Synthesis, anti-inflammatory activity and ulcerogenic liability of novel nitric oxide donating/chalcone hybrids

Article abstract of DOI:10.1016/j.bmc.2011.11.012

A group of novel nitric oxide (NO) donating chalcone derivatives was prepared by binding various amino chalcones with different NO donating moieties including; nitrate ester, oximes and furoxans. Most of the prepared compounds showed significant anti-inflammatory activity using carrageenan-induced rat paw edema method compared with indomethacin. The prepared compounds exhibited more protection than indomethacin in regard to gastric toxicity. Histopathological investigation confirmed the beneficial effects of the NO releasing compounds in reducing ulcer formation. The incorporation of the NO-donating group into the parent chalcone derivatives caused a moderate increase in the anti-inflammatory activity with a marked decrease in gastric ulcerations compared to their parent chalcone derivatives.

Full text of DOI:10.1016/j.bmc.2011.11.012

Bioorganic & Medicinal Chemistry 20 (2012) 195–206
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, anti-inflammatory activity and ulcerogenic liability of novel
nitric oxide donating/chalcone hybrids
Gamal El-Din A.A. Abuo-Rahma a, Mohamed Abdel-Aziz a, , Mai A.E. Mourad a, Hassan H. Farag b
⇑
a Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt
b Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, 71526 Assiut, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of novel nitric oxide (NO) donating chalcone derivatives was prepared by binding various amino
chalcones with different NO donating moieties including; nitrate ester, oximes and furoxans. Most of the
prepared compounds showed significant anti-inflammatory activity using carrageenan-induced rat paw
edema method compared with indomethacin. The prepared compounds exhibited more protection than
indomethacin in regard to gastric toxicity. Histopathological investigation confirmed the beneficial
effects of the NO releasing compounds in reducing ulcer formation. The incorporation of the NO-donating
group into the parent chalcone derivatives caused a moderate increase in the anti-inflammatory activity
with a marked decrease in gastric ulcerations compared to their parent chalcone derivatives.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 6 September 2011
Revised 1 November 2011
Accepted 7 November 2011
Available online 13 November 2011
Keywords:
Chalcones
Nitric oxide donors
Anti-inflammatory
Gastric ulceration
Histopathology
1. Introduction
interest because of their diverse pharmacological activities such
as anti-inflammatory,21–23 antimicrobial,24,25 antifungal,26,27 anti-
Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the
most useful clinical therapies for the treatment of pain, fever,
and inflammation.1 The major mechanism by which NSAIDs exert
their anti-inflammatory activity through inhibition of cyclooxy-
genase-derived prostaglandin synthesis, which is also responsible
for the gastrointestinal,2–6 renal,7–9 and hepatic side effects,10 that
are observed mainly in chronic use of NSAIDs. Therefore, the chal-
lenge still exists for the pharmaceutical industry to develop, effec-
tive anti-inflammatory agents with enhanced safety profile. One of
the most important strategies used to overcome NSAIDs side ef-
fects is to design nitric oxide-donating NSAIDs (NO–NSAIDs) which
are capable of generating the radical biomediator and gastropro-
tective agent NO.11,12 NO contributes to the modulation of several
key physiological functions in the digestive system,13 it has the
ability to increase the mucosal blood flow,14 resulting in enhanced
mucosal resistance to ulceration,15 NO also prevents adherence of
leukocytes to the vascular endothelium,16 it is known to modulate
gastroduodenal secretion of mucus,17 and bicarbonate,18 NO can
profoundly influence the mucosal immune system,13 it also in-
creases the ability of mucosal cells to undergo healing and repair
of the existing ulcers.19 NO is also known to spare the renal system
mainly through stimulating the renal blood flow.20 Moreover, nat-
urally occurring and synthetic chalcone derivatives are of current
viral,28 antituberculosis,29 antimalarial,30–33 antioxidant,34–36 anti-
mitotic,37 antileishmanial,38 antiplatelet,39 and anticancer
activities.40–43 Herencia et al.,44–47 tested a series of chalcone
derivatives for possible anti-inflammatory effect, chalcone
I
(Fig. 1) was significantly active as a scavenger of superoxide anion
generated by stimulated human neutrophils or by the hypoxan-
thine/xanthine oxidase system. Chalcone I inhibited also the induc-
ible NO synthase (iNOS) expression through
a superoxide-
dependent mechanism in stimulated mouse peritoneal macro-
phages and protected cells against oxidant stress. Additionally
chalcone I significantly reduced tumor necrosis factor-
a (TNF-a)
levels, a crucial mediator of the anti-inflammatory process, espe-
cially in chronic inflammatory conditions. 2,4,6-Trimethoxy-20-
tifluoromethyl chalcone (II),48 (Fig. 1) inhibited the production
prostaglandin E2 in lipopolysaccharaide-stimulated RAW 264.7
macrophage cells.
Promoted with the above-mentioned studies and as a continu-
ation of our research interest in the synthesis and biological activ-
ities of novel NO–NSAIDs derivatives,49,50 the present study aimed
at gathering the two bioactive entities (chalcone and NO) in one
compact structure for the purpose of synergism and/or decreasing
the expected ulcerogenic side effects. The prepared NO–chalcone
hybrids were evaluated for their anti-inflammatory activity using
carrageenan-induced rat paw edema and compared to a well-
known NSAID, indomethacin. The ability of the prepared com-
pounds to induce gastric toxicity has been evaluated. Moreover,
⇑
Corresponding author. Tel.: +20 103311327; fax: +20 86 2369075.
E-mail address: abulnil@hotmail.com (M. Abdel-Aziz).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.012

196
G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
CH3
H3CO
OCH3
N
CH3
H3CO
H3CO
OCH3
CF3
O
O
Chalcone I
Chalcone II
Figure 1. Structures of chalcones I and II.
histopathological investigation has been carried out to asses the
beneficial effects of the NO releasing chalcone hybrids in decreas-
ing ulcer formation.
attached to tertiary carbon. Also, the chemical shift value of the
CH-ONO2 showed a downfield shift by d 0.90–0.74 ppm, caused
by the deshielding effect of the nitrate group, comparable with
the data of their corresponding bromo-derivatives 4a–d.
The reaction of bromo-derivatives 2a–d with p-hydroxyaceto-
phenone in the presence of potassium carbonate was successfully
applied to prepare the ketones 6a–d. Heating at reflux of com-
pounds 6a–d with hydroxylamine HCl in EtOH yielded the target
ketoxime derivatives 7a–d (Scheme 3). In the 1H NMR spectra of
oximes 7a and 7b, the CH3 protons appeared upfield shifted by d
0.47–0.40 ppm than the CH3 protons of the corresponding ketones
due to the low electronegativity of the neighboring nitrogen atom
relative to oxygen atom. Mass spectrum for the oxime 7a indicated
that the base peak recorded at an abundant [Mꢀ15]+ ion, which
was attributed to the result of a process occurring prior to ioniza-
tion in analogy with other oximes,55 due to the loss of oxygen and
abstraction of hydrogen. Kallury and Rao,56 reported that the abun-
dances of some oximes are very low (less than 4%) of the respective
base peaks, which further support the respective mass results.
The synthesis of novel chalcone hybrids formed by linking dif-
ferent chalcone derivatives to the furoxan substructure 8 is out-
lined in Scheme 4. Synthesis of 3-phenyl-4-furoxanmethanol 8
was done by treatment of cinnamyl alcohol with sodium nitrite
in glacial acetic acid.57 Coupling of furoxan derivative 8 with bro-
mo-derivatives 2a–d in the presence of anhydrous potassium car-
bonate in acetone afforded the desired NO-donating furoxan
derivatives 9a–d in moderate yields.
2. Results and discussion
2.1. Chemistry
Chalcone derivatives 1a–d were synthesized by a base catalyzed
Claisen–Schmidt condensation of 4-aminoacetophenone with
substituted benzaldehyde 1a–c or furfural 1d.51–53 Treatment of
compounds 1a–d with bromoacetyl bromide in the presence of
potassium carbonate afforded the corresponding 2-bromo-N-{4-
[3-arylacryloyl]phenyl}acetamides 2a–d in high yields (Scheme
1). Conversion of the bromo-derivatives 2a–d into the target titled
NO-donating nitrate esters 3a–d has occurred in moderate yields
(52–62%), upon heating the bromo-derivatives 2a–d with silver ni-
trate in acetonitrile via simple nucleophilic attack of nitrate ion on
the carbon atom of the CH2Br group (Scheme 1). It is worth noting
that the 1H NMR spectra of the novel nitrate ester derivatives 3
compared to their corresponding bromo-derivatives 2 showed that
replacement of a bromine atom by a nitrate group clearly caused a
remarkable downfield effect on the observed chemical shift values
of the methylene protons by d 1.12 ppm. Mass spectra showed a
prominent peak at [M+-46(NO2)], this phenomenon is in agreement
with that reported.54
Acylation of the amino chalcone derivatives 1a–d with 2-
bromopropionic acid through mixed anhydride intermediate using
ethyl chloroformate in the presence of triethylamine afforded the
methylated analogues 4a–d in moderate yields (Scheme 2). Heat-
ing at reflux of compounds 4a–d with silver nitrate in acetonitrile
for 12 h yielded the corresponding NO-donating methylated ana-
logues 5a–d in moderate yields. In compounds 5a–d, it is worth
to mention that CHONO2 proton is more downfield shifted by d
0.17 ppm than the corresponding CH2ONO2, because its position
2.2. Measurement of nitric oxide release
The NO releasing properties of the prepared NO donors includ-
ing; NO-donating nitrate esters 3a–d, their methylated analogues
5a–d, NO-donating oximes 7a–d and NO-donating furoxans 8 and
9a–d were assessed. The produced nitrite which is a convenient in-
dex of nitric oxide production trend was determined in both
O
NaOH (10 %)
COCH3
BrCH2COBr
K2CO3
Ar
Ar
CHO + H2N
EtOH
NH2
1a-d
O
O
O
Ar
O
Ar
AgNO3
CH3CN
Br
O
NH
NO2
NH
2a-d
3a-d
Scheme 1. Synthesis of N-{4-[3-arylacryloyl]phenyl}-2-nitroxyacetamides 3a–d. Ar: (a) 4-Cl–C6H4; (b) 4-CH3O–C6H4; (c) 30,40-OCH2O–C6H3; (d) 2-furanyl.

G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
197
Br
O
O
O
Br
CH3CH C
CHCl3, 0-5°C
Et3N
1a-d
CH3CHCOOH + ClCOOC2H5
C2H5O
C
Hβ
O
O
Ar
O
Ar
O
AgNO3
Br
O
NH
Hα
CH3CN
NH
NO2
CH3
CH3
4a-d
5a-d
Scheme 2. Synthesis of N-{4-[3-arylacryloyl]phenyl}-2-nitrooxypropionamides 5a–d. Ar: (a) 4-Cl–C6H4; (b) 4-CH3O–C6H4; (c) 30,40-OCH2O–C6H3; (d) 2-furanyl.
O
H
N
CH3
Hα
H
N
K2CO3
Acetone, reflux
Br
Ar
+
HO
COCH3
O
Ar
O
O
O
2a-d
O
6a-d
Hβ
OH
N
CH3
NH2OH.HCl
EtOH, reflux
H
N
O
Ar
O
O
7a-d
Scheme 3. Synthesis of N-{4-[3-arylacyloyl]phenyl}-2-[4-(1-hydroxyiminoethyl)phenoxy]acetamides 7a–d. Ar: (a) 4-Cl–C6H4; (b) 4-CH3O–C6H4; (c) 30,40-OCH2O–C6H3; (d)
2-furanyl.
H
N
C6H5
N
CH2OH
Br
H
N
C6H5
O
K2CO3
Acetone, reflux
O
Ar
N
N
N
+
O
Ar
O
O
O
O
9a-d
O
O
2a-d
8
Scheme 4. Synthesis of N-[4-(arylacryloyl)phenyl]-2-(2-oxy-4-phenylfurazan-3-ylmethoxy)acetamides 9a–d. Ar: (a) 4-Cl–C6H4; (b) 4-CH3O–C6H4; (c) 30,40-OCH2O–C6H3; (d)
2-furanyl.
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
tested compounds could only release NO at pH 7.4. Nitrate esters
3a–d achieved maximum NO release after 5 h, while the methyl-
ated analogues 5a–d did after 3 h. The amount of NO released from
compounds 5a–d was lower than that released from compounds
3a–d. This might be attributed to the steric effect of the methyl
group in the propionic acid spacer present in compounds which
may decrease the dissociation of the nitrite group giving lower
amount of NO compared to the acetic acid spacer present in com-
pounds 3a–d.49 NO-donating oximes 7a–d gave the maximum
amount of NO released after 4 h, while NO-donating furoxans 8
and 9a–d exhibited maximum NO release after 5 h. On contrary,
the tested compounds were not able to release NO at pH 1 which
suggest that these compounds are weakly hydrolyzed in the gastric
lumen and this confirms that the suggested gastro protective ac-
tion of NO is mediated systemically.58
2.3. Biological investigations
2.3.1. Screening of anti-inflammatory activity
The synthesized compounds 1a–d, 2a–d, 3a–d, 4a–d, 5a–d,
6a–d, 7a–d, 8 and 9a–d were evaluated for their anti-inflammatory
activity using carrageenan-induced paw edema method in rats de-
scribed by Winter et al.59 The tested compounds and indomethacin
(reference drug) were administered orally at a dose level of
100 mg/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:

198
G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
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 3a–9d in the
presence of 0.5 mM N-acetylcysteine in phosphate buffer of pH 7.4
Compd No.
Amount of NO released (mol/mol) SEM
1h
2h
3h
4h
0.73 0.035
5h
6h
3a
3b
3c
3d
5a
5b
5c
5d
7a
7b
7c
7d
8
0.32 0.015
0.27 0.052
0.39 0.021
0.15 0.010
0.13 0.009
0.11 0.005
0.10 0.003
0.09 0.005
0.13 0.017
0.11 0.009
0.14 0.011
0.10 0.004
0.35 0.024
0.55 0.031
0.11 0.005
0.50 0.024
0.16 0.010
0.37 0.012
0.31 0.026
0.45 0.030
0.28 0.011
0.16 0.014
0.12 0.010
0.14 0.009
0.11 0.004
0.19 0.013
0.12 0.006
0.16 0.013
0.12 0.007
0.51 0.031
0.98 0.074
0.27 0.012
0.71 0.050
0.23 0.018
0.54 0.030
0.48 0.022
0.60 0.031
0.37 0.014
0.46 0.026
0.37 0.019
0.40 0.025
0.38 0.021
0.22 0.016
0.14 0.011
0.25 0.017
0.12 0.006
0.68 0.034
1.22 0.091
0.47 0.027
0.86 0.038
0.40 0.015
0.75 0.034
0.68 0.019
0.73 0.035
0.63 0.041
0.28 0.009
0.15 0.014
0.15 0.012
0.11 0.008
0.37 0.028
0.16 0.013
0.26 0.021
0.14 0.010
0.94 0.048
1.50 0.095
0.52 0.017
1.26 0.081
0.65 0.025
0.73 0.035
0.59 0.021
0.67 0.040
0.59 0.033
0.16 0.013
0.14 0.010
0.12 0.011
0.09 0.002
0.36 0.026
0.15 0.012
0.24 0.018
0.13 0.011
0.68 0.040
0.87 0.051
0.30 0.025
0.75 0.048
0.54 0.037
0.66 0.022
0.64 0.037
0.59 0.019
0.35 0.023
0.17 0.012
0.28 0.016
0.13 0.010
0.41 0.028
0.30 0.013
0.31 0.020
0.28 0.011
0.72 0.041
1.295 0.099
0.482 0.021
0.890 0.057
0.499 0.033
9a
9b
9c
9d
ðVR ꢀ VLÞcontrol ꢀ ðVR ꢀ VLÞtreated
ðVR ꢀ VLÞcontrol
starting chalcones 1a–d (Table 2). The NO-donating nitrate esters
3a–d showed significant increase in the anti-inflammatory activity
when compared to their starting chalcones 1a–d and bromoacetyl
derivatives 2a–d. The same results were obtained upon comparing
the anti-inflammatory activity of the NO-donating methylated
analogues 5a–d, oximes 7a–d and furoxans 9a–d with their corre-
sponding chalcones 1a–d and their bromoacetyl derivatives 4a–d.
This might suggest that NO potentiate the activity of the synthe-
sized compounds. The role of NO was also confirmed by the higher
anti-inflammatory activity achieved by the NO-donating furoxan
derivatives 9a–d and the NO-donating nitrate esters 3a–d com-
pared to the NO-donating methylated analogues 5a–d and NO-
donating oxime derivatives 7a–d (Table 2) that was coupled with
high NO release rates (Table 1) for the aforementioned compounds.
% of edema inhibition ¼
ꢁ 100
where VR represents the mean right paw thickness and VL repre-
sents the mean left paw thickness.
(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.
The results listed in Table 2 show the percentage of edema inhi-
bition induced by carrageenan for all of the tested compounds and
indomethacin versus time in h. Most of the tested compounds
showed a significant anti-inflammatory activity against carra-
geenan-induced paw edema in rats (p <0.01). The reference drug,
indomethacin showed an inhibitory activity of 78% against carra-
geenan-induced paw edema after 3 h, which is the time required
reaching the maximum activity for most of the tested compounds,
then the activity decreased gradually for the next 2 h for the major-
ity of the synthesized compounds. The synthesized compounds
exhibited considerable anti-inflammatory activity relative to indo-
methacin that increased significantly to a maximum after 3 h. The
nitrate ester 3a exhibited 69% anti-inflammatory activity which
represents 89% of indomethacin activity after 3 h. The nitrate ester
3b–d showed an anti-inflammatory activity of 53%, 61% and 48%,
respectively, which is equal to 69%, 79% and 61% of indomethacin
activity, respectively. Moreover, the anti-inflammatory activity of
the NO-donating methylated analogues 5a–d was 62%, 50%, 59%
and 54%, respectively, after 3 h that represents 80%, 65%, 77% and
69% of indomethacin activity, respectively. The anti-inflammatory
activity of the NO-donating oxime derivatives 7a–d was also eval-
uated and the results showed that the anti-inflammatory activity
of compounds 7a–d was 66%, 46%, 59% and 49% after 3 h, respec-
tively, that represents 85%, 59%, 77% and 64% of indomethacin
activity, respectively. For NO-donating furoxan derivatives 8 and
9a–d, the results showed that compounds 8, 9a and 9c exhibited
the strongest anti-inflammatory activity among the synthesized
compounds with 69%, 75% and 72% of edema inhibition, respec-
tively, after 4 h, which is equal to 84%, 91% and 87% of indometh-
acin activity, respectively. Furoxans 9b and 9d exhibited 56% and
59% of edema inhibition, respectively, after 3 h, which represents
73% and 76% of indomethacin activity, respectively.
2.3.2. Screening of ulcerogenic liability
The in vivo ulcerogenic liability was evaluated for the synthe-
sized compounds 1a–d, 2a–d, 3a–d, 4a–d, 5a–d, 6a–d, 7a–d, 8
and 9a–d relative to indomethacin according to the reported pro-
cedure.60 Ulcers were classified into levels, level I, ulcer area less
than 1 mm2, level II, ulcer area is 1–3 mm2 and level III, ulcer area
more than 3 mm2, and the following parameters were calculated:
1- Ulcer index (UI) was calculated as follows: 1 ꢁ (number of
ulcers level I) + 2 ꢁ (number of ulcers level II) + 3 ꢁ (number of
ulcers level III), etc.
2- Cure ratio = 100ꢀ(UItreated ꢁ 100/UIprotype
)
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 trea-
ted with the starting and the intermediate derivatives.
The UI of compounds 1a–d, 2a–d, 3a–d, 4a–d, 5a–d, 6a–d,
7a–d, 8 and 9a–d were calculated and were listed in Table 2 as
(mean S.E.). The results of ulcerogenic liability revealed that
indomethacin caused significant ulcerogenic toxicity with UI of
45.5, whereas an equal dose of the majority of the synthesized
compounds exhibited much lower UI. The NO-donating nitrate es-
ters 3a–d and NO-donating methylated analogues 5a–d exhibited
lower ulcerogenicity relative to indomethacin, with UIs of 2.5,
5.8, 3 and 7 for the nitrate esters 3a–d, and 3.5, 8, 4 and 5.7 for
NO-donating esters 5a–d (Fig. 2A). The NO-donating oximes
7a–d also exhibited lower toxicity relative to indomethacin where
the UIs were 2.2, 5, 4 and 4.8, respectively (Fig. 2B).
The anti-inflammatory activity of the bromoacetyl derivatives
2a–d and 4a–d did not significantly decrease compared to their

G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
199
Table 2
Anti-inflammatory activity at different time intervals and ulcer indices of compounds 1a–d, 2a–d, 3a–d, 4a–d, 5a–d, 6a–d, 7a–d, 8 and 9a–d using carrageenan-induced paw
edema in rats
Compd No.
% of edema inhibition (% mean SEM)
Ulcer index (UI) (Mean SEM)
1h
2h
3h
4h
5h
Control
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8
9a
9b
0
0
0
0
0
0.5 0.06
41 1.55⁄⁄⁄
42 1.28⁄⁄⁄
32 2.17⁄⁄⁄
33 1.80⁄⁄⁄
44 1.72⁄⁄⁄
37 1.07⁄⁄⁄
40 1.16⁄⁄⁄
30 1.12⁄⁄⁄
42 2.38⁄⁄⁄
33 1.26⁄⁄⁄
38 2.16⁄⁄⁄
34 1.41⁄⁄⁄
39 1.81⁄⁄⁄
39 1.38⁄⁄⁄
37 1.69⁄⁄⁄
30 1.70⁄⁄
45 2.13⁄⁄⁄
43 1.81⁄⁄⁄
45 1.44⁄⁄⁄
41 1.60⁄⁄⁄
43 1.74⁄⁄⁄
28 1.60⁄⁄
37 1.45⁄⁄⁄
28 1.25⁄⁄
39 2.05⁄⁄⁄
33 1.66⁄⁄⁄
40 3.45⁄⁄⁄
36 2.76⁄⁄⁄
40 1.20⁄⁄⁄
38 1.96⁄⁄⁄
30 1.74⁄⁄⁄
34 2.14⁄⁄⁄
30 1.31⁄⁄
56 0.78⁄⁄⁄
50 2.07⁄⁄⁄
48 2.62⁄⁄⁄
44 1.12⁄⁄⁄
38 1.07⁄⁄⁄
52 1.47⁄⁄⁄
42 1.23⁄⁄⁄
45 1.63⁄⁄⁄
37 2.16⁄⁄⁄
57 2.11⁄⁄⁄
49 2.02⁄⁄⁄
45 1.47⁄⁄⁄
36 1.56⁄⁄⁄
45 2.25⁄⁄⁄
40 3.08⁄⁄⁄
43 1.49⁄⁄⁄
37 1.79⁄⁄⁄
55 2.22⁄⁄⁄
46 1.71⁄⁄⁄
40 2.41⁄⁄⁄
48 3.05⁄⁄⁄
52 3.10⁄⁄⁄
35 1.59⁄⁄⁄
43 2.08⁄⁄⁄
32 1.96⁄⁄⁄
54 2.76⁄⁄⁄
41 1.52⁄⁄⁄
49 1.68⁄⁄⁄
44 1.88⁄⁄⁄
53 1.64⁄⁄⁄
48 1.37⁄⁄⁄
42 2.42⁄⁄⁄
55 3.84⁄⁄⁄
45 2.33⁄⁄⁄
70 2.23⁄⁄⁄
60 1.18⁄⁄⁄
51 2.31⁄⁄⁄
55 1.24⁄⁄⁄
42 1.98⁄⁄⁄
59 2.10⁄⁄⁄
46 2.44⁄⁄⁄
53 3.41⁄⁄⁄
41 2.60⁄⁄⁄
69 1.71⁄⁄⁄
53 2.05⁄⁄⁄
61 1.89⁄⁄⁄
48 1.52⁄⁄⁄
59 3.54⁄⁄⁄
40 1.65⁄⁄⁄
56 2.71⁄⁄⁄
42 2.39⁄⁄⁄
62 1.43⁄⁄⁄
50 1.70⁄⁄⁄
59 2.06⁄⁄⁄
54 2.60⁄⁄⁄
61 3.41⁄⁄⁄
42 2.38⁄⁄⁄
57 3.13⁄⁄⁄
45 1.84⁄⁄⁄
66 2.60⁄⁄⁄
46 2.92⁄⁄⁄
59 3.68⁄⁄⁄
49 1.66⁄⁄⁄
62 1.28⁄⁄⁄
66 2.50⁄⁄⁄
56 1.42⁄⁄⁄
62 1.73⁄⁄⁄
59 2.15⁄⁄⁄
78 1.14⁄⁄⁄
58 2.62⁄⁄⁄
48 1.16⁄⁄⁄
50 1.24⁄⁄⁄
33 2.53⁄⁄⁄
59 2.75⁄⁄⁄
41 1.94⁄⁄⁄
48 2.31⁄⁄⁄
33 1.20⁄⁄⁄
67 1.08⁄⁄⁄
51 1.57⁄⁄⁄
55 1.47⁄⁄⁄
40 2.03⁄⁄⁄
54 2.23⁄⁄⁄
38 1.86⁄⁄⁄
53 4.47⁄⁄⁄
36 2.44⁄⁄⁄
61 2.32⁄⁄⁄
43 1.52⁄⁄⁄
54 1.90⁄⁄⁄
49 1.08⁄⁄⁄
58 1.53⁄⁄⁄
33 1.68⁄⁄⁄
53 2.46⁄⁄⁄
39 3.63⁄⁄⁄
55 3.79⁄⁄⁄
42 1.27⁄⁄⁄
52 1.29⁄⁄⁄
48 1.45⁄⁄⁄
69 3.67⁄⁄⁄
75 3.17⁄⁄⁄
55 2.13⁄⁄⁄
72 3.59⁄⁄⁄
58 2.02⁄⁄⁄
83 2.36⁄⁄⁄
54 2.20⁄⁄⁄
40 1.13⁄⁄⁄
47 1.20⁄⁄⁄
30 2.24⁄⁄⁄
54 2.51⁄⁄⁄
34 2.32⁄⁄⁄
46 1.73⁄⁄⁄
25 1.16⁄⁄⁄
60 3.69⁄⁄⁄
46 1.80⁄⁄⁄
54 2.13⁄⁄⁄
39 2.16⁄⁄⁄
50 3.09⁄⁄⁄
31 1.18⁄⁄⁄
50 1.64⁄⁄⁄
34 2.06⁄⁄⁄
56 1.12⁄⁄⁄
35 1.06⁄⁄⁄
53 2.89⁄⁄⁄
49 3.05⁄⁄⁄
55 2.13⁄⁄⁄
30 0.84⁄⁄
50 1.52⁄⁄⁄
36 1.61⁄⁄⁄
49 1.81⁄⁄⁄
37 1.41⁄⁄⁄
44 2.10⁄⁄⁄
42 2.85⁄⁄⁄
67 3.62⁄⁄⁄
62 2.01⁄⁄⁄
49 1.40⁄⁄⁄
70 1.40⁄⁄⁄
49 2.70⁄⁄⁄
87 1.47⁄⁄⁄
18 1.04⁄⁄
22.5 0.28⁄⁄
20 0.86⁄⁄⁄
29 2.78⁄⁄
21.4 0.95⁄⁄⁄
28 1.32⁄⁄
24 1.73⁄⁄
25.8 0.96⁄⁄
2.5 0.28⁄⁄⁄
5.8 0.47⁄⁄⁄
3
7
0.28⁄⁄⁄
0.50⁄⁄⁄
16.7 0.90⁄⁄⁄
30.1 1.49⁄⁄
18 1.0⁄⁄⁄
25.3 2.0⁄⁄
3.5 0.28⁄⁄⁄
8
4
0.76⁄⁄⁄
0.17⁄⁄⁄
5.7 0.51⁄⁄⁄
8.1 0.55⁄⁄⁄
6
0.58⁄⁄⁄
11.2 0.72⁄⁄⁄
5
0.76⁄⁄⁄
2.2 0.11⁄⁄⁄
5
4
0.28⁄⁄⁄
0.23⁄⁄⁄
4.8 0.47⁄⁄⁄
1.5 0.12⁄⁄⁄
1
2
0.05⁄⁄⁄
0.11⁄⁄⁄
9c
9d
1.3 0.10⁄⁄⁄
2.5 0.23⁄⁄⁄
45.5 1.75
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.
The cure ratio of the target NO-donating derivatives 3a–d, 5a–d,
7a–d and 9a–d compared to their starting chalcone derivatives 1a–
d and their intermediates 2a–d, 4a–d, 6a–d and 8 has been calcu-
lated and the results revealed that the NO-donating nitrate esters
3a–d achieved 74–86% reduction of ulcers than their starting
chalcones 1a–d and 73–88% reduction of ulcers than their corre-
sponding bromoacetyl intermediates 2a–d. On the other hand,
the NO-donating methylated analogues 5a–d exhibited 64–81%
reduction of ulcers than their starting chalcones 1a–d and 73–
79% reduction of ulcers than their corresponding bromoacetyl
intermediates 4a–d. The NO-donating oximes 7a–d exhibited
Figure 2A. UI of compounds 3a–d, 5a–d compared to indomethacin expressed as
78–88% reduction of ulcers than their starting chalcones and 81–
mean S.E.
90% reduction of ulcers than their corresponding bromoacetyl
intermediates 2a–d and 4–73% reduction of ulcers than their
corresponding ketone intermediates 6a–d. Additionally, the results
revealed that NO-donating furoxans 9a–d exhibited the highest
cure ratio in comparison with the other synthesized NO-donors
(Fig. 2C). NO-donating furoxans 9a–d exhibited 91–94% reduction
of ulcers compared to their starting chalcones 1a–d and 90–95%
reduction of ulcers compared to their corresponding bromoacetyl
intermediates 2a–d and these results are in a good agreement with
the NO release data (Table 1). The decreased gastric toxicity of the
targeted NO-chalcone hybrids 3a–d, 5a–d, 7a–d and 9a–d
compared to their starting chalcones 1a–d and their intermediates
2a–d, 4a–d, 6a–d and 8 can be attributed to the release of NO that
increases mucosal blood flow resulting in enhanced mucosal resis-
Figure 2B. UI of compounds 7a–d compared to indomethacin expressed as
mean S.E.
tance to ulceration,61,62 and/or an enhanced ability of the intact

200
G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
50
40
30
20
10
0
8
9a
9b
9c
9d
Indomethacin
8
9a
9b
9c
9d Indomethacin
Figure 2C. UI of compounds 8 and 9a–d compared to indomethacin expressed as
mean S.E.
Figure 3C. Photomicrograph of mucosa of fundic stomach of furoxan 9a.
NO-donating derivatives to cross the gastric mucosal lining prior to
the subsequent release of NO and starting chalcone derivatives
(Fig. 2).63
including loss of mucosal membrane at the areas of ulceration
and loss of the morphology of certain glandular structures was ob-
served by the bromoacetyl derivative 2a associated with high UI of
21.4. In comparison to compound 3a, the NO-donating methylated
analogue 5a showed less protective effect against the ulcerative
changes that affect the gastric mucosa with partial loss of mucosal
membrane at the areas of ulceration with UI of 3.5. Partial healing
of the ulcers with remnants of the structural damage was still
found induced by the NO-donating oxime 7c associated with low
UI of 4. The ketonic intermediate 6c exhibited marked capillary
dilatation in the lumina propria just below the fundic glands, cap-
illary inflammatory cells were also found and edema fluid leads to
flattening of the above mucosal membrane which also confirmed
by the UI of 11.2. Complete protective effect against ulcers was
induced by the NO-donating furoxan 9a (Fig. 3C) where complete
healing of ulcers was observed with absence of capillary inflamma-
tory cells, the ulcerative damage of the gastric mucosa was mark-
edly decreased which was approved by the very low UI of 1.
2.3.3. Histopathological investigation
Different stomach sections of the ulcers including the control
and the treated groups were stained by standard hematoxylin
and eosin stains. The produced slides were subjected to microscop-
ical examination and pictures were picked for these slides.
The control (Fig. 3A) showed no lesions and characterized by
the presence of continuous mucosal layer. Indomethacin (Fig. 3B)
exhibited marked loss of mucosal membrane at the areas of ulcer-
ation where certain areas of fundic glands were totally degener-
ated and cellular details were lost. Capillary inflammatory cells
were also found and apoptotic glandular epithelial cells could be
detected. The stomach sections of the ulcers treated with
NO-donating nitrate ester 3a showed normal morphology for the
fundic glands and the results are in consistence with the previous
results.49,50 The edema was very minimal and also the vasodilata-
tion of blood capillaries was not marked and this was confirmed by
the low UI of 2.5. Changes in the structure of gastric mucosa
3. Conclusions
A group of novel chalcone–NO hybrids was prepared and char-
acterized by different spectroscopic and elemental analysis tech-
niques. Most of the synthesized compounds showed significant
anti-inflammatory activity using carrageenan-induced rat paw
edema method. The NO-donating furoxans exhibited maximum
amount of NO released among the synthesized NO-donating hy-
brids. The prepared chalcone/NO hybrids either nitrate ester;
oxime or furoxan derivatives showed pronounced gastroprotective
activity that might attribute to the release of NO. Histopathological
examination of some intermediates and their NO hybrids indicated
that the NO donating moiety reduced greatly the incidence of gas-
tric ulceration. In conclusion the use of hybrid molecules contain-
ing NO-donating moieties looks as a promising approach to
improve the safety of NSAIDs without altering their effectiveness.
Figure 3A. Photomicrograph of the mucosa of fundic stomach of control.
4. Experimental section
4.1. Chemistry
Reactions were monitored by TLC: Pre-coated plastic sheets,
0.2 mm silica gel with fluorescent indicator (Macherey–Nagel).
Column chromatography: Silica Gel 60 (Merck) for column chro-
matography. Melting points were determined on Stuart electro-
thermal melting point apparatus and were uncorrected. IR
spectra were recorded as KBr disks on a Shimadzu IR-408 spectro-
photometer. 1H NMR spectra were carried out on Bruker apparatus
at DRX400 MHz, AV300 MHz, DPX 200 MHz (Braunschweig Uni-
versity, Germany); Geol EX-270 MHz spectrometers (National
Research Center, Dokki, Giza, Egypt); using TMS as internal
Figure 3B. Photomicrograph of the mucosa of fundic stomach of indomethacin.

G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
201
reference. Chemical shifts (d values are given in parts per million
(ppm) using CDCl3 (7.29) or DMSO-d6 (2.5) as solvents and cou-
pling constants (J) in Hertz. Splitting patterns are designated as fol-
lows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of
doublet; m, multiplet; bs, broad singlet. EI-MS: Finnigan MAT
4515 and Jeol JMS 600 spectrometers. Elemental analysis was per-
formed on Perkin–Elmer 2400 CHN Elemental analyzer, Faculty of
Science, Cairo University, Egypt.
120 (38), 112 (11), 91 (16). Anal. Calcd for C18H16BrNO3 (373.03):
C, 57.77; H, 4.31; N, 3.74. Found: C, 57.49; H, 4.57; N, 3.99.
4.1.5. N-[4-(3-Benzo[1,3]dioxol-5-ylacryloyl)phenyl]-2-
bromoacetamide (2c)
Yellow crystals (methanol) in (1.99 g, 82% yield); mp 202–
203 °C; IR (KBr)
mmax = 3340 (NH), 1670 (br, CO–NH, CO–C@C),
1596 cmꢀ1 (C@C); 1H NMR (200 MHz, DMSO-d6, d = ppm)
d = 10.72 (s, 1H, NH), 8.18 (d, 2H, J = 8.03 Hz, ArH), 7.81 (d, 1H,
J = 15.30 Hz, Hb), 7.75 (d, 2H, J = 8.04 Hz, ArH), 7.60 (d, 1H,
4.1.1. General procedure for the synthesis of 1-(4-
aminophenyl)-3-arylprop-2-en-1-one 1a–d
J = 1.83 Hz, ArH), 7.51 (d, 1H, J = 15.30 Hz, H ), 7.29 (dd, 1H,
a
p-Aminoacetophenone (0.1 mol) and the aldehydes (0.1 mol)
were dissolved in a minimum amount of methanol, then aqueous
NaOH (0.25 mol, 10%) was added. The reaction mixture was stirred
at rt until a precipitate was formed (within 4–24 h). The precipitate
was filtered off and washed thoroughly with distilled water and
cold methanol (2 ꢁ 20 mL). The product was recrystallized from
the appropriate solvent. The structure of chalcone derivatives
1a–d was confirmed by mp and IR spectroscopy.
J = 8.04 Hz, J = 1.83 Hz, ArH), 6.93 (d, 1H, J = 8.03 Hz, ArH), 6.10 (s,
2H, OCH2O), 4.08 (s, 2H, CH2Br); EI-MS (70 eV) m/z (%) 389
(M++2, 57), 388 (M++1, 35), 387 (M+, 60), 359 (8), 309 (27), 308
(100), 266 (25), 240 (11), 238 (19), 180 (17), 175 (21), 152 (14),
145 (37), 120 (31), 91 (28). Anal. Calcd for C18H14BrNO4 (387.01):
C, 55.69; H, 3.63; N, 3.61. Found: C, 55.75; H, 3.39; N, 3.80.
4.1.6. 2-Bromo-N-[4-(3-furan-2-ylacryloyl)phenyl]acetamide
(2d)
1-(4-Aminophenyl)-3-(4-chlorophenyl)prop-2-en-1-one (1a). Mp
159–160 °C [Ref. 51; 160 °C].
Pale yellow crystals (ethanol) in (1.57 g, 75% yield); mp 138–
1-(4-aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (1b).
Mp 113–114 °C [Ref. 53; 114–115 °C].
1-(4-aminophenyl)-3-(1,3-benzodioxol-5-yl)prop-2-en-1-one
(1c). Mp 197–198 °C [Ref. 53; 199 °C].
1-(4-aminophenyl)-3-(2-furyl) prop-2-en-1-one (1d). Mp 119–
120 °C [Ref. 52; 118–119 °C].
139 °C; IR (KBr)
C@C), 1600 cmꢀ1 (C@C); 1H NMR (200 MHz, CDCl3, d = ppm)
d = 10.75 (s, 1H, NH), 8.45–6.43 (m, 9H: 3furanyl, 4ArH,
CH@CH–), 4.23 (s, 2H, CH2Br). Anal. Calcd for C15H12BrNO3
(333.00): C, 53.91; H, 3.62; N, 4.19. Found: C, 53.79; H, 3.25; N,
4.49.
mmax = 3460 (NH), 1695 (CO–NH), 1682 (CO–
–
4.1.2. General procedure for the synthesis of 2-bromo-N-{4-[3-
arylacryloyl] phenyl}acetamides 2a–d
4.1.7. General procedure for the synthesis of N-{4-[3-
arylacryloyl]phenyl}-2-nitrooxyacetamides 3a–d
To a stirred mixture of chalcones 1a–d (6.30 mmol) in dichloro-
methane (20 mL) and potassium carbonate (1.302 mmol) in
100 mL water cooled in an ice bath, bromoacetyl bromide
(1.856 g, 9.20 mmol) in 30 mL dichloromethane was added in a
dropwise manner with stirring over 30 min. Stirring was continued
for 2 h at 0 °C, and at rt overnight. The reaction mixture was ex-
tracted with (2 ꢁ 60 mL) dichloromethane, and the organic layer
was washed with distilled water (2 ꢁ 40 mL), dried over anhydrous
sodium sulphate, filtered, evaporated on a rotary evaporator and
the residue was recrystallized from appropriate solvent.
A solution of the appropriate bromoacetyl derivatives 2a–d
(2.7 mmol) in dry acetonitrile (5 mL) was treated portion wise with
a solution of silver nitrate (0.68 g, 4 mmol) in dry acetonitrile
(10 mL). The mixture was heated under reflux temperature for
12 h. The formed precipitate was filtered off, and the filtrate was
evaporated till dryness. The residue was dissolved in dichloro-
methane, washed with distilled water (2 ꢁ 25 mL) and brine
(2 ꢁ 25 mL), dried over sodium sulphate, filtered and the organic
layer was evaporated till dryness, and the residue was recrystal-
lized from the appropriate solvent.
4.1.3. 2-Bromo-N-{4-[3-(4-
chlorophenyl)acryloyl]phenyl}acetamide (2a)
Pale yellow crystals (ethanol) in (1.81 g, 76% yield); mp 190–
4.1.8. N-{4-[3-(4-Chlorophenyl)acryloyl]phenyl}-2-
nitrooxyacetamide (3a)
Yellow crystals (methanol) in (0.53 g, 55% yield); mp 180–
192 °C; IR (KBr)
m
max = 3300 (NH), 1675 (CO–NH), 1655 (CO–
181 °C; IR (KBr) mmax = 3350 (NH), 1655 (CO–NH), 1648 (CO–
C@C), 1600 cmꢀ1 (C@C); 1H NMR (400 MHz, DMSO-d6, d = ppm)
d = 10.78 (s, 1H, NH), 8.19 (d, 2H, J = 8.18 Hz, ArH), 7.98 (d, 1H,
J = 15.58 Hz, Hb), 7.94 (d, 2H, J = 8.18 Hz, ArH), 7.79 (d, 2H,
C@C), 1605 (C@C), 1275 cmꢀ1 (ONO2); 1H NMR (400 MHz, DMSO-
d6, d = ppm) d = 10.79 (s, 1H, NH), 8.20 (d, 2H, J = 8.15 Hz, ArH),
7.98 (d, 1H, J = 15.44 Hz, Hb), 7.94 (d, 2H, J = 8.15 Hz, ArH), 7.77
J = 8.18 Hz, ArH), 7.72 (d, 1H, J = 15.58 Hz, H ), 7.53 (d, 2H,
(d, 2H, J = 8.15 Hz, ArH), 7.73 (d, 1H, J = 15.44 Hz, H ), 7.54 (d,
a
a
J = 8.18 Hz, ArH), 4.11 (s, 2H, CH2Br); EI-MS (70 eV) m/z (%) 381
(M++4, 31), 379 (M++2, 92), 377 (M+, 59), 350 (20), 298 (100),
256 (18), 242 (33), 229 (31), 165 (56), 137 (29), 120 (47), 101
(31). Anal. Calcd for C17H13BrClNO2 (376.98): C, 53.92; H, 3.46; N,
3.70. Found: C, 54.26; H, 3.63; N, 4.00.
2H, J = 8.15 Hz, ArH), 5.28 (s, 2H, CH2); EI-MS (70 eV) m/z (%) 362
(M++2, 4), 360 (M+, 10), 314 (49), 313 (100), 284 (37), 256 (27),
241 (19), 228 (36), 176 (30), 164 (59), 146 (28), 136 (51), 110
(36), 101 (37). Anal. Calcd for C17H13ClN2O5 (360.05): C, 56.60; H,
3.63; N, 7.77. Found: C, 56.86; H, 4.05; N, 7.45.
4.1.4. 2-Bromo-N-{4-[3-(4-
methoxyphenyl)acryloyl]phenyl}acetamide (2b)
Pale orange crystals (chloroform) in (1.85 g, 79% yield); mp
4.1.9. N-{4-[3-(4-Methoxyphenyl)acryloyl]phenyl}-2-
nitrooxyacetamide (3b)
Yellow crystals (acetonitrile) in (0.57 g, 59% yield); mp 144–
160–162 °C; IR (KBr)
m
max = 3295 (NH), 1695 (CO–NH), 1685 (CO–
145 °C; IR (KBr) mmax = 3395 (NH), 1672 (CO–NH), 1660 (CO–
C@C), 1595 cmꢀ1 (C@C); 1H NMR (200 MHz, CDCl3, d = ppm)
d = 10.75 (s, 1H, NH), 8.04 (d, 2H, J = 8.44 Hz, ArH), 7.79 (d, 1H,
J = 15.60 Hz, Hb), 7.70 (d, 2H, J = 8.43 Hz, ArH), 7.60 (d, 2H,
C@C), 1600 (C@C), 1254 cmꢀ1 (ONO2); 1H NMR (400 MHz, DMSO-
d6, d = ppm) d = 10.77 (s, 1H, NH), 8.17 (d, 2H, J = 8.12 Hz, ArH),
8.86 (d, 1H, J = 15.40 Hz, Hb), 7.82 (d, 2H, J = 8.12 Hz, ArH), 8.76
J = 8.44 Hz, ArH), 7.41 (d, 1H, J = 15.60 Hz, H ), 6.94 (d, 2H,
(d, 2H, J = 8.12 Hz, ArH), 7.75 (d, 1H, J = 15.40 Hz, H ), 7.03 (d,
a
a
J = 8.44 Hz, ArH), 4.05 (s, 2H, CH2Br), 3.84 (s, 3H, OCH3); EI-MS
(70 eV) m/z (%) 375 (M++2, 71), 374 (M++1, 32), 373 (M+, 74), 294
(100), 257 (13), 252 (11), 240 (41), 234 (20), 161 (43), 133 (24),
2H, J = 8.12 Hz, ArH), 5.28 (s, 2H, CH2), 3.85 (s, 3H, OCH3); EI-MS
(70 eV) m/z (%) 356 (M+,3), 309 (100), 308 (27), 299 (12), 280
(13), 254 (11), 252 (33), 237 (13), 224 (23), 161 (40), 135 (14),

202
G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
133 (20), 120 (12), 108 (8). Anal. Calcd for C18H16N2O6 (356.10): C,
60.67; H, 4.53; N, 7.86. Found: C, 61.09; H, 4.45; N, 7.39.
J = 8.80 Hz, ArH), 7.79 (d, 1H, J = 15.53 Hz, Hb), 7.68 (d, 1H,
J = 15.53 Hz, H ), 7.64 (d, 2H, J = 8.80 Hz, ArH), 7.02 (d, 2H,
a
J = 8.80 Hz, ArH), 4.78 (q, 1H, J = 7.08 Hz, CHBr), 3.83 (s, 3H,
OCH3), 1.79 (d, 3H, J = 7.08 Hz, CH3); EI-MS (70 eV) m/z (%) 389
(M++2, 1), 388 (M++1, 2), 387 (M+, 1), 325 (100), 310 (5), 297
(15), 296 (36), 280 (6), 279 (11), 251 (9), 224 (15), 192 (9), 161
(21), 133 (10), 120 (7), 92 (8). Anal. Calcd for C19H18BrNO3
(387.05): C, 58.78; H, 4.67; N, 3.61. Found: C, 58.75; H, 4.39; N,
3.81.
4.1.10. N-[4-(3-Benzo[1,3]dioxol-5-ylacryloyl)phenyl]-2-
nitooxyacetamide (3c)
Yellow crystals (acetonitrile) in (0.62 g, 62% yield); mp 160–
162 °C; IR (KBr)
mmax = 3290 (NH), 1690 (CO–NH), 1665 (CO–
C@C), 1595 (C@C), 1250 cmꢀ1 (ONO2); 1H NMR (400 MHz, DMSO-
d6, d = ppm) d = 10.78 (s, 1H, NH), 8.19 (d, 2H, J = 8.06 Hz, ArH),
7.83 (d, 1H, J = 15.38 Hz, Hb), 7.76 (d, 2H, J = 8.06 Hz, ArH), 7.67
(d, 1H, J = 15.38 Hz, H ), 7.66 (d, 1H, J = 1.88 Hz, ArH), 7.33 (dd,
4.1.15. N-[4-(3-Benzo[1,3]dioxol-5-ylacryloyl)phenyl]-2-
bromopropionamide (4c)
a
1H, J = 8.06 Hz, J = 1.88 Hz, ArH), 7.00 (d, 1H, J = 8.06 Hz, ArH),
6.12 (s, 2H, OCH2O), 5.28 (s, 2H, OCH2ONO2); EI-MS (70 eV) m/z
(%) 324 (M+ꢀ46, 25), 323 (100), 322 (40), 295 (10), 293 (20), 268
(15), 266 (38), 238 (15), 176 (12), 175 (26), 149 (11), 147 (24),
Pale yellow powder (chloroform) in (2.77 g, 69% yield); mp 158–
159 °C; IR (KBr)
m
max = 3292 (NH), 1665 (CO–NH), 1650 (CO–C@C),
;
1595 (C@C) cmꢀ1
1H NMR (DMSO-d6, d = ppm) d = 10.69 (s, 1H,
146 (15), 120 (17), 104 (10). Anal. Calcd For
(370.08): C, 58.38; H, 3.81; N, 7.56. Found: C, 58.76; H, 3.62; N,
7.82.
C
18H14N2O7
NH), 8.21–6.97 (m, 9 H: 7 ArH, –CH@CH–), 6.12 (s, 2H, CH2), 4.74
(q, 1H, J = 6.69 Hz, CHBr), 1.78 (d, 3H, J = 6.67 Hz, CH3); EI-MS
(70 eV) m/z (%) 403 (M++2, 20), 402 (M++1, 67), 401 (M+, 33), 339
(100), 327 (66), 310 (41), 394 (17), 238 (42), 207 (45), 192 (45),
175 (59), 153 (28), 120 (41), 117 (26). Anal. Calcd for C19H16BrNO4
(401.03): C, 56.73; H, 4.01; N, 3.48. Found: C, 57.19; H, 4.25; N, 3.49.
4.1.11. N-[4-(3-Furan-2-ylacryloyl)phenyl]-2-
nitrooxyacetamide (3d)
Dark brown crystals (methanol) in (0.44 g, 52% yield);
mp > 300 °C; IR (KBr)
m
max = 3370 (NH), 1671 (CO–NH), 1652
4.1.16. 2-Bromo-N-[4-(3-furan-2-
ylacryloyl)phenyl]propionamide (4d)
(CO–C@C), 1598 (C@C), 1251 cmꢀ1 (ONO2); 1H NMR (200 MHz,
CDCl3, d = ppm) d = 10.79 (s, 1H, NH), 8.14–8.42 (m, 9H: 3furanyl,
4ArH, –CH@CH–), 5.35 (s, 2H, CH2). Anal. Calcd for C15H12N2O6
(316.07): C, 56.96; H, 3.82; N, 8.86. Found: C, 56.60; H, 4.00; N,
8.80.
Dark brown crystals (ethanol) in (2.26 g, 65% yield); mp 121–
122 °C; IR (KBr) mmax = 3297 (NH), 2927 (Aliph.–CH), 1671 (CO–
NH), 1655 (CO–C@C), 1596 (C@C) cmꢀ1; 1H NMR (200 MHz, CDCl3,
d = ppm) d = 10.82 (s, 1H, NH), 8.35–6.52 (m, 9H: 3furanyl, 4ArH, –
CH@CH–), 4.55 (q, 1H, J = 6.95 Hz, CHBr), 1.86 (d, 3H, J = 6.95 Hz,
CH3). Anal. Calcd for C16H14BrNO3 (347.02): C, 55.19; H, 4.05; N,
4.02. Found: C, 55.00; H, 4.10; N, 3.79.
4.1.12. General procedure for the synthesis of 2-bromo-N-{4-[3-
arylacryloyl]phenyl}propionamides 4a–d
To a stirred solution of 2-bromopropionic acid (1.53 g, 10 mmol)
in 50 mL chloroform cooled at 0 °C, triethylamine (1.0 g, 10 mmol)
was added, followed by ethyl chloroformate (1.08 g, 10 mmol) in
drop wise manner. Stirring was continued for further 1 h at 0 °C,
then chalcones 1a–d (10 mmol) were added in portion wise manner,
and the reaction mixture was then stirred for additional 12 h at rt
Chloroform (2 ꢁ 40 mL) was then added, and the organic layer was
separated, washed with distilled water (2 ꢁ 40 mL), 5% sodium
bicarbonate(2 ꢁ 40 mL), 1 N hydrochloric acid (2 ꢁ 40 mL), distilled
water (2 ꢁ 40 mL) and finally with brine (2 ꢁ 40 mL). The organic
layer was then dried over sodium sulphate, filtered, evaporation of
the organic layer and the obtained crude product was then recrystal-
lized from the appropriate solvent.
4.1.17. General procedure for the synthesis of N-{4-[3-
arylacryloyl]phenyl}-2-nitrooxypropionamides 5a–d
The titled compounds were prepared according to the general
procedure used in the synthesis of nitrate esters 3a–d, using the
same molar ratios.
4.1.18. N-{4-[3-(4-Chlorophenyl)acryloyl]phenyl}-2-
nitrooxypropionamide (5a)
Pale yellow crystals (acetonitrile) in (0.62 g, 61% yield); mp
192–193 °C; IR (KBr)
m
max = 3310 (NH), 1650 (CO–NH, CO–C@C),
1595 (C@C), 1225 (ONO2) cmꢀ1
;
1H NMR (200 MHz, DMSO-d6,
d = ppm) d = 10.89 (s, 1H, NH), 8.20 (d, 2H, J = 8.40 Hz, ArH), 7.94
(d, 2H, J = 8.40 Hz, ArH), 7.80 (d, 2H, J = 8.40 Hz, ArH), 7.78 (d, 1H,
J = 16.01 Hz, Hb), 7.53 (d, 2H, J = 8.40 Hz, ArH), 7.35 (d, 1H,
4.1.13. 2-Bromo-N-{4-[3-(4-
chlorophenyl)acryloyl]phenyl}propionamide (4a)
White crystals (methanol) in (2.82 g, 72% yield); mp 188–
J = 16.01 Hz, H ), 5.52 (q, 1H, J = 6.91 Hz, CHONO2), 1.54 (d, 3H,
a
J = 6.91 Hz, CH3); EI-MS (70 eV) m/z (%) 376 (M++2, 4), 374 (M+,
9), 328 (100), 284 (10), 267 (50), 256 (15), 239 (23), 231 (9), 204
(65), 190 (23), 164 (52), 155 (16), 136 (20), 110 (8), 101 (96). Anal.
Calcd for C18H15ClN2O5 (374.07): C, 57.69; H, 4.03; N, 7.47. Found:
C, 58.03; H, 4.45; N, 7.17.
190 °C; IR (KBr)
mmax = 3279 (NH), 1670 (br, CO–NH, CO–C@C),
1595 cmꢀ1 (C@C); 1H NMR (200 MHz, CDCl3, d = ppm) d = 10.70
(s, 1H, NH), 8.09 (d, 2H, J = 8.20 Hz, ArH), 7.71 (d, 1H,
J = 15.20 Hz, Hb), 7.59 (d, 2H, J = 8.17 Hz, ArH), 7.43 (d, 2H,
J = 8.20 Hz, ArH), 7.30 (d, 1H, J = 15.20 Hz, H ), 7.01 (d, 2H,
a
J = 8.17 Hz, ArH), 4.55 (q, 1H, J = 7.11 Hz, CHBr), 1.98 (d, 3H,
J = 7.11 Hz, CH3); EI-MS (70 eV) m/z (%) 395 (M++4, 12), 393
(M++2, 54), 391 (M+, 44), 312 (100), 302 (26), 267 (28), 229 (36),
204 (17), 165 (52), 137 (34), 120 (42), 101 (39). Anal. Calcd for
4.1.19. N-{4-[3-(4-Methoxyphenyl)acryloyl]phenyl}-2-
nitrooxypropionamide (5b)
Pale yellow crystals (acetonitrile) in (0.56 g, 56% yield); mp
144–145 °C; IR (KBr)
mmax = 3321 (NH), 2998 (Aliph.–CH), 1676
C18H15BrClNO2 (391.00): C, 55.06; H, 3.85; N, 3.57. Found: C,
(CO–NH), 1654 (CO–C@C), 1598 (C@C), 1220 cmꢀ1 (ONO2); 1H
NMR (400 MHz, CDCl3, d = ppm) d = 10.78 (s, 1H, NH), 8.02 (d,
2H, J = 8.09 Hz, ArH), 7.79 (d, 1H, J = 15.42 Hz, Hb), 7.60 (d, 2H,
J = 8.09 Hz, ArH), 7.52 (d, 2H, J = 8.09 Hz, ArH), 7.43 (d, 1H,
55.30; H, 3.46; N, 3.46.
4.1.14. 2-Bromo-N-{4-[3-(4-
methoxyphenyl)acryloyl]phenyl}propionamide (4b)
J = 15.42 Hz, H ), 6.94 (d, 2H, J = 8.09 Hz, ArH), 5.51 (q, 1H,
a
Buff crystals (methanol) in (2.55 g, 66% yield); mp 142–143 °C;
J = 7.11 Hz, CHONO2), 3.86 (s, 3H, OCH3), 1.61 (d, 3H, J = 7.12 Hz,
CH3); EI-MS (70 eV) m/z (%) 324 (M+ꢀ46, 100), 310 (7), 296 (47),
252 (15), 237 (8), 224 (64), 209 (9), 180 (14), 165 (22), 161 (41),
153 (16), 146 (19), 133 (29), 118 (19), 101 (10), 90 (31). Anal. Calcd
IR (KBr)
mmax = 3287 (NH), 1655 (CO–NH), 1645 (CO–C@C),
1589 cmꢀ1 (C@C); 1H NMR (400 MHz, DMSO-d6, d = ppm)
d = 10.68 (s, 1H, NH), 8.11 (d, 2H, J = 8.80 Hz, ArH), 7.84 (d, 2H,

G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
203
for C19H18N2O6 (370.12): C, 61.62; H, 4.90; N, 7.56. Found: C, 61.33;
H, 5.12; N, 7.50.
for C26H23NO5 (429.16): C, 72.71; H, 5.40; N, 3.26. Found: C, 72.55;
H, 5.23; N, 3.34.
4.1.20. N-[4-(3-Benzo[1,3]dioxol-5-ylacryloyl)phenyl]-2-
nitooxypropionamide (5c)
4.1.25. 2-(4-Acetylphenoxy)-N-[4-(3-benzo[1,3]dioxol-5-
ylacryloyl)phenyl]acetamide (6c)
Yellowish-green crystals (acetonitrile) in (0.69 g, 67% yield); mp
Yellow powder (chloroform) in (1.66 g, 75% yield); mp 244–
165–166 °C; IR (KBr)
m
max = 3310 (NH), 2850 (Aliph.–CH), 1662
246 °C; IR (KBr) mmax = 3408 (NH), 1692 (CO–NH), 1675 (CO–CH3),
(CO–NH), 1650 (CO–C@C), 1600 (C@C), 1250 cmꢀ1 (ONO2); 1H
NMR (400 MHz, DMSO-d6, d = ppm) d = 10.79 (s, 1H, NH), 8.19 (d,
2H, J = 8.11 Hz, ArH), 7.83 (d, 1H, J = 15.53 Hz, Hb), 7.78 (d, 2H,
1649 (CO–C@C), 1600 cmꢀ1 (C@C); 1H NMR (200 MHz, DMSO-d6,
d = ppm) d = 10.57 (s, 1H, NH), 8.20 (d, 2H, J = 8.73 Hz, ArH), 7.98
(d, 2H, J = 8.73 Hz, ArH), 7.86 (d, 1H, J = 15.42 Hz, Hb), 7.84 (d, 2H,
J = 8.11 Hz, ArH), 7.67 (d, 1H, J = 15.53 Hz, H ), 7.65 (s, 1H, ArH),
J = 8.78 Hz, ArH), 7.69 (d, 1H, J = 15.42 Hz, H ), 7.68 (d, 1H,
a
a
7.33 (dd, 1H, Jo = 8.11 Hz, Jm = 1.85 Hz, ArH), 7.00 (d, 1H,
J = 8.11 Hz, ArH), 6.12 (s, 2H, OCH2O), 5.49 (q, 1H, J = 7.07 Hz,
CHONO2), 1.54 (d, 3H, J = 7.07 Hz, CH3); EI-MS (70 eV) m/z (%)
338 (M+ꢀ46, 20), 310 (10), 239 (13), 238 (68), 224 (8), 181 (12),
175 (10), 165 (12), 152 (32), 146 (41), 120 (30), 117 (18), 95
(11), 89 (100). Anal. Calcd for C19H16N2O7 (384.10): C, 59.38; H,
4.20; N, 7.29. Found: C, 59.00; H, 4.25; N, 7.01.
Jo = 1.15 Hz, ArH), 7.36 (dd, 1H, Jo = 1.15, Jm = 8.78 Hz, ArH), 7.15
(d, 2H, J = 8.78 Hz, ArH), 7.03 (d, 1H, J = 8.78 Hz, ArH), 6.10 (s, 2H,
OCH2O), 4.92 (s, 2H, CH2), 2.53 (s, 3H, CH3); EI-MS (70 eV) m/z
(%) 444 (M++1, 24), 443 (M+, 100), 415 (8), 400 (4), 307 (44), 266
(20), 250 (8), 214 (8), 185 (5). Anal. Calcd for
C26H21NO6
(443.14): C, 70.42; H, 4.77; N, 3.16. Found: C, 70.64; H, 4.55; N,
3.25.
4.1.21. N-[4-(3-Furan-2-ylacryloyl)phenyl]-2-
nitrooxypropionamide (5d)
4.1.26. 2-(4-Acetylphenoxy)-N-[4-(3-furan-2-
ylacryloyl)phenyl]acetamide (6d)
Brown crystals (acetonitrile) in (0.52 g, 58% yield); mp 200–
Pale brown crystals (acetone) in (1.28 g, 66% yield); mp 218–
202 °C; IR (KBr)
m
max = 3306 (NH), 2922 (Aliph.–CH), 1660 (CO–
220 °C; IR (KBr) mmax = 3415 (NH), 1691 (CO–NH), 1672 (CO–CH3),
NH), 1647 (CO–C@C), 1597 (C@C), 1225 cmꢀ1 (ONO2); 1H NMR
(200 MHz, DMSO-d6, d = ppm) d = 10.72 (s, 1H, NH), 8.22–6.65
(m, 9H: 3furanyl, 4ArH, –CH@CH–), 5.45 (q, 1H, J = 7.15 Hz, CHON-
O2), 1.82 (d, 3H, J = 7.15 Hz, CH3). Anal. Calcd for C16H14N2O6
(330.09): C, 58.18; H, 4.27; N, 8.48. Found: C, 58.48; H, 4.48; N,
8.75.
1652 (CO–C@C), 1600 cmꢀ1 (C@C); 1H NMR (200 MHz, DMSO-d6,
d = ppm) d = 10.65 (s, 1H, NH), 8.22–6.65 (m, 13H: 3furanyl,
8ArH, –CH@CH–), 4.92 (s, 2H, CH2), 2.60 (s, 3H, CH3); EI-MS
(70 eV) m/z (%) 390 (M++1, 23), 389 (M+, 100), 361 (4), 335 (6),
296 (41), 254 (6), 226 (12), 198 (5), 160 (4), 140 (20), 121 (32).
Anal. Calcd for C23H19NO5 (389.13): C, 70.94; H, 4.92; N, 3.60.
Found: C, 71.12; H, 4.69; N, 3.45.
4.1.22. General procedure for the synthesis of 2-(4-
acetylphenoxy)-N-{4-[3-arylacryloyl]phenyl}acetamides 6a–d
To a solution of p-hydroxyacetophenone (0.68 g, 5 mmol),
bromoacetyl derivatives 2a–d (5 mmol) in 100 mL acetone, anhy-
drous potassium carbonate (0.50 g, 3.60 mmol) was added. The
reaction mixture was heated on a water-bath for 12 h. The solvent
was then evaporated, and the obtained residue was washed with
distilled water and recrystallized from the appropriate solvent.
4.1.27. General procedure for the synthesis of N-{4-[3-
arylacryloyl]phenyl}-2-[4-(1-hydroxyiminoethyl phenoxy]
acetamides 7a–d
A mixture of equimolar amounts of the appropriate ketone
derivatives 6a–d (2.3 mmol) and hydroxylamine hydrochloride in
absolute ethanol (30 mL) was heated under reflux for 20 h and
then left to cool. The separated solid was filtered, washed with
dil. ammonia solution and distilled water, dried, and recrystallized
from the appropriate solvent.
4.1.23. 2-(4-Acetylphenoxy)-N-{4-[3-(4-
chlorophenyl)acryloyl]phenyl}acetamide (6a)
Pale yellow crystals (chloroform) in (1.52 g, 70% yield); mp
4.1.28. N-{4-[3-(4-Chlorophenyl)acryloyl]phenyl}-2-[4-(1-
hydroxyiminoethyl)phenoxy]acetamide (7a)
224–226 °C; IR (KBr)
mmax = 3410 (NH), 1690 (br, CO–NH, CO–
CH3), 1675 (CO–C@C), 1600 cmꢀ1 (C@C); 1H NMR (200 MHz,
DMSO-d6, d = ppm) d = 10.60 (s, 1H, NH), 8.21 (d, 2H, J = 8.71 Hz,
ArH), 8.02 (d, 1H, J = 15.73 Hz, Hb), 7.99 (d, 2H, J = 8.71 Hz, ArH),
7.94 (d, 2H, J = 8.71 Hz, ArH), 7.86 (d, 2H, J = 8.73 Hz, ArH), 7.25
Yellow crystals (methanol) in (0.92 g, 89% yield); mp 200–
202 °C; IR (KBr)
mmax = 3600–3100 (OH), 3400 (NH), 1690 (CO–
NH), 1662 (CO–C@C), 1600 cmꢀ1 (C@C); 1H NMR (200 MHz,
DMSO-d6, d = ppm) d = 11.04 (s, 1H, OH), 10.52 (s, 1H, NH), 8.18
(d, 2H, J = 8.18 Hz, ArH), 7.95–7.16 (m, 10H: 8ArH, –CH@CH–),
7.03 (d, 2H, J = 6.41 Hz, ArH), 4.81 (s, 2H, CH2), 2.13 (s, 3H, CH3);
EI-MS (70 eV) m/z (%) 448 (M+, 38), 450 (M++2, 13), 433 (100),
417 (97), 404 (62), 369 (15), 297 (95), 279 (36), 270 (75), 257
(42), 191 (22), 165 (86), 121 (72). Anal. Calcd for C25H21ClN2O4
(448.12): C, 66.89; H, 4.72; N, 6.24. Found: C, 66.74; H, 4.60; N,
6.39.
(d, 1H, J = 15.70 Hz, H ) 7.55 (d, 2H, J = 8.71 Hz, ArH), 7.14 (d, 2H,
a
J = 8.73 Hz, ArH), 4.92 (s, 2H, CH2), 2.53 (s, 3H,, CH3); EI-MS
(70 eV) m/z (%) 435 (M++2, 31), 433 (M+, 100), 418 (8), 405 (12),
375 (33), 360 (17), 332 (8), 296 (51), 270 (87), 257 (88), 238
(37), 192 (44), 165 (90), 137 (75), 121 (96), 102 (51). Anal. Calcd
for C25H20ClNO4 (433.11): C, 69.20; H, 4.65; N, 3.23. Found: C,
68.87; H, 4.86; N, 3.40.
4.1.24. 2-(4-Acetylphenoxy)-N-{4-[3-(4-
4.1.29. 2-[4-(1-Hdroxyiminoethyl)phenoxy]-N-{4-[3-(4-
methoxyphenyl)acryloyl]phenyl}acetamide (7b)
methoxyphenyl)acryloyl]phenyl}acetamide (6b)
Pale yellow crystals (methanol) in (1.57 g, 73% yield); mp 216–
Buff crystals (methanol) in (0.92 g, 90% yield); mp 198–200 °C;
218 °C; IR (KBr)
mmax = 3398 (NH), 1678 ((br, CO–NH, CO–CH3),
1655 (CO–C@C), 1600 cmꢀ1 (C@C); 1H NMR (200 MHz, DMSO-d6,
d = ppm) d = 10.55 (s, 1H, NH), 8.16 (d, 2H, J = 8.68 Hz, ArH), 7.97
(d, 2H, J = 8.68 Hz, ArH), 7.88–7.79 (m, 5H: 4 ArH, Hb), 7.70 (d,
IR (KBr)
mmax = 3700–3200 (OH), 3398 (NH), 2923 (Aliph.–CH),
1685 (CO–NH), 1679 (CO–C@C), 1592 cmꢀ1 (C@C); 1H NMR
(270 MHz, DMSO-d6, d = ppm) d = 11.03 (s, 1H, OH), 10.53 (s, 1H,
NH), 8.14–7.01 (m, 14H: 12 ArH, –CH@CH–), 4.88 (s, 2H, CH2),
3.81 (s, 3H, OCH3), 2.10 (s, 3H, CH3); EI-MS (70 eV) m/z (%) 444
(M+, 3), 429 (100), 401 (8), 414 (8), 398 (4), 294 (22), 279 (10),
269 (12), 252 (13), 207 (7), 161 (22), 133 (14), 121 (15), 107
1H, J = 15.68 Hz, H ) 7.12 (d, 2H, J = 8.76 Hz, ArH), 7.02 (d, 2H,
a
J = 8.76 Hz, ArH), 4.90 (s, 2H, CH2), 4.19 (s, 3H, OCH3), 2.50 (s, 3H,
CH3); EI-MS (70 eV) m/z (%) 430 (M++1, 23), 429 (M+, 100),
401(8), 293 (22), 266 (18), 207 (8), 161 (24), 121 (16). Anal. Calcd

204
G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
(10). Anal. Calcd for C26H24N2O5 (444.17): C, 70.26; H, 5.44; N, 6.30.
Found: C, 70.40; H, 5.33; N, 6.35.
NH), 8.24–6.99 (m, 15H: 13H, ArH, –CH@CH–), 4.75 (s, 2H, Ar–
CH2–O), 4.68 (s, 2H, O–CH2–CO), 3.83 (s, 3H, OCH3); EI-MS
(70 eV) m/z (%) 486 (M++1, 4), 485 (M+, 12), 469 (12), 455 (5),
366 (6), 350 (13), 311 (4), 264 (10), 252 (20), 237 (13), 224 (8),
4.1.30. N-[4-(3-Benzo[1,3]dioxol-5-ylacryloyl)phenyl]-2-[4-(1-
hydroxyiminoethyl)phenoxy]acetamide (7c)
161 (24), 133 (15), 103 (100). Anal. Calcd for
C27H23N3O6
Yellow powder in (0.96 g, 91% yield); mp > 300 °C; IR (KBr)
(485.16): C, 66.80; H, 4.78; N, 8.66. Found: C, 67.00; H, 4.50; N,
8.29.
m
max = 3700–3300 (OH), 3408 (NH), 1670 (CO–NH), 1651 (CO–
C@C), 1600 cmꢀ1 (C@C); 1H NMR (insoluble in NMR deuterated
solvents); EI-MS (70 eV) m/z (%) 458 (M+, 5), 443 (100), 415 (10),
400 (4), 307 (44), 280 (18), 266 (11), 250 (8), 213 (10), 185 (8),
175 (11), 145 (17), 121 (17). Anal. Calcd for C26H22N2O6 (458.15):
C, 68.11; H, 4.84; N, 6.11. Found: C, 68.35; H, 4.70; N, 6.37.
4.1.36. N-[4-{3-Benzo[1,3]dioxol-5-ylacryloyl)phenyl]-2-(2-oxy-
4-phenylfurazan-3-ylmethoxy)acetamide (9c)
Pale yellow powder (DMF) in (0.33 g, 67% yield); mp 148–
150 °C; IR (without solvent) mmax = 3357 (NH), 2908 (Aliph. –CH),
1700 (CO–NH), 1659 (CO–C@C), 1590 (furoxan), 1243 cmꢀ1 (O–
CH2); 1H NMR (400 MHz, DMSO-d6, d = ppm) d = 10.45 (s, 1H,
NH), 8.25–6.93 (m, 14H: 12H, ArH, –CH@CH–), 6.15 (s, 2H, OCH2O),
4.65 (s, 2H, Ar–CH2–O), 4.55 (s, 2H, O–CH2–CO); EI-MS (70 eV) m/z
(%) 499 (M+, 16), 483 (18), 453 (5), 397 (4), 380 (7), 364 (19), 323
(28), 293 (24), 267 (52), 238 (21), 209 (7), 180 (10), 175 (20), 145
(36), 103 (100). Anal. Calcd for C27H21N3O7 (499.14): C, 64.93; H,
4.24; N, 8.41. Found: C, 65.25; H, 3.86; N, 8.30.
4.1.31. N-[4-(3-Furan-2-ylacryloyl)phenyl]-2-[4-(1-
hydroxyiminoethyl)phenoxy]acetamide (7d)
Pale yellow powder in (0.82 g, 88% yield); mp 270–272 °C; IR
(KBr) mmax = 3600–3200 (OH), 3389 (NH), 2925 (Aliph.–CH), 1668
(CO-NH), 1652 (CO–C@C), 1596 cmꢀ1 (C@C); 1H NMR (insoluble
in NMR deuterated solvents); EI-MS (70 eV) m/z (%) 405 (M++1,
7), 404 (M+, 4), 389 (100), 361 (7), 335 (13), 296 (41), 254 (5),
226 (16), 212 (5), 198 (8), 160 (9), 140 (20), 121 (33). Anal. Calcd
for C23H20N2O5 (404.14): C, 68.31; H, 4.98; N, 6.93. Found: C,
68.59; H, 5.23; N, 6.80.
4.1.37. N-[4-(3-Furan-2-ylacryloyl)phenyl]-2-(2-oxy-4-
phenylfurazan-3-ylmethoxy)acetamide (9d)
Pale yellow powder (DMF) in (0.24 g, 53% yield); mp > 250 °C;
4.1.32. Synthesis of 3-phenyl-4-furoxanmethanol (8)
IR (without solvent) mmax = 3326 (NH), 3096 (Ar–CH), 1655 (br,
To a stirred solution of cinnamyl alcohol (6.7 g, 0.05 mol) in
10 mL glacial acetic acid heated at 70 °C, a solution of sodium ni-
trite (6.9 g, 0.1 mol) in 5 mL water was added in a drop-wise man-
ner. The reaction mixture was stirred at rt for 2 h. The organic layer
was extracted with dichloromethane, washed with water
(2 ꢁ 40 mL), dried over anhydrous calcium chloride and evapo-
rated on a rotary evaporator giving an oily brown product, which
was purified by column chromatography using pentane/ethylace-
tate (4:1) as an eluent to give the titled compound (3.84 g, 40%)
as pale yellow crystals, mp 67 °C.
C@O), 1586 (furoxan), 1227 cmꢀ1 (O–CH2); 1H NMR (400 MHz,
DMSO-d6, d = ppm) d = 10.71 (s, 1H, NH), 8.39–6.62 (m, 14H, 9
ArH, 3 Furanyl, –CH@CH–), 4.55 (s, 2H, Ar–CH2–O), 4.46 (s, 2H,
O–CH2–CO); EI-MS (70 eV) m/z (%) 446 (M++1, 8), 445 (M+, 12),
413 (100), 385 (8), 369 (9), 291 (4), 237 (4), 219 (7), 225 (10),
213 (11), 197 (14), 160 (15), 132 (41), 121 (16), 105 (12), 77
(13). Anal. Calcd for C24H19N3O6 (445.13): C, 64.72; H, 4.30; N,
9.43. Found: C, 64.35; H, 4.45; N, 9.06.
4.2. Nitric oxide release
4.1.33. General procedure for the synthesis of N-[4-
(arylacryloyl)phenyl]-2-(2-oxy-4-phenylfurazan-3-
ylmethoxy)acetamides 9a–d
4.2.1. Measurement64
Different solutions of the tested compounds 3a–d, 5a–d, 7a–d, 8
and 9a–d (20 lL) in DMF was added to 2 mL of 1:1 v/v mixture of
To
a
solution of 3-phenyl-4-furoxanmethanol
8
(0.192 g,
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 °C, 1 mL of the reaction mixture was
1 mmol), bromoacetyl derivatives 2a–d (1 mmol) in 15 mL ace-
tone, anhydrous potassium carbonate (0.138 g, 1 mmol) was
added. The reaction mixture was heated on a water-bath for
12 h. The solvent was evaporated, and the residue was washed
with distilled water and recrystallized from the appropriate
solvent.
treated with 250 lL of Griess reagent [sulfanilamide (2 g), N-naph-
thylethylenediamine 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 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 expressed as the percentage of NO re-
leased relative to a theoretical maximum release of 1 mol NO/
mol of test compound.
4.1.34. N-{4-[3-(4-Chlorophenyl)acryloyl]phenyl}-2-(2-oxy-4-
phenylfurazan-3-ylmethoxy)acetamide (9a)
Yellow crystals (chloroform) in (0.39 g, 79% yield); mp > 250 °C;
IR (without solvent) mmax = 3359 (NH), 3063 (Ar –CH), 1700 (CO–
NH), 1657 (CO–C@C), 1592 (furoxan), 1269 cmꢀ1 (O–CH2); 1H
NMR (200 MHz, DMSO-d6, d = ppm) d = 10.73 (s, 1H, NH), 8.28–
7.29 (m, 15H: 13H, ArH, –CH@CH–), 4.19 (s, 2H, Ar–CH2–O), 4.05
(s, 2H, O–CH2–CO); EI-MS (70 eV) m/z (%) 491 (M++2, 33), 489
(M+, 100), 379 (42), 342 (10), 298 (88), 283 (6), 257 (28), 240
(10), 257 (22), 240 (10), 229 (23), 193 (11), 178 (10), 165 (30),
137 (17), 120 (33). Anal. Calcd for C26H20ClN3O5 (489.11): C,
63.74; H, 4.11; N, 8.58. Found: C, 63.50; H, 4.10; N, 8.33.
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 1a–d, 2a–d, 3a–d,
4a–d, 5a–d, 6a–d, 7a–d, 8 and 9a–d and reference drug (indomrth-
acin) in carboxy methyl cellulose (CMC) solution (0.5% w/v in
4.1.35. N-{4-[3-(4-Methoxyphenyl)acryloyl]phenyl}-2-(2-oxy-4-
phenylfurazan-3-ylmethoxy)acetamide (9b)
Yellow powder (chloroform) in (0.27 g, 55% yield); mp 182–
184 °C; IR (without solvent)
mmax = 3368 (NH), 3004 (Ar –CH),
1686 (CO–NH), 1653 (CO–C@C), 1589 (furoxan), 1254 cmꢀ1 (O–
CH2); 1H NMR (400 MHz, DMSO-d6, d = ppm) d = 10.54 (s, 1H,

G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
205
12. Bhandari, V. S.; Parikh, K. J.; Bothara, G. K.; Chitre, S. T.; Lokwani, K. D.; Devale,
L. T.; Modhave, S. N.; Pawar, S. V.; Panda, S. J. Enzyme Inhib. Med. Chem. 2010,
25, 520.
13. Wallace, J. L.; Miller, M. J. Gastroenterol. 2000, 119, 512.
14. Takeuchi, K.; Yasuhiro, T.; Asada, Y.; Sugawa, Y. Digestion 1998, 59, 298.
15. Holzer, P.; Pabst, M. A.; Lipp, I. T.; Peskar, B. M.; Livingston, E. H.; Guth, P. H.
Gastroenterology 1990, 98, 838.
16. Wallace, J. L.; Vergnolle, N.; Muscara, M. N.; Asfaha, S.; Chapman, K.;
McKnight, W.; Del Soldato, P.; Morelli, A.; Fiorucci, S. Gastroenterology 1999,
117, 557.
17. Brown, J. F.; Keates, A. C.; Hanson, P. J.; Whittle, B. J. Am. J. Physiol. Gastrointest.
Liver Physiol. 1993, 265, G418.
18. Holm, M.; Johansson, B.; Petterssson, A.; Fandrisk, L. Acta Physiol. Scand. 1998,
162, 461.
19. Iwata, F.; Leung, F. W. Am. J. Physiol. Gastrointest. Liver Physiol. 1995, 268, G153.
20. Fujihara, C. K.; Malheiros, D. M.; Donato, J. L.; Poli, A.; De Nucci, G.; Zatz, R. Am.
J. Physiol. Renal Physiol. 1998, 274, F573.
21. Lee, S. H.; Seo, G. S.; Kim, J. Y.; Jin, X. Y.; Kim, H. D.; KimSohn, D. H. Eur. J.
Pharmacol. 2006, 532, 178.
22. Nowakowska, Z. Eur. J. Med. Chem. 2007, 42, 125.
23. Rajendra, P. Y.; Srinivasa, R. A.; Rambabu, R. Asian J. Chem. 2009, 21, 907.
24. Tomar, V.; Bhattacharjee, G.; Kamaluddina; Kumar, A. Bioorg. Med. Chem. Lett.
2007, 17, 5321.
25. Nowakowska, Z.; Kedzia, B.; Shroeder, G. Eur. J. Med. Chem. 2008, 43, 707.
26. Lopez, S. N.; Castelli, M. V.; Zacchino, S. A.; Dominguez, J. N.; Lobo, G.; Charris-
Charris, J.; Cortes, J. C. J.; Ribas, J. C.; Devia, C.; Rodriguez, A. M.; Enriz, R. D.
Bioorg. Med. Chem. 2001, 9, 1999.
27. Lahtchev, K. L.; Batovska, D. I.; Parushev, P., St; Ubiyvovk, V. M.; Sibirny, A. A.
Eur. J. Med. Chem. 2008, 43, 2220.
28. Trivedi, J. C.; Bariwal, J. B.; Upadhyay, K. D.; Naliapara, E. D.; Joshi, S. K.;
Pannecouque, C. C.; Clercq, E. D.; Shah, A. K. Tetrahedron Lett. 2007, 48, 8472.
29. Lin, Y. M.; Zhou, Y.; Flavin, M. T.; Zhow, L. M.; Nie, W.; Chen, F. C. Bioorg. Med.
Chem. 2002, 10, 2795.
30. Dominguez, J. N.; León, C.; Rodrigues, J.; Domínguez, N. G.; Gut, J.; Rosenthal, P.
J. IL Farmaco. 2005, 60, 307.
31. Motta, L. F.; Gaudio, A. C.; Takahata, Y. Int. Elect. J. Mol. Des. 2006, 5, 555.
32. Dominguez, J. N.; Charris, J. E.; Lobo, G.; Gamboan, N.; Dominguez, N. G.;
Moreno, M. M.; Riggione, F.; Sanchez, E.; Olson, J.; Rosenthal, P. J. Eur. J. Med.
Chem. 2001, 36, 555.
33. Liu, M.; Wilairat, P.; Go, M. L. J. Med. Chem. 2002, 45, 1735.
34. Yayli, N.; Ucuncu, O.; Aydin, E.; Gok, Y.; Yasar, A.; Baltaci, C.; Yildirim, N.;
Kucuk, M. J. Photbio. A: Chem. 2005, 169, 229.
35. Vogel, S.; Ohmayer, S.; Brunner, G.; Heilmann, J. Bioorg. Med. Chem. 2008, 16,
4286.
water) was administered orally in a dose level of (100 mg/kg). Con-
trol 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 Ver-
nier celiper (SMIEC) directly before and after 1, 2, 3, 4 and 5 h inter-
vals 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.
4.3.2. Ulcerogenic liability
After measuring the anti-inflammatory activity the rats were
sacrified 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 le-
vel III, in which the ulcer area more than 3 mm2 and this rated
according to their areas in mm2.
The data are expressed as mean SEM, one way ANOVA test
was applied to determine the significance of the difference be-
tween the control group and rats treated with the tested
compounds.
4.3.3. Histopathological investigation
The histological slides were prepared according to the reported
procedures for examination of ulcers under light microscope.65
Identify site of the slide on which the section was applied by
scratching wax around section with a needle. Dewax hydrated sec-
tions by using graded alcohols to water. Slides were stained with
haematoxylin for 5–7 min, washed with tap water until sectioning
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 running tap
water for 1–5 min. The slide was then dehydrated using alcohols,
cleaned by using xylene, covered by glass cover using Canda bal-
sam then examined under microscope.
36. Gacche, R. N.; Dhole, N. A.; Kamble, S. G.; Bandgar, B. P. Enz. Inhib. Med. Chem.
2008, 23, 28.
37. Ducki, S.; Forrest, R.; Hadfield; Kendall, A.; Lawrence, R. J.; McGown, A. T.;
Rennison, D. Bioorg. Med. Chem. Lett. 1998, 8, 1051.
38. Boeck, P.; Falco, C. A. B.; Leal, P. C.; Yunes, R. A.; Filho, V. C.; Torres-Santos, E. C.;
Rossi-Bergmann, B. Bioorg. Med. Chem. 2006, 14, 1538.
39. Zhao, L.; Jin, H.; Sun, L.; Piaoa, H.; Quana, Z. Bioorg. Med. Chem. Lett. 2005, 15,
5027.
40. Won, S. J.; Liu, C. T.; Tsao, L. T.; Weng, J. R.; Ko, H. H.; Wang, J. P.; Lin, C. N. Eur. J.
Med. Chem. 2005, 40, 103.
41. Achanta, G.; Modzelewska, A.; Feng, L.; Khan, S. R.; Huang, P. Mol. Pharmacol.
2006, 70, 426.
42. Modzelewska, A.; Pettit, C.; Achanta, G.; Davidson, N. E.; Huang, P.; Khan, S. R.
Bioorg. Med. Chem. 2006, 14, 3491.
Acknowledgement
43. Ducki, S. IDrugs 2007, 10, 42.
44. Herencia, F.; Ferrandiz, M. L.; Ubeda, A.; Dominguez, J. N.; Charris, J. E.; Lobo, G.
M.; Alcaraz, M. J. Bioorg. Med. Chem. Lett. 1998, 8, 1169.
45. Herencia, F.; Ferrandiz, M. L.; Ubeda, A.; Guillen, I.; Dominguez, J. N.; Charris, J.
E.; Lobo, G. M.; Alcaraz, M. J. Free Radic. Biol. Med. 2001, 30, 43.
46. Herencia, F.; Ferrandiz, M. L.; Ubeda, A.; Guillen, I.; Dominguez, J. N.; Charris, J.
E.; Lobo, G. M.; Alcaraz, M. J. FEBS Lett. 1999, 453, 129.
47. Herencia, F.; Lopez-Garcia, M. P.; Ubeda, A.; Ferrandiz, M. L. Nitric Oxide 2002, 6,
242.
48. Rojas, J.; Dominguez, J. N.; Ferrandiz, M. L. Eur. J. Pharmacol. 2003, 465, 183.
49. Shoman, M. E.; Abdel-Aziz, M.; Aly, O. M.; Farag, H. H.; Morsy, M. A. Eur. J. Med.
Chem. 2009, 44, 3068.
50. Abdel-Hafez, M. N.; El-SM; Abuo-Rahma, A. A.; Gel-D; Abdel-Aziz, M.; Radwan,
M. F.; Farag, H. H. Bioorg. Med. Chem. 2009, 17, 3829.
Authors introduce their great thanks to Dr. Ahmed A. Mourad,
Pharmacology Department, Faculty of Pharmacy, Minia University
for his valuable help in measuring the anti-inflammatory activity
and ulcerogenic liability. Authors also introduce their great thanks
to Prof. Dr. Entesar Ali Saber and Dr. Sohaa Abdel-Kawi, Histology
Department, Faculty of Medicine, Minia University for their great
help in performing the histopathological investigation.
References and notes
1. Singh, G.; Triadafilopoulos, G. J. Rheumatol. 1999, 18.
2. Botting, R. M. J. Therm. Biol. 2006, 3, 208.
3. Naesdal, J.; Brown, K. Drug Saf. 2006, 29, 119.
51. Fengping, Y.; Yangping, P.; Gonghua, S.; Jizong, L. J. Chem. Res. 2005, 311.
52. Nakaya, K.; Funabiki, K.; Shibata, K.; Muramatsu, H.; Matsul, M. Bull. Chem. Soc.
Jpn. 1996, 69, 2961.
4. Cryer, B. Am. J. Gastroenterol. 2005, 100, 1694.
5. Lazzaroni, M.; Bianchi, P. G. Aliment. Pharmacol. Ther. 2004, 20, 48.
6. James, M. W.; Hawkey, C. J. Br. J. Clin. Pharmacol. 2003, 56, 146.
7. Schneider, V.; Levesque, L. E.; Zhang, B.; Hutchinson, T.; Brophy, J. M. Am. J.
Epidemiol. 2006, 164, 881.
53. Ansari, F. L.; Umbreen, S.; Hussain, L.; Makhmoor, T.; Nawaz, A. S.; Lodhi, M. A.;
Khan, S. N.; Shamsun, N.; Shaheen, F.; Choudhary; Atta.-ur.-Rahman, M. I.
Chem. Biodivers. 2005, 2, 487.
54. Silverstein, R. M; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of
Organic Compounds, 5th ed.; Wiley: New York, 1991.
8. Mounier, G.; Guy, C.; Berthoux, F.; Beyens, M. N.; Ratrema, M.; Ollagnier, M.
Therapie 2006, 61, 255.
55. Bowie, J. W.; Simons, B. K.; Donaghue, P. F.; Kallury, P. K. M. R. Tetrahedron
1969, 25, 3696.
9. Zadrazil, J. Vnitr. Lek. 2006, 52, 686.
10. Adebayo, D.; Bjarnason, I. Postgrad. Med. J. 2006, 82, 186.
56. Kallury, R. K. M. R.; Rao, P. L. K. M. Org. Mass Spectr. 1977, 12, 411.
57. Gasco, A. M.; Fruttero, R.; Sorba, G.; Gasco, A. Liebigs Ann. Chem. 1991, 1211.
58. Lolli, M. L.; Cena, C.; Medana, C.; Lazzarato, L.; Morini, G.; Coruzzi, G.; Manarini,
S.; Fruttero, R.; Gasco, A. J. Med. Chem. 2001, 44, 3463.
´
11. Velazquez, C. A.; Praveen Rao, P. N.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. Bioorg.
Med. Chem. 2007, 15, 4767.

206
G.E.-D.A. A. Abuo-Rahma et al. / Bioorg. Med. Chem. 20 (2012) 195–206
59. Winter, C. A.; Risely, E. A.; Nuss, G. W. Proc. Soc. Biol. Med. 1962, 111, 544.
60. De Andrade, S. F.; Lemosa, M.; Comunello, E.; Noldin, V. F.; Filho, V. C.; Niero, R.
J. Ethnopharmacol. 2007, 113, 252.
63. Vela´zquez, C. A.; Praveen Rao, P. N.; Citro, M. L.; Keefera, L. K.; Knaus, E. Bioorg.
Med. Chem. 2007, 15, 4767.
64. Tsikas, D. J. Chromatogr., B 2007, 851, 51.
61. Perini, R.; Fiorucci, S.; Wallace, J. L. Can. J. Gastroenterol. 2004, 18, 229.
62. Bastaki, S. M.; Wallace, J. L. Can. J. Gastroenterol. 1999, 13, 123.
65. Gartner, L. P.; Haitt, J. L. Color Textbook of Histology, 2nd ed.; W. B. Saunders
Company: Philadelphia, London, New York, 2001.