, 130.2, 131.0,
134.9, 137.1, 138.9, 168.6. MS (EI) m/z 306 (M++1). Anal. Calcd. for C22H24N4O: C, 73.31; H,
6.71; N, 15.54. Found: C, 73.54; H, 6.76; N, 15.72.
Ethyl-2-(4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)acetate (17b). Yield 67% as off-white
solid, m.p. 169-170 oC. IR (cm-1): 3100 (CH aromatic), 2900 (CH aliphatic), 1755 (C=O), 1666,
1
1611 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 1.21 (t, J = 6.7 Hz, 3H, CH3), 3.80 (s, 3H,
OCH3), 3.81(s, 3H, OCH3), 4.16 (q, J = 8 Hz, 2H, CH2), 4.48 (s, 2H, CH2), 6.76-7.43 (m, 8H,
aromatic), 8.00 (s, 1H, CH). 13C NMR (101 MHz, CDCl3) δ 14.0, 46.2, 55.1, 55.3, 61.9, 113.5,
114.5, 121.9, 127.1, 127.4, 127.6, 132.2, 137.1, 137.7, 158.2, 160.0, 167.8. MS (EI) m/z 366
(M++1). Anal. Calcd. for C21H22N2O4: C, 68.84; H, 6.05; N, 7.65. Found: C, 68.67; H, 6.80; N,
13.45.
2-(4,5-diphenyl-1H-imidazol-1-yl)-1-morpholinoethanone (18a). Yield 71% brown solid, m.p.
o
109-110 C. IR (cm-1): 3116 (CH aromatic), 2970, 2917 (CH aliphatic), 1643 (amide C=O),
1607, 1507 (C=C, C=N). 1H NMR (400 MHz, CDCl3) δ 3.19 (t, J = 4 Hz, 2H, CH2), 3.48 (t, J =
20

4 Hz, 2H, CH2), 3.57 (t, J = 4 Hz, 2H, CH2), 3.61 (t, J = 4 Hz, 2H, CH2), 4.53 (s, 2H, CH2),
7.14-7.51 (m, 10H, aromatic), 7.66 (s, 1H, CH).13C NMR (100 MHz, CDCl3) δ 42.5, 45.3, 45.5,
66.1, 66.7, 126.4, 126.6, 128.1, 128.3, 128.9, 129.1, 129.3, 130.3, 130.9, 134.4, 138.04, 165.1.
MS (EI) m/z 347 (M++1). Anal. Calcd. for C21H21N3O2: C, 72.60; H, 6.09; N, 12.10. Found: C,
72.69; H, 6.17; N, 12.34.
2-(4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)-1morpholinoethanone (18b). Yield 80% off-
o
white solid, m.p. 97-98 C. IR (cm-1): 3020 (CH aromatic), 2930, 2835 (CH aliphatic), 1643
1
(amide C=O), 1614, 1577 (C=C, C=N). H NMR (400 MHz, CDCl3): δ 3.21 (t, J = 4 Hz, 2H,
CH2), 3.52 (t, J = 4 Hz, 2H, CH2), 3.59 (t, J = 4 Hz, 2H, CH2), 3.64 (t, J = 4 Hz, 2H, CH2), 3.76
(s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.51 (s, 2H, CH2), 6.76-7.42 (m, 8H, aromatic), 7.62 (s, 1H,
CH).13C NMR (100 MHz, CDCl3) 42.5, 45.3, 45.5, 55.1, 55.3, 66.2, 66.7, 113.5, 114.5, 122.3,
127.1, 127.3, 127.7, 132.2, 137.5, 137.6, 158.1, 159.9, 165.2. MS (EI) m/z 407 (M++1). Anal.
Calcd. For C23H25N3O4: C, 67.80; H, 6.18; N, 10.31. Found: C, 67.96; H, 6.24; N, 10.44.
2-(4,5-diphenyl-1H-imidazol-1-yl)-1-(piperidin-1-yl)ethanone (18c). Yield 84% off-white solid,
m.p. 120-122 oC. IR (cm-1): 3116 (CH aromatic), 2937, 2865 (CH aliphatic), 1646 (amide C=O),
1
1604, 1506 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 1.34-1.37 (m, 2H, CH2), 1.50-1.53 (m,
2H, CH2), 1.59-1.62 (m, 2H, CH2), 3.15 – 3.17 (t, J = 4 Hz, 2H, CH2), 3.51-3.53 (t, J = 4 Hz,
2H, CH2), 4.53 (s, 2H, CH2), 7.16-7.49 (m, 10H, aromatic), 7.66 (s, 1H, CH).13C NMR (100
MHz, CDCl3) δ 24.2, 25.4, 26.1, 43.5, 45.9, 46.0, 126.2, 126.6, 128.0, 128.5, 128.8, 129.0,
130.4, 131.0, 134.5, 137.8, 138.0, 164.5. MS (EI) m/z 345 (M++1). Anal. Calcd. for C22H23N3O:
C, 76.49; H, 6.71; N, 12.16. Found: C, 76.56; H, 6.78; N, 12.31.
2-(4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)-1-(piperidin-1-yl)ethanone (18d). Yield 86%
o
off-white solid, m.p. 123-125 C. IR (cm-1): 3129 (CH aromatic), 2935, 2858 (CH aliphatic),
1
1650 (amide C=O), 1612, 1577 (C=C, C=N). H NMR (400 MHz, CDCl3): δ 1.37 (s, 2H, CH2),
1.52 (s, 2H, CH2), 1.60 (s, 2H, CH2), 3.18 (s, 2H, CH2), 3.53 (s, 2H, CH2), 3.76 (s, 3H, OCH3),
3.86 (s, 3H, OCH3), 4.50 (s, 2H, CH2), 6.75-7.43 (m, 8H, aromatic), 7.61 (s, 1H, CH).13C NMR
(100 MHz, CDCl3): 24.2, 25.4, 26.1, 43.5, 45.8, 46.0, 55.1, 55.3, 113.5, 114.5, 122.3, 127.1,
127.3, 127.7, 132.2, 137.5, 137.6, 158.1, 159.9, 165.2. MS (EI) m/z 405 (M++1). Anal. Calcd.
for C24H27N3O3: C, 71.09; H, 6.71; N, 10.36. Found: C, 71.31; H, 6.80; N, 10.51.
2-(4,5-diphenyl-1H-imidazol-1-yl)-1-(4-methylpiperazin-1-yl)ethanone (18e). Yield 71%
o
brown solid, m.p. 145-146 C. IR (cm-1): 3115 (CH aromatic), 2973-2942 (CH aliphatic), 1643
1
(amide C=O), 1600, 1506 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 2.19-2.21 (t, J = 4 Hz,
2H, CH2), 2.27 (s, 3H, CH3), 2.32-2.35 (t, J = 4 Hz, 2H, CH2), 3.21-3.23 (t, J = 4 Hz, 2H, CH2)
21

3.59-3.61 (t, J = 4 Hz, 2H, CH2), 4.53 (s, 2H, CH2), 7.14-7.47 (m, 10H, aromatic), 7.66 (s, 1H,
CH).13C NMR (100 MHz, CDCl3): δ 42.3, 44.8, 45.6, 45.9, 54.4, 54.6, 126.3, 126.6, 128.0,
128.3, 128.9, 129.1, 130.4, 131.0, 134.5, 137.9, 138.0, 164.8. MS (EI) m/z 360 (M++1). Anal.
Calcd. for C22H24N4O: C, 73.31; H, 6.71; N, 15.54. Found: C, 73.54; H, 6.76; N, 15.72.
2-(4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)-1-(4-methylpiperazin-1-yl)ethanone (18f).
o
Yield 79% brown solid, m.p. 163-165 C. IR (cm-1): 3100 (CH aromatic), 2930, 2835 (CH
1
aliphatic), 1657 (amide C=O), 1666, 1611 (C=C, C=N). H NMR (400 MHz, CDCl3) δ 2.21-
2.23 (t, J = 4 Hz, 2H, CH2), 2.27 (s, 3H, CH3), 2.33-2.35 (t, J = 4 Hz, 2H, CH2), 3.24-3.26 (t, J =
4 Hz, 2H, CH2 ), 3.59-3.61 (t, J = 4 Hz, 2H, CH2), 3.76 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.51
(s, 2H, CH2), 6.76-7.43 (m, 8H, aromatic), 7.62 (s, 1H, CH).13C NMR (100 MHz, CDCl3) δ
42.2, 44.8, 45.6, 45.9, 54.4, 54.6, 55.1, 55.3, 113.5, 114.5, 122.3, 127.2, 127.7, 130.9, 132.3,
137.5, 137.6, 158.1, 159.9, 165.0. MS (EI) m/z 420 (M++1). Anal. Calcd. for C24H28N4O3: C,
68.55; H, 6.71; N, 13.32. Found: C, 68.67; H, 6.80; N, 13.45.
2-(4,5-diphenyl-1H-imidazol-1-yl)acetic acid (19a). Yield 77% yellow solid, m.p. 125-128 oC.
IR (cm-1): 3112 (CH aromatic), 2972 (CH aliphatic), 1753 (acidic C=O), 1625, 1546 (C=C,
1
C=N). H NMR (400 MHz, CDCl3) δ 4.65 (s, 2H, CH2), 6.75-7.30 (m, 8H, aromatic), 8.68 (s,
13
1H, CH), 10.40 (s, 1H, COOH exchangeable with D2O). C NMR (100 MHz, DMSO-d6) δ
46.4, 126.3, 126.6, 128.5, 128.9, 129.3, 129.5, 130.4, 131.0, 134.9, 136.8, 138.9, 170.1. MS (EI)
m/z 278 (M+). Anal. Calcd. for C17H14N2O2: C, 73.37; H, 5.07; N, 10.07. Found: C, 73.52; H,
5.14; N, 10.31.
2-(4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)acetic acid (19b). Yield 70% yellow solid,
o
m.p. 154-155 C. IR (cm-1): 3300 (OH acidic), 3100 (CH aromatic), 2900 (CH aliphatic), 1724
(C=O), 1629, 1575 (C=C, C=N). 1H NMR (400 MHz, CDCl3) δ 3.73 (s, 3H, OCH3) 3.83 (s, 3H,
OCH3), 4.76 (s, 2H, CH2), 6.75-7.30 (m, 8H, aromatic), 8.68 (s, 1H, CH), 9.40 (s, 1H, COOH
exchangeable with D2O). 13C NMR (100 MHz, CDCl3) δ 47.3, 55.6, 55.7, 114.5, 115.21, 119.63,
123.08, 128.42, 128.47, 132.68, 137.29, 159.36, 160.55, 169.18. MS (EI) m/z 338 (M+). Anal.
Calcd. For C19H18N2O4: C, 67.44; H, 5.36; N, 8.28. Found: C, 67.59; H, 5.41; N, 8.37.
4.2. Biological screening
4.2.1. In vitro COXs inhibitory assay
The ability of the newly synthesized compounds to inhibit COX subtypes activity was evaluated
using COXs Colorimetric Inhibitor Screening Kit from Cayman Chemical Company (Ann
Arbor, MI) according manufacturer’s directions and as mentioned before.24,25 This assay is based
on measuring the peroxidase component of COXs colorimetrically by monitoring the production
22

of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) at 590 nm. The assay includes
both ovine COX-1 and human recombinant COX-2. The enzymes were pre-incubated for 5 min
o
at 25 C with the test compounds prior to addition of arachidonic acid (final concentration 1.1
o
mM) and TMPD and incubation for 5 min at 25 C. Sixteen compounds were screened for their
COX-1/COX-2 affinity and selectivity. All assays were performed in triplicates and IC50 values
are the average of three determinations for each compound.
4.2.2. Acetic acid-induced writhing test
The analgesic activity of the tested compounds was estimated using acetic acid-induced writhing
method as described by Koster et al..27 The writhing test was performed in groups of five mice
each. One hour after the administration of the test compound, 0.01 ml/g of 0.6% acetic acid
solution was injected intra-peritoneal in each mouse. The writhing movements of each animal
were counted for 15 min (between the fifth and 20th min after the injection of the irritant).
Diclofenac (6 mg/kg) was used as reference drug. The number of abdominal constrictions was
cumulatively counted over a period of 20 min.
4.2.2. Carrageenan-induced rat paw edema assay
The tested compounds were evaluated for their ant-inflammatory activity using carrageenan-
induced rat paw edema model described by Winter et al..28,29 Albino rats of either sex weighing
120-150 g were divided into 15 groups of five animals each. Rats were uniformly hydrated by
giving 3 mL water/rat through gastric inoculation to reduce variability to edema response. The
control group was given 10% DMSO aqueous solution (v/v). Diclofenac sodium was taken as a
reference standard (60 mg/kg) while the tested compounds in the form of 10% DMSO aqueous
solutions were administered orally to the rest groups at a dose of 100 mg/kg body weight,
treatments began 1 h before induction of inflammation. Paw edema was induced by
subcutaneous injection of 50 µl of 1% carrageenan-sodium gel (Sigma-Aldrich, USA), into the
subplantar region of the right hind paw. The thickness of the right and left hind paw of each rat
was measured using a pair of dial thickness gauge calipers accurate to 0.001 cm 1, 3 and 7 hours
after induction of inflammation. The left hind paw diameter served as a control for the degree of
inflammation in the right hind paw.
4.2.3. Acute ulcerogenicity study
The ulcerogenic potential of the most active compounds 15a, 15b, 19a and 19b and
indomethacin as a reference drug was examined in rats as reported by Cioli et al..30 Adult albino
rats of both sexes weighing 120-150 g were used. Rats randomly divided into 6 groups each of
five animals. Fasted animals administered the tested compounds and indomethacin orally in dose
23

of 20 mmol/Kg suspended in 1% tween while one group received vehicle. Fasting rats for 2
hours, feeding for another 2 hours followed by fasting for another 20 hours. Two doses were
given to rats in second and third days. Finally, rats were scarified to remove stomach and rinsing
it in 0.9% saline. The mucosal damage was determined according to the following scores:
-
-
-
-
-
Zero for normal (no injury).
1 latent small red spot.
2 wide red spots.
3 slight injuries.
4 severe injuries.
The average of number of ulcers was determined by dividing the number of ulcers in the group
to the total number of rats in the group. The ulcer index is the sum of % incidence, average
severity and average of number of ulcer. To determine the ulcer index, we calculated the %
incidence/10 and average severity as shown in the following equations:
4.2.4. Statistical analysis
All data are presented as means ± SEM (standard error of the mean). Statistical analysis was
done using Statistical Package for Social Science (SPSS) software (version 22). One-way
analysis of variance (ANOVA) test was used to detect significance among group means,
followed by Tukey’s post-hoc test for pair-wise comparison between means of groups.
Differences were considered significant at p < 0.05 (*) or p < 0.001(**).
4.3. Molecular docking study
This study was performed using LIGANDFIT imbedded into Discovery Studio Software where
the binding site was generated from the co-crystallized ligands IMM within COX-1 protein
structure (PDB codes: 1PGF).31 Two selected compounds, 14f and 15b were sketched and
energy minimized using CHARMm Force Field and then docked into the aforementioned
prepared protein active site. The number of Monte Carlo search trials in the utilized docking
protocol was set to 15000 and torsional step size for polar hydrogens was equal to 30o.
Moreover, the Root Mean Square Difference (RMS) threshold for ligand-to-binding site shape
24

match was set to 2.0 employing a maximum of 1.0 binding site partitions and 1.0 site partition
seed. In addition, the Force Field used for evaluating and calculating ligand-receptor interaction
energies was set to DREIDING force field with a dielectric constant of 1.0 and a non-bonded
cutoff distance of 10.0 Å. An energy grid extending 3.0 Å from the binding site was
implemented. Furthermore, a maximum of 10 diverse docked conformations/poses of optimal
interaction energies were saved. The saved conformers/ poses were further energy-minimized
within the binding site for a maximum of 1000 rigid-body iterations. Finally, docking results
were analyzed and the pictures were generated using Discover Studio Visualizer software.
Acknowledgments
The authors are thankful to Dr. Ahmed Mansour, Head of the Department of Pharmacology,
Faculty of Pharmacy, Al-Azhar University for his great efforts in performing the in vivo studies.
Also, the authors thank Dr. Mohammed Ragab, assistant lecturer of pharmacology, Faculty of
Pharmacy, Beni-Suef University for his kind assistance in doing the statistical analysis.
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