1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3- carboxamides: Synthesis, molecular modeling, evaluation of their anti-inflammatory activity and ulcerogenicity

Article abstract of DOI:10.1016/j.ejmech.2014.03.001

A series of novel 1-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4- triazole-3-carboxamides were synthesized and confirmed with different spectroscopic techniques. The prepared compounds exhibited remarkable anti-inflammatory activity that represen

Full text of DOI:10.1016/j.ejmech.2014.03.001

European Journal of Medicinal Chemistry 77 (2014) 155e165
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-
3-carboxamides: Synthesis, molecular modeling, evaluation of their
anti-inflammatory activity and ulcerogenicity
Mohamed Abdel-Aziz a, Eman A. Beshr a, Islam M. Abdel-Rahman a, Keriman Ozadali b,
,
Oya Unsal Tan b, Omar M. Alya
*
a Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sihhiye, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamides
were synthesized and confirmed with different spectroscopic techniques. The prepared compounds
exhibited remarkable anti-inflammatory activity that represents 38%e100% of indomethacin activity and
44%e115% of celecoxib activity after 3 h. The anilides 5ael and hydrazide 6 exhibit low incidence of
gastric ulceration compared to indomethacin which was confirmed with histopathological investigation.
Received 17 September 2013
Received in revised form
26 February 2014
Accepted 2 March 2014
Available online 3 March 2014
In vitro COX-1/COX-2 inhibition studies showed compounds 4b (COX-1 IC50
¼
45.9 mM; COX-2
IC50 ¼ 68.2
m
M) and 6 (COX-1 IC50 ¼ 39.8
m
M; COX-2 IC50 ¼ 46.3
m
M) are the most potent COX in-
Keywords:
1,2,4-Triazole
Anti-inflammatory
Ulcerogenicity
hibitors in the tested compounds. The binding mode for some of the tested compounds to the enzymes
was predicted using docking studies.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Histopathological investigation
Cyclooxygenase activity
Docking study
1. Introduction
has stimulated a lot of interest,1,2,4-triazoles and their heterocyclic
derivatives have a wide range of therapeutic properties like anti-
Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the
most useful clinical therapies for the treatment of pain, fever, and
inflammation [1]. The major mechanism by which NSAIDs exert
their anti-inflammatory activity is through inhibition of
cyclooxygenase-derived prostaglandin synthesis, which is also
responsible for the gastrointestinal [2e6], renal [7e9], and hepatic
side effects [10] that are observed mainly in chronic use of NSAIDs.
Diaryl-heterocycles have become the major class of selective COX-2
inhibitors, such as celecoxib, rofecoxib, parecoxib and valdecoxib
(Fig. 1), which display improved gastrointestinal safety profile
compared to the traditional NSAIDs [11e13]. However, the cardio-
vascular risk associated with “-coxibs” has become a concern since
rofecoxib and valdecoxib were withdrawn from the market [14].
Therefore, the challenge still exists for the pharmaceutical industry
to develop, effective anti-inflammatory agents with enhanced
safety profile. The considerable biological importance of triazoles
bacterial [15e17], antifungal [16e18] antitubercular [19e21], anti-
viral [22,23], analgesic [24,25], anti-inflammatory [25e27],
anticonvulsant [28,29], antidepressant [30], anticancer [31,32],
antihypertensive [33], hypoglycemic [34], insecticidal [35,36] and
plant growth activities [37]. Besides, studies that described simple
chemical derivatization (esterification or amidation) of the car-
boxylic function of representative NSAIDs showed that this
approach resulted not only in the reduction of the ulcerogenic ef-
fect but also in an increased anti-inflammatory activity [38e40].
Various oxadiazole, triazole, thiadiazole and triazine derivatives of
indomethacin have been synthesized and tested for anti-
inflammatory activity. The test compounds inhibited the induc-
tion of gastric mucosal lesions which may be related to inhibition of
lipid peroxidation in gastric mucosa [41]. Promoted with the above-
mentioned studies and as a continuation of our research interest in
the synthesis and biological activities of novel NSAIDs derivatives
with lower ulcerogenic liability [42e44], the present study is
concerned with the synthesis of novel 1-(4-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamides with
the objective of discovering novel and potent anti-inflammatory
* Corresponding author.
E-mail addresses: omarsokkar@yahoo.com, omarsokkar@mu.edu.eg (O.M. Aly).
http://dx.doi.org/10.1016/j.ejmech.2014.03.001
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

156
M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 77 (2014) 155e165
Fig. 1. Selective COX-2 inhibitors.
agents. The ability of the prepared compounds to induce gastric
toxicity has been also evaluated. Among the synthesized series,
some selected derivatives were tested for their inhibitory activity
against COX-2 and COX-1 enzymes and their binding mode to the
enzymes were predicted using docking studies.
were administered orally at a dose level of (0.28 mmol/kg) 30 min
before carrageenan injection at the right hind paw of Albino male
rats. The thickness of both paws was measured at different time
intervals of 1, 2, 3, 4 and 5 h after carrageenan injection. The anti-
inflammatory activity of the tested compounds, celecoxib and
indomethacin was calculated as the percentage decrease in edema
thickness induced by carrageenan and was determined using the
following formula:
2. Results and discussions
2.1. Chemistry
ðVR ꢀ VLÞcontrol ꢀ ðVR ꢀ VLÞtreated
% of edema inhibition ¼
The synthetic route used to synthesized 1-(4-methoxyphenyl)-
5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamides
4aec, 5ael and 6 is outlined in Scheme 1. Key starting compound,
2-(3,4,5-trimethoxybenzamido)acetic acid 1 was prepared in
good yield (85%) by the reaction of glycine with 3,4,5-
trimethoxybenzoyl chloride in 10% NaOH. Heating of compound
1 with acetic anhydride afforded the corresponding compound 2.
The synthesis of the key intermediate 3 was carried out using
Kuskov like reaction through coupling of the diazonium salt of 4-
methoxyaniline with the active methylene of 2 in presence of
sodium acetate. According to Sawdey rearrangement [45], reac-
tion of 3 with ammonia or appropriate amine in methanol gave
the corresponding amides 4aec in 55e71% yield. Also, treatment
of 3 with primary aromatic amines in acidic medium formed the
target anilides 5ael in 35e62% yield. Moreover, reaction of 3 with
hydrazine monohydrate in methanol afforded hydrazide 6 in 65%
yield. The physical and spectral data are listed in the experimental
part.
ðVR ꢀ VLÞcontrol
ꢁ 100
where VR represents the mean right paw thickness and VL repre-
sents the mean left paw thickness.
(VReVL)control represents the mean increase in paw thickness in
the control group of rats.
(VReVL)treated represents the mean increase in paw thickness in
rats treated with the test compounds.
The results listed in Table 1 show the percentage of edema in-
hibition induced by carrageenan for all of the test compounds and
indomethacin versus time in h. The obtained results listed in
Table 1 indicated that most of the test compounds showed signif-
icant (p < 0.01) inhibition against carrageenan-induced paw edema
in rats and the maximum anti-inflammatory activity was obtained
after 3 h which is the time required for reaching the maximum
activity and then the activity decreased gradually for the next 2 h.
Indomethacin showed an inhibitory activity of 78% against
2.2. Biological investigations
carrageenan-induced paw edema after
3 h while celecoxib
2.2.1. Screening of anti-inflammatory activity
exhibited an inhibitory activity of 68% against carrageenan-induced
paw edema after 3 h. Compounds 4aec exhibited good anti-
inflammatory activity of 72%, 74% and 69%, respectively, which
represents 92%, 95% and 88% of indomethacin activity and 106%,
109% and 101% of celecoxib activity, respectively, after 3 h (Table 2).
The synthesized compounds 4aec, 5ael and 6 were evaluated
for their anti-inflammatory activity using carrageenan-induced
paw edema method in rats described by Winter et al [46]. The
test compounds, celecoxib and indomethacin (reference drugs)

M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 77 (2014) 155e165
157
Scheme 1. Synthesis of the target compounds 4aec, 5ael and 6.
The anti-inflammatory activity of amide derivatives 4aec revealed
that the unsubstituted amide 4a was found to possess good anti-
inflammatory activity (72%). The substitution on the amide nitro-
gen by methyl group 4b lead to a slight increase of the anti-
inflammatory activity (74%), while substitution on the amide ni-
trogen by ethyl group 4c lead to a slight decrease of the anti-
inflammatory activity (69%). The anilides 5ael showed a variable
anti-inflammatory activity ranging from 30% to 78% and these re-
sults represent 38%e100% of indomethacin activity and 44%e115%
of celecoxib activity, respectively (Table 2). The results indicated
that substitution on the amide nitrogen by phenyl 5l, 4-
methoxyphenyl 5a, 2-methoxyphenyl 5b, 4-methylphenyl 5c, 2-
methylphenyl 5d, 4-bromophenyl 5e, 3-trifluoromethylphenyl 5h
and 4-hydroxyphenyl 5k moiety resulted in a dramatic decrease of
the anti-inflammatory activity (30%e67%), while substitution on
the amide nitrogen by 4-fluorophenyl moiety 5g and 4-
carboxyphenyl 5j moieties increases the anti-inflammatory activ-
ity (72%e74%). Moreover, substitution on the amide nitrogen by 4-
chlorophenyl 5f, 3,4-dimethoxyphenyl 5i moieties resulted in an
increase of the anti-inflammatory activity of the test compounds
and resulted in equipotent activity compared to indomethacin
(78%). The hydrazide derivative 6 showed an anti-inflammatory
activity of 57% which represents 73% and 84% of indomethacin
and celecoxib activity, respectively.

158
M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 77 (2014) 155e165
Table 1
The anti-inflammatory activity at different time intervals and ulcer indices of compounds 4aec, 5ael and 6 compared to celecoxib and indomethacin.
Compound
% Of edema inhibition (% mean ꢂ S.E.M)
UI mean ꢂ S.E.M
1 h
2 h
3 h
4 h
5 h
Control
4a
4b
4c
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
0
0
0
0
0
0.5 ꢂ 0.06
9.0 ꢂ 1.03**
7.5 ꢂ 0.18**
9.0 ꢂ 0.79***
4.5 ꢂ 0.92***
5.0 ꢂ 1.31**
5.5 ꢂ 1.63**
1.5 ꢂ 0.86**
1.8 ꢂ 0.18***
5.0 ꢂ 0.47***
13 ꢂ 0.25***
5.0 ꢂ 0.50***
7.0 ꢂ 0.70***
12 ꢂ 1.46**
1.0 ꢂ 1.0***
4.0 ꢂ 2.0**
40.00 ꢂ 1.45***
56.00 ꢂ 1.18***
60.00 ꢂ 2.07***
39.00 ꢂ 1.72***
52.00 ꢂ 1.07***
38.00 ꢂ 1.16***
19.00 ꢂ 1.12***
40.00 ꢂ 2.38***
55.00 ꢂ 1.26***
48.00 ꢂ 2.16***
48.00 ꢂ 1.41***
57.00 ꢂ 1.41***
50.00 ꢂ 1.38***
36.00 ꢂ 1.69***
45.00 ꢂ 1.60**
36.00 ꢂ 1.80***
42.00 ꢂ 1.60***
55.00 ꢂ 0.78***
59.00 ꢂ 2.07***
74.00 ꢂ 2.62***
68.00 ꢂ 1.22***
45.00 ꢂ 1.47***
57.00 ꢂ 1.13***
60.00 ꢂ 1.63***
30.00 ꢂ 2.16***
50.00 ꢂ 2.11***
70.00 ꢂ 2.02***
67.00 ꢂ 1.47***
55.00 ꢂ 1.46***
68.00 ꢂ 2.25***
57.00 ꢂ 3.08***
35.00 ꢂ 1.49***
51.00 ꢂ 1.79***
47.00 ꢂ 1.07***
48.00 ꢂ 0.60***
68.00 ꢂ 2.23***
72.00 ꢂ 1.08***
74.00 ꢂ 2.31***
69.00 ꢂ 1.24***
67.00 ꢂ 2.10***
30.00 ꢂ 2.44***
60.00 ꢂ 3.41***
50.00 ꢂ 2.60***
66.00 ꢂ 1.71***
78.00 ꢂ 2.05***
72.00 ꢂ 1.89***
65.00 ꢂ 1.42***
78.00 ꢂ 3.34***
74.00 ꢂ 1.65***
50.00 ꢂ 2.71***
54.00 ꢂ 2.39***
57.00 ꢂ 1.98***
68.00 ꢂ 1.20***
78.00 ꢂ 1.14***
71.00 ꢂ 2.52***
65.00 ꢂ 1.16***
58.00 ꢂ 1.24***
63.00 ꢂ 2.75***
24.00 ꢂ 1.94***
58.00 ꢂ 2.31***
46.00 ꢂ 1.20***
59.00 ꢂ 1.08***
75.00 ꢂ 1.57***
70.00 ꢂ 1.47***
56.00 ꢂ 2.03***
70.00 ꢂ 2.23***
69.00 ꢂ 1.86***
37.00 ꢂ 4.47***
50.00 ꢂ 2.44***
54.00 ꢂ 2.53***
57.00 ꢂ 0.80***
81.00 ꢂ 2.36***
64.00 ꢂ 2.21***
50.00 ꢂ 1.13***
52.00 ꢂ 1.20***
51.00 ꢂ 2.51***
10.00 ꢂ 2.32***
42.00 ꢂ 1.73***
47.00 ꢂ 1.16***
57.00 ꢂ 3.69***
70.00 ꢂ 1.80***
53.00 ꢂ 2.11***
50.00 ꢂ 2.16***
61.00 ꢂ 3.09***
60.00 ꢂ 1.18***
27.00 ꢂ 1.64***
46.00 ꢂ 2.06***
56.00 ꢂ 2.24***
51.00 ꢂ 1.40***
86.00 ꢂ 1.47***
6
2.0 ꢂ 2.67**
0.50 ꢂ 0.60***
41 ꢂ 0.38***
Celecoxib
Indomethacin
**Significantly different from control group at P < 0.01.
***Significantly different from control group at P < 0.001.
Note. One way ANOVA test was applied to determine the significance of the difference between the control group and rats treated with the tested compounds. (n ¼ 4),
**p < 0.01,***p < 0.001, significant difference from control group.
2.2.2. Screening of ulcerogenicity
UIs compared to indomethacin. Compounds 4aec exhibited lower
ulcerogenic liability relative to indomethacin with UIs of 9, 7.5 and
9, respectively, (Fig. 2A). The anilides 5ael also exhibited lower
gastric toxicity relative to indomethacin with UI ranging from 1 to
13 (Fig. 2BeD). The hydrazide derivative 6 showed lower gastric
toxicity relative to indomethacin with UI of 2 (Fig. 2A). The obtained
results revealed that all of the tested compounds exhibit safer ul-
cerogenic liability relative to indomethacin.
The in vivo ulcerogenic liability was evaluated for the synthe-
sized compounds 4aec, 5ael and 6 relative to both celecoxib and
indomethacin according to a reported procedure [47]. Ulcers were
classified into levels, level I, ulcer area less than 1 mm2, level II,
ulcer area from 1 to 3 mm2 and level III, ulcer area more than
3 mm2, and the ulcer index (UI) was calculated as follows:
UI ¼ 1 ꢁ ðnumber of ulcers level IÞ þ 2
ꢁ ðnumber of ulcers level IIÞ þ 3
2.2.3. Histopathological investigation
Different stomach sections of the ulcers including the control
and the treated groups were stained by standard hematoxylin and
eosin stains. The produced slides were subjected to microscopical
examination and pictures were picked for these slides.
ꢁ ðnumber of ulcers level IIIÞ; etc.
The UI of compounds 4aec, 5ael and 6 were calculated and
listed in Table 1 as (mean ꢂ S.E.M). The results of ulcerogenic lia-
bility revealed that indomethacin caused significant ulcerogenic
toxicity with UI of 41, while celecoxib exhibited very low UI of 0.5.
The majority of the synthesized compounds exhibited much lower
The control (Fig. 3A) showed no lesions and characterized by the
presence of continuous mucosal layer. Indomethacin (Fig. 3B)
exhibited marked loss of mucosal membrane at the areas of ul-
ceration where certain areas of fundic glands were totally degen-
erated and cellular details were lost. Capillary inflammatory cells
were also found and apoptotic glandular epithelial cells could be
detected. The stomach sections of the ulcers treated with anilide 5d
showed normal morphology for the fundic glands and the results
are in consistence with the previous results [42e44]. The edema
was very minimal and also the vasodilatation of blood capillaries
was not marked and this was confirmed by the low UI of 1.5
(Fig. 3C). A very low incidence of gastric ulceration was induced by
the anilide 5k (Fig. 3D) where a continuous mucosal layer was
observed with the absence of capillary inflammatory cells and the
ulcerative damage of the gastric mucosa was markedly decreased
which was proved by the very low UI of 1.
Table 2
% Activity of the tested compounds 4aec, 5ael and 6 relative to celecoxib and
indomethacin after 3 h.
Compound
% Activity relative to
% Activity relative to
celecoxib after 3 h
indomethacin after 3 h
Control
0
0
4a
4b
4c
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6
92.00
95.00
88.00
86.00
38.00
77.00
64.00
85.00
100.00
92.00
83.00
100.00
95.00
64.00
69.00
73.00
106.00
109.00
101.00
99.00
44.00
88.00
74.00
97.00
115.00
106.00
96.00
115.00
109.00
74.00
79.00
84.00
100.00
2.3. Cyclooxygenase inhibitory activity
Compounds 4b, 5e, 5i, 5f and 6 were evaluated for their ability
to inhibit COX-1 and COX-2 enzymatic activity using COX Inhibitor
Screening Assay Kit. The potency (IC50 values) of test compounds
was determined and compared to that of reference molecules NS-
398 and indomethacin; their IC50 values and selectivity indices
(SI) are given in Table 3.
Celecoxib
Indomethacin
100.00

M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 77 (2014) 155e165
159
Fig. 2. A. UI of compounds 4aec and 6 compared to indomethacin and celecoxib expressed as mean ꢂ S.E.M. B. UI of compounds 5aed compared to indomethacin and celecoxib
expressed as mean ꢂ S.E.M. C. UI of compounds 5eeh compared to indomethacin and celecoxib expressed as mean ꢂ S.E.M. D. UI of compounds 5iel compared to indomethacin
and celecoxib expressed as mean ꢂ S.E.M.
In vitro COX-1/COX-2 inhibition studies showed that the most
potent COX inhibitors in this series were 4b (COX-1 IC50 ¼ 45.9 M;
COX-2 IC50 ¼ 68.2 M) and 6 (COX-1 IC50 ¼ 39.8 M; COX-2
IC50 ¼ 46.3 M) whereas they did not have selectivity (SI w 1).
Compounds 5e, 5f and 5i did not inhibit cyclooxygenase enzymes at
On the other hand, it was seen that 5e, 5i and 5f oriented in a
different way compared to 4a and 6. These compounds were unable
to fill the adjunct pocket because of their bulky groups (Fig. 4B).
This may explain why 4b and 6 are the active derivatives whereas
5e, 5i and 5f are not.
m
m
m
m
the highest test compound concentration (100
m
M).
In COX-1 enzyme, it was observed that the methoxy group, at
meta position of trimethoxyphenyl ring of 4a and 6, formed strong
hydrogen bonds with hydroxyl group of Ser530. It can be assumed
that the increase in COX-1 potency of 4a and 6 (Fig. 4C) is due to the
presence of this interaction.
2.4. Docking studies
With the aim of getting insights into the structural basis for their
activities, compound 4b, 5e, 5i, 5f and 6 were docked into the active
site of COX-1 and COX-2 enzyme by using MOE software program.
Docking studies in COX-2 showed that the trimethoxyphenyl ring
of all the compounds fitted into the hydrophobic cavity formed by
Phe381, Leu384, Tyr385, Trp387, Phe518 and Ser530. In the crystal
structure of COX-2, this hydrophobic cavity was occupied by bro-
mophenyl ring of SC-558. 4b and 6 were oriented so that their
methoxyphenyl group fitted into the adjunct pocket which is
responsible for COX-2 inhibitory activity (Fig. 4A). The oxygen atom
of methoxy group of 4b and 6 formed weak bonds with Arg513 and
3. Conclusions
A
group
of
novel
1-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide derivatives
was prepared and characterized by different spectroscopic and
elemental analysis techniques. Most of the synthesized compounds
showed significant anti-inflammatory activity using carrageenan-
induced rat paw edema method and the tested compounds
exhibit safer UI relative to indomethacin. Histopathological exam-
ination indicated that the novel anilides 5ael have very low inci-
dence of gastric ulceration. Considering the COX inhibitory activity
and docking study together, it is observed that substitution of less
ꢀ
His90 approximately at distances of 3.8 and 3.1 A respectively.
Furthermore, hydrogen bonds were observed between Arg120 and
C]O group of 4a and 6 at distances of 2.24 and 2.12 A respectively.
ꢀ

160
M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 77 (2014) 155e165
Fig. 3. A. Photomicrograph of the mucosa of fundic stomach of control. B. Photomicrograph of the mucosa of fundic stomach of indomethacin. C. Photomicrograph of the mucosa of
fundic stomach of compound 5d. D. Photomicrograph of the mucosa of fundic stomach of compound 5k.
bulky groups such as CH3 and NH2 on the amide nitrogen of 1-(4-
methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-
carboxamide increased the COX activity. In conclusion the prepared
1-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-
triazole-3-carboxamide derivatives looks as a promising approach
to improve the safety of NSAIDs without altering their
effectiveness.
recorded on Nicolet iS5 FT-IR spectrometer at Minia University.
NMR spectra were carried out using a Bruker Advance 300 MHz
NMR spectrometer and a Bruker Advance 400 MHz NMR spec-
trometer using TMS as internal reference. Chemical shifts (d) values
are given in parts per million (ppm) relative to CDCl3 (7.29 for
proton and 76.9 for carbon) or DMSO-d6 (2.50 for proton and 39.50
for carbon) and coupling constants (J) in Hertz. Splitting patterns
are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet;
dd, doublet of doublet; m, multiplet. Accurate masses were ob-
tained on a Bruker Daltonics BioTOF Mass Spectrometer, in high
resolution mode using cesium acetate solution (1 mM) in methanol
as internal standard in a positive ion mode with flow injection
4. Experimental
4.1. Chemistry
electron spray ionization (ESI) with a flow rate of 120 mL/h and the
Melting points were determined on Stuart electrothermal
melting point apparatus and were uncorrected. IR spectra were
EI-MS was carried out using Jeol JMS 600 spectrometers. High
resolution mass spectra (HRMS) were obtained on a JEOL JMS D-
300 mass spectrometer. The progress of reactions and the purity of
the prepared compounds were monitored by thin-layer chroma-
tography (TLC) using Merck 9385 pre-coated aluminum plate silica
gel (Kieselgel 60) 5 ꢁ 20 cm plates with a layer thickness of 0.2 mm.
Table 3
In vitro COX-1 and COX-2 enzyme inhibition data for compounds 4b, 5e, 5f, 5i and 6
compared to NS-398 and indomethacin.
Compound
R
COX-1 IC50
(
m
M)a COX-2 IC50 M)a SIb
(m
The spots were detected by exposure to UV-lamp at
l
¼ 254 nm.
4b
5e
5f
5i
CH3
4-Br-C6H4
4-Cl-C6H4
3,4-diOCH3-C6H4 >100
NH2 39.8
45.9
>100
>100
68.2
>100
>100
>100
46.3
0.67
ndc
Elemental analysis was performed on Vario El Elementar CHN
Elemental analyzer, organic microanalysis section, national
research center, Giza, Egypt and the results were within ꢂ0.4% of
the theoretical values.
ndc
ndc
6
0.86
101.52
0.03
NS-398
Indomethacin
213.2
0.6
2.1
18.5
4.1.1. Synthesis of 4-[(4-methoxyphenyl)hydrazono]-2-(3,4,5-
trimethoxyphenyl)-4H-oxazol-5-one 3
Trimethoxyhippuric acid 1 (0.13 mol, 35.00 g) in acetic anhy-
dride (75 mL) was heated until a clear solution of 2 was obtained;
this solution was cooled to room temperature (solution A). To a cold
solution of p-methoxyaniline (0.1 mol, 12.31 g) in 5N HCl (35 mL) in
The result (IC50, mM) is the mean of two determinations acquired using the COX
Inhibitor Screening Assay Kit (Catalog No. 560131, Cayman Chemicals Inc., Ann
Arbor, MI, USA).
a
The in vitro test compound concentration required to produce 50% inhibition of
enzymatic activity.
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
Not determined.
c

M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 77 (2014) 155e165
161
(0.18 mol, 15 g). The reaction mixture was stirred at 0e10 ꢃC for 2 h,
the formed precipitate was filtered off and dried (yield 70%).
4.1.2. General procedure for the synthesis of 1-(4-methoxyphenyl)-
5-(3,4,5-trimethoxy-phenyl)-1H-1,2,4-triazole-3-carboxamides
(4aec)
A suspension of compound 3 (0.01 mol, 3.85 g) in methanol
(30 mL) was treated with aqueous solution of ammonium hy-
droxide (25%, 50 mL) or methyl amine solution 41% (0.02 mol,
0.62 g) or ethyl amine (0.02 mol, 0.90 g) was refluxed for 30 min.
The solvent was evaporated in vacuo. The formed precipitate solid
was crystallized from ethanol.
4.1.2.1. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid amide 4a. Pale yellow crystals (2.53 g;
65.90% yield); mp 180 ꢃC; IR (KBr, cmꢀ1): 3329 (NH2), 1669 (C]O)
1588 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm): 3.69 (s, 6H,
2OCH3), 3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.65 (br.s, 2H,
CONH2), 6.78 (s, 2H, AreH), 6.98 (d, 2H, J ¼ 9.00 Hz, AreH), 7.35 (d,
2H, J ¼ 9.00 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d (ppm): 57.68
(CH3), 58.04 (2CH3), 62.85 (CH3), 107.74 (CH), 115.88 (CH), 122.81
(C), 128.36 (CH), 131.79 (C), 140.85 (C), 153.91 (C), 155.67 (C), 156.18
(C), 161.08 (C), 162.00 (CO); EI-MS (70 eV) m/z (%): 384 (100) (Mþ),
372 (13), 371 (31), 340 (54), 339 (30), 324 (30); Anal. Calcd for
C19H20N4O5 (384.14): C, 59.37; H, 5.24; N, 14.58. Found: C, 59.40; H,
5.22; N, 14.33.
4.1.2.2. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid methylamide 4b. Yellowish brown pow-
der (2.82 g; 70.85% yield); mp 175 ꢃC; IR (KBr, cmꢀ1): 3359 (NH),
1676 (C]O) 1584 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm): 3.04
(d, 3H, J ¼ 5.1 Hz, NHeCH3), 3.66 (s, 6H, 2OCH3), 3.83 (s, 3H, OCH3),
3.84 (s, 3H, OCH3), 6.73 (s, 2H, AreH), 6.95 (d, 2H, J ¼ 9.00 Hz, Are
H), 7.31 (d, 2H, J ¼ 9.00 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d
(ppm): 28.46 (CH3), 57.65 (CH3), 57.66 (CH3), 57.98 (CH3), 62.82
(CH3), 107.63 (CH), 115.81 (CH), 122.99 (C), 128.37 (CH), 131.85 (C),
140.74 (C), 153.86 (C), 155.34 (C), 156.75 (C), 160.50 (C), 161.00 (C]
O); EI-MS (70 eV) m/z (%): 398 (17) (Mþ), 388 (40), 386 (100), 370
(15), 367 (14), 340 (33); Anal. Calcd for C20H22N4O5 (398.16): C,
60.29; H, 5.57; N, 14.06. Found: C, 59.90; H, 5.60; N, 13.80.
4.1.2.3. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid ethylamide 4c. Brown powder (2.27 g;
55.10% yield); mp 155 ꢃC; IR (KBr, cmꢀ1): 3350 (NH), 1675 (CO),
1586 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm): 1.29 (t, 3H,
J ¼ 7.50 Hz, CH3), 3.56 (p, 2H, CH2), 3.69 (s, 6H, 2OCH3), 3.85 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 6.75 (s, 2H, AreH), 6.97 (d, 2H,
J ¼ 8.50 Hz, A-H), 7.20 (t, 1H, J ¼ 8.00 Hz, NH), 7.33 (d, 2H,
J ¼ 9.00 Hz, ArH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.25
(CH3), 34.04 (CH2), 56.09 (CH3), 56.16 (CH3), 60.59 (CH3), 106.70
(CH), 115.08 (CH), 122.50 (C), 128.32 (CH), 131.00 (C), 139.36 (C),
153.07 (C), 154.68 (C), 156.53 (C), 158.98 (C), 160.42 (C]O); HRMS
(FAB) m/z Calcd for C21H25N4O5 (Mþ þ 1): 413.1825; found:
413.1846; Anal. Calcd for C21H24N4O5 (412.17): C, 61.15; H, 5.87; N,
13.58. Found: C, 61.00; H, 5.70; N, 13.40.
Fig. 4. A. The orientation of compounds 4b, 6 and SC-558 (shown as blue, yellow and
purple respectively) in COX-2 active site. B. The orientation of 5e, 5f, 5i and SC-558
(shown as yellow, blue, green and purple respectively) in COX-2 active site. C. The
orientation of 4b and 6 (shown as blue and yellow respectively) in COX-1 active site.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
4.1.3. General procedure for the synthesis of 1-(4-methoxyphenyl)-
5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxyanilides
(5ael)
A mixture of compound 3 (3.85 g, 0.01 mol) and appropriate
primary aromatic amine (0.01 mol) was refluxed in acetic acid
(50 mL) in the presence of anhydrous sodium acetate (1.5 g,
0.018 mol) for 2 h. The reaction mixture was cooled and poured into
ice water (50 mL). The formed precipitate was filtered off, dried and
recrystallized from aqueous methanol.
an ice-salt bath ꢀ5 to 0 ꢃC, a solution of sodium nitrite (0.13 mol,
8.97 g) in water (15 mL) was added in a dropwise manner. The
reaction mixture was left for 10 min (solution B). Solution A was
added to solution B in presence of anhydrous sodium acetate

162
M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 77 (2014) 155e165
4.1.3.1. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid (4-methoxyphenyl)amide 5a. Dark brown
crystals (2.40 g, 48.98% yield); mp 190 ꢃC; IR (KBr, cmꢀ1): 3380
4.1.3.5. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid (4-bromophenyl)amide 5e. Brown powder
(3.18 g, 59.10% yield); mp 135 ꢃC; IR (KBr, cmꢀ1): 3310 (NH), 1692
(NH), 1686 (CO), 1588 (C]N); 1H NMR (300 MHz, CDCl3)
d
(ppm):
(CO), 1589 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm): 3.74 (s, 6H,
3.75 (s, 6H, 2OCH3), 3.86 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.92 (s,
3H, OCH3), 6.83 (s, 2H, AreH), 6.94e7.04 (m, 4H, AreH), 7.41 (d, 2H,
J ¼ 9.00 Hz, AreH), 7.71 (d, 2H, J ¼ 9.00 Hz, AreH), 9.03 (s, 1H, NH);
2OCH3), 3.91 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.82 (s, 2H, AreH),
7.02 (d, 2H, J ¼ 7.20 Hz, AreH), 7.39 (d, 2H, J ¼ 7.20 Hz, AreH), 7.51
(d, 2H, J ¼ 8.10 Hz, AreH), 7.72 (d, 2H, J ¼ 8.40 Hz, AreH), 9.17 (s, 1H,
13C NMR (75 MHz, CDCl3)
d
(ppm): 57.40 (CH3), 58.10 (CH3), 62.90
NH); 13C NMR (75 MHz, CDCl3)
d (ppm): 57.75 (CH3), 58.14 (CH3),
(CH3), 107.78 (CH), 115.56 (CH), 115.91 (CH), 122.80 (CH), 128.41
(CH), 131.72 (C), 131.81 (C), 141.50 (C), 154.01 (C), 156.50 (C), 157.10
(C), 157.41 (C), 161.13 (C]O); EI-MS (70 eV) m/z (%): 490 (15) (Mþ),
451 (14), 444 (20), 422 (15), 421 (29), 403 (31), 389 (100), 374 (16);
Anal. Calcd for C26H26N4O6 (490.19): C, 63.66; H, 5.34; N, 11.42.
Found: C, 63.54; H, 5.60; N, 11.20.
62.92 (CH3), 107.83 (CH), 115.62 (C), 115.97 (CH), 118.55 (C), 122.70
(CH), 128.41 (CH), 131.65 (C), 133.15 (CH), 137.55 (C), 141.11 (C),
154.03 (C), 155.64 (C), 157.31 (C), 157.42 (C), 161.22 (C]O); EI-MS
(70 eV) m/z (%): 540 (10) (Mþ þ 2), 538 (9) (Mþ), 403 (96), 387
(100); Anal. Calcd for C25H23BrN4O5 (538.09): C, 55.67; H, 4.30; N,
10.39. Found: C, 55.40; H, 4.20; N, 10.00.
4.1.3.6. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid (4-chlorophenyl)amide 5f. Dark brown
powder (3.06 g, 61.94% yield); mp 120e121 ꢃC; IR (KBr, cmꢀ1): 3360
(NH), 1691 (CO), 1588 (C]N); 1H NMR (300 MHz, DMSO-d6)
4.1.3.2. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid (2-methoxyphenyl)amide 5b. Golden yel-
low crystals (2.69 g, 54.90% yield); mp 131e132 ꢃC; IR (KBr, cmꢀ1):
3381 (NH), 1696 (CO), 1588 (C]N); 1H NMR (300 MHz, CDCl3)
d
(ppm): 3.75 (s, 6H, 2OCH3), 3.91 (s, 3H, OCH3), 3.92 (s, 3H, OCH3),
d
(ppm): 3.75 (s, 6H, 2OCH3), 3.91 (s, 3H, OCH3), 3.92 (s, 3H, OCH3),
6.84 (s, 2H, AreH), 7.02 (d, 2H, J ¼ 8.70 Hz, AreH), 7.39e7.45 (m, 4H,
3.98 (s, 3H, OCH3), 6.84 (s, 2H, AreH), 6.97e7.17 (m, 5H, AreH), 7.41
(d, 2H, J ¼ 9.00 Hz, AreH), 8.63 (dd, 1H, J ¼ 7.80 and 1.80 Hz, AreH),
AreH), 7.81 (d, 2H, J ¼ 7.80 Hz, AreH), 9.20 (s, 1H, NH); 13C NMR
9.62 (s, 1H, NH); 13C NMR (75 MHz, CDCl3)
d (ppm): 57.71 (CH3),
(75 MHz, DMSO-d6)
d (ppm): 57.73 (CH3), 58.12 (CH3), 62.91 (CH3),
107.84 (CH), 115.95 (CH), 121.20 (CH), 122.47 (C), 125.85 (C), 128.42
(C),130.21 (CH),131.68 (CH),138.45 (C),141.11 (C),154.03 (C),155.50
(C), 156.44 (C), 157.19 (C), 161.20 (C]O); ESI-MS: m/z (%) 495 (100)
(Mþ þ 1). Anal. Calcd for C25H23ClN4O5 (494.14): C, 60.67; H, 4.68;
N, 11.32. Found: C, 60.40; H, 4.40; N, 11.10.
57.83 (CH3), 58.08 (CH3), 62.89 (CH3), 107.87 (CH), 111.38 (CH),
115.86 (CH), 121.51 (CH), 122.37 (CH), 123.14 (C), 125.40 (C), 128.39
(CH), 128.48 (CH), 131.97 (C), 140.85 (C), 149.23 (C), 153.94 (C),
155.69 (C), 157.23 (C), 157.50 (C), 161.07 (C]O); EI-MS (70 eV) m/z
(%): 490 (12) (Mþ), 403 (67), 388 (22), 387 (100), 371 (13), 339 (12);
Anal. Calcd for C26H26N4O6 (490.19): C, 63.66; H, 5.34; N, 11.42.
Found: C, 63.50; H, 5.20; N, 11.10.
4.1.3.7. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid (3-fluorophenyl)amide 5g. Pale brown
powder (2.96 g, 61.92% yield); mp 126 ꢃC; IR (KBr, cmꢀ1): 3310
(NH), 1693 (CO), 1588 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm):
4.1.3.3. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid p-tolylamide 5c. Pale brown powder
(2.60 g, 54.85% yield); mp 167 ꢃC; IR (KBr, cmꢀ1): 3351 (NH), 1691
3.74 (s, 6H, 2OCH3), 3.90 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.81 (s,
2H, AreH), 7.01 (d, 2H, J ¼ 8.70 Hz, AreH), 7.10 (t, 2H, J ¼ 8.70 Hz,
AreH), 7.39 (d, 2H, J ¼ 9.00 Hz, AreH), 7.74e7.79 (m, 2H, AreH),
(CO), 18589 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm): 2.39 (s, 3H,
9.14 (s, 1H, NH); 13C NMR (75 MHz, CDCl3)
d (ppm): 57.71 (CH3),
CH3), 3.74 (s, 6H, 2OCH3), 3.90 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.82
(s, 2H, AreH), 7.01 (d, 2H, J ¼ 8.70 Hz, AreH), 7.21 (d, 2H, J ¼ 8.10 Hz,
AreH), 7.40 (d, 2H, J ¼ 8.70 Hz, AreH), 7.68 (d, 2H, J ¼ 8.10 Hz, Are
58.08 (CH3), 62.89 (CH3), 107.76 (CH), 115.92 (CH), 116.90 (C), 117.19
(C), 122.82 (CH), 122.92 (CH), 128.37 (CH), 131.77 (C), 134.53 (C),
140.98 (C), 154.01 (C), 155.71 (C), 156.68 (C), 157.50 (C), 161.15 (CO),
161.88 (C); EI-MS (70 eV) m/z (%): 480 (5) (Mþ þ 2), 478 (10) (Mþ),
405 (10), 386 (87), 371 (100), 352 (24), 332 (22), 325 (41), 275 (18);
Anal. Calcd for C25H23FN4O5 (478.17): C, 62.76; H, 4.85; N, 11.71.
Found: C, 62.50; H, 5.20; N, 11.30.
H), 9.01 (s, 1H, NH); 13C NMR (75 MHz, CDCl3)
d (ppm): 23.37 (CH3),
57.71 (CH3), 58.09 (CH3), 62.89 (CH3), 107.77 (CH), 115.89 (CH),
121.15 (CH), 122.95 (C), 128.40 (CH), 130.69 (CH), 131.86 (C), 135.31
(C), 135.96 (C), 140.91 (C), 153.99 (C), 155.62 (C), 156.92 (C), 157.39
(C), 161.09 (C]O); EI-MS (70 eV) m/z (%): 474 (16) (Mþ), 469 (16),
450 (10), 424 (21), 421 (49), 390 (35), 389 (74), 375 (34), 324 (65);
Anal. Calcd for C26H26N4O5 (474.19): C, 65.81; H, 5.52; N, 11.81.
Found: C, 65.50; H, 5.20; N, 11.60.
4.1.3.8. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid (4-trifluoromethylphenyl)amide 5h.
Yellowish brown crystals (2.42 g, 45.83% yield); mp 151 ꢃC; IR (KBr,
cmꢀ1): 3350 (NH), 1696 (CO), 1588 (C]N); 1H NMR (300 MHz,
4.1.3.4. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid o-tolylamide 5d. Yellowish brown powder
(2.80 g, 59.10% yield); mp 173 ꢃC; IR (KBr, cmꢀ1): 3386 (NH), 1700
CDCl3) d (ppm): 3.75 (s, 6H, 2OCH3), 3.91 (s, 3H, OCH3), 3.93 (s, 3H,
OCH3), 6.82 (s, 2H, AreH), 7.03 (d, 2H, J ¼ 8.70 Hz, AreH), 7.39e7.58
(CO), 1588 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm): 2.40 (s, 3H,
(m, 4H, AreH), 8.00 (s,1H, AreH), 8.10 (d, 1H, J ¼ 8.40 Hz, AreH),
CH3), 3.70 (s, 6H, 2OCH3), 3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.79
(s, 2H, AreH), 6.99 (d, 2H, J ¼ 8.50 Hz, AreH), 7.12 (t, 1H, J ¼ 7.50 Hz,
AreH), 7.23e7.30 (m, 2H, AreH), 7.37 (d, 2H, J ¼ 9.00 Hz, AreH),
8.13 (d, 1H, J ¼ 8.00 Hz, AreH), 8.95 (s, 1H, NH); 13C NMR (100 MHz,
9.19 (s, 1H, NH); 13C NMR (75 MHz, CDCl3)
d (ppm): 57.72 (CH3),
58.09 (CH3), 62.90 (CH3), 107.77 (CH), 115.94 (CH), 117.78 (C), 122.79
(CH), 124.20 (CH), 128.37 (CH), 130.86 (C), 131.75 (C), 138.97 (C),
141.01 (C), 154.02 (C), 155.84 (C), 156.46 (C), 157.74 (C),161.19 (C]
O); EI-MS (70 eV) m/z (%): 528 (14) (Mþ), 403 (42), 387 (40), 370
(12), 300 (16); Anal. Calcd for C26H23F3N4O5 (528.16): C, 59.09; H,
4.39; N, 10.60. Found: C, 58.80; H, 4.10; N, 10.30.
DMSO-d6)
d (ppm): 20.98 (CH3), 56.13 (CH3), 56.21 (CH3), 60.62
(CH3), 106.83 (CH), 115.12 (CH), 121.09 (CH), 122.39 (C), 128.41 (CH),
129.52 (CH), 130.91 (C), 133.63 (C), 136.20 (C), 139.45 (C), 153.10 (C),
154.99 (C), 156.47 (C), 157.66 (C), 160.51 (C]O); EI-MS (70 eV) m/z
(%): 474 (12) (Mþ), 469 (18), 450 (20), 424 (18), 421 (49), 390 (40),
389 (80), 375 (34), 324 (55); HRMS (FAB) m/z Calcd for C26H27N4O5
(Mþ þ 1): 475.1981; found: 475.1972; Anal. Calcd for C26H26N4O5
(474.19): C, 65.81; H, 5.52; N, 11.81. Found: C, 65.70; H, 5.20; N,
11.80.
4.1.3.9. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid (3,4-dimethoxyphenyl)amide 5i. Pale
brown powder (2.91 g, 55.96% yield); mp 175 ꢃC; IR (KBr, cmꢀ1):
3320 (NH), 1686 (CO), 1588 (C]N); 1H NMR (300 MHz, CDCl3)
d
(ppm): 3.75 (s, 6H, 2OCH3), 3.90 (s, 3H, OCH3), 3.91 (s, 3H, OCH3),

M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 77 (2014) 155e165
163
3.92 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 6.83 (s, 2H, AreH), 6.89 (d,1H,
J ¼ 8.70 Hz, AreH), 7.02 (d, 2 H, J ¼ 8.70 Hz, AreH), 7.22 (dd, 1H,
J ¼ 6.90 and 1.80 Hz, AreH), 7.40 (d, 2H, J ¼ 9.00 Hz, AreH), 7.60 (d,
1H, J ¼ 2.40 Hz, AreH), 9.08 (s, 1H, NH); 13C NMR (75 MHz, CDCl3)
solvent was evaporated and the obtained product was recrystal-
lized from ethanol give compound 6 as pale yellow powder (7.78 g,
65.00% yield); mp 210 ꢃC; IR (KBr, cmꢀ1): 3380 and 3150 (NH and
NH2), 1661 (CO), 1590 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm):
d
(ppm): 57.71 (CH3), 58.04 (CH3), 58.10 (CH3), 61.90 (CH3), 62.89
4.01 (s, 6H, 2OCH3), 4.02 (s, 6H, 2OCH3), 7.09 (d, 2H, J ¼ 9.00 Hz, Are
(CH3), 106.21 (CH), 107.80 (CH), 112.73 (CH), 113.25 (CH), 115.93
(CH), 122.62 (C), 128.37 (CH), 131.74 (C), 132.17 (C), 141.05 (C), 147.03
(C), 149.98 (C), 154.02 (C), 155.50 (C), 156.61 (C), 157.14 (C), 161.16
(C]O); EI-MS (70 eV) m/z (%): 520 (14) (Mþ), 403 (100), 387 (91),
371 (14); Anal. Calcd for C27H28N4O7 (520.20): C, 62.30; H, 5.42; N,
10.76. Found: C, 62.10; H, 5.40; N, 10.60.
H), 7.54 (s, 2H, AreH), 8.18 (d, 2H, J ¼ 9.00 Hz, AreH); 13C NMR
(75 MHz, CDCl3)
d (ppm): 58.31 (CH3), 58.49 (CH3), 63.01 (CH3),
107.10 (CH), 115.89 (CH), 119.86 (C), 127.94 (CH), 143.52 (C), 148.14
(C), 154.44 (C), 165.28 (C), 176.07 (C), 177.28 (C); ESI-MS: m/z (%)
400 (100) [Mþ þ 1]; Anal. Calcd for C19H21N5O5 (399.15): C, 57.14; H,
5.30; N, 17.53. Found: C, 56.80; H, 5.21; N, 17.38.
4.1.3.10. 4-{[1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-
[1,2,4]triazole-3-carbonyl]amino}benzoic acid 5j. Brown powder
(2.72 g, 53.97% yield); mp 259 ꢃC; IR (KBr, cmꢀ1): 3301 (OH), 1696
4.2. Biology
4.2.1. Screening of the anti-inflammatory activity
(CO), 1593 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm): 3.70 (s, 6H,
The experiments were performed on adult male albino rats,
weighing (120e140 g), obtained from the animal house, Minia
University. The animals were housed in stainless steel cages,
divided into groups of four animals each and deprived of food but
not water 24 h before the experiment. The anti-inflammatory ac-
tivity of the compounds under investigation was studied using
carrageenan. A suspension of the tested compounds 4aec, 5ael
and 6, celecoxib and indomethacin in carboxy methyl cellulose
(CMC) solution (0.5% w/v in water) was administered orally in a
dose level of (0.28 mmol/kg). Control animals were similarly
treated with CMC solution (0.5% w/v in water). After 30 min, 0.1 mL
of freshly prepared 1% carrageenan solution in normal saline was
injected into the subplantar region of the right hind paw of rats
according to the method of Winter et al. An equal volume of saline
was injected into the left hind paw of each rat. The right paw
thickness was measured by a Vernier caliper (SMIEC) directly
before and after 1, 2, 3, 4 and 5 h intervals after carrageenan in-
jection. The anti-inflammatory activity of the tested compounds,
celecoxib and indomethacin was calculated as the percentage
decrease in edema thickness induced by carrageenan.
2OCH3), 3.86 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.78 (s, 2H, AreH),
6.86 (d, 2H, J ¼ 9.00 Hz, AreH), 6.99 (d, 2H, J ¼ 9.00 Hz, AreH), 7.37
(d, 2H, J ¼ 9.50 Hz, AreH), 7.62 (d, 2H, J ¼ 9.00 Hz, AreH), 8.90 (s,
1H, NH), 10.22 (s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6)
d
(ppm): 56.13 (CH3), 56.22 (CH3), 60.63 (CH3), 105.76 (CH), 106.86
(CH), 115.13 (CH), 120.36 (CH), 122.29 (C), 126.50 (C), 128.43 (C),
130.67 (CH), 130.84 (CH), 139.50 (C), 142.81 (C), 153.11 (C), 153.93
(C), 155.19 (C), 156.12 (C), 158.12 (C), 160.56 (C]O), 167.42 (C]O);
HRMS (FAB) m/z Calcd for C26H25N4O7 (Mþ þ 1): 505.1723; found:
505.1751; Anal. Calcd for C26H24N4O7 (504.16): C, 61.90; H, 4.80; N,
11.11. Found: C, 61.70; H, 5.20; N, 11.00.
4.1.3.11. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4]
triazole-3-carboxylic acid (4-hydroxyphenyl)amide 5k. Black pow-
der (2.62 g, 55.04% yield); mp 205 ꢃC; IR (KBr, cmꢀ1): 3316 (OH),
1683 (CO), 1588 (C]N); 1H NMR (300 MHz, CDCl3)
d (ppm): 3.69 (s,
6H, OCH3), 3.85 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.76 (s, 2H, AreH),
6.98 ( d, 2H, J ¼ 9.00 Hz, AreH), 7.36 (d, 2H, J ¼ 8.70 Hz, AreH), 7.88
(d, 2H, J ¼ 8.40 Hz, AreH), 8.13 (d, 2H, J ¼ 8.70 Hz, AreH), 9.19 (s,1H,
NH),10.33 (s,1H, OH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 56.12
(CH3), 56.21 (CH3), 60.62 (CH3), 106.83 (CH), 115.12 (CH), 121.13
(CH), 122.37 (C), 124.61 (C), 126.09 (C), 128.42 (CH), 129.13 (CH),
130.89 (C), 138.71 (C), 139.47 (C), 153.10 (C), 155.05 (C), 156.39 (C),
157.83 (C), 160.52 (C]O); HRMS (FAB) m/z Calcd for C25H25N4O6
(Mþ þ 1): 477.1774; found: 477.1760. Anal. Calcd for C25H24N4O6
(476.17): C, 63.02; H, 5.08; N,11.76. Found: C, 62.90; H, 5.10; N,11.70.
4.2.2. Screening of ulcerogenicity
After measuring the anti-inflammatory activity the rats were
sacrificed by decapitation. The stomachs were removed, collected,
opened along the greater curvature, washed with distilled water
and cleaned gently by dipping in saline. The mucosal damage for
each stomach was examined with a magnifying lens for the pres-
ence of macroscopically visible lesions. The number of lesions in
each stomach, if any, was counted and recorded. Ulcers were clas-
sified into levels, level I, in which the ulcer area is less than 1 mm2,
level II, in which ulcer area is in the range from 1 to 3 mm2 and level
III, in which the ulcer area more than 3 mm2 and this rated ac-
cording to their areas in mm2.
4.1.3.12. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-
[1,2,4]triazole-3-carboxylic acid phenylamide 5l. Brown powder
(1.61 g, 35.00% yield); mp 135 ꢃC; IR (KBr, cmꢀ1): 3312 (NH), 1691
(CO), 1593 (C]N); 1H NMR (300 MHz, DMSO-d6)
d (ppm): 3.65 (s,
6H, 2OCH3), 3.72 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 6.84 (s, 2H, Are
H), 7.12e7.17 (m, 3H, AreH), 7.37e7.56 (m, 4H, AreH), 7.86 (d, 2H,
J ¼ 8.10 Hz, AreH), 10.50 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6)
The data are expressed as mean ꢂ S.E.M, one way ANOVA test
was applied to determine the significance of the difference be-
tween the control group and rats treated with the tested
compounds.
d
(ppm): 57.73 (2CH3), 62.07 (2CH3), 107.76 (CH), 115.91 (CH), 121.83
(CH), 123.32 (C), 125.29 (C), 129.01 (CH), 129.73 (CH), 131.47 (CH),
139.16 (C), 140.01 (C),153.42 (C), 155.33 (C), 156.65 (C), 158.08 (C),
160.75 (C]O); ESI-MS: m/z (%) 461 (100) (Mþ þ 1); Anal. Calcd for
C
25H24N4O5 (460.17): C, 65.21; H, 5.25; N, 12.17. Found: C, 65.00; H,
4.2.3. Histopathological investigation
5.20; N, 12.10.
The histological slides were prepared according to the reported
procedures for examination of ulcers under light microscope [48].
Identify site of the slide on which the section was applied by
scratching wax around section with a needle. Dewax hydrated
sections by using graded alcohols to water. Slides were stained with
haematoxylin for 5e7 min, washed with tap water until sectioning
for 5 min and immersed for 5e10 s in solution of (1% HCl in 70%
alcohol), then washed well with tap water for 10e15 min followed
by staining with 1% Eosin for 10 min, and washed with running tap
water for 1e5 min. The slide was then dehydrated using alcohols,
4.1.4. General procedure for the synthesis of 1-(4-methoxyphenyl)-
5-(3,4,5-trimethoxy-phenyl)-1H-[1,2,4]triazole-3-carboxylic acid
hydrazide 6
To a stirred suspension of compound 3 (11.55 g, 0.03 mol) in
methanol (100 mL), hydrazine hydrate 95% (4.5 g, 0.09 mol) was
added gradually, stirring was continued overnight at 25 ꢃC, solvent
was evaporated and the obtained oily product was then purified
using silica gel column eluted with chloroform/methanol (9.5:0.5),

164
M. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 77 (2014) 155e165
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then examined under microscope.
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4.4. Docking studies
Molecular modeling studies were performed with MOE (The
Molecular Operating Environment) Version 2011.10, software
available from Chemical Computing Group Inc., 1010 Sherbrooke
Street West, Suite 910, Montreal, Canada H3A2R7, http://www.
chemcomp.com. The ligands were built using the builder tool of
the MOE program and subjected to energy minimization
(MMFF94x, gradient: 0.05). The X-ray crystallographic structures of
COX-1 (PDB: 3KK6) [50] and COX-2 (PDB: 1CX2) [51] were obtained
from the Protein Data Bank. The errors of the protein were cor-
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hydrogens were added and partial charges (Gasteiger methodol-
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method was used for docking. GBVI/WSA dG scoring function
which estimates the free energy of binding of the ligand from a
given pose was used to rank the final poses. The ligandeenzyme
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.001.
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