1,2,4-Triazole/oxime hybrids as new strategy for nitric oxide donors: Synthesis, anti-inflammatory, ulceroginicity and antiproliferative activities

Article abstract of DOI:10.1016/j.ejmech.2013.11.006

A series of novel nitric oxide (NO) donating triazole/oxime hybrids was prepared and evaluated for their anti-inflammatory activity and antiproliferative 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 ketone intermediates and indomethacin. The NO-donating oximes 7i and 7k achieved remarkable cell growth inhibition activity against most of the tested cell lines. Compound 7k was found to be with high selectivity against CNS subpanel with selectivity ratio of 11.99 at GI ₅₀ level.

Full text of DOI:10.1016/j.ejmech.2013.11.006

European Journal of Medicinal Chemistry 71 (2014) 185e198
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1,2,4-Triazole/oxime hybrids as new strategy for nitric oxide donors:
Synthesis, anti-inflammatory, ulceroginicity and antiproliferative
activities
*
Gamal El-Din A.A. Abuo-Rahma, Mohamed Abdel-Aziz , 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 and antiproliferative activity. Most of the tested compounds showed signifi-
cant anti-inflammatory activity using carrageenan-induced rat paw edema method compared to indo-
methacin. Calculation of the ulcer indices and histopathological investigation indicated that the prepared
NO-donating oximes exhibited less ulcerogenicity compared to their ketone intermediates and indo-
methacin. The NO-donating oximes 7i and 7k achieved remarkable cell growth inhibition activity against
most of the tested cell lines. Compound 7k was found to be with high selectivity against CNS subpanel
with selectivity ratio of 11.99 at GI50 level.
Received 10 June 2013
Received in revised form
30 October 2013
Accepted 2 November 2013
Available online 12 November 2013
Keywords:
1,2,4-Triazole
Nitric oxide donors
Anti-inflammatory
Ulcerogenicity
Antiproliferative
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
renal system through increasing the mucosal blood flow [25].
Additionally, it was reported that alkylthio-3-(3,4-dimethoxy-
1,2,4-Triazole derivatives represent an interesting class of het-
erocyclic compounds, they possess many biological activities such
as antimicrobial [1,2], anti-tubercular [3], anti-inflammatory [4e
10], analgesic [10] and anticancer activities [11e15]. In recent de-
cades, NO has attracted a tremendous interest in a broad field of
basic and applied research as one of the most significant physio-
logical signaling molecule in the body. One of the most important
strategies used to overcome NSAIDs side effects is designing nitric
oxide-donating NSAIDs (NO-NSAIDs), which are capable of gener-
ating the radical biomediator and gastroprotective NO [16,17]. It
was reported that NO plays several physiological functions in the
digestive system [18] such as; increasing the mucosal blood flow
[19] which results in enhancement of the mucosal resistance to
ulceration [20], preventing adherence of leukocytes to the vascular
endothelium [21] and modulating gastroduodenal secretion of both
mucus [22] and bicarbonate [23]. Moreover, NO can profoundly
influences the mucosal immune system [18] and increases the
ability of ulcerated mucosal cells to undergo healing and repair
[24]. Also, the vasodilatation effect of NO is known to spare the
phenyl)-4H-1,2,4-triazole derivatives exhibits high anti-inflamm-
tnqh_0009;atory activity with low acute toxicity [26]. Y.-P. Hou
et al. [11] reported that the triazole derivative I (Fig. 1) exhibited
potent inhibitory activity against HEPG2 cancer cell line growth. X.
Ouyang et al. [12] reported that the triazole derivative II (Fig. 1)
exhibited 100% inhibition of tubulin polymerization in vitro and
induced G2/M arrest of A431 human cancer cells with EC50 similar
to Combretastatin A4.
The application of NO donors as cancer therapeutics is a new
venue; the literature provides evidence that metabolic NO release
mediates the cytotoxic activities against different cancer cell lines
[27e30]. Moreover, NO can prevent tumor cells from metastasizing
and assist macrophage to kill tumor cells [31]. Several targets have
been reported for the combination between NO and cancer therapy
including either synergistic effect between the anticancer drugs
and NO [32,33], increasing the influx of the anticancer therapy by
NO into intracellular compartments [34], or increasing the effi-
ciency of cytostatic therapy and retard the development of drug
resistance to anticancer agents [35].
NO-NSAIDs are considered promising anticancer agents, in vitro
and in vivo studies indicated that NCX 4040 (Fig. 2) shows a
promising anticancer activity, compared to its parent aspirin [36].
Moreover, the NO-ketoprofen hybrid (Fig. 2) exhibits significant
* Corresponding author. Tel.: þ20 1003311327; fax: þ20 862369075.
E-mail addresses: abulnil@hotmail.com, abulnil2002@yahoo.com (M. Abdel-
Aziz).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.006

186
G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
N
O
N
N
N
N
O
N
H
N
N
H
O
S
O
O
F
OCH3
H3CO
I
II
Fig. 1. Structure of some 1,2,4-triazole derivatives that have strong cell growth inhibition.
anti-proliferative activity against PC-3 cells. Additionally, several
reports indicated that oximation of the carbonyl group in some
compounds enhances the anticancer activity several folds
compared to their corresponding ketones [37,38].
the corresponding ketone intermediates 6ael in 62e85% yield. In
the 1H NMR spectra for compounds 6ael; three singlet peaks are
common and appeared at
d
10.68e10.70 ppm related to (NH)
2.49 ppm
proton, at 4.19e4.24 ppm related (SeCH2eCO) and at
d
d
Promoted with the above-mentioned studies and as a continu-
ation of our research interest in the field of synthesis and biological
evaluation of NO-NSAIDs hybrids [39e41]. 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 struc-
ture for the purpose of synergism and/or minimizing the expected
ulcerogenic side effects. A new linker has been designed aiming for
improvement of the amount of NO released from these hybrids
compared to our recent research work; by avoiding the expected
hydrogen bonding in the previous linker [42]. The prepared tri-
azole/NO hybrids are evaluated for their anti-inflammatory activity
using carrageenan-induced rat paw edema and compared to the
well-known NSAID, indomethacin. Calculation of ulcer indices and
histopathological investigation was carried out to assess the
beneficial effects of the NO in decreasing ulcer formation. The
prepared triazole/NO hybrids were also evaluated for their anti-
proliferative activity using different cancer cell lines in order to
investigate the possibility of contributing synergistically to their
potential antiproliferative effect.
which related to (COeCH3). The target NO releasing oxime de-
rivatives 7ael were prepared in high yield by heating at reflux
ketone intermediates 6ael and hydroxylamine hydrochloride in
ethanol. The chemical structure of the prepared compounds was
elucidated on the basis of their IR, 1H NMR, 13C NMR, mass spectra
as well as the elemental analyses. A characteristic feature of the 1H
NMR spectra for oximes 7ael is the appearance of downfield sin-
glets in the range of
d 7.76e11.10 ppm, related to the hydroxyl
group. The CH3 protons appeared to be more upfield shifted by
0.23e0.42 ppm than the CH3 protons of the corresponding ketones
due to the low electronegativity of N atom of the oxime relative to O
atom of the ketone. The 13C NMR spectra of compounds 7d, 7g and
7j showed one carbonyl group of the amide at
and disappearance of the ketonic carbonyl due to its conversion to
ketoxime group (C]NeOH) which appear at 155.98e160.89 ppm.
d 166.41e166.72 ppm
d
A characteristic feature of the mass spectra of the oximes 7ael is
the appearance of a very weak abundance for the molecular ion
peaks from 0.1 to 17% of the respective base peak. Kallury and Rao
[44] reported that the abundances of some oximes are very low
(less than 4%) of the corresponding base peak.
2. Results and discussion
2.2. Measurement of nitric oxide release
2.1. Chemistry
The NO releasing properties of the prepared NO-donating ox-
imes 7ael 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 N 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 Table 1.
The results of measurement of NO release revealed that the NO-
donating oximes 7ael release NO at pH of 7.4 after 4 h. Com-
pound 7g that contains 3,4-dimethoxyphenyl moiety released the
highest amount of this group (0.43 mol/mol). The results of NO
4-Allyl/ethyl/phenyl-5-aryl-4H-1,2,4-triazole-3-thiol
rivatives 4ael were synthesized as outlined in Scheme 1 according
to the reported procedure [5].
The synthesis of the target compounds 1-phenyl-2-((4-allyl/
ethyl/phenyl-5-aryl-4H-1,2,4-triazol-3-yl)thio)ethanone
7ael is illustrated in Scheme 2.
de-
oxime
N-(4-Acetylphenyl)-2-bromoacetamide
5 was prepared in
high yield according to the reported procedure [43] through
treatment of p-aminoacetophenone with bromoacetyl bromide in
the presence of potassium carbonate. Heating at reflux of the 1,2,4-
triazole-3-thiol derivatives 4ael with N-(4-acetylphenyl)-2-
bromoacetamide 5 in acetonitrile in the presence of TEA afforded
O
O
CH3
ONO2
O2NO
O
O
O
O
ONO2
CH3
O
NCX 4040
Ketoprofen-NO hybrid
Fig. 2. NCX 4040 and ketoprofeneNO hybrids.

G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
187
O
O
O
EtOH
NH2NH2. H2O
EtOH
RNCS
EtOH
H2SO4
Ar
O
Ar
NH
Ar
OH
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
3a-l
R
4a-l
Compound
1a, 2a
1b, 2b
1c, 2c
Ar
Ph
Compound
3e, 4e
3f, 4f
R
Ar
Ph
---
---
---
---
Et
Et
4-OCH3-Ph
4-OCH3-Ph
3,4-di-OCH3-Ph
3,4,5-tri-OCH3-Ph
Ph
3g, 4g
3h, 4h
3i, 4i
Et
3,4-di-OCH3-Ph
3,4,5-tri-OCH3-Ph
Ph
1d, 2d
3a, 4a
3b, 4b
3c, 4c
Et
allyl
allyl
allyl
Ph
Ph
Ph
Ph
4-OCH3-Ph
3j, 4j
4-OCH3-Ph
3,4-di-OCH3-Ph
3k, 4k
3l, 4l
3,4-di-OCH3-Ph
3,4,5-tri-OCH3-Ph
3d, 4d
allyl 3,4,5-tri-OCH3-Ph
Scheme 1. Synthesis of 4-allyl/ethyl/phenyl-5-aryl-4H-1,2,4-triazole-3-thiol derivatives 4ael.
release indicated that the target compounds released several folds
amount of NO compared to the previously prepared NO-triazole
hybrids [42].
The result also indicated that NO-donating compounds were not
able to release NO at pH 1, which may support the fact that these
compounds are weakly hydrolyzed in the gastric lumen and con-
firms that the suggested gastroprotective action of NO is mediated
systemically [45].
derivatives 7ael and indomethacin versus time in hours. Most of
the tested compounds showed a significant anti-inflammatory ac-
tivity against carrageenan-induced paw edema in rats (p < 0.01).
Indomethacin showed an inhibitory activity of 83% against
carrageenan-induced paw edema after 4 h, which is the time
required to reach the maximum activity for most of the tested
compounds, then the activity decreased in the next hour. Com-
pounds 6ael exhibited from 62% to 70% anti-inflammatory activity
after the fourth hour representing from 74% to 85% of indomethacin
activity.
2.3. Biological investigations
Furthermore, oxime derivatives 7ael exhibited high anti-
inflammatory activity after the fourth hour ranging from 67% to
79% representing 81%e96% of indomethacin activity, respectively.
Several conclusions could be deduced from the above mentioned
results; slight improvement for the anti-inflammatory activity was
achieved by the oxime derivatives 7ael compared to their ketone
intermediates 6ael that may attributed to the synergistic effect of
NO and/or change of the physichochemical properties of the final
oximes 7ael than their corresponding ketone intermediates 6ael.
Compounds 7c (R ¼ ethyl, Ar ¼ 3,4-di-OCH3ePh) and 7k (R ¼ Ph,
Ar ¼ 3,4-di-OCH3ePh) exhibited the highest anti-inflammatory
activity among the tested compounds that may explain the
impact of the presence of the 3,4-di-OCH3ePh moiety on the anti-
inflammatory activity of the tested compounds which is in agree-
ment with the scope of this work. The results also showed that
there is no great difference between allyl, ethyl and phenyl sub-
stituents on the triazole nucleus on the anti-inflammatory activity
of these compounds.
2.3.1. Screening of anti-inflammatory activity
The synthesized compounds 6ael and 7ael were evaluated for
their anti-inflammatory activity using carrageenan-induced paw
edema in rats described by Winter et al. [46]. The tested com-
pounds 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
ðVR ꢀ VLÞcontrol
ꢁ 100
where VR represents the mean right paw thickness and VL repre-
sents the mean left paw thickness.
2.3.2. Screening of ulcerogenicity
The ulcerogenicity was evaluated according to the reported
procedure [47] for the synthesized compounds 6ael and 7ael
relative to indomethacin. Ulcerogenicity was evaluated in rats after
oral administration of the tested compounds and the reference
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.
(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
induced by carrageenan for the ketone intermediates 6ael, oxime

188
G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
Compound
6a, 7a
6b, 7b
6c, 7c
R
Ar
Ph
Compound
6g, 7g
6h, 7h
6i, 7i
R
Ar
Allyl
Allyl
Allyl
Et
3,4-di-OCH3-Ph
3,4,5-tri-OCH3-Ph
Ph
4-OCH3-Ph
3,4-di-OCH3-Ph
Et
Ph
Ph
Ph
Ph
Allyl 3,4,5-tri-OCH3-Ph
4-OCH3-Ph
6d, 7d
6e, 7e
6j, 7j
Et
Et
Ph
3,4-di-OCH3-Ph
3,4,5-tri-OCH3-Ph
6k, 7k
6l, 7l
4-OCH3-Ph
6f, 7f
Scheme 2. Preparation of N-(4-(1-(hydroxyimino)ethyl)phenyl)-2-[(4-allyl/ethyl/phenyl-5-aryl-4H-1,2,4-triazol-3-yl)thio]acetamide 7ael.
Ulcers were classified into levels, level I, in which the ulcer area
The UI of compounds 6ael and 7ael were calculated and listed
in Table 3 (mean ꢂ SEM). The results revealed that indomethacin
caused significant ulcerogenic toxicity with UI of 63.34, whereas an
equal dose of most of the synthesized compounds exhibited much
safer UI compared to indomethacin. For example, the ketone de-
rivatives 6ael exhibited UI ranging from 2.45 to 10.52, while their
corresponding NO-donating oxime analogs 7ael exhibited a very
low significant ulcerogenicity compared to both indomethacin and
their ketone intermediates with UI ranging from of 0.25e3.43
(Table 3 and Fig. 3AeC).
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,
where the following parameters were calculated [48]:
1 -The ulcer index (UI) was calculated as follows:
1 ꢁ (number of ulcers level I) þ 2 ꢁ (number of ulcers level
II) þ 3 ꢁ (number of ulcers level III), etc.
2 -Cure ratio ¼ 100 ꢀ (UItreated ꢁ 100/UIprotype
)
The cure ratio of the target NO-donating oximes 7ael relative
to their ketone intermediates 6ael was calculated and listed in
Table 3. The results revealed that the NO-donating oximes 7ael
achieved a great reduction of ulcers than their corresponding
ketone intermediates 6ael. Several conclusions could be deduced
from the above mentioned results; all the tested compounds
where, UItreated: means the average of the UI of the groups of rats
treated with the NO-donating derivatives.
UIprotype: means the average of the UI of the groups of rats
treated with the starting and the intermediate derivatives.
Table 1
Amount of NO released (n ¼ 4, number of reaction mixtures assayed for each compound) determined by Griess reagent using 0.1 mM of the tested compounds7ael 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
7a
7b
7c
7d
7e
7f
7g
7h
7i
0.15 ꢂ 0.015
0.15 ꢂ 0.052
0.13 ꢂ 0.021
0.11 ꢂ 0.010
0.11 ꢂ 0.009
0.16 ꢂ 0.005
0.15 ꢂ 0.003
0.12 ꢂ 0.005
0.11 ꢂ 0.017
0.13 ꢂ 0.009
0.15 ꢂ 0.011
0.11 ꢂ 0.004
0.20 ꢂ 0.012
0.17 ꢂ 0.026
0.16 ꢂ 0.030
0.15 ꢂ 0.011
0.17 ꢂ 0.014
0.20 ꢂ 0.010
0.19 ꢂ 0.009
0.13 ꢂ 0.004
0.15 ꢂ 0.013
0.17 ꢂ 0.006
0.20 ꢂ 0.013
0.14 ꢂ 0.007
0.22 ꢂ 0.030
0.23 ꢂ 0.022
0.32 ꢂ 0.031
0.19 ꢂ 0.014
0.20 ꢂ 0.026
0.24 ꢂ 0.019
0.25 ꢂ 0.025
0.14 ꢂ 0.021
0.20 ꢂ 0.016
0.24 ꢂ 0.011
0.23 ꢂ 0.017
0.16 ꢂ 0.006
0.24 ꢂ 0.035
0.32 ꢂ 0.022
0.35 ꢂ 0.037
0.24 ꢂ 0.019
0.21 ꢂ 0.023
0.29 ꢂ 0.012
0.43 ꢂ 0.016
0.22 ꢂ 0.010
0.23 ꢂ 0.028
0.29 ꢂ 0.013
0.39 ꢂ 0.020
0.25 ꢂ 0.011
0.16 ꢂ 0.034
0.23 ꢂ 0.019
0.27 ꢂ 0.035
0.18 ꢂ 0.041
0.14 ꢂ 0.009
0.24 ꢂ 0.014
0.36 ꢂ 0.012
0.15 ꢂ 0.008
0.14 ꢂ 0.028
0.24 ꢂ 0.013
0.35 ꢂ 0.021
0.15 ꢂ 0.010
0.15 ꢂ 0.035
0.16 ꢂ 0.021
0.26 ꢂ 0.040
0.16 ꢂ 0.033
0.12 ꢂ 0.013
0.23 ꢂ 0.010
0.32 ꢂ 0.011
0.14 ꢂ 0.002
0.16 ꢂ 0.026
0.23 ꢂ 0.012
0.31 ꢂ 0.018
0.14 ꢂ 0.011
7j
7k
7l

G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
189
Table 2
The anti-inflammatory activity at different time intervals for the ketone intermediates 6ael and their corresponding oximes 7ael using carrageenan-induced paw edema in
rats intermediates 6ael and their corresponding oximes 7ael using carrageenan-induced paw edema in rats.
Compound number
% Of edema inhibition (% mean ꢂ SEM)
1 h
2 h
3 h
4 h
5 h
Control
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
0
0
0
0
0
48 ꢂ 1.43***
50 ꢂ 1.04***
56 ꢂ 2.41***
48 ꢂ 1.94***
50 ꢂ 1.84***
51 ꢂ 2.48***
56 ꢂ 1.64***
46 ꢂ 1.93***
41 ꢂ 1.70***
29 ꢂ 1.69**
28 ꢂ 1.38**
49 ꢂ 1.81***
61 ꢂ 1.15***
54 ꢂ 2.31***
53 ꢂ 1.14***
49 ꢂ 0.51***
54 ꢂ 1.25***
54 ꢂ 1.34***
53 ꢂ 1.45***
29 ꢂ 1.73**
50 ꢂ 1.65***
45 ꢂ 1.47***
57 ꢂ 1.52***
59 ꢂ 2.43***
51 ꢂ 0.78***
52 ꢂ 2.41***
51 ꢂ 1.53***
57 ꢂ 3.13***
54 ꢂ 1.12***
53 ꢂ 1.59***
54 ꢂ 3.54***
59 ꢂ 0.84***
50 ꢂ 1.71***
49 ꢂ 2.25***
42 ꢂ 1.49***
43 ꢂ 3.08***
54 ꢂ 2.13***
63 ꢂ 1.70***
59 ꢂ 1.27***
56 ꢂ 3.06***
53 ꢂ 1.17***
57 ꢂ 2.11***
58 ꢂ 3.15***
65 ꢂ 3.25***
49 ꢂ 1.02***
52 ꢂ 1.89***
56 ꢂ 2.73***
63 ꢂ 2.92***
62 ꢂ 1.70***
67 ꢂ 1.36***
54 ꢂ 2.89***
52 ꢂ 2.74***
58 ꢂ 1.89***
55 ꢂ 3.42***
56 ꢂ 1.41***
57 ꢂ 2.22***
62 ꢂ 1.45***
54 ꢂ 1.84***
62 ꢂ 2.60***
58 ꢂ 3.13***
51 ꢂ 2.46***
55 ꢂ 1.90***
73 ꢂ 2.31***
63 ꢂ 1.28***
70 ꢂ 0.76***
62 ꢂ 1.27***
65 ꢂ 4.17***
60 ꢂ 2.16***
66 ꢂ 0.45***
70 ꢂ 1.98***
63 ꢂ 2.79***
57 ꢂ 2.84***
77 ꢂ 3.02***
66 ꢂ 0.74***
74 ꢂ 2.17***
70 ꢂ 2.61***
68 ꢂ 3.21***
70 ꢂ 3.22***
68 ꢂ 2.61***
69 ꢂ 0.45***
65 ꢂ 1.86***
69 ꢂ 2.91***
68 ꢂ 1.61***
67 ꢂ 2.44***
63 ꢂ 1.06***
62 ꢂ 4.47***
68 ꢂ 1.12***
73 ꢂ 3.06***
75 ꢂ 1.15***
79 ꢂ 0.31***
72 ꢂ 2.54***
77 ꢂ 0.15***
73 ꢂ 2.56***
75 ꢂ 2.27***
70 ꢂ 1.47***
67 ꢂ 0.28***
69 ꢂ 1.92***
79 ꢂ 0.62***
75 ꢂ 2.03***
83 ꢂ 1.41***
63 ꢂ 2.45***
66 ꢂ 1.18***
64 ꢂ 2.46***
64 ꢂ 1.49***
66 ꢂ 2.86***
62 ꢂ 0.42***
66 ꢂ 0.86***
64 ꢂ 0.21***
63 ꢂ 1.64***
60 ꢂ 2.97***
55 ꢂ 1.86***
63 ꢂ 2.03***
71 ꢂ 1.33***
72 ꢂ 1.47***
79 ꢂ 2.31***
64 ꢂ 1.39***
66 ꢂ 0.35***
65 ꢂ 3.19***
73 ꢂ 2.65***
61 ꢂ 3.45***
66 ꢂ 2.34***
58 ꢂ 0.73***
76 ꢂ 2.32***
70 ꢂ 3.33***
87 ꢂ 0.47***
7l
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 ¼ 4),
**p < 0.01, ***p < 0.001, significant difference from control group.
achieved a very low significant ulcerogenicity compared to indo-
methacin that may be attributed to the beneficial effect of
the triazole nucleus as less ulcerogenic bioisostere of the carboxyl
function. The oxime derivatives produce lower UI than their
corresponding ketones that may be attributed to the synergistic
effect of the NO and the triazole nucleus. The NO donating oxime
and leukocytes infiltration. The gastric glands restore their normal
arrangement and this was confirmed by its low UI, which was 0.25.
In contrast the group treated with its ketone derivative 6c (Fig. 4C)
exhibited changes in the structure of gastric mucosa through the
loss of mucosal membrane at the areas of ulceration and the
morphology of certain glandular structures were lost.
7c (R
¼
allyl, Ar
¼
3,4-di-OCH3ePh) achieved high anti-
In conclusion, the histopathological investigation confirms the
previously mentioned ulcerogenicity results of the tested com-
pounds and supports the effect of NO as a gastroprotective agent
inflammatory activity after 4 h (79%) and in the same time
exhibited lower ulcerogenicity (0.25) among the synthesized ox-
imes 7ael. The decreased gastric toxicity of the targeted NO-
triazole hybrids 7ael compared to their starting ketone in-
termediates 6ael may be attributed to the release of NO that
increases mucosal blood flow resulting in enhanced mucosal
resistance to ulceration [49,50].
Table 3
Ulcer indices of compounds 6ael and 7ael compared to indomethacinexpressed as
mean ꢂ SEM and cure ratio (%) of oximes relative to its starting ketones expressed as
mean ꢂ SEM and cure ratio (%) of oximes relative to its starting ketones.
Compound
number
Ulcer index (UI) Compound Ulcer index (UI) Cure ratio (%)
2.3.3. Histopathological investigation
(mean ꢂ SEM)
number
(mean ꢂ SEM)
of oximes
After assessment of gastric mucosal ulcerogenicity and deter-
mination of UI, stomach sections of the ulcers for the control and
the treated groups were stained by standard hematoxylin and eosin
stain. The produced slides were subjected to microscopical exam-
ination and pictures were picked for these slides (Fig. 4AeD).
The control group (Fig. 4A) showed no lesions and characterized
by continuous mucosal layer while the one treated with indo-
methacin (Fig. 4B) showed that gastric mucosa was decreased in
thickness 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 was noticed. Apoptotic glan-
dular epithelial cells could be detected.
relative to
its ketones
intermediates
Control
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
0.60 ꢂ 0.05
9.30 ꢂ 1.19*** 7a
8.66 ꢂ 1.28*** 7b
2.45 ꢂ 0.46*** 7c
6.49 ꢂ 1.09*** 7d
7.12 ꢂ 0.58*** 7e
10.52 ꢂ 0.55*** 7f
3.60 ꢂ 1.06*** 7g
3.34 ꢂ 1.67*** 7h
3.38 ꢂ 0.64*** 7i
5.53 ꢂ 1.19*** 7j
8.64 ꢂ 1.26*** 7k
4.38 ꢂ 0.42*** 7l
0.64 ꢂ 0.05***
2.69 ꢂ 0.55***
0.25 ꢂ 0.04***
0.75 ꢂ 0.06***
1.18 ꢂ 0.10***
1.04 ꢂ 0.11***
3.43 ꢂ 0.56***
0.83 ꢂ 0.05***
0.95 ꢂ 0.14***
1.61 ꢂ 0.39***
1.11 ꢂ 0.48***
2.21 ꢂ 1.19***
93.12
68.94
89.80
88.44
83.43
90.11
4.72
75.15
71.89
70.89
87.15
49.54
Indomethacin 63.34 ꢂ 1.19
The group treated with NO-donating oxime 7c (Fig. 4D) showed
no ulcers. The histological appearance of gastric mucosa showed
improvement e.g. disappearance of the capillary dilatation, edema
Note. One way ANOVA test was applied to determine the significance of the differ-
ence between the control group and rats treated with the tested compounds. (n ¼ 4),
***p < 0.001, significant difference from control group.

190
G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
A
B
80
60
40
20
0
80
60
40
20
***
***
###
****
###
***
###
###
###
###
###
****
###
0
6a 6b 6c 6d 7a 7b 7c 7d Indo
6e 6f 6g 6h 7e 7f 7g 7h Indo
80
C
60
40
20
0
****
*
###
###
###
###
6i 6j 6k 6l 7i 7j 7k 7l Indo
Fig. 3. A. UI of compounds 6aed and 7aed compared to indomethacin (Indo) expressed as mean ꢂ SEM. B. UI of compounds 6eeh and 7eeh compared to indomethacin (Indo)
expressed as mean ꢂ SEM. C. UI of compounds 6iel and 7iel compared to indomethacin (Indo) expressed as mean ꢂ SEM.
through increasing the mucosal blood flow, mucus and bicarbonate
secretion.
TK-10, breast cancer MDA-MB-231/ATCC and HS 578T cell lines
(Table 4).
Compounds 7c, 7e and 7g exhibited a weak cell growth inhibi-
tion against most of the tested cell lines. The obtained results
indicate that the NO-donating oximes 7i and 7k exhibited the
highest ability to inhibit the proliferation of different cancer cell
lines (Table 4) compared to others 7c, 7e and 7g. The presence of
N(4)ephenyl is essential for the antiproliferative activity of the test
compounds over ethyl or allyl (compounds 7i and 7k has the
highest ability to inhibit the proliferation of different cancer cell
lines over compounds 7c, 7e and 7g). In the group of N(4)ephenyl
triazole derivatives, the presence of 3,4-dimethoxyphenyl sub-
stituents on C(5) of triazole nucleus is preferable over the unsub-
stituted phenyl moiety (NO-donating oxime 7k has significant
antiproliferative activity against different cancer cell lines over 7i).
2.3.4. Screening of antiproliferative activity
Compounds 7c, 7e, 7g, 7i and 7k were selected by the National
Cancer Institute (NCI) according to the protocol of the Drug Eval-
uation Branch of the National Cancer Institute [51] Bethesda, USA
for in vitro anticancer screening. Primary in vitro one dose anti-
cancer 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 protein 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 un-
treated control cells.
2.3.5. In vitro five dose full NCI 60 cell panel assay
The NO-donating oxime 7i exhibited remarkable cell growth
inhibition activity against non-small cell lung cancer HOP-92, Colon
cancer HCT-116, ovarian cancer SK-OV-3, and breast cancer BT-549.
A complete cell death was recorded for the non-small cell lung
cancer NCI-H522 cell line where the growth percent was ꢀ34.71. A
moderate cell growth inhibition was achieved against colon cancer
COLO 205, CNS cancer SF-539, SNB-75 and melanoma SK-MEL-5
cell lines (Table 3).
The NO-donating oxime 7k achieved remarkable cell growth
inhibition activity against most of the tested cell lines including
non-small cell lung cancer NCI-H522, CNS cancer SF-268, SF-295,
U251, ovarian cancer SK-OV-3, renal cancer RXF 393 cell lines. A
complete cell death was recorded for the non-small cell lung cancer
HOP-62, HOP-92 and CNS cancer SNB-75 where the growth percent
were ꢀ26.44, ꢀ8.56 and ꢀ16.53, respectively. Compound 7k
revealed moderate cell growth inhibition against CNS cancer SF-
539, melanoma SK-MEL-2, ovarian cancer OVACR-8, renal cancer
Compound 7k was selected for advanced five dose testing
against the full panel of 60 human tumor cell lines. All the 60 cell
lines representing nine tumor subpanels were incubated at five
different concentrations (0.01, 0.1, 1, 10, and 100 mM). The outcomes
were used to create log concentration versus % growth inhibition
curves and three response parameters (GI50, TGI, and LC50) were
calculated for each cell line. The GI50 value (growth inhibitory ac-
tivity) corresponds to the concentration of the compound causing
50% decrease in net cell growth, the TGI value (cytostatic activity) is
the concentration of the compound resulting in total growth inhi-
bition (TGI) and LC50 value (cytotoxic activity) is the concentration
of the compound causing net 50% loss of initial cells at the end of
the incubation period of 48 h. Compound 7k exhibited remarkable
antiproliferative activity against most of the tested cell lines rep-
resenting nine different subpanels. Compound 7k showed high
activity against most of the tested cell lines with GI50 ranging from
2.06 to >100
mM (Table 5). The criterion for selectivity of a

G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
191
Fig. 4. A. Photomicrograph of the mucosa of fundic stomach of control. B. Photomicrograph of the mucosa of fundic stomach treated with indomethacin. C. Photomicrograph of the
mucosa of fundic stomach treated with compound 6c. D. Photomicrograph of the mucosa of fundic stomach treated with compound 7c.
compound depends upon the ratio obtained by dividing the full
panel MID (the average sensitivity of all cell lines toward the test
NSAIDs without altering their effectiveness and may be a challenge
in the field of antiproliferative therapy.
agent) (mM) by their individual subpanel MID (mM). Ratios between
3 and 6 refer to moderate selectivity; ratios >6 indicate high
selectivity toward the corresponding cell line, while compounds
not meeting either of these criteria rated nonselective [52]. In this
context, compound 7k was found to have broad-spectrum anti-
tumor activity against most of the tumor subpanels tested with
selectivity ratios ranging between 0.51 and 11.99 at the GI50 level.
Compound 7k was found to be broad-spectrum antitumor activity
against most of the tested tumor subpanels cell lines with high
selectivity against CNS subpanel with selectivity ratio of 11.99 at
GI50 level (Table 5). Compound 7k exhibited a moderate selectivity
against non-small lung subpanel with selectivity ratio of 2.55 at
GI50 level (Table 5).
4. Experimental section
4.1. Chemistry
Reactions were monitored by TLC, pre-coated plastic sheets,
0.2 mm silica gel F254 with fluorescent indicator (MachereyeNagel).
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). 13
C
3. Conclusions
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
A series of novel triazole-NO hybrids was prepared and charac-
terized by different spectroscopic techniques and elemental analysis.
Most of the synthesized compounds showed significant anti-
inflammatory activity using carrageenan-induced rat paw edema
method. The NO-donating oximes 7ael could release NO at pH 7.4
where the maximum amount was released after 4 h. The prepared
NO-donating oximes hybrids showed pronounced gastroprotective
activity better than their corresponding ketone intermediates that
may be attributed to the release of NO. Histopathological examina-
tion indicated that the NO donating moiety reduced greatly the
incidence of gastric ulceration. The NO-donating oximes 7i and 7k
achieved remarkable cell growth inhibition activity against most of
the tested cell lines. Compound 7k was found to be highly selective
against CNS subpanel with a selectivity ratio of 11.99 at GI50 level. In
summary, the use of hybrid molecules containing NO-donating
moieties looks as a promising approach to improve the safety of
reference. 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
patterns are designated as follows: s, singlet; d, doublet; t, triplet; q,
quartet; dd, doublet of doublet; m, multiplet; bs, broad singlet. EI-
MS was performed on JEOL JMS 600 spectrometers. Elemental
analyses were recorded on Perkin Elmer 2400 CHN, Microanalytical
unit, Faculty of Science, Cairo University, Egypt.
4.1.1. Substituted ethylbenzoate 1aed, benzohydrazides 2aed, 2-
benzoyl-N-allyl/ethyl/phenylthiosemicarbazides 3ael and 4-allyl/
ethyl/phenyl-5-aryl-4H-1,2,4-triazole-3-thiol derivatives 4ael
Substituted ethylbenzoate 1aed, benzohydrazides 2aed, 2-
Benzoyl-N-allyl/ethyl/phenylthiosemicarbazides 3ael and 4-Allyl/

192
G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
ethyl/phenyl-5-aryl-4H-1,2,4-triazole-3-thiol derivatives 4ael was
prepared according to the reported procedure [5].
dropwise manner with stirring over 30 min. Stirring was
continued for 2 h at 0 ꢃC, then at room temperature overnight.
The reaction mixture was extracted with (2 ꢁ 50 ml) dichloro-
methane, and the organic layer was washed with distilled water
(2 ꢁ 50 ml), dried over anhydrous sodium sulfate, filtered,
evaporated on a rotary evaporator and the residue was recrys-
tallized from 95% ethanol to give 1.53 g (95%) of compound 5,
m.p. ¼ 155e156 ꢃC.
4.1.2. Synthesis of N-(4-acetylphenyl)-2-bromoacetamide 5
To a stirred mixture of p-aminoacetophenone (6.30 mmol) in
dichloromethane (20 ml) and potassium carbonate (1.30 mmol)
in 100 ml water cooled in an ice bath, bromoacetyl bromide
(1.86 g, 9.20 mmol) in 30 ml dichloromethane was added in a
4.1.3. General procedure for the synthesis of N-(4-acetylphenyl)-2-
[(4-allyl/ethyl/phenyl-5-aryl-4H-1,2,4-triazol-3-yl)thio]acetamide
6ael
Table 4
Single concentration mean graph growth percent of nine different cancer cell types
for compounds 7i and 7k cell types for compounds 7i and 7k.
An equimolar mixture of 4ael, compound 5 (1 mmol) and TEA
(1.2 mmol) in acetonitrile (50 ml) was heated at reflux for 4e8 h.
The reaction mixture was evaporated to dryness. The residue was
crystallized from aqueous ethanol affording the pure white prod-
ucts 6ael. The structure of compounds 6ael was confirmed by mp,
Rf, IR and 1H NMR spectroscopy.
Panel
Cell line
Growth percent
for compound 7i
Growth percent
for compound 7k
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
92.03
90.42
95.84
95.18
91.16
106.36
81.91
91.89
64.67
30.06
72.38
76.56
66.70
ꢀ34.71
53.29
91.14
28.61
97.47
84.04
73.30
83.11
99.81
92.20
52.61
97.20
45.78
59.23
63.61
82.92
91.75
100.10
118.22
97.74
48.64
94.49
81.85
76.73
81.42
87.28
86.16
84.71
90.76
39.26
80.55
92.72
63.71
85.65
99.90
72.45
76.21
73.73
90.62
104.11
67.90
62.48
20.85
98.47
77.33
105.08
91.71
91.56
95.41
70.71
71.08
ꢀ26.44
ꢀ8.56
81.87
68.75
75.74
102.05
35.30
99.10
94.02
69.23
98.60
91.21
106.14
114.80
30.60
39.07
43.04
75.80
ꢀ16.53
34.38
80.07
61.84
89.91
103.56
57.60
87.31
94.63
91.79
82.83
70.90
82.00
103.49
97.05
42.75
90.56
4.74
4.1.3.1. N-(4-Acetylphenyl)-2-(4-allyl-5-phenyl-4H-[1,2,4]triazol-3-
ylsulfanyl)- acetamide 6a. White crystals (ethanol) in (0.345 g, 88%
yield); mp 196e197 ꢃC; Rf value using CHCl3:CH3OH (9:1) as an
eluent was 0.63; FT-IR (nmax): 3239 (br, NH), 1678 (COeNH, COe
CH3), 1597 (C]C), 1538 cmꢀ1 (C]N); 1H NMR (400 MHz, DMSO-d6,
Non-small
cell lung
cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF-393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
d
¼ ppm) ¼ 2.49 (s, 3H, CH3), 4.21 (s, 2H, SCH2), 4.62e4.63 (m, 2H,
d
NCH2), 4.81 (d, 1H, CH]CH2, Jtrans ¼ 17.6 Hz), 5.21 (d, 1H, CH]CH2,
Jcis ¼ 10.8 Hz), 5.92e5.99 (m, 1H, CH]CH2), 7.51e7.59 (m, 5H, Are
H), 7.68 (d, 2H, J ¼ 8 Hz, AreH), 7.91 (d, 2H, J ¼ 8 Hz, AreH), 10.68 (s,
1H, NH).
Colon cancer
4.1.3.2. N-(4-Acetylphenyl)-2-[4-allyl-5-(4-methoxyphenyl)-4H-
[1,2,4]triazol-3-ylsulfanyl]acetamide 6b. White crystals (ethanol) in
(0.376 g, 89% yield); mp 208e209 ꢃC; Rf value using CHCl3:CH3OH
(9:1) as an eluent was 0.63; FT-IR (nmax): 3256 (br, NH), 1673 (COe
NH, COeCH3), 1595 (C]C), 1536 (C]N), 1250 cmꢀ1 (CeO); 1H NMR
CNS cancer
Melanoma
(400 MHz, DMSO-d6,
d
¼ ppm) ¼ 2.49 (s, 3H, COeCH3), 3.78 (s, 3H,
d
OCH3), 4.19 (s, 2H, SCH2), 4.60e4.61 (m, 2H, NCH2), 4.81 (d, 1H,
CHCH3CH2, Jtrans ¼ 17.2 Hz), 5.20 (d, 1H, CHCH3CH2, Jcis ¼ 10.8 Hz),
5.91e6.00 (m, 1H, CH]CH2), 7.05 (d, 2H, J ¼ 8.8 Hz, AreH), 7.51 (d,
2H, J ¼ 8.4 Hz, AreH), 7.68 (d, 2H, J ¼ 8.4 Hz, AreH), 7.91 (d, 2H,
J ¼ 8.4 Hz, AreH), 10.68 (s, 1H, NH).
4.1.3.3. N-(4-Acetylphenyl)-2-[4-allyl-5-(3,4-dimethoxyphenyl)-4H-
[1,2,4]triazol-3-ylsulfanyl]acetamide 6c. White crystals (ethanol) in
(0.384 g, 85% yield); mp 171e172 ꢃC; Rf value using CHCl3:CH3OH
(9:1) as an eluent was 0.61; FT-IR (nmax): 3258 (br, NH), 1674 (COe
NH, COeCH3), 1595 (C]C), 1535 (C]N), 1266 cmꢀ1 (CeO); 1H NMR
Ovarian cancer
Renal cancer
(400 MHz, DMSO-d6,
d
¼ ppm) ¼ 2.49 (s, 3H, COeCH3), 3.74 (s, 3H,
d
OCH3), 3.78 (s, 3H, OCH3), 4.19 (s, 2H, SCH2), 4.63e4.64 (m, 2H,
NCH2), 4.85 (d, 1H, CH]CH2, Jtrans ¼ 17.2 Hz), 5.23 (d, 1H, CH]CH2,
Jcis ¼ 10.8 Hz), 5.94e6.03 (m, 1H, CH]CH2), 7.05e7.12 (m, 3H, Are
H), 7.68 (d, 2H, J ¼ 8.4 Hz, AreH), 7.91 (d, 2H, J ¼ 8.8 Hz, AreH),
10.68 (s, 1H, NH).
64.48
91.78
75.54
87.36
32.35
71.81
41.08
104.17
109.75
88.28
69.24
47.86
4.1.3.4. N-(4-Acetylphenyl)-2-[4-allyl-5-(3,4,5-trimethoxyphenyl)-
4H-[1,2,4]- triazol-3-ylsulfanyl]acetamide 6d. White crystals
(ethanol) in (0.434 g, 90% yield); mp 196e197 ꢃC; Rf value using
CHCl3:CH3OH (9:1) as an eluent was 0.58; FT-IR (nmax): 3247 (br,
NH), 1672 (COeNH, COeCH3), 1600, 1585 (C]C), 1547 (C]N),
Prostate cancer
Breast cancer
1120 cmꢀ1 (CeO); 1H NMR (400 MHz, DMSO-d6,
d
¼ ppm) ¼ 2.49
d
MDA-MB-231/
ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
(s, 3H, COeCH3), 3.70 (s, 3H, OCH3), 3.78 (s, 6H, 2 OCH3), 4.23 (s, 2H,
SCH2), 4.68e4.69 (m, 2H, NCH2), 4.91 (d, 1H, CH]CH2,
Jtrans ¼ 17.2 Hz), 5.27 (d, 1H, CH]CH2, Jcis ¼ 10.8 Hz), 5.99e6.08 (m,
1H, CH]CH2), 6.86 (s, 2H, AreH), 7.70 (d, 2H, J ¼ 8.8 Hz, AreH), 7.93
(d, 2H, J ¼ 8.8 Hz, AreH), 10.70 (s, 1H, NH).
79.56
75.35
92.03
90.42
42.83
65.18
67.95
78.51

G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
193
Table 5
NCI in vitro testing results of compound 7k at five dose level in
mM.
Panel
Cell line
GI50
TGI
LC50
Conc. per cell line
Subpanel MIDb
Selectivity ratio (MIDa/MIDb)
0.51
Conc. per cell line
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
A549/ATCC
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
U251
>100
>100
>100
>100
>100
>100
9.73
2.06
2.12
7.28
12.90
4.41
>100
21.4
>100
>100
11.3
>100
>100
>100
>100
3.07
3.39
4.77
6.43
3.62
>100
17.20
97.4
>100
5.04
>100
>100
>100
>100
>100
>100
>100
>100
6.80
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Non-small cell lung cancer
19.99
87.33
2.55
0.58
6.42
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
20.3
37.7
38.8
>100
19.6
>100
>100
>100
>100
28.8
Colon cancer
CNS cancer
Melanoma
4.26
11.99
0.64
LOX IMVI
MALME-3M
M14
79.96
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF-393
SN12C
TK-10
UO-31
PC-3
DU-145
>100
>100
>100
>100
3.74
>100
>100
>100
>100
29.4
>100
>100
>100
>100
>100
13.7
>100
46.1
>100
>100
7.11
>100
52.70
>100
>100
>100
>100
23.8
>100
>100
>100
>100
Ovarian cancer
Renal cancer
45.96
33.22
1.11
1.54
7.87
>100
>100
7.36
>100
2.73
14.90
2.72
8.08
>100
2.56
26.20
11.30
>100
>100
24.90
>100
4.26
3.75
13.70
9.45
Prostate cancer
Breast cancer
62.45
24.59
0.82
2.08
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
16.4
MIDa
51.07
a
MID: Average sensitivity of all cell lines in
MID: Average sensitivity of all cell lines of a particular subpanel in
m
M.
b
mM.
4.1.3.5. N-(4-Acetylphenyl)-2-(4-ethyl-5-phenyl-4H-[1,2,4]triazol-3-
ylsulfanyl)-acetamide 6e. White crystals (ethanol) in (0.331 g, 87%
yield); mp 190e191 ꢃC; Rf value using CHCl3:CH3OH (9:1) as an
eluent was 0.41; FT-IR (nmax): 3239 (br, NH), 1666 (COeNH, COe
CH3), 1596 (C]C), 1539 (C]N); 1H NMR (400 MHz, DMSO-d6,
(m, 5H, AreH), 7.68 (d, 2H, J ¼ 8.4 Hz, AreH), 7.91 (d, 2H, J ¼ 8.8 Hz,
AreH), 10.69 (s, 1H, NH).
4.1.3.6. N-(4-Acetylphenyl)-2-[4-ethyl-5-(4-methoxyphenyl)-4H-
[1,2,4]triazol-3-ylsulfanyl]acetamide 6f. White crystals (ethanol) in
(0.353 g, 86% yield); mp 199e200 ꢃC; Rf value using CHCl3:CH3OH
(9:1) as an eluent was 0.36; FT-IR (nmax): 3243 (br, NH), 1675 (COe
d
¼ ppm)
d
¼ 1.19 (t, 3H, NeCH2eCH3, J ¼ 7.2 Hz), 2.49 (s, 3H, CH3),
3.99 (q, 2H, NeCH2eCH3, J ¼ 7.2 Hz), 4.23 (s, 2H, SCH2), 7.49e7.60

194
G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
NH, COeCH3), 1596 (C]C), 1539 (C]N), 1250 cmꢀ1 (CeO); 1H NMR
(400 MHz, DMSO-d6,
J ¼ 7 Hz), 2.49 (s, 3H, COeCH3), 3.79 (s, 3H, OCH3), 3.97 (q, 2H, Ne
CH2eCH3, J ¼ 7 Hz), 4.21 (s, 2H, SCH2), 7.06 (d, 2H, J ¼ 8.8 Hz, AreH),
7.52 (d, 2H, J ¼ 8 Hz, AreH), 7.68 (d, 2H, J ¼ 8.4 Hz, AreH), 7.91 (d,
2H, J ¼ 8.8 Hz, AreH), 10.69 (s, 1H, NH).
SCH2), 6.59 (s, 2H, AreH), 7.44e7.55 (m, 5H, AreH), 7.68 (d, 2H,
J ¼ 8.4 Hz, AreH), 7.91 (d, 2H, J ¼ 8.4 Hz, AreH), 10.70 (s, 1H, NH).
d
¼ ppm)
d
¼ 1.18 (t, 3H, NeCH2eCH3,
4.1.4. General procedure for the synthesis of N-(4-(1-
(hydroxyimino)ethyl)phenyl)-2-((4-allyl/ethyl/phenyl-5-aryl-4H-
1,2,4-triazol-3-yl)thio)acetamide 7ael
A mixture of equimolar amounts of the appropriate ketones 6ae
l (0.3 mmol) and hydroxylamine hydrochloride in absolute ethanol
(30 ml) was heated under reflux for 8e12 h and then left to cool.
The separated solid was filtered off, washed with dil. ammonia
solution (10%) and distilled water, dried, and recrystallized from
aqueous ethanol affording the pure white products 7ael.
4.1.3.7. N-(4-Acetylphenyl)-2-[5-(3,4-dimethoxyphenyl)-4-ethyl-4H-
[1,2,4]triazol-3-ylsulfanyl]acetamide 6g. White crystals (ethanol) in
(0.374 g, 85% yield); mp 172e173 ꢃC; Rf value using CHCl3:CH3OH
(9:1) as an eluent was 0.31; FT-IR (nmax): 3243 (br, NH), 1675 (COe
NH, COeCH3), 1595 (C]C), 1535 (C]N), 1269 cmꢀ1 (CeO); 1H NMR
(400 MHz, DMSO-d6,
d
¼ ppm)
d
¼ 1.19 (t, 3H, NeCH2eCH3,
J ¼ 7.0 Hz), 2.49 (s, 3H, COeCH3), 3.76 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3), 3.99 (q, 2H, NeCH2eCH3, J ¼ 7.0 Hz), 4.21 (s, 2H, SCH2),
7.06e7.12 (m, 3H, AreH), 7.68 (d, 2H, J ¼ 8.4 Hz, AreH), 7.91 (d, 2H,
J ¼ 8 Hz, AreH), 10.69 (s, 1H, NH).
4.1.4.1. 2-(4-Allyl-5-phenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-N-[4-(1-
hydroxyimino-ethyl)phenyl]acetamide 7a. White crystals (ethanol)
in (0.11 g, 90% yield); mp 194e195 ꢃC; Rf value using CHCl3:CH3OH
(9:1) as an eluent was 0.48; FT-IR (nmax): 3500e3000 (br, OH, NH),
1685 (COeNH), 1598 (C]C), 1534 (C]N); 1H NMR (400 MHz,
4.1.3.8. N-(4-Acetylphenyl)-2-[4-ethyl-5-(3,4,5-trimethoxyphenyl)-
4H-[1,2,4]- triazol-3-ylsulfanyl]acetamide 6h. White crystals
(ethanol) in (0.423 g, 90% yield); mp 215e216 ꢃC; Rf value using
CHCl3:CH3OH (9:1) as an eluent was 0.28; FT-IR (nmax): 3246 (br,
NH), 1675 (COeNH, COeCH3), 1600, 1586 (C]C), 1546 (C]N),
DMSO-d6,
d
¼ ppm)
d
¼ 2.07 (s, 3H, HOeN]CeCH3). 4.15 (s, 2H,
SCH2), 4.61e4.62 (m, 2H, NCH2), 4.79 (d, 1H, CH]CH2,
Jtrans ¼ 17.2 Hz), 5.19 (d, 1H, CH]CH2, Jcis ¼ 10.8 Hz), 5.89e5.97 (m,
1H, CH]CH2), 7.48e7.57 (m, 9H, AreH), 10.40 (s, 1H, NH), 11.04 (s,
1H, OH); EI-MS (70 eV) m/z (%): 408 (Mþ, 4), 257 (48), 217 (41), 216
(59), 202 (45), 201 (33), 184 (100), 150 (97), 104 (39), 103 (51). Anal.
Calcd for C21H21N5O2S (407.14): C, 61.90; H, 5.19; N, 17.19; S, 7.87.
Found: C, 61.95; H, 5.24; N, 16.95; S, 7.65.
1128 cmꢀ1 (CeO); 1H NMR (400 MHz, DMSO-d6,
d
¼ ppm) ¼ 1.22
d
(t, 3H, NeCH2eCH3, J ¼ 6.8 Hz), 2.49 (s, 3H, COeCH3), 3.69 (s, 3H,
OCH3), 3.79 (s, 6H, 2 OCH3), 4.02 (q, 2H, NeCH2eCH3, J ¼ 6.8 Hz),
4.24 (s, 2H, SeCH2), 6.84 (s, 2H, AreH), 7.69 (d, 2H, J ¼ 8.4 Hz, Are
H), 7.91 (d, 2H, J ¼ 8.4 Hz, AreH), 10.70 (s, 1H, NH).
4.1.4.2. 2-[4-Allyl-5-(4-methoxyphenyl)-4H-[1,2,4]triazol-3-
ylsulfanyl]-N-[4-(1-hydroxyiminoethyl)phenyl]acetamide
7b.
4.1.3.9. N-(4-Acetylphenyl)-2-(4,5-diphenyl-4H-[1,2,4]triazol-3-
ylsulfanyl)-acetamide 6i. White crystals (ethanol) in (0.377 g, 88%
yield); mp 245e246 ꢃC; Rf value using CHCl3:CH3OH (9:1) as an
eluent was 0.67; FT-IR (nmax): 3243 (br, NH), 1685 (COeNH), 1661
(COeCH3), 1595 (C]C), 1534 cmꢀ1 (C]N); 1H NMR (400 MHz,
White crystals (ethanol) in (0.108 g, 82% yield); mp 219e220 ꢃC; Rf
value using CHCl3:CH3OH (9:1) as an eluent was 0.43; FT-IR (nmax):
3200e3000 (br, OH, NH), 1647 (COeNH), 1596 (C]C), 1536 (C]N),
1179 (CeO); 1H NMR (400 MHz, CDCl3 þ CD3OD,
d
¼ ppm) ¼ 2.25
d
(s, 3H, HOeN]CeCH3), 3.87 (s, 3H, OCH3), 4.03 (s, 2H, SCH2), 4.55e
4.56 (m, 2H, NCH2), 5.09 (d, 1H, CH]CH2, Jtrans ¼ 17.2 Hz), 5.38 (d,
1H, CH]CH2, Jcis ¼ 10.4 Hz), 5.89e5.96 (m, 1H, CH]CH2), 7.02 (d,
2H, J ¼ 8.8 Hz, AreH), 7.57 (d, 4H, J ¼ 8.8 Hz, AreH), 7.67 (d, 2H,
J ¼ 8.8 Hz, AreH), 10.77 (s, 1H, NH). Anal. Calcd for C22H23N5O3S
(437.15): C, 60.39; H, 5.30; N, 16.01; S, 7.33. Found: C, 60.51; H, 5.35;
N, 16.31; S, 7.23.
DMSO-d6,
d
¼ ppm)
d
¼ 2.49 (s, 3H, COeCH3), 4.22 (s, 2H, SCH2),
7.30e7.53 (m, 10H, AreH), 7.68 (d, 2H, J ¼ 8.8 Hz, AreH), 7.91 (d, 2H,
J ¼ 8.8 Hz, AreH), 10.70 (s, 1H, NH).
4.1.3.10. N-(4-Acetylphenyl)-2-[5-(4-methoxyphenyl)-4-phenyl-4H-
[1,2,4]triazol-3-ylsulfanyl]acetamide 6j. White crystals (ethanol) in
(0.398 g, 87% yield); mp 256e257 ꢃC; Rf value using CHCl3:CH3OH
(9:1) as an eluent was 0.63; FT-IR (nmax): 3244 (br, NH), 1680 (COe
NH), 1664 (COeCH3), 1610 (C]C), 1539 (C]N), 1245 cmꢀ1 (CeO);
4.1.4.3. 2-[4-Allyl-5-(3,4-dimethoxyphenyl)-4H-[1,2,4]triazol-3-
ylsulfanyl]-N-[4-(1-hydroxyiminoethyl)phenyl]acetamide
7c.
1H NMR (400 MHz, DMSO-d6,
d
¼ ppm)
d
¼ 2.49 (s, 3H, COeCH3),
White crystals (ethanol) in (0.119 g, 85% yield); mp 140e141 ꢃC; Rf
value using CHCl3:CH3OH (9:1) as an eluent was 0.41; FT-IR (nmax):
3700e3000 (br, OH, NH), 1689 (COeNH), 1599 (C]C), 1535 (C]N),
3.68 (s, 3H, OCH3), 4.19 (s, 2H, SCH2), 6.86 (d, 2H, J ¼ 8.8 Hz, AreH),
7.24 (d, 2H, J ¼ 8.8 Hz, AreH), 7.37e7.53 (m, 5H, AreH), 7.68 (d, 2H,
J ¼ 8.8 Hz, AreH), 7.91 (d, 2H, J ¼ 8.4 Hz, AreH), 10.70 (s, 1H, NH).
1266 (CeO); 1H NMR (400 MHz, CDCl3 þ CD3OD,
d
¼ ppm) ¼ 2.25
d
(s, 3H, HOeN]CeCH3), 3.91 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.96
(s, 2H, SCH2), 4.57e4.58 (m, 2H, NCH2), 5.11 (d, 1H, CH]CH2,
Jtrans ¼ 17.4 Hz), 5.39 (d, 1H, CH]CH2, Jcis ¼ 10.5 Hz), 5.91e6.00 (m,
1H, CH]CH2), 6.97 (d, 2H, J ¼ 8.4 Hz, AreH), 7.14e7.26 (m, 3H, Are
H), 7.58 (d, 2H, J ¼ 8.4 Hz, AreH), 7.67 (d, 2H, J ¼ 9 Hz, AreH), 10.74
(s, 1H, NH). EI-MS (70 eV) m/z (%): 467 (Mþ, 17), 311 (96), 247 (70),
232 (43), 214 (59), 161 (61), 150 (82), 134 (50), 133 (100), 118 (49),
107 (50), 100 (49), 77 (54). Anal. Calcd for C23H25N5O4S: (467.16) C,
59.08; H, 5.39; N, 14.98; S, 6.86. Found: C, 59.10; H, 5.72; N, 15.17; S,
6.63.
4.1.3.11. N-(4-Acetylphenyl)-2-[5-(3,4-dimethoxyphenyl)-4-phenyl-
4H-[1,2,4]triazol-3-ylsulfanyl]acetamide
6k. White
crystals
(ethanol) in (0.44 g, 90% yield); mp 219e220 ꢃC; Rf value using
CHCl3:CH3OH (9:1) as an eluent was 0.61; FT-IR (nmax): 3179 (br,
NH), 1674 (COeNH, COeCH3), 1595 (C]C), 1535 (C]N), 1255 cmꢀ1
(CeO); 1H NMR (400 MHz, DMSO-d6,
d
¼ ppm) ¼ 2.49 (s, 3H, COe
d
CH3), 3.48 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 4.19 (s, 2H, SCH2), 6.83e
6.88 (m, 3H, AreH), 7.39e7.54 (m, 5H, AreH), 7.68 (d, 2H, J ¼ 8 Hz,
AreH), 7.91 (d, 2H, J ¼ 8 Hz, AreH), 10.70 (s, 1H, NH).
4 .1. 3 .12 . N - ( 4 - A c e t yl p h e n yl ) - 2 - [ 4 - p h e n yl - 5 - ( 3 , 4 , 5 -
4.1.4.4. 2-[4-Allyl-5-(3,4,5-trimethoxyphenyl)-4H-[1,2,4]triazol-3-
trimethoxyphenyl)-4H-[1,2,4]triazol-3-ylsulfanyl]acetamide
6l.
ylsulfanyl]-N-[4-(1-hydroxyiminoethyl)phenyl]acetamide
7d.
White crystals (ethanol) in (0.462 g, 89% yield); mp 229e230 ꢃC; Rf
value using CHCl3:CH3OH (9:1) as an eluent was 0.61; FT-IR (nmax):
3172 (br, NH), 1675 (COeNH, COeCH3), 1589 (C]C), 1538 (C]N),
White crystals (ethanol) in (0.113 g, 76% yield); mp 189e190 ꢃC; Rf
value using CHCl3:CH3OH (9:1) as an eluent was 0.39; FT-IR (nmax):
3600e3000 (br, OH, NH), 1677 (COeNH), 1588 (C]C), 1533 (C]N),
1122 cmꢀ1 (CeO); 1H NMR (400 MHz, DMSO-d6,
d
¼ ppm)
d
¼ 2.49
1125 cmꢀ1 (CeO); 1H NMR (300 MHz, CDCl3, d ¼ ppm)
d
¼ 2.24 (s,
(s, 3H, COeCH3), 3.49 (s, 6H, 2 OCH3), 3.59 (s, 3H, OCH3), 4.21 (s, 2H,
3H, HOeN]CeCH3), 3.84 (s, 6H, 2 OCH3), 3.89 (s, 3H, OCH3), 4.02 (s,

G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
195
2H, SCH2), 4.58e4.59 (m, 2H, NCH2), 5.12 (d, 1H, CH]CH2,
Jtrans ¼ 17.4 Hz), 5.42 (d, 1H, CH]CH2, Jcis ¼ 10.5 Hz), 5.94e6.03 (m,
1H, CH]CH2), 6.85 (s, 2H, AreH), 7.54 (d, 2H, J ¼ 8.4 Hz, AreH), 7.62
1123 cmꢀ1 (CeO); (400 MHz, CDCl3 þ CD3OD,
d
¼ ppm)
d
¼ 1.40 (t,
3H, NeCH2eCH3, J ¼ 6.8 Hz), 2.24 (s, 3H, HOeN]CeCH3), 3.92 (s,
6H, 2 OCH3), 3.93 (s, 3H, OCH3), 4.03 (q, 2H, NeCH2eCH3,
J ¼ 7.2 Hz), 4.06 (s, 2H, SCH2), 6.84 (s, 2H, AreH), 7.57 (d, 2H,
J ¼ 8.8 Hz, AreH), 7.66 (d, 2H, J ¼ 8.8 Hz, AreH), 10.71 (s, 1H, NH).
Anal. Calcd for C23H27N5O5S (485.17): C, 56.89; H, 5.60; N, 14.42; S,
6.60. Found: C, 56.58; H, 5.46; N, 14.60; S, 6.40.
(d, 2H, J ¼ 8.7 Hz, AreH), 9.59 (bs, 1H, OH), 10.64 (s, 1H, NH). 13
C
NMR (75 MHz, CDCl3,
d
¼ ppm)
d
¼ 11.97 (CH3), 36.57 (CH2), 47.28
(CH2), 56.23 (CH3), 60.97 (CH3), 105.60 (CH), 118.74 (CH2), 119.45
(CH), 120.93 (C), 126.53 (CH), 130.88 (C), 132.41 (CH), 138.94 (C),
139.83 (C), 152.87 (C), 153.59 (C), 155.10 (C), 156.49 (C), 166.62 (CO).
EI-MS (70 eV) m/z (%): 497 (Mþ, 8), 318 (52), 284 (61), 277 (67), 261
(56), 224 (42), 186 (47), 150 (100), 149 (69), 105 (78), 83 (42), 69
(41), 55 (47). Anal. Calcd for C24H27N5O5S (497.17): C, 57.93; H, 5.47;
N, 14.08; S, 6.44. Found: C, 58.22; H, 5.56; N, 14.33; S, 6.31.
4.1.4.9. 2-(4,5-Diphenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-N-[4-(1-
hydroxyimino-ethyl)phenyl]acetamide 7i. White crystals (ethanol)
in (0.116 g, 87% yield); mp 213e215 ꢃC; Rf value using CHCl3:CH3OH
(9:1) as an eluent was 0.53; FT-IR (nmax): 3600e3000 (br, OH, NH),
1670 (COeNH), 1598 (C]C), 1535 (C]N); (400 MHz, DMSO-d6,
4.1.4.5. 2-(4-Ethyl-5-phenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-N-[4-(1-
hydroxy-iminoethyl)phenyl]acetamide 7e. White crystals (ethanol)
in (0.101 g, 85% yield); mp 224e225 ꢃC; Rf value using CHCl3:CH3OH
(9:1) as an eluent was 0.33; FT-IR (nmax): 3300e3100 (br, OH, NH),
1667 (COeNH), 1600 (C]C), 1537 (C]N); 1H NMR (400 MHz,
d
¼ ppm) ¼ 2.10 (s, 3H, HOeN]CeCH3), 4.21 (s, 2H, SCH2), 7.34 (s,
d
5H, AreH), 7.41e7.62 (m, 9H, AreH), 10.49 (s, 1H, NH), 11.10 (s, 1H,
OH). EI-MS (70 eV) m/z (%): 443 (Mþ, 0.3), 404 (22), 403 (42), 388
(100), 387 (87), 370 (24), 150 (22), 133 (24), 121 (75), 106 (15). Anal.
Calcd for C24H21N5O2S (443.14): C, 64.99; H, 4.77; N, 15.79; S, 7.23.
Found: C, 65.18; H, 4.96; N, 15.45; S, 7.11.
DMSO-d6,
d
¼ ppm) ¼ 1.21 (t, 3H, NeCH2eCH3, J ¼ 7.4 Hz), 2.10 (s,
d
3H, HOeN]CeCH3), 4.02 (q, 2H, NeCH2eCH3, J ¼ 7.4 Hz), 4.21 (s,
2H, SCH2), 7.54e7.63 (m, 9H, AreH), 10.47 (s, 1H, NH), 11.10 (s, 1H,
OH). EI-MS (70 eV) m/z (%): 396 (Mþ, 6), 378 (100), 297 (49), 265
(68), 150 (46), 133 (63), 77 (57). Anal. Calcd for C20H21N5O2S
(395.14): C, 60.74; H, 5.35; N, 17.71; S, 8.11. Found: C, 61.04; H, 5.50;
N, 17.48; S, 7.86.
4.1. 4.10. N-[4-(1-Hydroxyiminoethyl)phenyl]-2-[5-(4-
methoxyphenyl)-4-phenyl-4H-[1,2,4]triazol-3-ylsulfanyl]acetamide
7j. White crystals (ethanol) in (0.125 g, 88% yield); mp 181e182 ꢃC;
Rf value using CHCl3:CH3OH (9:1) as an eluent was 0.47; FT-IR
(nmax): 3300e3050 (br, OH, NH), 1677 (COeNH), 1599 (C]C),
1535 (C]N), 1237 (CeO); (400 MHz, CDCl3 þ CD3OD, ¼ ppm)
d
4.1.4.6. 2-[4-Ethyl-5-(4-methoxyphenyl)-4H-[1,2,4]triazol-3-
d
¼ 2.25 (s, 3H, HOeN]CeCH3), 3.78 (s, 3H, OCH3), 3.95 (s, 2H,
ylsulfanyl]-N-[4-(1-hydroxyiminoethyl)phenyl]acetamide
7f.
SCH2), 6.81 (d, 2H, J ¼ 8.8 Hz, AreH), 7.33e7.24 (m, 2H, AreH), 7.34
(d, 2H, J ¼ 8.8 Hz, AreH), 7.49e7.56 (m, 3H, AreH), 7.59 (d, 2H,
J ¼ 8.8 Hz, AreH), 7.67 (d, 2H, J ¼ 8.8 Hz, AreH), 8.01 (bs, 1H, OH),
White crystals (ethanol) in (0.101 g, 79% yield); mp 254e255 ꢃC; Rf
value using CHCl3:CH3OH (9:1) as an eluent was 0.27; FT-IR (nmax):
3700e3100 (br, OH, NH), 1673 (COeNH), 1599 (C]C), 1534 (C]N),
10.64 (s, 1H, NH). 13C NMR (100 MHz, CDCl3 þ CD3OD,
d
¼ ppm)
1247 (CeO); 1H NMR (400 MHz, CDCl3 þ CD3OD,
d
¼ ppm)
d
¼ 1.37
d
¼ 11.96 (CH3), 36.27 (CH2), 55.11 (CH3), 114.01 (CH), 117.70 (C),
(t, 3H, NeCH2eCH3, J ¼ 7.4 Hz), 2.24 (s, 3H, HOeN]CeCH3), 3.88 (s,
3H, OCH3), 4.00 (q, 2H, NeCH2eCH3, J ¼ 7.2 Hz), 4.07 (s, 2H, SCH2),
7.05 (d, 2H, J ¼ 8.8 Hz, AreH), 7.54 (d, 2H, J ¼ 8.8 Hz, AreH), 7.57 (d,
2H, J ¼ 8.4 Hz, AreH), 7.67 (d, 2H, J ¼ 8.8 Hz, AreH), 10.81 (s, 1H,
NH). EI-MS (70 eV) m/z (%): 425 (Mþ, 3), 344 (12), 343 (100), 328
(16), 327 (36), 307 (33), 295 (29), 235 (19), 219 (17), 150 (29), 133
(14). Anal. Calcd for C21H23N5O3S (425.15): C, 59.28; H, 5.45; N,
16.46; S, 7.54. Found: C, 59.33; H, 5.70; N, 16.43; S, 7.51.
119.30 (CH), 126.52 (CH), 126.98 (CH), 129.53 (CH), 130.07 (CH),
130.24 (C), 132.62 (C), 133.27 (C), 138.62 (C), 152.82 (C), 155.16 (C),
160.89 (C), 166.41 (CO). EI-MS (70 eV) m/z (%): 474 (Mþ, 0.1), 307
(18), 291 (25), 150 (100), 133 (43), 118 (29), 93 (20), 92 (33), 65 (18).
Anal. Calcd for C25H23N5O3S (473.15): C, 63.41; H, 4.90; N, 14.79; S,
6.77. Found: C, 63.50; H, 5.04; N, 14.57; S, 6.49.
4.1.4.11. 2-[5-(3,4-Dimethoxyphenyl)-4-phenyl-4H-[1,2,4]triazol-3-
ylsulfanyl]-N-[4-(1-hydroxyiminoethyl)phenyl]acetamide
7k.
4.1.4.7. 2-[5-(3,4-Dimethoxyphenyl)-4-ethyl-4H-[1,2,4]triazol-3-
White crystals (ethanol) in (0.113 g, 75% yield); mp 214e215 ꢃC; Rf
value using CHCl3:CH3OH (9:1) as an eluent was 0.43; FT-IR (nmax):
3450e3150 (br, OH, NH), 1680 (COeNH), 1603 (C]C), 1544 (C]N),
ylsulfanyl]-N-[4-(1-hydroxyiminoethyl)phenyl]acetamide
7g.
White crystals (ethanol) in (0.126 g, 92% yield); mp 225e226 ꢃC; Rf
value using CHCl3:CH3OH (9:1) as an eluent was 0.23; FT-IR (nmax):
3450e3000 (br, OH, NH), 1685 (COeNH), 1598 (C]C), 1535 (C]N),
1260 (CeO); (400 MHz, CDCl3 þ CD3OD,
d
¼ ppm)
d
¼ 2.25 (s, 3H,
HOeN]CeCH3), 3.69 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.95 (s, 2H,
SCH2), 6.76e7.02 (m, 3H, AreH), 7.28e7.55 (m, 5H, AreH), 7.68 (d,
2H, J ¼ 8.8 Hz, AreH), 7.68 (d, 2H, J ¼ 8.6 Hz, AreH), 10.63 (s, 1H,
NH). Anal. Calcd for C26H25N5O4S (503.16): C, 62.01; H, 5.00; N,
13.91; S, 6.37. Found: C, 61.93; H, 5.23; N, 13.93; S, 6.30.
1255 (CeO); (300 MHz, CDCl3,
d
¼ ppm)
d
¼ 1.37 (t, 3H, NeCH2e
CH3, J ¼ 7.2 Hz), 2.23 (s, 3H, HOeN]CeCH3), 3.90 (s, 3H, OCH3),
3.93 (s, 3H, OCH3), 3.98 (q, 2H, NeCH2eCH3, J ¼ 7.2 Hz), 4.04 (s, 2H,
SCH2), 6.95e7.17 (m, 3H, AreH), 7.53 (d, 2H, J ¼ 9 Hz, AreH), 7.61 (d,
2H, J ¼ 9 Hz, AreH), 9.58 (bs, 1H, OH), 10.66 (s, 1H, NH). 13C NMR
(75 MHz, CDCl3,
d
¼ ppm)
d
¼ 11.99 (CH3), 15.29 (CH3), 36.42 (CH2),
4.1.4.12. N-[4-(1-Hydroxyiminoethyl)phenyl]-2-[4-phenyl-5-(3,4,5-
trimethoxy- phenyl)-4H-[1,2,4]triazol-3-ylsulfanyl]acetamide 7l.
White crystals (ethanol) in (0.134 g, 84% yield); mp 227e228 ꢃC;
Rf value using CHCl3:CH3OH (9:1) as an eluent was 0.40; FT-IR
40.14 (CH2), 55.97 (CH3), 56.01 (CH3), 111.04 (CH), 111.81 (CH),
118.55 (CH), 119.46 (CH), 120.86 (C), 126.54 (CH), 132.41 (C), 138.94
(C), 149.32 (C), 150.79 (C), 151.76 (C), 155.16 (C), 155.98 (C), 166.72
(CO). EI-MS (70 eV) m/z (%): 455 (Mþ, 0.2), 410 (69), 392 (62), 391
(37), 264 (100), 237 (60), 164 (43), 163 (86), 103 (70). Anal. Calcd for
(
nmax): 3600e3100 (br, OH, NH), 1671 (COeNH), 1593 (C]C),
1536 (C]N), 1126 cmꢀ1 (CeO); (400 MHz, CDCl3 þ CD3OD,
C
22H25N5O4S (455.16): C, 58.01; H, 5.53; N, 15.37; S, 7.04. Found: C,
d
¼ ppm)
d
¼ 2.26 (s, 3H, HOeN]CeCH3), 3.62 (s, 6H, OCH3),
58.30; H, 5.59; N, 15.32; S, 6.81.
3.84 (s, 3H, OCH3), 3.96 (s, 2H, SCH2), 6.64 (s, 2H, AreH), 7.29e
7.57 (m, 5H, AreH), 7.60 (d, 2H, J ¼ 8.8 Hz, AreH), 7.68 (d, 2H,
J ¼ 8.4 Hz, AreH), 7.76 (bs, 1H, OH), 10.56 (s, 1H, NH). EI-MS
(70 eV) m/z (%): 515 (Mþ ꢀ H2O, 0.2), 327 (57), 311 (83), 224
(45), 150 (100), 133 (81), 108 (47), 92 (54), 65 (23). Anal. Calcd for
4.1.4.8. 2-[4-Ethyl-5-(3,4,5-trimethoxyphenyl)-4H-[1,2,4]triazol-3-
ylsulfanyl]-N-[4-(1-hydroxyiminoethyl)phenyl]acetamide
7h.
White crystals (ethanol) in (0.137 g, 94% yield); mp 219 ꢃC; Rf value
using CHCl3:CH3OH (9:1) as an eluent was 0.20; FT-IR (nmax): 3500e
3000 (br, OH, NH), 1680 (COeNH), 1589 (C]C), 1532 (C]N),
C
27H27N5O5S (533.17): C, 60.77; H, 5.10; N, 13.12; S, 6.01. Found:
C, 61.00; H, 5.05; N, 13.36; S, 5.85.

196
G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
4.2. Nitric oxide release measurement [53]
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, washed with running tap
water for 1e5 min. The slide was then dehydrated using alcohols,
cleaned by using xylene, covered by glass cover using Canada bal-
sam then examined under microscope.
Different solutions of the tested compounds 6ael (20 mL) 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-acetylcysteine. The
final concentration of drug was 10ꢀ4 M. After 1 h at 37 ꢃC, 1 ml of
the reaction mixture was treated with 250
mL of Griess reagent
[sulfanilamide (2 g), N-naphthylethylenediamine dihydrochloride
(0.2 g), 85% phosphoric acid (10 ml) in distilled water (final volume:
100 ml)]. After 10 min at room temperature, the absorbance was
4.3.4. Antiproliferative activity
The methodology of the NCI anticancer screening has been
described in detail elsewhere (http://www.dtp.nci.nih.gov).
Briefly, the primary anticancer assay was performed at approxi-
mately 60 human tumor cell lines panel derived from nine
neoplastic diseases, 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. Re-
sults 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 spectropho-
tometrically versus controls not treated with test agents. The
cytotoxic and/or growth inhibitory effects of the most active
selected compound were tested in vitro against the full panel of
about 60 human tumor cell lines at 10-fold dilutions of five
concentrations ranging from 10ꢀ4 to 10ꢀ8 M. A 48-h continuous
drug exposure protocol was followed and an SRB protein assay
was used to estimate cell viability or growth. Using the seven
absorbance measurements [time zero (Tz), control growth in the
absence of drug (C), and test growth in the presence of drug at
the five concentration levels (Ti)], the percentage growth was
calculated at each of the drug concentrations levels. Percentage
growth inhibition was calculated as: [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢀ 100 for
concentrations for which Ti > Tz, and [(Ti ꢀ Tz)/Tz] ꢀ 100 for
concentrations for which Ti < Tz. Three doseeresponse param-
eters were calculated for each compound. Growth inhibition of
50% (GI50) was calculated from [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢀ 100 ¼ 50,
which is the drug concentration resulting in a 50% lower net
protein increase in the treated cells (measured by SRB staining)
as compared to the net protein increase seen in the control cells.
The drug concentration resulting in TGI was calculated from
Ti ¼ Tz. The LC50 (concentration of drug resulting in a 50%
reduction in the measured protein at the end of the drug treat-
ment as compared to that at the beginning) indicating a net loss
of cells following treatment was calculated from [(Ti ꢀ Tz)/
Tz] ꢀ 100 ¼ ꢀ50. Values were calculated for each of these three
parameters if the level of activity is reached; however, if the
effect was not reached or was exceeded, the value for that
parameter was expressed as more or less than the maximum or
minimum concentration tested. The log GI50, log TGI, and log
LC50 were then determined, defined as the mean of the logs of
the individual GI50, TGI, and LC50 values. The lowest values are
obtained with the most sensitive cell lines. Compound having
log GI50 values ꢀ4 and <ꢀ4 was declared to be active.
measured at
l 546 nm. Sodium nitrite standard solutions (10e
80 nmol/ml) were used to construct the calibration curve. The same
procedure was repeated using different solutions of the test com-
pounds under the same conditions using 0.1 N HCl of pH 1 instead
of phosphate buffer of pH 7.4. The results were expressed as the
percentage of NO released relative to a theoretical maximum
release of 1 mol NO/mol of test compound.
4.3. Biological evaluation
4.3.1. Anti-inflammatory activity
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 activity of the compounds under investigation
was studied using carrageenan. A suspension of the tested
compounds 6ael and 7ael and reference drug (indomethacin) in
carboxy methyl cellulose (CMC) 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 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 injection. The anti-inflammatory ac-
tivity of the tested compounds and indomethacin was calculated
as the percentage decrease in edema thickness induced by
carrageenan.
4.3.2. 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.
The data are expressed as mean ꢂ 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.
Acknowledgments
Authors introduce their great thanks to Prof. Dr. Entesar Ali
Saber and Dr. Sarah M. N. Abdel-Hafez, Histology Department,
Faculty of Medicine, Minia University for their great help in per-
forming the histopathological investigation. Authors thank also the
Development Therapeutics Program of the National Cancer Insti-
tute, Bethesda, MD, USA, for in vitro evaluation of antiproliferative
activity.
4.3.3. Histopathological investigation
The histological slides were prepared according to the reported
procedures for examination of ulcers under light microscope [54].
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

G.El-DinA.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 71 (2014) 185e198
197
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