New nitric oxide donating 1,2,4-triazole/oxime hybrids: Synthesis, investigation of anti-inflammatory, ulceroginic liability and antiproliferative activities

Article abstract of DOI:10.1016/j.bmc.2013.04.022

A series of novel nitric oxide (NO) donating triazole/oxime hybrids was prepared and evaluated for their anti-inflammatory activity. Most of the tested compounds showed significant anti-inflammatory activity using carrageenan-induced rat paw edema method compared to indomethacin. Calculation of the ulcer indices and histopathological investigation indicated that the prepared NO-donating oximes exhibited less ulcerogenicity compared to their intermediate ketones and indomethacin. The NO-donating oxime 6i revealed significant activity against renal cancer A498 cell lines with 50.52 cell growth inhibition.

Full text of DOI:10.1016/j.bmc.2013.04.022

Bioorganic & Medicinal Chemistry 21 (2013) 3839–3849
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New nitric oxide donating 1,2,4-triazole/oxime hybrids: Synthesis,
investigation of anti-inflammatory, ulceroginic liability and anti-
proliferative activities
⇑
Mohamed Abdel-Aziz, Gamal El-Din A. A. Abuo-Rahma , Eman A. M. Beshr, Taha F. S. Ali
Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt.
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 nitric oxide (NO) donating triazole/oxime hybrids was prepared and evaluated for their
anti-inflammatory activity. Most of the tested compounds showed significant anti-inflammatory activity
using carrageenan-induced rat paw edema method compared to indomethacin. Calculation of the ulcer
indices and histopathological investigation indicated that the prepared NO-donating oximes exhibited
less ulcerogenicity compared to their intermediate ketones and indomethacin. The NO-donating oxime
6i revealed significant activity against renal cancer A498 cell lines with 50.52 cell growth inhibition.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 29 January 2013
Revised 30 March 2013
Accepted 8 April 2013
Available online 18 April 2013
Keywords:
1,2,4-Triazole
Nitric oxide donors
Anti-inflammatory
Gastric ulceration
Antiproliferative
1. Introduction
repair.13 Also, the vasodilatation effect of NO is known to spare
the renal system through increasing the mucosal blood flow.14
Conventional NSAIDs are the most commonly prescribed medi-
cations all over the world. Although the free carboxylic group in
most of these compounds is critical for their anti-inflammatory
activity,1 it is responsible for the potential unwanted gastrointesti-
nal discomfort associated with these compounds.2 Modification of
the carboxyl function by the less ulcerogenic bioisosters such as
triazoles and oxadiazoles may help to minimize the gastrointesti-
nal upset.3 Amir et al. reported that cyclization of carboxyl group
of diclofenac into the 1,2,4-triazole analogues (Fig. 1) increases
both the anti-inflammatory and the analgesic activities with reduc-
tion of ulcerogenic liability compared to the parent diclofenac.4
One of the most important strategies used to overcome NSAIDs
side effects is designing nitric oxide-donating NSAIDs (NO-NSA-
IDs), which are capable of generating the radical biomediator and
gastro protective NO.5,6 It was reported that NO plays several phys-
iological functions in the digestive system7 such as; increasing the
mucosal blood flow8 which results in enhancement of the mucosal
resistance to ulceration,9 preventing adherence of leukocytes to
the vascular endothelium10 and modulating gastroduodenal secre-
tion of both mucus11 and bicarbonate.12 Moreover, NO can pro-
foundly influences the mucosal immune system7 and increases
the ability of ulcerated mucosal cells to undergo healing and
On the other hand, NO and reactive oxygen species exert multiple
modulating effects on inflammation and play a key role in the reg-
ulation of immune responses.15,16
Additionally, NO can prevent tumor cells from metastasizing and
assists macrophage to kill tumor cells.17 Several targets have been
reported for the combination between NO and cancer therapy
including; synergistic effect,18,19 increasing the influx of the anti-
cancer therapy,20 increasing the efficiency of cytostatic therapy
and retardation of drug resistance to anticancer agents.21
1,2,4-Triazole derivatives represent an interesting class of het-
erocyclic compounds, they possess many biological activities such
as antimicrobial,22,23 anti-tubercular,24 anti-inflammatory,3,4,25–27
analgesic4 and anticancer28–32 activities. Additionally, it was re-
ported that alkylthio-3-(3,4-dimethoxyphenyl)-4H-1,2,4-triazole
derivatives exhibits high anti-inflammatory activity with low acute
toxicity.33
NO-NSAIDs are considered promising anticancer agents, in vitro
and in vivo studies indicated that NCX 4040 (Fig. 1) shows a prom-
ising anticancer activity, compared to its parent aspirin.34 More-
over, the NO–profen hybrid (Fig. 1) exhibits significant anti-
proliferative activity against PC-3 cells. Additionally, several re-
ports indicated that oximation of the carbonyl group in some com-
pounds enhances the anticancer activity several folds compared to
their corresponding ketones.35,36
Promoted with the above-mentioned studies and as a continu-
ation of our research interest in the synthesis and biological
⇑
Corresponding author. Tel.: +20 1003069431; fax: +20 862369075.
E-mail address: gamalaburahma@yahoo.com (G.E.-D.A.A. Abuo-Rahma).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.022

3840
M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
CH3
HS
N
N
Cl
N
H
N
Cl
1,2,4-triazole analogues of diclofenac.
O
O
O
CH3
ONO2
O2NO
O
O
O
O
ONO2
CH3
NCX 4040
Ketoprofen-NO hybrid
Figure 1. The structure of 1,2,4-triazole analogues of diclofenac, NCX 4040 and ketoprofen–NO hybrids.
evaluation of NO-NSAIDs derivatives.37–40 The aim of the present
study is gathering the two bioactive entities, the less acidic 1,2,4-
triazole-3-thiol and oxime as a NO donor in one compact structure
for the purpose of synergism and/or minimizing the expected
ulcerogenic side effects. The prepared triazole/NO hybrids are eval-
uated for their anti-inflammatory activity using carrageenan-in-
duced rat paw edema and compared to the well-known NSAID,
indomethacin. Calculation of ulcer indices and histopathological
investigation were carried out to assess the beneficial effects of
the NO in decreasing ulcer formation. The prepared triazole/NO hy-
brids were also evaluated for their antiproliferative activity using
different cancer cell lines.
2.2. Measurement of nitric oxide release
The NO releasing properties of the prepared NO-donating
oximes 6a–l were assessed. The produced nitrite which is a conve-
nient index of nitric oxide production trend was determined in
both phosphate buffer of pH 7.4 and 0.1 M HCl buffer of pH 1 by
using Griess colorimetric method. The reaction was carried out in
the presence of N-acetylcysteine as a source of the SH group. The
amount of NO released from the tested compounds, was measured
relative to NO released from standard sodium nitrite solution and
calculated as amount of NO released (mol/mol) and listed in Ta-
ble 1. The results of measurement of NO release revealed that
the NO-donating oximes 6a–l release NO at pH of 7.4 after 5 h.
Compound 6g that contains 3,4-dimethoxyphenyl moiety released
the highest amount of NO among this group (0.25 mol/mol). The
results also indicated that NO-donating compounds were not able
to release NO at pH 1, which may support the fact that these com-
pounds are weakly hydrolyzed in the gastric lumen and confirms
that the suggested gastroprotective action of NO is mediated
systemically.42
2. Results and discussion
2.1. Chemistry
4-R-5-Aryl-4H-1,2,4-triazole-3-thiol derivatives 4a–l were syn-
thesized as outlined in Scheme IA according to the reported
procedure.24
Coupling of 1,2,4-triazole-3-thiol derivatives 4a–l with phena-
cyl bromide was achieved in acetonitrile in the presence of TEA
afforded the corresponding ketone intermediates 5a–l in 62–85%
yield. Heating at reflux of the ketone intermediates 5a–l with
hydroxylamine HCl in ethanol gave the corresponding oximes
6a–l in 56–93% yields. The chemical structure of the prepared com-
pounds was elucidated on the basis of their IR, 1H NMR, 13C NMR,
mass spectra as well as the elemental analyses.
The data of Table 1 indicated that the released amount of NO
from oximes 6a–l was relatively small compared to the previously
reported data from other NO-donating hybrids37–39 and this may
be attributed to the expected intramolecular hydrogen bonding be-
tween the oxime OH group and the sulfur atom (Fig. 2).
2.3. Biological investigations
A characteristic feature of the 1H NMR spectra for oximes 6a–l is
the appearance of a downfield singlet at d 8.08–11.84 ppm corre-
sponding to the hydroxyl group. The high downfield shifted OH
proton may be attributed to the expected intramolecular hydrogen
bonding with the sulfur atom. The CH2 protons appears upfield
shifted by d 0.28–0.63 ppm compared to the CH2 protons of the
corresponding ketones that may be attributed to the low electro-
negativity of N atom relative to O atom. The 13C NMR spectra of
oximes 6d, 6f, 6h, 6k and 6l showed the disappearance of the
ketonic carbonyl due to its conversion to ketoxime group
(C@N–OH). A characteristic feature of the mass spectra of the oxi-
mes 6a–l is the appearance of a very weak abundance for the
molecular ion peaks from 0.1% to 11.5% of the respective base peak.
Kallury and Rao41 reported that the abundances of some oximes
are very low (less than 4%) of the corresponding base peak.
2.3.1. Screening of anti-inflammatory activity
The synthesized compounds 5a–l and 6a–l were evaluated for
their anti-inflammatory activity using carrageenan-induced paw
edema in rats described by Winter et al.,43 The tested compounds
and the reference drug indomethacin 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 and indomethacin was calculated as the percentage
decrease in edema thickness induced by carrageenan and was
determined using the following formula:
ðVR ꢀ VLÞcontrol ꢀ ðVR ꢀ VLÞtreated
% of edema inhibition ¼
ꢁ 100
ðVR ꢀ VLÞcontrol

M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
3841
O
O
O
EtOH
H2SO4
NH2NH2. H2O
EtOH
RNCS
EtOH
Ar
NH
Ar
OH
Ar
O
NH2
2a-d
1a-d
S
H
N
1) NaOH
2) HCl
N
N
Ar
R
SH
N
H
N
H
N
O
Ar
R
4a-l
3a-l
Compound
1a, 2a
1b, 2b
1c, 2c
1d, 2d
3a, 4a
3b, 4b
3c, 4c
R
---
---
---
---
allyl
allyl
allyl
Ar
Ph
Compound
3e, 4e
3f, 4f
3g, 4g
3h, 4h
3i, 4i
3j, 4j
3k, 4k
3l, 4l
R
Ar
Ph
Et
Et
Et
Et
Ph
Ph
Ph
Ph
4-OCH3-Ph
3,4-di-OCH3-Ph
3,4,5-tri-OCH3-Ph
Ph
4-OCH3-Ph
3,4-di-OCH3-Ph
4-OCH3-Ph
3,4-di-OCH3-Ph
3,4,5-tri-OCH3-Ph
Ph
4-OCH3-Ph
3,4-di-OCH3-Ph
3,4,5-tri-OCH3-Ph
3d, 4d
allyl 3,4,5-tri-OCH3-Ph
Scheme IA. Synthesis of 4-R-5-aryl-4H-1,2,4-triazole-3-thiol derivatives 4a–l. The synthesis of the target compounds 1-phenyl-2-((4-R-5-aryl-4H-1,2,4-triazol-3-
yl)thio)ethanone oxime 6a–l is illustrated in Scheme IB.
N
N
1) Ph-CO-CH2Br
2) TEA, CH3CN
Ar
NH2OH. HCl
EtOH
4a-l
N
S
R
O
5a-l
N
N
Ar
N
S
R
N
HO
6a-l
Compound
5a, 6a
5b, 6b
5c, 6c
5d, 6d
5e, 6e
R
Ar
Ph
Compound
5g, 6g
5h, 6h
5i, 6i
5j, 6j
5k, 6k
5l, 6l
R
Et
Et
Ph
Ph
Ph
Ph
Ar
allyl
allyl
allyl
3,4-Di-OCH3-Ph
3,4,5-Tri-OCH3-Ph
Ph
4-OCH3-Ph
3,4-Di-OCH3-Ph
3,4,5-Tri-OCH3-Ph
4-OCH3-Ph
3,4-Di-OCH3-Ph
allyl 3,4,5-Tri-OCH3-Ph
Et
Et
Ph
5f, 6f
4-OCH3-Ph
Scheme IB. Synthesis of 1-phenyl-2-((4-R-5-aryl-4H-1,2,4-triazol-3-yl)thio)ethanone oxime 6a–l.
where VR represents the mean right paw thickness and VL repre-
sents the mean left paw thickness.
<0.01). Indomethacin showed an inhibitory activity of 83% against
carrageenan-induced paw edema after 4 h, which is the time re-
quired to reach the maximum activity for most of the tested com-
pounds, then the activity decreased in the next hour. Compounds
5a–l exhibited from 63% to 73% anti-inflammatory activity after
the fourth hour representing 80%, 81%, 87%, 80%, 76%, 84%, 88%,
87%, 82%, 82%, 78% and 88% of indomethacin activity, respectively.
Furthermore, oxime derivatives 6a–l exhibited high anti-
inflammatory activity after the fourth hour ranging from 73% to
82% representing 93%, 92%, 94%, 88%, 88%, 94%, 99%, 95%, 95%,
96%, 98% and 92% of indomethacin activity, respectively.
(VR ꢀ VL) control represents the mean increase in paw thickness
in the control group of rats.
(VR ꢀ VL) treated represents the mean increase in paw thickness
in rats treated with the tested compounds.
Results in Table 2 show the percentage of edema inhibition in-
duced by carrageenan for the ketone intermediates 5a–l, oxime
derivatives 6a–l and indomethacin versus time in hours. Most of
the tested compounds showed a significant anti-inflammatory
activity against carrageenan-induced paw edema in rats (p

3842
M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
Table 1
Amount of NO released (n = 4, number of reaction mixtures assayed for each compound) determined by Griess reagent using 0.1 mM of the tested compounds 6a–l in the
presence of 0.5 mM N-acetylcysteine in phosphate buffer of pH 7.4
Compound number
Amount of NO released (mol/mol)
1 h
2 h
3 h
4 h
5 h
6 h
6a
6b
6c
6d
6e
6f
6g
6h
6i
0.06 0.010
0.09 0.091
0.09 0.048
0.08 0.017
0.06 0.005
0.08 0.003
0.06 0.005
0.11 0.024
0.06 0.031
0.08 0.005
0.15 0.024
0.07 0.010
0.10 0.099
0.12 0.024
0.15 0.074
0.10 0.013
0.08 0.010
0.11 0.009
0.13 0.004
0.12 0.031
0.10 0.074
0.08 0.012
0.17 0.050
0.11 0.018
0.13 0.031
0.12 0.010
0.17 0.099
0.12 0.016
0.12 0.019
0.14 0.025
0.2 0.021
0.12 0.034
0.12 0.091
0.09 0.027
0.18 0.038
0.12 0.015
0.16 0.095
0.15 0.027
0.17 0.012
0.13 0.028
0.13 0.012
0.19 0.016
0.24 0.010
0.21 0.041
0.13 0.099
0.15 0.099
0.19 0.057
0.14 0.033
0.22 0.048
0.15 0.040
0.24 0.099
0.17 0.028
0.14 0.014
0.14 0.012
0.25 0.008
0.18 0.048
0.15 0.095
0.15 0.017
0.23 0.081
0.16 0.025
0.17 0.091
0.14 0.048
0.13 0.081
0.17 0.026
0.13 0.010
0.12 0.011
0.20 0.002
0.14 0.040
0.09 0.051
0.12 0.025
0.17 0.048
0.15 0.037
6j
6k
6l
N
N
of the presence of the 3,4-di-OCH3-Ph moiety on the anti-inflam-
matory activity of the tested compounds which is in agreement
with the scope of this work. The results also showed that there is
no great difference between allyl, ethyl and phenyl substituents
on the triazole nucleus on the anti-inflammatory activity of these
compounds.
Ar
N
S
R
H
O
N
Figure 2. Expected intramolecular hydrogen bonding between the oxime OH group
and the sulfur atom.
2.3.2. Screening of ulcerogenic liability
The ulcerogenic liability was evaluated according to the re-
ported procedure44 for the synthesized compounds 5a–l and 6a–l
relative to indomethacin. Ulcerogenic liability was evaluated in
rats after oral administration of the tested compounds and the ref-
erence indomethacin at a dose level of 0.28 mmol/kg suspended in
0.5% aqueous CMC. 0.5% Aqueous CMC was used as a control.
Ulcers were classified into levels, level I, in which the ulcer area
is less than 1 mmꢀ2, 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,
where the following parameters were calculated:45
Several conclusions could be deduced from the above men-
tioned results; slight improvement for the anti-inflammatory
activity was achieved by the oxime derivatives 6a–l compared to
their ketone intermediates 5a–l that may attributed to the syner-
gistic effect of NO and/or the enhanced physicochemical properties
of oxime derivatives compared to their corresponding ketones.
Compounds 6g (R = ethyl, Ar = 3,4-di-OCH3-Ph) and 6k (R = Ph,
Ar = 3,4-di-OCH3-Ph) exhibited the highest anti-inflammatory
activity among the tested compounds that may explain the impact
Table 2
The anti-inflammatory activity at different time intervals for the ketone intermediates 5a–l and their corresponding oximes 6a–l using carrageenan-induced paw edema in rats
Compound number
% of edema inhibition (% mean SEM)
3 h
1 h
2 h
4 h
5 h
Control
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
0
0
0
0
0
43 1.16⁄⁄⁄
49 4.38⁄⁄⁄
61 3.49⁄⁄⁄
49 2.75⁄⁄⁄
52 2.81⁄⁄⁄
53 1.42⁄⁄⁄
42 0.19⁄⁄⁄
29 1.02⁄⁄
53 3.36⁄⁄⁄
52 1.17⁄⁄⁄
56 4.92⁄⁄⁄
44 2.47⁄⁄⁄
58 1.36⁄⁄⁄
61 1.07⁄⁄⁄
53 4.36⁄⁄⁄
61 1.20⁄⁄⁄
65 1.93⁄⁄⁄
69 1.70⁄⁄⁄
61 1.83⁄⁄⁄
63 2.72⁄⁄⁄
60 1.70⁄⁄⁄
58 0.22⁄⁄⁄
55 4.36⁄⁄⁄
61 2.38⁄⁄⁄
51 0.78⁄⁄⁄
58 1.55⁄⁄⁄
53 4.70⁄⁄⁄
63 0.42⁄⁄⁄
62 3.87⁄⁄⁄
55 2.44⁄⁄⁄
60 2.89⁄⁄⁄
55 1.46⁄⁄⁄
41 3.03⁄⁄⁄
60 0.40⁄⁄⁄
57 5.63⁄⁄⁄
57 0.83⁄⁄⁄
51 2.92⁄⁄⁄
60 2.57⁄⁄⁄
66 2.26⁄⁄⁄
57 3.05⁄⁄⁄
68 1.81⁄⁄⁄
69 2.04⁄⁄⁄
72 1.35⁄⁄⁄
67 1.36⁄⁄⁄
67 1.23⁄⁄⁄
63 0.76⁄⁄⁄
63 1.76⁄⁄⁄
61 2.42⁄⁄⁄
61 2.81⁄⁄⁄
67 1.36⁄⁄⁄
70 3.39⁄⁄⁄
63 4.16⁄⁄⁄
64 1.85⁄⁄⁄
66 2.79⁄⁄⁄
61 2.26⁄⁄⁄
67 1.88⁄⁄⁄
61 3.36⁄⁄⁄
49 0.69⁄⁄⁄
64 3.94⁄⁄⁄
58 2.42⁄⁄⁄
59 1.75⁄⁄⁄
68 1.89⁄⁄⁄
65 2.29⁄⁄⁄
67 2.74⁄⁄⁄
58 2.93⁄⁄⁄
70 3.29⁄⁄⁄
72 4.38⁄⁄⁄
75 3.72⁄⁄⁄
71 2.81⁄⁄⁄
69 4.76⁄⁄⁄
65 1.93⁄⁄⁄
63 3.29⁄⁄⁄
67 0.99⁄⁄⁄
64 2.47⁄⁄⁄
74 2.17⁄⁄⁄
66 0.99⁄⁄⁄
67 2.82⁄⁄⁄
72 4.77⁄⁄⁄
66 2.03⁄⁄⁄
63 1.49⁄⁄⁄
70 3.50⁄⁄⁄
73 5.35⁄⁄⁄
72 3.04⁄⁄⁄
68 4.89⁄⁄⁄
68 2.36⁄⁄⁄
65 1.27⁄⁄⁄
73 3.00⁄⁄⁄
77 4.31⁄⁄⁄
76 1.37⁄⁄⁄
78 1.26⁄⁄⁄
73 4.29⁄⁄⁄
73 2.80⁄⁄⁄
78 2.21⁄⁄⁄
82 0.92⁄⁄⁄
79 1.79⁄⁄⁄
79 2.71⁄⁄⁄
80 1.74⁄⁄⁄
81 1.69⁄⁄⁄
76 2.84⁄⁄⁄
83 1.41⁄⁄⁄
66 1.48⁄⁄⁄
61 4.10⁄⁄⁄
62 2.78⁄⁄⁄
60 0.59⁄⁄⁄
51 2.72⁄⁄⁄
61 5.34⁄⁄⁄
69 3.87⁄⁄⁄
59 1.66⁄⁄⁄
63 1.83⁄⁄⁄
61 3.37⁄⁄⁄
59 5.09⁄⁄⁄
72 3.11⁄⁄⁄
68 1.84⁄⁄⁄
57 0.56⁄⁄⁄
69 1.06⁄⁄⁄
77 0.24⁄⁄⁄
71 1.23⁄⁄⁄
71 0.30⁄⁄⁄
71 3.14⁄⁄⁄
74 0.92⁄⁄⁄
75 1.62⁄⁄⁄
73 1.48⁄⁄⁄
78 3.53⁄⁄⁄
68 2.65⁄⁄⁄
87 0.47⁄⁄⁄
6l
Indomethacin
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 = 4), ⁄⁄p
<0.01, ⁄⁄⁄p <0.001, significant difference from control group.

M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
3843
1. The ulcer index (UI) was calculated as follows:
80
60
40
20
0
1 ꢁ (number of ulcers level I) + 2 ꢁ (number of ulcers level
II) + 3 ꢁ (number of ulcers level III), etc. . .. . ..
2. Cure ratio = 100 ꢀ (UItreated ꢁ 100/UIprotype
)
where, UItreated: means the average of the UI of the groups of rats
treated with the NO-donating derivatives; UI protype: means the
average of the UI of the groups of rats treated with the starting
and the intermediate derivatives.
###
*
###
###
###
The UI of compounds 5a–l and 6a–l were calculated and listed
in Table 3 (mean SEM). The results revealed that indomethacin
caused significant ulcerogenic toxicity with UI of 63.45, whereas
an equal dose of most of the synthesized compounds exhibited
much safer UI compared to indomethacin. For example, the ketone
derivatives 5a–l exhibited UI ranging from 2.44 to 9.58, while their
corresponding NO-donating oxime analogues 6a–l exhibited a very
low significant ulcerogenic liability compared to indomethacin and
their starting ketone derivatives with UI ranging from of 1.14 to
7.57 (Table 3 and Figs. 3A–C).
The cure ratio of the target NO-donating derivatives 6a–l rela-
tive to their ketone intermediates 5a–l was calculated and listed
in Table 3. The results revealed that the NO-donating oxime deriv-
atives 6a–g and 6i–k achieved a great reduction of ulcers than their
corresponding ketone intermediates 5a–g and 5i–k, respectively.
On the other hand, compounds 6h and 6l showed a negative cure
ratio of ꢀ7.02% and ꢀ4.35%, respectively, than their corresponding
ketone intermediate 5h and 5l.
5a 5b 5c 5d 6a 6b 6c 6d Indo
Figure 3A. UI of compounds 5a–d and 6a–d compared to indomethacin (Indo)
expressed as mean SEM.
80
60
40
20
***
**
###
###
###
###
0
5e 5f 5g 5h 6e 6f 6g 6h Indo
Several conclusions could be deduced from the above men-
tioned results; all the tested compounds achieved a very low sig-
nificant ulcerogenic liability compared to indomethacin that may
be attributed to the beneficial effect of the triazole nucleus as less
ulcerogenic bioisoster of the carboxyl function. The oxime deriva-
tives produce lower UI than their corresponding ketone that may
be attributed to the beneficial effect of the NO and the triazole nu-
cleus. The NO donating oxime 6b (R = allyl, Ar = 4-OCH3-Ph)
showed the lowest UI (1.14) among all the synthesized oximes
6a–l. The decreased gastric toxicity of the targeted NO–triazole hy-
brids 6a–l compared to their starting ketone intermediates 5a–l
may be attributed to the release of NO that increases mucosal
blood flow resulting in enhanced mucosal resistance to
ulceration.46,47
Figure 3B. UI of compounds 5e–h and 6e–h compared to indomethacin (Indo)
expressed as mean SEM.
80
60
40
20
*
###
###
###
****
###
0
2.3.3. Histopathological investigation
5i 5j 5k 5l 6i 6j 6k 6l Indo
After assessment of gastric mucosal ulcerogenicity and determi-
nation of UI, stomach sections of the ulcers for the control and the
treated groups were stained by standard hematoxylin and eosin
Figure 3C. UI of compounds 5i–l and 6i–l compared to indomethacin (Indo)
expressed as mean SEM.
Table 3
Ulcer indices of compounds 5a–l and 6a–l compared to indomethacin expressed as mean SEM and cure ratio (%) of oximes relative to its starting ketones
Compound number
Ulcer index (UI)
(mean SEM)
Compound number
Ulcer index (UI)
(mean SEM)
Cure ratio (%) of oximes relative to its ketones intermediates
Control
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
0.60 0.05⁄⁄⁄
2.44 0.82⁄⁄⁄
5.34 1.16⁄⁄⁄
4.26 0.62⁄⁄⁄
6.39 1.67
6a
6b
6c
6d
6e
6f
6g
6h
6i
2.30 0.67⁄⁄⁄
1.14 0.65⁄⁄⁄
1.82 0.81⁄⁄⁄
5.52 1.15⁄⁄⁄
1.78 0.54⁄⁄⁄
2.53 1.05⁄⁄⁄
1.58 0.90⁄⁄⁄
3.66 0.46⁄⁄⁄
2.53 0.70⁄⁄⁄
7.57 0.54⁄⁄⁄
5.71 0.82⁄⁄⁄
5.52 0.63⁄⁄⁄
5.74
78.65
57.28
13.62
60.36
61.61
78.53
ꢀ7.02
73.59
13.39
39.32
ꢀ4.35
4.49 0.62⁄⁄⁄
6.59 1.17⁄⁄⁄
7.36 0.65⁄⁄⁄
3.42 0.10⁄⁄⁄
9.58 1.13⁄⁄⁄
8.74 0.53⁄⁄⁄
9.41 0.54⁄⁄⁄
6j
6k
6l
⁄⁄⁄
5l
5.29 1.24
Indomethacin
63.45 2.75
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 = 4), ⁄⁄⁄p
<0.001, significant difference from control group.

3844
M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
stain. The produced slides were subjected to microscopical exami-
nation and pictures were picked for these slides (Figs. 4A–D).
The control group (Fig. 4A) showed no lesions and characterized
by continuous mucosal layer while the one treated with indometh-
acin (Fig. 4B) showed that gastric mucosa was decreased in thick-
ness with marked loss of mucosal membrane at the areas of
ulceration. Lamina propria showed capillary vasodilatation associ-
ated with accumulation of edema fluid and acute inflammatory cell
infiltrating its whole thickness and apparently destroying the sur-
face epithelium and the epithelial lining of gastric pits. Severe
atrophic gastritis and gland atrophy and was noticed. Apoptotic
glandular epithelial cells could be detected.
The group treated with NO-donating oxime 6g (Fig. 4D) indi-
cated partial healing of the ulcer but remnants of the structural
damage were still found and not completely regenerated that also
indicated by the low UI where it was 1.58. On the other hand, the
group treated with its ketone derivative 5g (Fig. 4C) exhibited
marked capillary dilatation in the lumina propria just below the
fundic glands, capillary inflammatory cells were also found and
edema fluid leads to flattening of the above mucosal membrane
which also confirmed by its high UI as it was 7.36.
Figure 4B. Photomicrograph of the mucosa of fundic stomach of indomethacin.
In conclusion, the histopathological investigation confirms the
previously mentioned ulcerogenic liability results of the tested
compounds and supports the effect of NO as a gastroprotective
agent through increasing the mucosal blood flow, mucus and bicar-
bonate secretion.
2.3.4. Screening of antiproliferative activity
Compounds 6c, 6e, 6g, 6i, 6j, 6k and 6l were selected by the Na-
tional Cancer Institute (NCI) according to the protocol of the Drug
Evaluation Branch of the National Cancer Institute, Bethesda, USA
forinvitroanticancerscreening. Primaryinvitroonedoseanticancer
assay was performed in full NCI 60 cell lines derived from nine tumor
subpanels, including leukemia, melanoma, lung, colon, CNS, ovarian,
renal, prostate, and breast cancer cell lines. The selected compounds
were added at a single concentration (10ꢀ5 M) and the culture was
incubated for 48 h. End point determinations were made with a pro-
tein binding dye sulforhodamine B (SRB). Results for each compound
were reported as a mean graph of the percent growth of the treated
cells when compared to the untreated control cells.
Figure 4C. Photomicrograph of the mucosa of fundic stomach of compound 5g.
The NO-donating oxime 6c achieved cell growth inhibition of
27.70 and 22.07, respectively, against both leukemia HL-60(TB)
and renal cancer UO-31, while the NO-donating oxime 6e
exhibited moderate cell growth inhibition activity against
non-small cell lung cancer HOP-92 and renal cancer A498 cell lines
Figure 4D. Photomicrograph of the mucosa of fundic stomach of compound 6g.
with cell growth inhibition of 37.52 and 30.67, respectively. In
addition, the NO-donating oxime 6i exhibited moderate
cell growth inhibition activity against both renal cancer A498
and breast cancer MDA-MB-231/ATCC cell lines with cell growth
Figure 4A. Photomicrograph of the mucosa of fundic stomach of control.

M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
3845
inhibition of 50.52 and 31.56, respectively and weak cell growth
inhibition activity against both leukemia K-562 and renal cancer
UO-31 cell lines with cell growth inhibition of 25.39 and 22.60,
respectively. Compound 6j exhibited cell growth inhibition of
29.72, 26.55, 24.12 and 26.15 against leukemia MOLT-4, RPMI-
8226, SR and renal cancer A498 cell lines, respectively. The NO-
donating oxime 6k exhibited moderate cell growth inhibition
activity against leukemia HL-60(TB), RPMI-8226 cell lines with cell
growth inhibition of 31.76 and 30.60, respectively. Additionally,
compound 6l exhibited 26.43 and 25.17, 21.04 and 30.48, respec-
tively, against leukemia RPMI-8226, SR, renal cancer CAKI-1 and
UO-31 cell lines. From the above mentioned results, it is clear that
the N-allyl and N-ethyl substituted triazoles exhibited weak anti-
proliferative activity while the N-phenyl substituted triazoles re-
vealed from moderate to weak activity against most of the tested
cell lines. It is obvious that the unsubstituted 1,2-diphenyl triazole
6i exhibited the most significant activity among the tested com-
pounds against renal cancer A498 cell lines.
4.1.1. Substituted ethylbenzoate 1a–d, benzohydrazides 2a–d, 2-
Benzoyl-N-Rthiosemicarbazides 3a–l and 4-R-5-aryl-4H-1,2,4-
triazole-3-thiol derivatives 4a–l was prepared according to the
reported procedure24
.
4.1.2. General procedure for the synthesis of 1-phenyl-2-((4-R-
5-aryl-4H-1,2,4-triazol-3-yl)thio)ethanone 5a–l
An equimolar mixture of 4a–l, phenacyl bromide (1 mmol) and
TEA (1.2 mmol) in acetonitril (50 mL) was heated at reflux for 4–
8 h. The reaction mixture was evaporated to dryness. The residue
was crystallized from aqueous ethanol affording the pure products
5a–l. The structure of compounds 5a–l was confirmed by mp, IR
and 1H NMR spectroscopy.
4.1.2.1.
phenylethanone 5a.
73% yield); mp 106–107 °C; FT-IR (
2-(4-Allyl-5-phenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-1-
White crystals (ethanol) in (0.244 g,
max): 3058 (CH aromatic),
m
2904 (CH aliphatic), 1686 (C@O), 1595 (C@C), 1576 cmꢀ1 (C@N);
1H NMR (400 MHz, DMSO-d6, d = ppm) d = 4.65–4.66 (m, 2H,
NCH2), 4.85 (d, 1H, CH@CH2, Jtrans = 17.6 Hz), 4.98 (s, 2H, SCH2),
5.24 (d, 1H, CH@CH2, Jcis = 10.4 Hz), 5.94–6.03 (m, 1H, CH@CH2),
7.53–7.70 (m, 8H, Ar-H), 8.03 (d, 2H, J = 7.6 Hz, Ar-H); 13C NMR
(75 MHz, DMSO-d6, d = ppm) d = 40.85 (CH2), 46.51 (CH2), 117.17
(@CH2), 126.88 (CH), 128.10 (CH), 128.39 (CH), 128.79 (CH),
128.94 (CH), 130.07 (CH), 132.40 (CH), 133.72 (C), 135.27 (C),
150.45 (C), 155.13 (C), 193.27 (C@O); FAB-MS (%) 336.2 (M+1, 100).
3. Conclusions
A series of novel triazole–NO hybrids was prepared and charac-
terized by different spectroscopic techniques and elemental analy-
sis. Most of the synthesized compounds showed significant anti-
inflammatory activity using carrageenan-induced rat paw edema
method. The NO-donating oximes 6a–l could release NO at pH
7.4 where the maximum amount was released after 5 h. The pre-
pared NO-donating oximes hybrids showed pronounced gastropro-
tective activity better than their corresponding ketones that might
be attributed to the release of NO. Histopathological examination
indicated that the NO donating moiety reduced greatly the inci-
dence of gastric ulceration. Additionally, the oxime 6i revealed
the most significant activity against renal cancer A498 cell lines
with 50.52 cell growth inhibition. In summary, the use of hybrid
molecules containing NO-donating moieties looks as a promising
approach to improve the safety of NSAIDs without altering their
effectiveness and may be a challenge in the field of anticancer
therapy.
4.1.2.2. 2-[4-Allyl-5-(4-methoxyphenyl)-4H-[1,2,4]triazol-3-yls-
ulfanyl]-1-phenylethanone 5b.
(0.296 g, 81% yield); mp 114–115 °C; FT-IR (
White crystals (ethanol) in
max): 3062 (CH aro-
m
matic), 2914, 2837 (CH aliphatic), 1682 (C@O), 1612 (C@C), 1580
(C@N), 1250 (C–O); 1H NMR (400 MHz, DMSO-d6, d = ppm)
d = 3.80 (s, 3H, OCH3), 4.62–4.63 (m, 2H, NCH2), 4.84 (d, 1H,
CH@CH2, Jtrans = 17.6 Hz), 4.95 (s, 2H, SCH2), 5.23 (d, 1H, CH@CH2,
Jcis = 10.8 Hz), 5.93–6.03 (m, 1H, CH@CH2), 7.08 (d, 2H, J = 8.8 Hz,
Ar-H), 7.80–7.53 (m, 5H, Ar-H), 8.04 (d, 2H, J = 8.8 Hz, Ar-H); 13C
NMR (75 MHz, DMSO-d6, d = ppm) d = 40.82 (CH2), 46.42 (CH2),
55.29 (CH3), 114.37 (CH), 117.07 (@CH2), 119.06 (C), 128.39 (CH),
128.79 (CH), 129.61 (CH), 132.51 (CH), 133.72 (CH), 135.27 (C),
149.92 (C), 154.80 (C), 160.52 (C), 193.32 (C@O); FAB-MS (%)
366.2 (M+1, 100).
4. Experimental section
4.1. Chemistry
4.1.2.3. 2-[4-Allyl-5-(3,4-dimethoxyphenyl)-4H-[1,2,4]triazol-3-
Reactions were monitored by TLC, pre-coated plastic sheets,
0.2 mm silica gel F254 with fluorescent indicator (Macherey-Nagel).
Melting points were determined on Stuart electrothermal melting
point apparatus and were uncorrected. IR spectra were recorded on
Nicolet iS5 (ATR) FT-IR spectrometer. 1H NMR spectra were run on
JEOL JNM-GX-300 spectrometer (300 MHz), JEOL JNM-GX-400
spectrometer (400 MHz), JEOL JNM-GX-500 spectrometer
(500 MHz) and JEOL JNM-LA-400 FT-spectrometer (400 MHz). 13C
NMR spectra were recorded on a JEOL JNM-GX-300 spectrometer
(75 MHz) or JEOL JNM-GX-400 spectrometer (100 MHz) or JEOL
JNM-GX-500 spectrometer (125 MHz) using TMS as internal refer-
ence. Chemical shifts (d) values are given in parts per million (ppm)
using CDCl3 (7.29 for proton and 76.98 for carbon), CD3OD (3.31
and 4.87 for protons and 49.00 for carbon) or DMSO-d6 (2.50 for
proton) as solvents and coupling constants (J) in Hz. Splitting pat-
terns are designated as follows: s, singlet; d, doublet; t, triplet; q,
quartet; dd, doublet of doublet; m, multiplet; br s, broad singlet.
EI-MS was performed on JEOL JMS 600 spectrometers and Micro-
mass LCT mass spectrometer which was recorded in the positive
ion mode. Elemental analyses were recorded on Perkin Elmer
2400 CHN, Microanalytical unit, Faculty of Science, Cairo Univer-
sity, Egypt.
ylsulfanyl]-1-phenylethanone 5c.
in (0.328 g, 83% yield); mp 123–124 °C; FT-IR (
White crystals (ethanol)
max): 3062 (CH aro-
m
matic), 2938, 2837 (CH aliphatic), 1680 (C@O), 1596 (C@C), 1534
(C@N), 1264 (C–O); 1H NMR (400 MHz, DMSO-d6, d = ppm)
d = 3.77 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 4.64–4.65 (m, 2H, N-
CH2), 4.88 (d, 1H, CH@CH2, Jtrans = 17.6 Hz), 4.95 (s, 2H, SCH2),
5.26 (d, 1H, CH@CH2, Jcis = 10.8 Hz), 5.97–6.06 (m, 1H, CH@CH2),
7.00–7.15 (m, 3H, Ar-H), 7.54–7.70 (m, 3H, Ar-H), 8.03 (d, 2H,
J = 8.4 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6, d = ppm) d = 40.82
(CH2), 46.57 (CH2), 55.54 (CH3), 55.57 (CH3), 111.47 (CH), 111.78
(CH), 117.06 (@CH2), 119.08 (C), 120.81 (CH), 128.44 (CH), 128.84
(CH), 132.70 (C), 133.78 (CH), 135.30 (CH), 148.73 (C), 150.03 (C),
150.20 (C), 155.17 (C), 193.33 (C@O); FAB-MS (%) 396.2 (M+1, 100).
4.1.2.4. 2-[4-Allyl-5-(3,4,5-trimethoxyphenyl)-4H-[1,2,4]triazol-
3-ylsulfanyl]-1-phenylethanone 5d.
nol) in (0.323 g, 76% yield); mp 73–74 °C; FT-IR (
White crystals (etha-
max): 3061 (CH
m
aromatic), 2937, 2833 (CH aliphatic), 1680 (C@O), 1586 (C@C),
1534 (C@N), 1125 (C–O); 1H NMR (400 MHz, DMSO-d6, d = ppm)
d = 3.71 (s, 3H, OCH3), 3.79 (s, 6H, 2 OCH3), 4.67–4.68 (m, 2H,
NCH2), 4.92 (d, 1H, CH@CH2, Jtrans = 18.8 Hz), 4.97 (s, 2H, SCH2),
5.28 (d, 1H, CH@CH2, Jcis = 10 Hz), 6.01–6.07 (m, 1H, CH@CH2),

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M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
6.87 (s, 2H, Ar-H), 7.55–7.71 (m, 3H, Ar-H), 8.03 (d, 2H, J = 8.4 Hz,
Ar-H); 13C NMR (75 MHz, DMSO-d6, d = ppm) d = 40.80 (CH2),
46.73 (CH2), 56.05 (CH3), 60.15 (CH3), 105.64 (CH), 117.15
(@CH2), 122.14 (C), 128.47 (CH), 128.89 (CH), 132.80 (C), 133.83
(CH), 135.33 (CH), 138.81 (C), 150.30 (C), 153.12 (C), 155.17 (C),
193.32 (C@O); EI-MS (70 eV) m/z (%) 425 (M+Å, 0.2), 347 (20), 307
(53), 292 (25), 194 (15), 193 (17), 105 (100), 77 (40). Anal. Calcd
for C22H23N3O4S (425.14): C, 62.10; H, 5.45; N, 9.88. Found: C,
61.95; H, 5.27; N, 9.64.
(CH aromatic), 2915 (CH aliphatic), 1676 (C@O), 1595 (C@C),
1534 (C@N), 1260 (C–O); 1H NMR (400 MHz, DMSO-d6, d = ppm)
d = 3.51 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 4.92 (s, 2H, SCH2),
6.88–6.92 (m, 3H, Ar-H), 7.42–7.71 (m, 8H, Ar-H), 8.02 (d, 2H,
J = 8.4 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6, d = ppm) d = 40.26
(–SCH2), 56.65 (CH3), 56.93 (CH3), 112.87 (CH), 113.07 (CH),
120.16 (C), 122.71 (CH), 129.40 (CH), 130.04 (CH), 130.49 (CH),
131.71 (CH), 131.84 (C), 135.47 (CH), 135.72 (CH), 137.00 (C),
150.16 (C), 151.94 (C), 153.02 (C), 156.29 (C), 194.82 (C@O); FAB-
MS (%) 332.2 (M+1, 15).
4.1.2.5.
phenylethanone 5e48
62% yield); mp 135–136 °C; FT-IR (
2-(4-Ethyl-5-phenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-1-
White crystals (ethanol) in (0.200 g,
max): 3064 (CH aromatic),
.
4.1.2.12.
[1,2,4]triazol-3-ylsulfanyl]-1-phenylethanone
crystals (ethanol) in (0.328 g, 71% yield); mp 152–153 °C; FT-IR
max): 3058 (CH aromatic), 2937, 2835 (CH aliphatic), 1681
2-[5-(3,4,5-Trimethoxyphenyl)-4-phenyl-4H-
m
5l. White
2977, 2918 (CH aliphatic), 1688 (C@O), 1594 (C@C), 1578 (C@N).
(m
4.1.2.6. 2-[4-Ethyl-5-(4-methoxyphenyl)-4H-[1,2,4]triazol-3-yls-
(C@O), 1587 (C@C), 1487 (C@N), 1124 (C–O); 1H NMR (400 MHz,
DMSO-d6, d = ppm) d = 3.52 (s, 3H, OCH3), 3.62 (s, 6H, 2 OCH3),
4.94 (s, 2H, SCH2), 6.61 (s, 2H, Ar-H), 7.42–7.71 (m, 8H, Ar-H),
8.02 (d, 2H, J = 7.2 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6,
d = ppm) d = 41.56 (–SCH2), 56.63 (CH3), 61.16 (CH3), 106.84 (CH),
122.71 (C), 129.04 (CH), 129.21 (CH), 129.73 (CH), 130.11 (CH),
131.42 (CH), 131.59 (CH), 135.07 (CH), 135.55 (C), 136.82 (C),
140.48 (C), 153.32 (C), 154.41 (C), 155.98 (C), 194.45 (C@O); FAB-
MS (%) 462.2 (M+1, 100).
ulfanyl]-1-phenyl-ethanone 5f48
.
White crystals (ethanol) in
(0.279 g, 79% yield); mp 83–84 °C; FT-IR ( max): 3060 (CH aro-
m
matic), 2839 (CH aliphatic), 1680 (C@O), 1612 (C@C), 1596
(C@N), 1249 (C–O).
4.1.2.7. 2-[4-Ethyl-5-(3,4-dimethoxyphenyl)-4H-[1,2,4]triazol-3-
ylsulfanyl]-1-phenylethanone 5g.
in (0.264 g, 69% yield); mp 121 °C; FT-IR (
White crystals (ethanol)
max): 3060 (CH aro-
m
matic), 2837 (CH aliphatic), 1680 (C@O), 1596 (C@C), 1533
(C@N), 1255 (C–O); 1H NMR (400 MHz, DMSO-d6, d = ppm)
d = 1.22 (t, 3H, N-CH2-CH3, J = 7.2 Hz), 3.79 (s, 3H, OCH3), 3.82 (s,
3H, OCH3), 4.02 (q, 2H, N-CH2-CH3, J = 7.2 Hz), 4.98 (s, 2H, SCH2),
7.09–7.15 (m, 3H, Ar-H), 7.54–7.70 (m, 3H, Ar-H), 8.03 (d, 2H,
J = 8.4 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6, d = ppm) d = 16.71
(–CH2CH3), 38.30 (–CH2CH3), 40.87 (–SCH2), 51.17 (CH3), 57.52
(CH3), 113.91 (CH), 114.11 (CH), 121.25 (C), 123.45 (CH), 130.54
(CH), 130.92 (CH), 135.90 (CH), 137.61 (C), 151.46 (C), 152.26 (C),
153.00 (C), 157.60 (C), 195.42 (C@O); FAB-MS (%) 384.2 (M+1, 100).
4.1.3. General procedure for the synthesis of 1-phenyl-2-((4-R-
5-aryl-4H-1,2,4-triazol-3-yl)thio)ethanone oxime 6a–l
A mixture of equimolar amounts of the appropriate ketone
derivatives 5a–l (0.3 mmol) and hydroxylamine hydrochloride in
absolute ethanol (30 mL) was heated under reflux for 8–12 h and
then left to cool. The separated solid was filtered, washed with
dil. ammonia solution and distilled water, dried, and recrystallized
from aqueous ethanol affording the pure novel products 6a–l.
4.1.3.1.
phenylethanone oxime 6a.
(0.097 g, 92% yield); mp 145–146 °C; FT-IR (
2-(4-Allyl-5-phenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-1-
White crystals (ethanol) in
max): 3600–3100
4.1.2.8.
zol-3-ylsulfanyl]-1-phenylethanone 5h.
(ethanol) in (0.331 g, 80% yield); mp 82–83 °C; FT-IR (
2-[4-Ethyl-5-(3,4,5-trimethoxyphenyl)-4H-[1,2,4]tria-
White crystals
max): 3060
m
m
(br, OH), 3021 (CH aromatic), 2849 (CH aliphatic), 1620 (C@C),
1570 (C@N); 1H NMR (400 MHz, DMSO-d6, d = ppm) d = 4.43 (s,
2H, SCH2), 4.52–4.53 (m, 2H, N-CH2), 4.70 (d, 1H, CH@CH2,
Jtrans = 16.8 Hz), 5.13 (d,1H, CH@CH2, Jcis = 10.8 Hz), 5.81–5.90 (m,
1H, CH@CH2), 7.37–7.68 (m, 10H, Ar-H), 11.83 (s, 1H, OH); 13C
NMR (75 MHz, DMSO-d6, d = ppm) d = 26.45 (–SCH2), 46.41 (CH2),
116.98 (@CH2), 125.89 (CH), 126.99 (CH), 128.21(CH), 128.53
(CH), 128.95 (CH), 129.15 (CH), 129.35 (CH), 130.17 (C), 132.59
(C), 134.31 (C), 152.19 (C), 155.50 (C); EI-MS (70 eV) m/z (%) 350
(M+Å, 0.3), 340 (18), 339 (23), 338 (82), 242 (27), 218 (25), 217
(61), 202 (45), 131 (32), 104 (80), 103 (100), 77 (49), 76 (22). Anal.
Calcd for C19H18N4OS (350.12): C, 65.12; H, 5.18; N, 15.99. Found:
C, 64.81; H, 4.96; N, 16.06.
(CH aromatic), 2937 (CH aliphatic), 1680 (C@O), 1585 (C@C),
1486 (C@N), 1124 (C–O); 1H NMR (400 MHz, DMSO-d6, d = ppm)
d = 1.24 (t, 3H, N-CH2-CH3, J = 7.2 Hz), 3.72 (s, 3H, OCH3), 3.82 (s,
6H, 2 OCH3), 4.04 (q, 2H, N-CH2-CH3, J = 7.2 Hz), 4.99 (s, 2H,
SCH2), 6.87 (s, 2H, Ar-H), 7.55–7.71 (m, 3H, Ar-H), 8.04 (d, 2H,
J = 8 Hz, Ar-H); 13C NMR (75 MHz, DMSO-d6, d = ppm) d = 15.07 (–
CH2CH3), 38.65 (–CH2CH3), 40.60 (–SCH2), 56.80 (CH3), 60.14
(CH3), 105.94 (CH), 122.49 (C), 128.44 (CH), 128.86 (CH), 133.78
(CH), 135.33 (C), 138.78 (C), 149.49 (C), 153.15 (C), 154.90 (C),
193.27 (C@O); FAB-MS (%) 414.2 (M+1, 100).
4.1.2.9. 2-(4,5-Diphenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-1-phe-
nylethanone 5i49
.
White crystals (ethanol) in (0.316 g, 85%
yield); mp 183–184 °C ([Ref. 48; 181 °C]); FT-IR ( max): 3056 (CH
m
4.1.3.2. 2-[4-Allyl-5-(4-methoxyphenyl)-4H-[1,2,4]triazol-3-yls-
aromatic), 2914 (CH aliphatic), 1676 (C@O), 1594 (C@C), 1578
(C@N).
ulfanyl]-1-phenylethanone oxime 6b.
nol) in (0.097 g, 85% yield); mp 141–142 °C; FT-IR (
White crystals (etha-
max): 3500–
m
3000 (br, OH), 3015 (CH aromatic), 2838 (CH aliphatic), 1612
(C@C), 1580 (C@N), 1252 cmꢀ1 (C–O); 1H NMR (300 MHz, CDCl3,
d = ppm) d = 3.85 (s, 3H, OCH3), 4.40–4.42 (m, 2H, NCH2), 4.59 (s,
2H, SCH2), 4.92 (d, 1H, CH@CH2, Jtrans = 17.1 Hz), 5.24 (d, 1H,
CH@CH2, Jcis = 10.5 Hz), 5.77–5.86 (m, 1H, CH@CH2), 6.98 (d, 2H,
J = 8.4 Hz, Ar-H), 7.34–7.37 (m, 3H, Ar-H), 7.52 (d, 2H, J = 8.4 Hz,
Ar-H), 7.66–7.69 (m, 2H, Ar-H); EI-MS (70 eV) m/z (%) 380 (M+Å,
0.4), 284 (10), 247 (100), 232 (54), 214 (18), 134 (80), 133 (59),
119 (17), 103 (45), 77 (22); 13C NMR (75 MHz, CDCl3, d = ppm)
d = 26.48 (–SCH2), 46.38 (CH2), 55.35 (CH3), 114.37 (CH), 114.41
(CH), 116.93 (@CH2), 119.00 (CH), 125.88 (CH), 128.52 (CH),
129.14 (CH), 129.74 (CH), 132.63 (C), 134.32 (C), 149.87 (C),
4.1.2.10. 2-[5-(4-Methoxyphenyl)-4-phenyl-4H-[1,2,4]triazol-3-
ylsulfanyl]-1-phenylethanone 5j50
.
White crystals (ethanol)
in (0.31 g, 77% yield); mp 159–160 °C; FT-IR ( max): 3057 (CH aro-
m
matic), 2916 (CH aliphatic), 1678 (C@O), 1609 (C@C), 1595 (C@N),
1253 (C–O); 1H NMR (400 MHz, DMSO-d6, d = ppm) d = 3.71 (s, 3H,
OCH3), 4.92 (s, 2H, SCH2), 6.89 (d, 2H, J = 8.8 Hz, Ar-H), 7.26 (d, 2H,
J = 8 Hz, Ar-H), 7.40–7.70 (m, 8H, Ar-H), 8.02 (d, 2H, J = 7.2 Hz, Ar-H).
4.1.2.11. 2-[5-(3,4-Dimethoxyphenyl)-4-phenyl-4H-[1,2,4]tria-
zol-3-ylsulfanyl]-1-phenylethanone 5k.
anol) in (0.285 g, 66% yield); mp 168–169 °C; FT-IR (
White crystals (eth-
max): 3056
m

M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
3847
152.21 (C), 155.15 (C), 160.61 (C); Anal. Calcd for C20H20N4O2S
(380.13): C, 63.14; H, 5.30; N, 14.73. Found: C, 63.14; H, 5.61; N,
14.64.
156.96 (C), 167.12 (C). EI-MS (70 eV) m/z (%) 368 (M+Å, 0.1), 235
(100), 210 (83), 161 (34), 134 (59), 133 (67), 104 (46), 103 (70),
90 (39), 77 (51). Anal. Calcd for C19H20N4O2S (368.13): C, 61.94;
H, 5.47; N, 15.21. Found: C, 62.09; H, 5.81; N, 15.27.
4.1.3.3. 2-[4-Allyl-5-(3,4-dimethoxyphenyl)-4H-[1,2,4]triazol-3-
ylsulfanyl]-1-phenylethanone oxime 6c.
(ethanol) in (0.069 g, 56% yield); mp 183 °C; FT-IR (
White crystals
max): 3450–
4.1.3.7. 2-[4-Ethyl-5-(3,4-Dimethoxyphenyl)-4H-[1,2,4]triazol-
m
3-ylsulfanyl]-1-phenylethanone oxime 6g.
(ethanol) in (0.079 g, 66% yield); mp 176–177 °C; FT-IR (
White crystals
max):
3000 (br, OH), 3139 (CH aromatic), 2838 (CH aliphatic), 1607
(C@C), 1534 (C@N), 1265 cmꢀ1 (C–O); 1H NMR1H NMR (300 MHz,
CDCl3 + CD3OD, d = ppm) d = 3.89 (s, 3H, OCH3), 3.93 (s, 3H,
OCH3), 4.43–4.45 (m, 2H, NCH2), 4.60 (s, 2H, SCH2), 4.94 (d, 1H,
CH@CH2, Jtrans = 17.1 Hz), 5.26 (d, 1H, CH@CH2, Jcis = 10.5 Hz),
5.79–5.88 (m, 1H, CH@CH2), 7.08–7.18 (m, 3H, Ar-H), 7.35–7.38
(m, 3H, Ar-H), 7.66–7.69 (m, 2H, Ar-H), 8.31 (br s, 1H, OH); 13C
NMR (75 MHz, CDCl3 + CD3OD, d = ppm) d = 26.51 (–SCH2), 46.45
(–CH2), 55.59 (2 CH3), 111.52 (CH), 111.75 (CH), 116.87 (@CH2),
119.18 (CH), 120.93 (CH), 125.90 (CH), 128.54 (CH), 128.57 (CH),
129.16 (CH), 132.83 (C), 134.34 (C), 148.70 (C), 149.85 (C), 150.22
(C), 152.27 (C), 155.34 (C); EI-MS (70 eV) m/z (%) 410 (M+Å, 0.3),
405 (18), 404 (21), 276 (100), 261 (24), 164 (44), 163 (54), 104
(19), 103 (42), 77 (27). Anal. Calcd for C21H22N4O3S (410.14): C,
61.44; H, 5.40; N, 13.65. Found: C, 61.08; H, 5.19; N, 13.36.
m
3300–3000 (br, OH), 3020 (CH aromatic), 2975, 2832 (CH ali-
phatic), 1607 (C@C), 1536 (C@N), 1257 cmꢀ1 (C–O); 1H NMR
(300 MHz, CDCl3 + CD3OD, d = ppm) d = 1.22 (t, 3H, N-CH2-CH3,
J = 7.2 Hz), 3.89 (q, 2H, N-CH2-CH3, J = 7.2 Hz), 3.91 (s, 3H, OCH3),
3.93 (s, 3H, OCH3), 4.61 (s, 2H, SCH2), 6.93–7.14 (m, 3H, Ar-H),
7.33–7.38 (m, 3H, Ar-H), 7.67–7.71 (m, 2H, Ar-H), 8.74 (br s, 1H,
OH); 13C NMR (75 MHz, CDCl3 + CD3OD, d = ppm) d = 15.13 (–
CH2CH3), 26.43 (–SCH2), 39.50 (–CH2CH3), 55.57 (CH3), 55.60
(CH3), 111.76 (CH), 115.80 (CH), 119.53 (CH), 121.04 (C), 125.86
(CH), 128.52 (CH), 129.14 (C), 134.34 (CH), 148.76 (C), 148.97 (C),
150.14 (C), 152.26 (C), 155.00 (C). EI-MS (70 eV) m/z (%) 398 (M+Å,
0.4), 246 (100), 218 (50), 204 (70), 104 (89), 77 (43). Anal. Calcd
for C20H22N4O3S (398.14): C, 60.28; H, 5.56; N, 14.06. Found: C,
60.50; H, 5.81; N, 14.09.
4.1.3.4. 2-[4-Allyl-5-(3,4,5-trimethoxyphenyl)-4H-[1,2,4]triazol-
4.1.3.8. 2-[4-Ethyl-5-(3,4,5-Trimethoxyphenyl)-4H-[1,2,4]tria-
3-ylsulfanyl]-1-phenylethanone oxime 6d.
(ethanol) in (0.12 g, 91% yield); mp 159–160 °C; FT-IR (
White crystals
max):
zol-3-ylsulfanyl]-1-phenylethanone oxime 6h.
tals (ethanol) in (0.108 g, 84% yield); mp 162–163 °C; FT-IR
max): 3500–3100 (br, OH), 3136 (CH aromatic), 2835 (CH ali-
White crys-
m
3600–3100 (br, OH), 3138 (CH aromatic), 2837 (CH aliphatic),
1587 (C@C, C@N), 1126 cmꢀ1 (C–O); 1H NMR (300 MHz, CDCl3,
d = ppm) d = 3.83 (s, 6H, 2 OCH3), 3.87 (s, 3H, OCH3), 4.44–4.45
(m, 2H, NCH2), 4.60 (s, 2H, SCH2), 4.95 (d, 1H, CH@CH2,
Jtrans = 17.4 Hz), 5.28 (d, 1H, CH@CH2, Jcis = 10.5 Hz), 5.81–5.99 (m,
1H, CH@CH2), 6.80 (s, 2H, Ar-H), 7.32–7.73 (m, 5H, Ar-H), 9.93
(br s, 1H, OH); 13C NMR (75 MHz, CDCl3, d = ppm) d = 25.92 (–
SCH2), 46.83 (CH2), 56.26 (CH3), 60.92 (CH3), 105.81 (CH), 117.96
(@CH2), 121.81 (C), 126.30 (CH), 128.55 (CH), 129.52 (C), 131.80
(CH), 133.83 (C), 139.70 (C), 151.46 (C), 153.46 (C), 156.00 (C). EI-
MS (70 eV) m/z (%) 440 (M+Å, 1.5), 422 (15), 357 (19), 344 (21),
343 (100), 328 (28), 327 (88), 311 (23), 292 (21), 193 (30), 150
(27), 104 (29), 103 (42), 91 (20), 77 (74). Anal. Calcd for
(m
phatic), 1586 (C@C, C@N), 1126 cmꢀ1 (C–O); 1H NMR (300 MHz,
CDCl3, d = ppm) d = 1.24 (t, 3H, N-CH2-CH3, J = 7.2 Hz), 3.72 (s, 3H,
OCH3), 3.82 (s, 6H, 2 OCH3), 4.04 (q, 2H, N-CH2-CH3, J = 7.2 Hz),
4.63 (s, 2H, S-CH2), 6.72 (s, 2H, Ar-H), 7.30–7.68 (m, 5H, Ar-H),
10.39 (s, 1H, OH); 13C NMR (75 MHz, CDCl3, d = ppm) d = 15.57 (–
CH2CH3), 26.79 (–SCH2), 39.85 (–CH2CH3), 56.23 (CH3), 60.95
(CH3), 105.82 (CH), 122.14 (C), 126.20 (CH), 128.54 (CH), 129.46
(CH), 133.83 (C), 139.47 (C), 150.74 (C), 153.44 (C), 154.05 (C),
155.60 (C). EI-MS (70 eV) m/z (%) 428 (M+Å, 11.5), 409 (8), 309
(14), 295 (100), 279 (26), 252 (13), 135 (89), 103 (91), 77 (9). Anal.
Calcd for C21H24N4O4S (428.15): C, 58.86; H, 5.65; N, 13.07. Found:
C, 58.86; H, 5.81; N, 13.21.
C22H24N4O4S (440.15): C, 59.98; H, 5.49; N, 12.72. Found: C,
59.74; H, 5.62; N, 13.02.
4.1.3.9. 2-(4,5-Diphenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-1-phe-
nylethanone oxime 6i.
86% yield); mp 175–176 °C; FT-IR (m
White crystals (ethanol) in (0.10 g,
max): 3500–3000 (br, OH),
4.1.3.5.
2-(4-Ethyl-5-phenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-1-
phenylethanone oxime 6e.
(0.090 g, 89% yield); mp 166–167 °C; FT-IR (
White crystals (ethanol) in
max): 3400–3100
3054 (CH aromatic), 2850 (CH aliphatic), 1590 (C@C), 1496
(C@N); 1H NMR (400 MHz, DMSO-d6, d = ppm) d = 4.40 (s, 2H,
SCH2), 7.66–7.32 (m, 15H, Ar-H), 11.80 (s, 1H, OH); 13C NMR
(75 MHz, DMSO-d6, d = ppm) d = 25.57 (–SCH2), 125.90 (CH),
126.66 (CH), 127.79 (CH), 127.97 (CH), 128.57 (CH), 128.60 (CH),
129.19 (CH), 129.84 (C), 129.87 (CH), 130.06 (C), 133.86 (C),
134.29 (CH), 151.09 (C), 151.91 (C), 154.72 (C). EI-MS (70 eV) m/z
(%) 386 (M+Å, 0.6), 378 (17), 361 (4), 253 (100), 252 (64), 237
(17), 180 (10), 104 (27), 103 (47), 77 (57). Anal. Calcd for
m
(br, OH), 3056 (CH aromatic), 2913, 2848 (CH aliphatic), 1600
(C@C, C@N); 1H NMR (400 MHz, DMSO-d6, d = ppm) d = 1.08 (t,
3H, N-CH2-CH3, J = 7.2 Hz), 3.90 (q, 2H, N-CH2-CH3, J = 7.2 Hz),
4.44 (s, 2H, SCH2), 7.37–7.68 (m, 10H, Ar-H), 11.84 (s, 1H, OH);
EI-MS (70 eV) m/z (%) 338 (M+Å, 0.5), 325 (67), 205 (53), 204 (52),
177 (33), 131 (34), 104 (100), 103 (73), 77 (57). Anal. Calcd for
C18H18N4OS (338.12): C, 63.88; H, 5.36; N, 16.56. Found: C,
64.03; H, 5.29; N, 16.86.
C22H18N4OS (386.12): C, 68.37; H, 4.69; N, 14.50. Found: C,
68.55; H, 5.04; N, 14.43.
4.1.3.6. 2-[4-Ethyl-5-(4-methoxyphenyl)-4H-[1,2,4]triazol-3-yls-
ulfanyl]-1-phenylethanone oxime 6f.
nol) in (0.087 g, 79% yield); mp 142–143 °C; FT-IR (
White crystals (etha-
max): 3500–
4.1.3.10. 2-[5-(4-Methoxyphenyl)-4-phenyl-4H-[1,2,4]triazol-3-
m
ylsulfanyl]-1-phenylethanone oxime 6j.
(ethanol) in (0.116 g, 93% yield); mp 184–185 °C; FT-IR (
White crystals
max):
3000 (br, OH), 3051 (CH aromatic), 2838 (CH aliphatic), 1612
(C@C), 1600 (C@N), 1252 cmꢀ1 (C–O); 1H NMR (300 MHz, CDCl3,
d = ppm) d = 1.16 (t, 3H, N-CH2-CH3, J = 7.2 Hz), 3.84 (q, 2H, N-
CH2-CH3, J = 7.2 Hz), 3.85 (s, 3H, OCH3), 4.60 (s, 2H, SCH2), 7.08
(d, 2H, J = 8.8 Hz, Ar-H) 7.53–7.80 (m, 5H, Ar-H), 8.03 (d, 2H,
J = 7.2 Hz, Ar-H), 10.66 (s, 1H, OH); 13C NMR (125 MHz, CDCl3,
d = ppm) d = 11.92 (–CH2CH3), 26.41 (–SCH2), 38.22 (CH2CH3),
56.64 (CH3), 106.50 (CH), 120.09 (CH), 121.83 (C), 127.14 (CH),
132.32 (CH), 133.72 (CH), 139.28 (C), 140.49 (C), 154.21 (C),
m
3500–3000 (br, OH), 3052 (CH aromatic), 2837 (CH aliphatic),
1611 (C@C), 1496 (C@N), 1253 cmꢀ1 (C–O); 1H NMR (300 MHz,
CDCl3 + CD3OD, d = ppm) d = 3.77 (s, 3H, OCH3), 4.62 (s, 2H, SCH2),
6.79 (d, 2H, J = 9 Hz, Ar-H), 7.12–7.15 (m, 2H, Ar-H), 7.30–7.49
(m, 8H, Ar-H), 7.71–7.74 (m, 2H, Ar-H), 8.08 (br s, 1H, OH); 13C
NMR (75 MHz, CDCl3 + CD3OD, d = ppm) d = 25.61 (–SCH2), 55.24
(CH3), 114.05 (CH), 118.88 (CH), 125.89 (CH), 127.82 (CH), 128.53
(CH), 129.16 (C), 129.43 (CH), 129.87 (CH), 129.98 (C), 133.99 (C),

3848
M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
134.30 (CH), 150.53 (C), 151.92 (C), 154.59 (C), 160.26 (C). EI-MS
(70 eV) m/z (%) 399 (M+ÅꢀH2O, 0.1), 315 (33), 282 (100), 266 (34),
133 (52), 103 (44), 77 (52). Anal. Calcd for C23H20N4O2S (416.13):
C, 66.33; H, 4.84; N, 13.45. Found: C, 65.97; H, 4.91; N, 13.68.
solution (0.5% w/v in water) was administered orally at 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 pre-
pared 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 celiper (SMIEC) directly before and after
1, 2, 3, 4 and 5 h intervals after carrageenan injection. The anti-
inflammatory activity of the tested compounds and indomethacin
was calculated as the percentage decrease in edema thickness in-
duced by carrageenan.
4.1.3.11. 2-[5-(3,4-Dimethoxyphenyl)-4-phenyl-4H-[1,2,4]tria-
zol-3-ylsulfanyl]-1-phenylethanone oxime 6k.
tals (ethanol) in (0.116 g, 87% yield); mp 182–183 °C; FT-IR
max): 3500–3100 (br, OH), 3144 (CH aromatic), 2837 (CH ali-
White crys-
(m
phatic), 1606 (C@C), 1534 (C@N), 1258 cmꢀ1 (C–O) cmꢀ1 (C–O);
1H NMR (400 MHz, CDCl3, CD3OD, d = ppm) d = 3.66 (s, 3H, OCH3),
3.84 (s, 3H, OCH3), 4.64 (s, 2H, SCH2), 6.69–7.73 (m, 13H, Ar-H),
8.77 (br s, 1H, OH); 13C NMR (100 MHz, CDCl3 + CD3OD, d = ppm)
d = 27.57 (–SCH2), 56.69 (CH3), 56.91 (CH3), 112.31 (CH), 112.34
(CH), 119.53 (CH), 122.72 (C), 127.46 (CH), 128.89 (CH), 129.67
(CH), 130.53 (CH), 131.20 (CH), 131.36 (CH), 135.17 (C), 135.62
(C), 149.94 (C), 151.78 (C), 153.20 (C), 154.06 (C), 156.59 (C). EI-
MS (70 eV) m/z (%) 446 (M+Å, 1.2), 316 (100), 315 (77), 298 (65),
103 (47), 77 (50). Anal. Calcd for C24H22N4O3S (446.14): C, 64.56;
H, 4.97; N, 12.55. Found: C, 64.92; H, 5.24; N, 12.27.
4.3.2. Ulcerogenic liability
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 mmꢀ2, level II, in which ulcer area is in the range from 1–
3 mm2 and level III, in which the ulcer area more than 3 mm2
and this rated according to their areas in mm2.
4.1.3.12. 2-[5-(3,4,5-Trimethoxyphenyl)-4-phenyl-4H-[1,2,4]
triazol-3-ylsulfanyl]-1-phenylethanone oxime 6l.
crystals (ethanol) in (0.107 g, 75% yield); mp 180–181 °C; FT-IR
max): 3600–3100 (br, OH), 3054 (CH aromatic), 2837 (CH ali-
White
(
m
phatic), 1588 (C@C), 1489 (C@N), 1125 cmꢀ1 (C–O); 1H NMR
(500 MHz, CDCl3, d = ppm) d = 3.57 (s, 6H, 2 OCH3), 3.81 (s, 3H,
OCH3), 4.66 (s, 2H, SCH2), 6.62 (s, 2H, Ar-H), 7.17–7.73 (m, 10H,
Ar-H), 9.47 (br s, 1H, OH); 13C NMR (125 MHz, CDCl3, d = ppm)
d = 25.66 (–SCH2), 55.80 (CH3), 60.87 (CH3), 105.19 (CH), 126.41
(C), 127.54 (CH), 128.58 (CH), 129.63 (CH), 129.96 (CH), 129.99
(C), 133.81 (C), 134.22 (C), 139.10 (C), 152.97 (C), 154.30 (C),
154.81 (C). EI-MS (70 eV) m/z (%) 476 (M+Å, 3), 343 (14), 327
(21), 251 (14), 232 (14), 169 (18), 135 (21), 103 (100), 77 (78).
Anal. Calcd for C25H24N4O4S (476.15): C, 63.01; H, 5.08; N,
11.76. Found: C, 62.85; H, 5.27; N, 11.73.
The data are expressed as mean SEM, one way ANOVA test was
applied to determine the significance of the difference between the
control group and rats treated with the tested compounds.
4.3.3. Histopathological investigation
The histological slides were prepared according to the reported
procedures for examination of ulcers under light microscope.52
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 5–7 min, washed with tap water until sec-
tioning for 5 min and immersed for 5–10 s in solution of (1% HCl
in 70% alcohol), then washed well with tap water for 10–15 min
followed by staining with 1% Eosin for 10 min, washed with run-
ning tap water for 1–5 min. The slide was then dehydrated using
alcohols, cleaned by using xylene, covered by glass cover using
Canda balsam then examined under microscope (magnification
ꢁ400).
4.2. Nitric oxide release measurement51
Different solutions of the tested compounds 6a–l (20 lL) in
DMF was added to 2 mL of 1:1 v/v mixture of 50 mM phosphate
buffer (pH 7.4) with MeOH, containing 5 ꢁ 10ꢀ4 M of N-acetylcys-
teine. The final concentration of drug was 10ꢀ4 M. After 1 h at
37 °C, 1 mL of the reaction mixture was treated with 250 lL of Gri-
ess reagent [sulfanilamide (2 g), N-naphthylethylenediamine dihy-
drochloride (0.2 g), 85% phosphoric acid (10 mL) in distilled water
(final volume: 100 mL)]. After 10 min at room temperature, the
absorbance was measured at k 546 nm. Sodium nitrite standard
solutions (10–80 nmol/mL) were used to construct the calibration
curve. The same procedure was repeated using different solutions
of the test compounds under the same conditions using 0.1 N HCl
of pH 1 instead of phosphate buffer of pH 7.4. The results were ex-
pressed as the percentage of NO released relative to a theoretical
maximum release of 1 mol NO/mol of test compound.
4.3.4. Antiproliferative activity
The methodology of the NCI anticancer screening has been de-
scribed in detail elsewhere (http://www.dtp.nci.nih.gov). Briefly,
the primary anticancer assay was performed at approximately 60
human tumor cell lines panel derived from nine neoplastic dis-
eases, in accordance with the protocol of the Drug Evaluation
Branch, National Cancer Institute, Bethesda. Tested compounds
were added to the culture at a single concentration (10ꢀ5 M) and
the cultures were incubated for 48 h. End point determinations
were made with a protein binding dye, SRB. Results for each tested
compound were reported as the percent of growth of the treated
cells when compared to the untreated control cells. The percentage
growth was evaluated spectrophotometrically versus controls not
treated with test agents.
4.3. Biological evaluation
4.3.1. Anti-inflammatory activity
The experiments were performed on adult male albino rats,
weighing (120–140 g), obtained from the animal house, Minia Uni-
versity. 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 activity
of the compounds under investigation was studied using carra-
geenan. A suspension of the tested compounds 5a–l and 6a–l and
reference drug (indomrthacin) in carboxy methyl cellulose (CMC)
Acknowledgments
Authors introduce their great thanks to Professor Dr. Entesar Ali
Saber and Dr. Sarah M. N. Abdel-Hafez, Histology Department, Fac-
ulty of Medicine, Minia University for their great help in perform-
ing the histopathological investigation. Authors thank also the
Development Therapeutics Program of the National Cancer

M. Abdel-Aziz et al. / Bioorg. Med. Chem. 21 (2013) 3839–3849
3849
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