Synthesis and investigation of anti-inflammatory activity and gastric ulcerogenicity of novel nitric oxide-donating pyrazoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.07.008

A group of 3,5-diaryl-2-pyrazoline derivatives were prepared via the reaction of various chalcones with hydrazine hydrate in ethanol. A group of NO-donating-2-pyrazoline derivatives were synthesized by carrying a nitrate ester group or an oxime group onto

Full text of DOI:10.1016/j.ejmech.2008.07.008

European Journal of Medicinal Chemistry 44 (2009) 3068–3076
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and investigation of anti-inflammatory activity and gastric
ulcerogenicity of novel nitric oxide-donating pyrazoline derivatives
,
a
b
c
Mai E. Shoman a, Mohamed Abdel-Aziz a , Omar M. Aly , Hassan H. Farag , Mohamed A. Morsy
*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia, Egypt
b Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt
c Department of Pharmacology, Faculty of Medicine, Minia University, Minia, 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 3,5-diaryl-2-pyrazoline derivatives were prepared via the reaction of various chalcones with
hydrazine hydrate in ethanol. A group of NO-donating-2-pyrazoline derivatives were synthesized by
carrying a nitrate ester group or an oxime group onto the prepared pyrazoline derivatives through
different spacers. The prepared compounds were evaluated for their anti-inflammatory activity using
carrageenan-induced rat paw edema and compared to a well-known NSAID, indomethacin as a reference
drug. The ability of the prepared compounds to induce gastric toxicity was also evaluated. Most of the
prepared compounds showed significant anti-inflammatory activity at the injected dose (100 mg/kg) but
they were safer than indomethacin in regard to gastric toxicity. The incorporation of the NO-donating
group into the parent pyrazoline derivatives caused a non-significant reduction in the anti-inflammatory
activity while a marked decrease in gastric ulcerations induced by their parent pyrazolines was observed.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Received 25 March 2008
Received in revised form 10 May 2008
Accepted 8 July 2008
Available online 16 July 2008
Keywords:
2-Pyrazoline
Nitric oxide
Anti-inflammatory
Gastric toxicity
1. Introduction
known to spare the renal system mainly through stimulating the
renal blood flow [19].
Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the
most useful clinical therapies for the treatment of pain, fever, and
inflammation [1]. The major mechanism by which NSAIDs exert
their anti-inflammatory activity is by the inhibition of cyclo-
oxygenase-derived prostaglandin synthesis, which is also respon-
sible for the gastrointestinal [2–6], renal [7–9], and hepatic [10] side
effects that are observed mainly in chronic use of NSAIDs. There-
fore, the challenge still exists for the pharmaceutical industry to
develop, effective anti-inflammatory agents with enhanced safety
profile. One of the most important strategies used to overcome
NSAIDs side effects is to design nitric oxide-donating NSAIDs (NO-
NSAIDs) which are capable of generating the radical biomediator
and gastroprotective agent NO [11]. NO contributes to the modu-
lation of several key physiological functions in the digestive system
[12], it has the ability to increase the mucosal blood flow [13],
resulting in enhanced mucosal resistance to ulceration [14], NO also
prevents adherence of leukocytes to the vascular endothelium [15],
it is known to modulate gastroduodenal secretion of mucus [16]
and bicarbonate [17], NO can profoundly influence the mucosal
immune system [12], it also increases the ability of mucosal cells to
undergo healing and repair of the existing ulcers [18]. NO is also
Moreover, 2-pyrazoline derivatives have been reported to
exhibit various pharmacological activities such as antimicrobial
[20–22], anti-inflammatory [23–25], and antihypertensive [26]. In
addition, 1-unsubstituted-3,5-diaryl-2-pyrazolines were reported
to exhibit human Acyl CoA cholesterol acyltransferase activity [27]
as well as activity of low-density lipoprotein oxidation inhibitors
[28]. In addition, 1,3,5-triaryl-2-pyrazolines were reported to
possess antidepressant properties [29], and monoamine oxidase
(MAO) inhibitory activities [30].
Promoted with the above-mentioned studies, the present study
aimed at gathering the two bioactive entities (pyrazoline and NO)
into one compact structure and evaluating their biological activities
as anti-inflammatory agents with reduced gastric toxicity.
2. Chemistry
Chalcones (6a–d, Scheme 1) were synthesized by a base cata-
lyzed Claisen–Schmidt condensation reaction of appropriately
substituted acetophenones (1–2) and substituted aromatic alde-
hydes (3–5) in the presence of 10% NaOH in ethanol. Heating at
reflux chalcones (6a–d) with hydrazine monohydrate 95% in
absolute ethanol afforded the corresponding pyrazolines (7a–d).
Treatment of pyrazolines (7a–d) with bromoacetyl bromide in
the presence of K2CO3 at 0 ꢀC afforded the corresponding 2-
bromoacyl pyrazolines (8a–d). Other 2-acyl pyrazoline derivatives
* Corresponding author. Tel./fax: þ2086 2369075.
E-mail address: abulnil@hotmail.com (M. Abdel-Aziz).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.07.008

M.E. Shoman et al. / European Journal of Medicinal Chemistry 44 (2009) 3068–3076
3069
Scheme 1. Synthesis of different NO-donating pyrazoline derivatives 9a–d, 11a–c, 13a and 13b.
(10a–c, Scheme 1) were prepared by the reaction of pyrazolines
(7a–c) with 2-bromopropionic acid mixed anhydride, which was
prepared by the reaction of 2-bromopropionic acid with ethyl
chloroformate in the presence of triethylamine in equimolar ratio in
dry chloroform at ꢁ10 ꢀC. The objective nitrate esters (9a–d and
11a–c) were prepared by stirring the corresponding bromoacyl pyr-
azolinederivatives(8a–dand10a–c)withsilvernitrateinacetonitrile
for 15 h at 80 ꢀC, which afforded the corresponding nitrate esters.
Heating at reflux of pyrazoline bromoacyl derivatives 8a and 8b
with p-amino acetophenone in acetone in the presence of K2CO3
afforded the corresponding coupling products 12a and 12b,
respectively. Heating at reflux of compounds 12a and 12b with
hydroxylamine hydrochloride in absolute ethanol gave the corre-
sponding oximes 13a and 13b, respectively. The structure of the
prepared compounds was confirmed on the basis of their IR, 1H
NMR, mass spectra and elemental analysis. The reaction sequences
are outlined in Scheme 1.
3. NO release measurement
The NO-releasing properties of the tested compounds using
different forms including: starting derivatives (7a and 8a), NO-
donating nitrates (9a–d and 11a–c) and NO-donating oximes (13a
and 13b) were assessed in both phosphate buffer of pH 7.4 and pH 1
using 0.1 N HCl with Griess reagent. 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,
calculated as the amount of NO (mol/mol) released and are listed in
Table 1.
Table 1
The amount of NO released from tested compounds 7a, 8a, 9a–d, 11a–c, 13a and 13b in phosphate buffer (pH ¼ 7.4)
Compound no.
Amount of NO released (mol/mol) ꢂ SEM
1 h
2 h
3 h
4 h
5 h
6 h
7a
–
–
–
–
–
–
8a
–
–
–
–
–
–
9a
9b
9c
9d
11a
11b
11c
13a
13b
0.126 ꢂ 0.050
0.329 ꢂ 0.036
0.147 ꢂ 0.031
0.195 ꢂ 0.034
0.078 ꢂ 0.013
0.098 ꢂ 0.023
0.123 ꢂ 0.017
0.118 ꢂ 0.021
0.137 ꢂ 0.030
0.211 ꢂ 0.029
0.391 ꢂ 0.020
0.395 ꢂ 0.045
0.295 ꢂ 0.068
0.190 ꢂ 0.021
0.160 ꢂ 0.027
0.190 ꢂ 0.024
0.128 ꢂ 0.023
0.114 ꢂ 0.030
0.281 ꢂ 0.037
0.433 ꢂ 0.044
0.516 ꢂ 0.047
0.356 ꢂ 0.015
0.398 ꢂ 0.041
0.438 ꢂ 0.037
0.501 ꢂ 0.027
0.138 ꢂ 0.016
0.148 ꢂ 0.082
0.483 ꢂ 0.079
0.523 ꢂ 0.047
0.555 ꢂ 0.080
0.451 ꢂ 0.060
0.200 ꢂ 0.040
0.128 ꢂ 0.028
0.255 ꢂ 0.041
0.167 ꢂ 0.032
0.194 ꢂ 0.043
0.614 ꢂ 0.082
0.780 ꢂ 0.088
0.738 ꢂ 0.086
0.656 ꢂ 0.071
0.125 ꢂ 0.012
0.065 ꢂ 0.005
0.145 ꢂ 0.015
0.277 ꢂ 0.021
0.302 ꢂ 0.024
0.535 ꢂ 0.100
0.588 ꢂ 0.012
0.625 ꢂ 0.065
0.593 ꢂ 0.074
0.087 ꢂ 0.009
0.090 ꢂ 0.007
0.114 ꢂ 0.012
0.267 ꢂ 0.030
0.268 ꢂ 0.010

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M.E. Shoman et al. / European Journal of Medicinal Chemistry 44 (2009) 3068–3076
4. Biological activity
and more than 3 mm2 and this is rated according to their areas in
mm2, and the following parameters were calculated [34]:
4.1. Anti-inflammatory activity
1. The ulcer index (UI) was calculated as 1 ꢃ (number of ulcers of
level I) þ 2 ꢃ (number of ulcers of level II) þ 3 ꢃ (number of
ulcers of level III), etc.
2. Cure ratio ¼ 100 ꢁ (UItreated ꢃ 100/UIprotype).
The synthesized new compounds 7a–d, 8a–d, 9a–d, 10a–c, 11a–
c, 13a and 13b were evaluated for their anti-inflammatory activity
using carrageenan-induced paw edema described by Winter et al.
[31]. The tested compounds and reference drug (indomethacin)
were administered orally at a dose level of 100 mg/kg, 0.5 h 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 with the
following formula [32].
Where UItreated means the average of ulcer index of groups treated
with the NO-donating derivatives and UIprotype means the average
of ulcer index of groups treated with the starting pyrazoline
derivatives. The ulcer indexes of the tested compounds were
calculated and listed in Table 2 and Fig. 2A–D.
5. Results and discussions
The IR spectra of the prepared pyrazolines (7a–d) showed
n
(C]N) stretching at 1611–1599 cmꢁ1 due to the ring closure. In
ðVR ꢁ VLÞcontrolꢁðVR ꢁ VLÞtreatedꢃ100
% of edema inhibition ¼
addition, absorption bands at 1115–1261 cmꢁ1 were attributed to
ðVR ꢁ VLÞcontrol
the
n (C–N) stretching vibrations, which also confirm the formation
where VR represents the mean right paw thickness, VL represents
the mean left paw thickness, (VR ꢁ VL)control represents the mean
increase in paw thickness in the control group of rats and
(VR ꢁ VL)treated represents the mean increase in paw thickness in
rats treated with the tested compounds. The results are listed in
Table 2 and shown in Fig. 1A–D.
of the desired pyrazoline ring in all prepared compounds. A sharp
band in the region of 3226–3334 cmꢁ1 due to the
n (NH) stretching
was also observed. 1H NMR spectra recorded for the prepared
compounds in CDCl3 clearly supported the proposed structures.
Protons of pyrazoline ring in compounds 7a and 7b showed
a prominent ABX system, with protons Ha, Hb and Hx seen as
doublets of doublets at
d 3.20–3.44, 3.40–3.53 and 5.04–5.27 ppm
4.2. Ulcerogenic liability
(JHa–Hb ¼ 16.50–16.70, JHa–Hx ¼ 7.30–7.90, JHb–Hx ¼ 9.90–10.20 Hz),
respectively. On the other hand, pyrazolines (7c and 7d) with
substituted benzaldehyde carrying highly electronegative groups
(2,6-dichloro) made Ha and Hb that appeared to be equivalent so
the pattern became AX system. The CH2 protons appeared as
The synthesized compounds 7a–d, 8a–d, 9a–d,10a–c,11a–c,13a
and 13b were evaluated for their ulcerogenic activity according to
a reported procedure [33]. Ulcerogenic activity was evaluated in
rats after oral administration of the tested compounds and indo-
methacin at a dose level of 100 mg/kg in 0.5% aqueous CMC, 0.5%
aqueous CMC was used as a control.
Ulcers were classified into levels: level I, in which the ulcer area
is less than 1 mm2; level II, in which the ulcer area is in the range
from 1 to 3 mm2; and level III, in which the ulcer area equals 3 mm2
doublet at
triplet at
d 3.24 and 3.44 ppm while the CH proton appeared as
5.80 ppm (J ¼ 11.90–12.40 Hz). The protons belonging to
d
the aromatic system and phenyl substituents were observed at the
expected chemical shifts and integral values.
Treatment of pyrazolines (7a–d) with bromoacetyl bromide in
the presence of K2CO3 afforded the corresponding 2-bromoacyl
pyrazolines (8a–d), the structure of the prepared compounds was
confirmed by the appearance of an additional strong absorption
band of (C]O) stretching at 1653–1665 cmꢁ1 and disappearance of
that of NH stretching in IR spectra, while 1H NMR spectra showed
an additional two doublet peaks each for one proton of the CH2
Table 2
The anti-inflammatory activity at different time intervals and ulcer indexes of
compounds 7a–d, 8a–d, 9a–d, 10a–c, 11a–c, 13a and 13b using carrageenan-induced
paw edema in rats compared to indomethacin
protons nearly at
(J ¼ 11.40–12.00 Hz) due to geminal coupling.
d 4.3 and 4.4 ppm with coupling constant
Compound
no.
Percent of edema inhibition (% mean ꢂ SEM)
Ulcer index
(mean ꢂ SEM)
1 h
2 h
3 h
Another form of 2-bromoacyl pyrazolines (10a–c) was prepared
by the reaction of pyrazolines (7a–c) with 2-bromopropionic acid
mixed anhydride. The IR spectra of the prepared compounds
showed an additional strong absorption band of (C]O) stretching
at 1656–1687 cmꢁ1 and disappearance of the NH stretching band of
pyrazoline. 1H NMR spectra showed prominent doublet signals
Control
Indomethacin
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
10a
10b
10c
11a
11b
11c
13a
13b
0
0
0
1 ꢂ 0.5
43.5 ꢂ 3.5
26.5 ꢂ 1.5*
7 ꢂ 0.6**
3 ꢂ 1.0**
31 ꢂ 3.0
13 ꢂ 1.0*
30 ꢂ 3.0
3 ꢂ 0.5**
16 ꢂ 1.0*
2 ꢂ 0.3**
5 ꢂ 0.5**
4 ꢂ 0.3**
0 ꢂ 0.0**
10 ꢂ 0.5**
23 ꢂ 2.0*
10 ꢂ 1.0**
7 ꢂ 1.0**
3 ꢂ 0.1**
7 ꢂ 0.0**
0 ꢂ 0.0**
2 ꢂ 1.0**
58 ꢂ 3.01***
45 ꢂ 4.61***
38 ꢂ 0.82***
26 ꢂ 4.45*
54 ꢂ 3.04***
38 ꢂ 4.44***
53 ꢂ 3.01***
48 ꢂ 6.01**
43 ꢂ 6.90**
32 ꢂ 4.32***
38 ꢂ 1.65***
40 ꢂ 4.61***
34 ꢂ 2.49**
22 ꢂ 1.65*
58 ꢂ 4.25***
51 ꢂ 7.52***
35 ꢂ 3.78***
44 ꢂ 5.51***
47 ꢂ 5.62***
38 ꢂ 0.82***
27 ꢂ 6.01
67 ꢂ 2.70***
53 ꢂ 5.40***
39 ꢂ 2.70***
49 ꢂ 2.70***
58 ꢂ 2.70***
46 ꢂ 1.60***
67 ꢂ 3.62***
58 ꢂ 5.26***
49 ꢂ 2.70**
37 ꢂ 2.34***
48 ꢂ 4.77***
49 ꢂ 6.03***
42 ꢂ 4.10***
36 ꢂ 3.62***
63 ꢂ 5.40***
56 ꢂ 3.77***
39 ꢂ 3.04***
61 ꢂ 5.80***
53 ꢂ 3.82***
46 ꢂ 3.63***
44 ꢂ 3.44**
82 ꢂ 4.86***
60 ꢂ 5.53***
42 ꢂ 2.58***
58 ꢂ 7.62***
60 ꢂ 2.58***
51 ꢂ 2.58***
71 ꢂ 1.29***
69 ꢂ 2.58***
47 ꢂ 0.44**
43 ꢂ 2.14***
58 ꢂ 3.95***
58 ꢂ 3.87***
52 ꢂ 2.98***
42 ꢂ 3.46***
69 ꢂ 2.58***
60 ꢂ 5.76***
42 ꢂ 4.46**
56 ꢂ 4.94***
55 ꢂ 2.30***
46 ꢂ 0.44***
46 ꢂ 7.29***
nearly at
d
1.69 ppm indicating the three protons of CH3, and
5.39 ppm.
quartet signals of the CH proton nearly at
d
The objective nitrate esters (9a–d and 11a–c) were prepared by
stirring the corresponding bromoacyl pyrazolines (8a–d and 10a–c)
with silver nitrate in acetonitrile. The IR spectra of the prepared
compounds showed additional absorption bands at 1536–
1653 cmꢁ1 due to asymmetric stretching of NO2 group and another
strong band at 1256–1276 cmꢁ1 due to NO2 group symmetrical
stretching. A characteristic downfield shift of the CH2 protons
attached to the nitrate ester was observed by nearly 1 ppm to
appear at
nitrates.
d
5.37–6.09 ppm in the 1H NMR spectra of the prepared
Heating at reflux of bromoacyl pyrazoline derivatives 8a and 8b
with p-amino acetophenone followed by refluxing with hydroxyl-
amine hydrochloride in absolute ethanol gave the corresponding
*Significantly different from control group at P ¼ 0.05; **significantly different from
control group at P ¼ 0.01; ***significantly different from control group at P ¼ 0.001.

M.E. Shoman et al. / European Journal of Medicinal Chemistry 44 (2009) 3068–3076
3071
Anti-inflammatory activity of
compounds 7a, 8a, 9a, 10a, 11a, 13a
Anti-inflammatory activity of
compounds 7b, 8b, 9b, 10b, 11b, 13b
B
A
80
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
8a
7a
9a
10a
11a
13a
8a
7a
9a
10a
11a
13a
1
2
3
Time (hr)
4
5
1
2
3
4
5
Time (hr)
Anti-inflammatory activity of
compounds 7c, 8c, 9c, 10c, 11c
Anti-inflammatory activity of
compounds 7d, 8d, 9d
C
D
80
70
60
50
40
30
20
10
0
11c
10c
8c
9c
7c
70
60
50
40
30
20
10
0
7d
8d
9d
1
2
3
Time (hr)
4
5
1
2
3
4
5
Time (hr)
Fig. 1. Anti-inflammatory activity of different tested compounds expressed as mean ꢂ S.E.
oximes (13a and 13b). The disappearance of ketonic (C]O)
stretching band and appearance of the (C]N) stretching band at
1606 cmꢁ1 in addition to the appearance of the(OH) stretching
band at 3491 cmꢁ1 in the IR spectra confirmed the proposed
structure. Mass spectra and elemental analysis are in agreement
with the proposed structure of the prepared compounds.
The results of NO-releasing properties of the tested compounds
7a, 8a, 9a–d, 11a–c, 13a and 13b indicated that the starting
indomethacin
indomethacin
B
A
50
45
40
35
30
25
20
15
10
5
50
45
40
35
30
25
20
15
10
5
8b
13b
11b
10b
9b
13a
11a
10a
9a
8a
7b
7a
indomethacin
8b
indomethacin
7a
10b
**
*
11a
*
8a
**
**
10a
*
7b
9b
11b
13b
9a
13a
0
0
Ulcer index of compounds
7a, 8a, 9a, 10a, 11a and 13a
Ulcer index of compounds
7b, 8b, 9b,10b,11b and 13b
indomethacin
indomethacin
C
D
50
45
40
35
30
25
20
15
10
5
50
40
11c
10c
9c
8c
9d
8d
7c
7d
7d
indomethacin
indomethacin
30
20
*
8d
**
10c
11c
10
0
7c
9c
8c
9d
0
Ulcer index of compounds
7c, 8c, 9c, 10c and 11c
Ulcer index of compounds
7d, 8d, 9d
* Significantly different fromparent pyrazoline at P < 0.001
** Significantly different fromits acetyl derivative at P < 0.001
Fig. 2. Ulcer indexes of different tested compounds compared to indomethacin expressed as mean ꢂ S.E.

3072
M.E. Shoman et al. / European Journal of Medicinal Chemistry 44 (2009) 3068–3076
pyrazoline derivative 7a and its bromoacyl derivative 8a did not
release any amount of NO neither at phosphate buffer of pH 7.4 nor
at pH 1 and this is a proof that the nitrates and oximes are the main
sources of NO release.
nitrate 11a showed 73% reduction of ulcers induced by its starting
pyrazoline derivative 7a, and only 30% reduction of ulcers induced
by its bromoacyl intermediate 10a. Also, oxime 13b indicated 71%
and 93% reduction of ulcers induced by its starting pyrazoline
derivative 7b and its bromoacyl intermediate 8b, respectively.
While NO-donating compound 11b exhibited 57% and 86% reduc-
tion of ulcers induced by its starting pyrazoline derivative 7b and its
bromoacyl intermediate 10b, respectively. On the other hand the
NO-donating derivative 9b indicated 28% and 83% reduction of
ulcers induced by its starting pyrazoline derivative 7b and its
bromoacyl intermediate 8b, respectively. The NO-donating nitrate
9d showed 96% and 93% reduction of ulcers induced by its starting
pyrazoline derivative 7d and its bromoacyl intermediate 8d,
respectively (Fig. 2A–D).
The decreased gastric toxicity of the targeted NO-donating
derivatives 9a–d, 11a–c, 13a and 13b relative to their starting
compounds 7a–d, 8a–d and 10a–c, could be explained by the
release of NO that increases mucosal blood flow resulting in
enhanced mucosal resistance to ulceration [37,38] and/or an
enhanced ability of the intact NO-donating derivatives to cross the
gastric mucosal lining prior to the subsequent release of NO and
starting pyrazoline derivatives [11].
Both forms of NO-donating derivatives including nitrate esters
(9a–d and 11a–c) and NO-donating oximes 13a and 13b were found
to release different amounts of NO at phosphate buffer of pH 7.4
(Table 1). For the first group of NO-donating nitrate esters 9a–d,
there was a regular increase in the amount of NO released reaching
their maximum after 5 h and then begin to decrease gradually in the
sixth hour. The NO-donating oximes 13a and 13b were found to
release fewer amount of NO compared to the previous nitrate esters
9a–d reaching their maximum after 5 h. The last group of nitrate
esters 11a–c were found to release a moderate amount of NO
reaching their maximum after 3 h and then begin to decrease
gradually in the fourth hour. This may be attributed to the electron
donating properties of the methyl group in the propionic acid
spacer present in compounds 11a–c which decrease the dissocia-
tion of the nitrite group giving lower amount of NO compared to the
acetic acid spacer present in compounds 9a–d. On the other hand
there is no release of NO at pH 1 and this may support the fact that
NO-donating moieties (nitrates and oximes) are weakly hydrolyzed
in the gastric lumen and this confirms that the suggested gastro-
protective action of NO is mediated systemically [35,36].
In conclusion the use of hybrid molecules containing NO-
releasing moieties looks as a promising approach to improve the
safety of NSAIDs without altering their effectiveness.
The synthesized new compounds 7a–d, 8a–d, 9a–d, 10a–c, 11a–
c, 13a and 13b were evaluated for their anti-inflammatory activity
using carrageenan-induced paw edema described by Winter et al.
The results show that most of the synthesized compounds showed
significant anti-inflammatory activity against carrageenan-induced
paw edema in rats after 3 h, which is the time necessary to reach the
maximum activity for the tested compounds. The tested pyrazoline
derivatives 7a–c showed considerable anti-inflammatory activity,
which is nearly equal to 75% of indomethacin activity. Introduction
of an acetyl moiety into compounds 8a–d and 10a–c was found to
increase the anti-inflammatory activity in most compounds.
Compounds 8b and 10b induced equipotent activity relative to
indomethacin for the first 2 h and reached about 86% after 3 h.
Compound 8c showed 84% of indomethacin activity while
compound 10c showed 86% in the first hour and began to decrease
gradually to reach to 73% after 3 h. On the other hand compounds 8a
and 8d showed inhibitory activity of about 70% of indomethacin
after 2 h and began to decrease gradually in the third hour to reach
about 58%. The anti-inflammatory activity of the designed NO
donors 9a–d, 11a–c, 13a and 13b indicated that NO donors 9b, 9c,
11b and 11c showed considerable anti-inflammatory activity, which
ranges from 67% to 70% of indomethacin activity. The anti-inflam-
matory activity of the oxime NO donors 13a and 13b was found to be
56% and 59% of indomethacin activity, respectively. These results
indicated that the incorporation of NO-donating group into the
pyrazoline nucleus had caused a non-significant reduction in the
anti-inflammatory activity (Fig. 1A–D).
6. Experimental
6.1. Chemistry
Reactions were monitored by TLC analysis using Merck
9385 pre-coated aluminum plate silica gel (Kieselgel 60) with
F254 indicator thin layer plates. Melting points were determined
on Stuart electrothermal melting point apparatus and were
uncorrected.
IR spectra were recorded as KBr disks on a Bruker Vector 22 IR
spectrophotometer. 1H NMR spectra were carried out on 300 MHz
Mercury 300BB NMR spectrophotometer, on 200 MHz GEMINI-200
NMR spectrophotometer and on 60 MHz Varian EM-360L NMR
spectrophotometer using TMS as an internal reference. Chemical
shift (d) values are given in parts per million (ppm) relative to CDCl3
(7.29) or DMSO-d6 (2.5) and coupling constants (J) in Hertz. Split-
ting patterns are designated as follows: s, singlet; d, doublet; t,
triplet; q, quartet; dd, doublet of doublet; m, multiplet.
Accurate masses were obtained on Micromass LCT mass spec-
trometer and on HP mass spectrometer. Elemental analysis was
performed on Perkin Elmer 2400 CHN Elemental Analyzer and on
Vario EL III, Elemental Analyzer, GmbH. D-63452 Hanau.
6.1.1. Chalcones (6a–d)
The synthesized compounds 7a–d, 8a–d, 9a–d,10a–c,11a–c,13a
and 13b were evaluated for their ulcerogenic activity. The results
indicated that there is a direct relationship between the anti-
inflammatory activity and the ulcerogenic toxicity of the tested
compounds. The starting pyrazoline derivatives and their bro-
Chalcones (6a–d) were prepared according to reported proce-
dure [39].
6.1.2. General procedure for the preparation of compounds
7a–d [40]
moacyl intermediates caused
a
milder ulcerogenic toxicity
A mixture of appropriate chalcones 6a–d (10.0 mmol) and
hydrazine monohydrate (95%) (1 g, 20 mmol) was heated at reflux
for 6–10 h in 50 mL of absolute ethanol. The solution was left to cool
at room temperature and the solid formed was filtered off, washed
with water, dried and recrystallized from absolute ethanol.
compared to indomethacin, while the NO-donating pyrazolines
were considered to be safe in regard to gastric toxicity having 0–7
range of ulcer index (Table 2). The percentage inhibition of ulcers
(cure ratio) was calculated and revealed that oxime 13a exhibited
96% and 92% reduction of ulcers induced by its starting pyrazoline
derivative 7a and its bromoacyl intermediate 8a, respectively, while
the nitrate ester 9a exhibited 92% and 84% reduction of ulcers
induced by its starting pyrazoline derivative 7a and its bromoacyl
intermediate 8a, respectively. On the other hand the NO-donating
6.1.2.1. 5-(2-Furyl)-4,5-dihydro-3-(3,4-dimethoxyphenyl)-1H-pyrazole
(7a). Pale yellow crystals (1.82 g, 66.8% yield), m.p. 150–151 ꢀC. IR
(KBr) nmax (cmꢁ1): 3226 (NH), 3045–3004 (Ar-CH), 2960–2830
(Aliph-CH), 1599 (C]N), 1567 (CfC). 1H NMR (300 MHz, CDCl3)

M.E. Shoman et al. / European Journal of Medicinal Chemistry 44 (2009) 3068–3076
3073
d
(ppm): 3.30 (dd,1H, J ¼ 16.50 and 9.86 Hz, CH2 of pyrazoline), 3.40
yield), m.p. 147–148 ꢀC. IR (KBr) nmax (cmꢁ1): 3068–3004 (Ar-CH),
2965–2842 (Aliph-CH), 1653 (C]O), 1597 (C]N), 1570 (CfC). 1H
(dd, 1H, J ¼ 16.50 and 7.32 Hz, CH2 of pyrazoline), 3.84 (s, 6H, 2
OCH3), 5.05 (dd, 1H, J ¼ 9.85 and 7.40 Hz, CH of pyrazoline), 6.26 (d,
2H, J ¼ 7.33 Hz, Ar-H), 6.79 (d, 1H, J ¼ 8.43 Hz, Ar-H), 7.04 (d, 1H,
J ¼ 8.43 Hz, Ar-H), 7.20 (s, 1H, NH), 7.30 (s, 1H, Ar-H), 7.38 (d, 1H,
J ¼ 1.47 Hz, Ar-H). MS: m/z (%): 272 (100) [Mþ], 271 (12), 154 (26),
137 (14), 136 (18). Anal. calcd for C15H16N2O3: C, 66.16; H, 5.92; N,
10.29. Found: C, 66.10; H, 5.92; N, 10.14.
NMR (300 MHz, CDCl3)
d
(ppm): 3.48 (dd, 1H, J ¼ 17.30 and 5.77 Hz,
CH2 of pyrazoline), 3.60 (dd, 1H, J ¼ 17.30 and 11.55 Hz, CH2 of
pyrazoline), 3.94 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 4.30 (d, 1H,
J ¼ 11.54 Hz, CH2), 4.40 (d, 1H, J ¼ 11.54 Hz, CH2), 5.67 (dd, 1H,
J ¼ 11.50 and 5.70 Hz, CH of pyrazoline), 6.29–7.45 (m, 6H, Ar-H).
MS: m/z (%): 394 (79) [M þ 2], 392 (80) [Mþ], 271 (100), 243 (27),
128 (30), 121 (23), 77 (20). Anal. calcd for C17H17BrN2O4: C, 51.92; H,
4.36; N, 7.12. Found: C, 51.92; H, 4.40; N, 7.06.
6.1.2.2. 4,5-Dihydro-5-(2,4-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-
1H-pyrazole (7b). Pale yellow crystals (2.44 g, 71.5% yield), m.p.
120–121 ꢀC. IR (KBr) nmax (cmꢁ1): 3334 (NH), 3076–3002 (Ar-CH),
2960–2834 (Aliph-CH), 1611 (C]N), 1566 (CfC). 1H NMR
6.1.3.2. 1-(2-Bromoacetyl)-4,5-dihydro-5-(2,4-dimethoxyphenyl)-3-
(3,4-dimethoxyphenyl)-1H-pyrazole (8b). Pale orange powder
(1.81 g, 92.5% yield), m.p. 148–149 ꢀC. IR (KBr) nmax (cmꢁ1): 3062–
2998 (Ar-CH), 2958–2835 (Aliph-CH), 1658 (C]O), 1598 (C]N),
(300 MHz, CDCl3)
d
(ppm): 3.20 (dd, 1H, J ¼ 16.68 and 7.90 Hz, CH2
of pyrazoline), 3.54 (dd, 1H, J ¼ 16.60 and 10.05 Hz, CH2 of pyr-
azoline), 3.76 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.89 (s, 6H, 2 OCH3),
5.27 (dd, 1H, J ¼ 9.90 and 8.00 Hz, CH of pyrazoline), 6.40 (d, 1H,
J ¼ 2.57 Hz, Ar-H), 6.43 (s, 1H, Ar-H), 6.83 (d, 1H, J ¼ 8.43 Hz, Ar-H),
7.11 (dd, 1H, J ¼ 8.25 and 2.02 Hz, Ar-H), 7.24 (d, 1H, J ¼ 8.10 Hz, Ar-
H), 7.47 (d, 1H, J ¼ 2.20 Hz, Ar-H). MS: m/z (%): 342 (100) [Mþ], 341
(21), 326 (7), 164 (7), 154 (5). Anal. calcd for C19H22N2O4: C, 66.65;
H, 6.48; N, 8.18. Found: C, 66.57; H, 6.44; N, 7.79.
1514 (CfC). 1H NMR (300 MHz, CDCl3)
d (ppm): 3.07 (dd, 1H,
J ¼ 16.36 and 5.45 Hz, CH2 of pyrazoline), 3.68 (dd, 1H, J ¼ 16.30 and
12.10 Hz, CH2 of pyrazoline), 3.77 (s, 3H, OCH3), 3.80 (s, 3H, OCH3),
3.92 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 4.32 (d, 1H, J ¼ 12.00 Hz, CH2),
4.43 (d, 1H, J ¼ 12.00 Hz, CH2), 5.74 (dd, 1H, J ¼ 12.00 and 5.50 Hz,
CH of pyrazoline), 6.38–7.44 (m, 6H, Ar-H); MS: m/z (%): 464 (35)
[M þ 2], 463 (9) [M þ 1], 462 (38) [Mþ], 342 (34), 341 (100), 121
(36.5), 139 (28), 93 (25), 77 (30). Anal. calcd for C21H23BrN2O5: C,
54.44; H, 5.00; N, 6.05. Found: C, 54.47; H, 5.05; N, 5.93.
6.1.2.3. 5-(2,6-Dichlorophenyl)-4,5-dihydro-3-(3,4-dimethoxyphenyl)-
1H-pyrazole (7c). Yellow crystals (2.42 g, 69% yield), m.p.130–131 ꢀC.
IR (KBr) nmax (cmꢁ1): 3291 (NH), 3076–2999 (Ar-CH), 2941–2837
(Aliph-CH), 1599 (C]N), 1568 (CfC). 1H NMR (300 MHz, CDCl3)
6.1.3.3. 1-(2-Bromoacetyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-3-(3,4-
dimethoxyphenyl)-1H-pyrazole (8c). White crystals (1.73 g, 87.5%
yield), m.p. 167–168 ꢀC. IR (KBr) nmax (cmꢁ1): 3070–3001 (Ar-CH),
2962–2840 (Aliph-CH), 1688 (C]O), 1599 (C]N), 1565 (CfC). 1H
d
(ppm): 3.46 (d, 2H, J ¼ 11.90 Hz, CH2 of pyrazoline), 3.91 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3), 5.80 (t, 1H, J ¼ 11.91 Hz, CH of pyrazoline),
6.86 (d, 1H, J ¼ 8.43 Hz, Ar-H), 7.03 (dd, 1H, J ¼ 8.25 and 2.01 Hz, Ar-
H), 7.15 (dd, 1H, J ¼ 8.80 and 1.47 Hz, Ar-H), 7.33 (d, 2H, J ¼ 8.06 Hz,
Ar-H), 7.47 (d, 1H, J ¼ 2.20 Hz, Ar-H), 7.60 (br s, 1H, NH). MS: m/z (%):
354 (11.0) [M þ 4], 352 (72) [M þ 2], 350 (100) [Mþ], 205 (86), 189
(32),163 (44), 77 (37). Anal. calcd for C17H16Cl2N2O2: C, 58.13; H, 4.59;
N, 7.98. Found: C, 58.19; H, 4.62; N, 7.87.
NMR (300 MHz, CDCl3)
d
(ppm): 3.45 (dd, 1H, J ¼ 18.00 and 9.00 Hz,
CH2 of pyrazoline), 3.81 (dd, 1H, J ¼ 18.00 and 12.00 Hz, CH2 of
pyrazoline), 3.95 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.33 (d, 1H,
J ¼ 11.44 Hz, CH2), 4.43 (d, 1H, J ¼ 11.40 Hz, CH2), 6.25 (dd, 1H,
J ¼ 12.00 and 9.00 Hz, CH of pyrazoline), 6.85–7.47 (m, 6H, Ar-H).
MS: m/z (%): 476 (15) [M þ 4], 475 (15) [M þ 3], 474 (43.0) [M þ 2],
473 (26.0) [M þ 1], 472 (100) [Mþ], 471 (17.0) [M ꢁ 1], 471 (60)
[M ꢁ 2], 437 (40), 350 (60), 271 (48), 205 (83), 163 (64), 121 (87), 93
(39), 77 (24). Anal. calcd for C19H17BrCl2N2O3: C, 48.33; H, 3.63; N,
5.93. Found: C, 48.39; H, 3.62; N, 5.81.
6.1.2.4. 5-(2,6-Dichlorophenyl)-4,5-dihydro-3-(4-methoxyphenyl)-
1H-pyrazole (7d). Brown crystals (2.03 g, 63.5% yield), m.p.
120–121 ꢀC. IR (KBr) nmax (cmꢁ1): 3264 (NH), 3075–3001 (Ar-CH),
2954–2832 (Aliph-CH), 1602 (C]N), 1561 (CfC). 1H NMR
(300 MHz, CDCl3)
d
(ppm): 3.44 (d, 2H, J ¼ 12.04 Hz, CH2 of
6.1.3.4. 1-(2-Bromoacetyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-3-(4-
methoxyphenyl)-1H-pyrazole (8d). Yellowish white crystals (1.45 g,
79.0% yield), m.p. 159–160 ꢀC. IR (KBr) nmax (cmꢁ1): 3084–2989 (Ar-
CH), 2964–2838 (Aliph-CH), 1656 (C]O), 1602 (C]N), 1562 (CfC).
pyrazoline), 3.84 (s, 3H, OCH3), 5.78 (t, 1H, J ¼ 11.90 Hz, CH of
pyrazoline), 6.90 (d, 2H, J ¼ 9.10 Hz, Ar-H), 7.12–7.17 (m, 2H, Ar-H),
7.32 (d, 1H, J ¼ 8.06 Hz, Ar-H), 7.63 (d, 2H, J ¼ 8.80 Hz, Ar-H), 8.00
(br s, 1H, NH). MS: m/z (%): 324 (6) [M þ 4], 322 (38) [M þ 2], 320
(63) [Mþ], 175 (100), 133 (66), 77 (25). Anal. calcd for C16H14Cl2N2O:
C, 59.83; H, 4.39; N, 8.72. Found: C, 59.88; H, 4.09; N, 8.50.
1H NMR (300 MHz, CDCl3)
d
(ppm): 3.32 (dd, 1H, J ¼ 18.00 and
9.00 Hz, CH2 of pyrazoline), 3.68 (dd, 1H, J ¼ 18.00 and 12.55 Hz,
CH2 of pyrazoline), 3.85 (s, 3H, OCH3), 4.30 (d, 1H, J ¼ 12.00 Hz,
CH2), 4.40 (d, 1H, J ¼ 12.00 Hz, CH2), 6.21 (dd, 1H, J ¼ 12.60 and
9.00 Hz, CH of pyrazoline), 6.90–7.85 (m, 7H, Ar-H). MS: m/z (%):
444 (29) [M þ 2], 443 (7) [M þ 1], 442 (73) [Mþ], 441 (19) [M ꢁ 1],
440 (47), 407 (72), 405 (46), 322 (56), 175 (100), 144 (92), 121 (63),
77 (32). Anal. calcd for C18H15BrCl2N2O2: C, 48.90; H, 3.42; N, 6.34.
Found: C, 48.87; H, 3.44; N, 6.28.
6.1.3. General procedure for the preparation of compounds
8a–d [41]
To a stirred solution of 7a–d (4.20 mmol) in dichloromethane in
an ice bath, a solution of K2CO3 (0.868 g, 6.30 mmol) in 100 mL
water was added. To this solution bromoacetyl bromide (0.928 g,
4.60 mmol) in 20 mL of dichloromethane was added in a drop wise
manner while stirring over 30 min; the mixture was left for stirring
for further 2 h at 0 ꢀC and 24 h at room temperature. The organic
layer was separated and the aqueous layer was extracted with
(2 ꢃ 30 mL) CH2Cl2, the combined organic layer was washed
consequently with distilled water (2 ꢃ 20 mL), 1 N HCl (2 ꢃ 20 mL)
and again with distilled water (2 ꢃ 20 mL); the organic layer was
dried over anhydrous sodium sulfate, evaporated under reduced
pressure and the obtained crude product was recrystallized from
absolute ethanol.
6.1.4. General procedure for the preparation of compounds
10a–c [42]
To a stirred solution of 2-bromopropionic acid (0.76 g, 5 mmol)
in 30 mL dry chloroform at ꢁ10 ꢀC, triethylamine (0.5 g, 5 mmol)
was added, followed by ethyl chloroformate (0.54 g, 5 mmol) in
a drop wise manner, the mixture was stirred for 30 min, and then
the corresponding pyrazolines 7a–c were added. The mixture was
stirred for additional 12 h at room temperature, the solvent was
evaporated under reduced pressure and the crude product was
dissolved in 30 mL chloroform and washed consequently with 1 N
HCl (2 ꢃ 20 mL), distilled water (2 ꢃ 30 mL), 5% NaHCO3 solution
(2 ꢃ 20 mL), distilled water (2 ꢃ 30 mL) and finally with brine
6.1.3.1. 1-(2-Bromoacetyl)-5-(2-furyl)-4,5-dihydro-3-(3,4-dimethox-
yphenyl)-1H-pyrazole (8a). Pale brown crystals (1.42 g, 86.2%

3074
M.E. Shoman et al. / European Journal of Medicinal Chemistry 44 (2009) 3068–3076
(2 ꢃ 30 mL). The organic layer was dried over anhydrous sodium
sulfate, and evaporated under reduced pressure; the product was
collected and recrystallized from methanol.
1H, J ¼ 4.00 Hz, Ar-H), 7.39 (s, 1H, Ar-H). MS: m/z (%): 375 (8) [Mþ],
374 (11) [M ꢁ 1], 272 (25), 162 (20), 122 (76), 121 (100), 93 (23), 77
(31). Anal. calcd for C17H17N3O7: C, 54.40; H, 4.57; N,11.20. Found: C,
54.33; H, 4.21; N, 10.61.
6.1.4.1. 1-(2-Bromopropionyl)-5-(2-furyl)-4,5-dihydro-3-(3,4-dimethoxy-
phenyl)-1H-pyrazole (10a). Yellowish brown crystals (1.40 g, 69.0%
yield), m.p. 135–136 ꢀC. IR (KBr) nmax (cmꢁ1): 3111–2965 (Ar-CH),
2937–2837 (Aliph-CH), 1687 (C]O), 1601 (C]N), 1516 (CfC). 1H
6.1.5.2. 4,5-Dihydro-5-(2,4-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-
1-(2-nitrooxyacetyl)-1H-pyrazole (9b). White powder (1.47 g,
65.80% yield), m.p. 153–154 ꢀC. IR (KBr) nmax (cmꢁ1): 3079–3000
(Ar-CH), 2962–2836 (Aliph-CH), 1682 (C]O), 1653 (NO2), 1617
NMR (60 MHz, CDCl3)
d
(ppm): 1.39 (d, 3H, J ¼ 8.40 Hz, CH3), 3.65–
3.95 (m, 2H, CH2 of pyrazoline), 4.30 (s, 6H, 2 OCH3), 4.60 (q, 1H,
J ¼ 8.40 Hz, CH), 5.95 (dd, 1H, J ¼ 11.00 and 6.20 Hz, CH of pyrazo-
line), 6.70–8.10 (m, 6H, Ar-H). MS: m/z (%): 408 (33) [M þ 2], 406
(34) [Mþ], 344 (64), 272 (100), 255 (52), 243 (62), 122 (34), 77 (24).
Anal. calcd for C18H19BrN2O4: C, 53.08; H, 4.70; N, 6.88. Found: C,
53.10; H, 4.71; N, 7.11.
(C]N), 1514 (CfC), 1256 (NO2). 1H NMR (60 MHz, CDCl3)
d (ppm):
3.35 (dd, 1H, J ¼ 17.10 and 7.10 Hz, CH2 of pyrazoline), 4.00 (dd, 1H,
J ¼ 17.10 and 11.40 Hz, CH2 of pyrazoline), 4.10 (s, 6H, 2 OCH3), 4.30
(s, 6H, 2 OCH3), 5.95 (s, 2H, CH2), 6.20 (dd, 1H, J ¼ 11.40 and 7.10 Hz,
CH of pyrazoline), 6.90–8.00 (m, 6H, Ar-H). MS: m/z (%): 445 (16)
[Mþ], 398 (25), 342 (38), 192 (100), 163 (26), 121 (24), 77 (18). Anal.
calcd for C21H23N3O8: C, 56.63; H, 5.20; N, 9.43. Found: C, 56.59; H,
4.63; N, 9.28.
6.1.4.2. 1-(2-Bromopropionyl)-4,5-dihydro-5-(2,4-dimethoxyphenyl)-
3-(3,4-dimethoxyphenyl)-1H-pyrazole (10b). White powder (1.61 g,
67.50% yield), m.p. 208–209 ꢀC. IR (KBr) nmax (cmꢁ1): 3066–2998
(Ar-CH), 2968–2838 (Aliph-CH), 1657 (C]O), 1599 (C]N), 1521
6.1.5.3. 5-(2,6-Dichlorophenyl)-4,5-dihydro-3-(3,4-dimethoxyphenyl)-
1-(2-nitrooxyacetyl)-1H-pyrazole (9c). Yellow crystals (1.30 g,
57.70% yield), m.p. 172–173 ꢀC. IR (KBr) nmax (cmꢁ1): 3080–3009
(Ar-CH), 2964–2837 (Aliph-CH), 1681 (C]O), 1642 (NO2), 1601
(CfC). 1H NMR (60 MHz, CDCl3)
d
(ppm): 1.85 (d, 3H, J ¼ 7.80 Hz,
CH3), 3.20–3.90 (m, 2H, CH2 of pyrazoline), 4.05 (s, 6H, 2 OCH3),
4.25 (s, 6H, 2 OCH3), 5.70–6.20 (m, 2H, CH(CH3) and CH of pyr-
azoline), 7.00–8.20 (m, 6H, Ar-H). MS: m/z (%): 478 (22) [M þ 2], 476
(26) [Mþ], 341 (100), 154 (35), 121 (22), 77 (34). Anal. calcd for
C22H25BrN2O5: C, 55.35; H, 5.28; N, 5.87. Found: C, 55.00; H, 5.31; N,
5.73.
(C]N), 1517 (CfC), 1276 (NO2). 1H NMR (300 MHz, CDCl3)
d (ppm):
3.44 (dd, 1H, J ¼ 17.40 and 7.80 Hz, CH2 of pyrazoline), 3.74 (dd, 1H,
J ¼ 17.35 and 13.20 Hz, CH2 of pyrazoline), 3.93 (s, 3H, OCH3), 3.97
(s, 3H, OCH3), 5.37 (d, 1H, J ¼ 15.00 Hz, CH2), 5.49 (d, 1H,
J ¼ 15.00 Hz, CH2), 6.27 (dd, 1H, J ¼ 13.20 and 7.87 Hz, CH of pyr-
azoline), 6.86–7.43 (m, 6H, Ar-H). MS: m/z (%): 455 (18) [M þ 2], 453
(27) [Mþ], 377 (53), 350 (70), 205 (100). Anal. calcd for
C19H17Cl2N3O6: C, 50.24; H, 3.77; N, 9.25. Found: C, 50.28; H, 3.75;
N, 9.17.
6.1.4.3. 1-(2-Bromopropionyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-3-
(3,4-dimethoxyphenyl)-1H-pyrazole (10c). White crystals (1.75 g,
72.30% yield), m.p. 150–151 ꢀC. IR (KBr) nmax (cmꢁ1): 3086–2974
(Ar-CH), 2946–2835 (Aliph-CH), 1656 (C]O), 1602 (C]N), 1515
(CfC). 1H NMR (200 MHz, CDCl3)
d
(ppm): 1.69 (d, 3H, J ¼ 6.80 Hz,
6.1.5.4. 5-(2,6-Dichlorophenyl)-4,5-dihydro-3-(4-methoxyphenyl)-1-
(2-nitrooxyacetyl)-1H-pyrazole (9d). Yellow crystals (1.33 g, 62.80%
yield), m.p. 165–166 ꢀC. IR (KBr) nmax (cmꢁ1): 3078–3013 (Ar-CH),
2964–2837 (Aliph-CH), 1686 (C]O), 1631 (NO2), 1602 (C]N), 1514
CH3), 3.35 (dd, 1H, J ¼ 17.10 and 8.90 Hz, CH2 of pyrazoline), 3.71
(dd, 1H, J ¼ 17.30 and 12.90 Hz, CH2 of pyrazoline), 3.91 (s, 3H,
OCH3), 3.96 (s, 3H, OCH3), 5.39 (q, 1H, J ¼ 6.70 Hz, CH), 6.17 (dd, 1H,
J ¼ 13.10 and 8.90 Hz, CH of pyrazoline), 6.83–7.55 (m, 6H, Ar-H).
MS: m/z (%): 488 (6) [M þ 4], 486 (13) [M þ 2], 484 (18) [Mþ], 422
(71), 350 (22), 205 (41), 163 (56), 77 (24), 63 (100). Anal. calcd for
C20H19BrCl2N2O3: C, 49.41; H, 3.94; N, 5.76. Found: C, 49.43; H, 3.95;
N, 6.06.
(CfC), 1265 (NO2). 1H NMR (60 MHz, CDCl3)
d (ppm): 3.20–4.00 (m,
2H, CH2 of pyrazoline), 4.20 (s, 3H, OCH3), 5.80 (s, 2H, CH2), 6.70
(dd, 1H, J ¼ 10.00 and 7.00 Hz, CH of pyrazoline), 7.35–8.40 (m, 7H,
Ar-H). MS: m/z (%): 427 (6) [M þ 4], 425 (26) [M þ 2], 423 (34) [Mþ],
349 (41), 321 (24), 200 (81),121 (52), 93 (8), 76 (100). Anal. calcd for
C18H15Cl2N3O5: C, 50.69; H, 3.56; N, 9.90. Found: C, 51.07; H, 3.14; N,
9.45.
6.1.5. General procedure for the preparation of compounds
9a–d and 11a–c [43]
To a stirred solution of the bromoacetyl derivatives 8a–d and
10a–c (10.00 mmol) in 20 mL acetonitrile, silver nitrate (6.80 g,
40.00 mmol) was added, the mixture was heated for 13–17 h at
80 ꢀC, the formed precipitate of silver bromide was filtered off.
The filtrate was evaporated till dryness, and dissolved in
dichloromethane; the organic layer was washed consequently
with distilled water (2 ꢃ 20 mL) and brine (2 ꢃ 20 mL). The organic
layer was dried over anhydrous sodium sulfate, evaporated under
reduced pressure and the crude product was recrystallized from
methanol.
6.1.5.5. 5-(2-Furyl)-4,5-dihydro-3-(3,4-dimethoxyphenyl)-1-(2-nitro-
oxypropionyl)-1H-pyrazole (11a). Yellow crystals (1.36 g, 67.0%
yield), m.p. 143–145 ꢀC. IR (KBr) nmax (cmꢁ1): 3121–2966 (Ar-CH),
2938–2838 (Aliph-CH), 1686 (C]O), 1636 (NO2), 1601 (C]N), 1517
(CfC), 1263 (NO2). 1H NMR (60 MHz, CDCl3)
d (ppm): 1.76 (d, 3H,
J ¼ 6.60 Hz, CH3), 3.60–4.00 (m, 2H, CH2 of pyrazoline), 4.30 (s, 6H, 2
OCH3), 5.80–6.30 (m, 1H, CH of pyrazoline), 6.6 (q, 1H, J ¼ 6.90 Hz,
CH), 6.80–8.20 (m, 6H, Ar-H). MS: m/z (%): 389 (19) [Mþ], 272 (26),
163 (33), 122 (100), 77 (30). Anal. calcd for C18H19N3O7: C, 55.53; H,
4.92; N, 10.79. Found: C, 55.59; H, 5.18; N, 10.69.
6.1.5.1. 5-(2-Furyl)-4,5-dihydro-3-(3,4-dimethoxyphenyl)-1-(2-nitro-
oxyacetyl)-1H-pyrazole (9a). White crystals (1.18 g, 63% yield), m.p.
155–156 ꢀC. IR (KBr) nmax (cmꢁ1): 3119–3020 (Ar-CH), 2967–2841
(Aliph-CH), 1678 (C]O), 1641 (NO2), 1601 (C]N), 1516 (CfC), 1258
6.1.5.6. 4,5-Dihydro-5-(2,4-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-
1-(2-nitrooxypropionyl)-1H-pyrazole (11b). White powder (1.45 g,
63.20% yield), m.p. 213–214 ꢀC. IR (KBr) nmax (cmꢁ1): 3101–3000
(Ar-CH), 2954–2834 (Aliph-CH), 1666 (C]O), 1641 (NO2), 1601
(NO2). 1H NMR (300 MHz, CDCl3)
d
(ppm): 3.49 (dd, 1H, J ¼ 16.00
(C]N), 1515 (CfC), 1268 (NO2). 1H NMR (60 MHz, CDCl3)
d (ppm):
and 12.00 Hz, CH2 of pyrazoline), 3.62 (dd, 1H, J ¼ 16.00 and
8.00 Hz, CH2 of pyrazoline), 3.94 (s, 3H, OCH3), 3.96 (s, 3H, OCH3),
5.38 (d, 1H, J ¼ 16.00 Hz, CH2), 5.44 (d, 1H, J ¼ 16.00 Hz, CH2), 5.68
(dd, 1H, J ¼ 12.00 and 8.00 Hz, CH of pyrazoline), 6.33 (d, 1H,
J ¼ 4.00 Hz, Ar-H), 6.37 (d, 1H, J ¼ 4.00 Hz, Ar-H), 6.90 (d, 1H,
J ¼ 8.00 Hz, Ar-H), 7.19 (dd, 1H, J ¼ 8.00 and 4.00 Hz, Ar-H), 7.31 (d,
1.87 (d, 3H, J ¼ 7.20 Hz, CH3), 3.00–3.80 (m, 2H, CH2 of pyrazoline),
4.10 (s, 6H, 2 OCH3), 4.25 (s, 6H, 2 OCH3), 5.80–6.35 (m, 2H, CH(CH3)
and CH of pyrazoline), 6.80–8.00 (m, 6H, Ar-H). MS: m/z (%): 461
(41) [M þ 2], 459 (26) [Mþ], 341 (100), 121 (32), 77 (22). Anal. calcd
for C22H25N3O8: C, 57.51; H, 5.48; N, 9.15. Found: C, 57.53; H, 5.41; N,
9.27.

M.E. Shoman et al. / European Journal of Medicinal Chemistry 44 (2009) 3068–3076
3075
6.1.5.7. 5-(2,6-Dichlorophenyl)-4,5-dihydro-3-(3,4-dimethoxyphenyl)-
1-(2-nitrooxypropionyl)-1H-pyrazole (11c). White crystals (1.66 g,
71.30% yield), m.p. 185–187 ꢀC. IR (KBr) nmax (cmꢁ1): 3097–2990
(Ar-CH), 2935–2838 (Aliph-CH), 1672 (C]O), 1635 (NO2), 1601
for C25H26N4O5: C, 64.92; H, 5.67; N, 12.11. Found: C, 64.90; H, 5.65;
N, 11.62.
6.1.7.2. 4,5-Dihydro-1-[2-(4-(1-hydroxyiminoethyl)phenylamino-
acetyl]-5-(2,4-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1H-
pyrazole (13b). White powder (1.84 g, 69.50% yield), m.p.
145–146 ꢀC. IR (KBr) nmax (cmꢁ1): 3490 (OH), 3388 (NH), 3056–
3012 (Ar-CH), 2960–2835 (Aliph-CH), 1658 (C]O), 1615 (C]N),
(C]N), 1517 (CfC), 1263 (NO2). 1H NMR (200 MHz, CDCl3)
d (ppm):
1.60 (d, 3H, J ¼ 6.40 Hz, CH3), 3.35 (dd, 1H, J ¼ 17.70 and 8.30 Hz,
CH2 of pyrazoline), 3.73 (dd, 1H, J ¼ 17.60 and 12.8 Hz, CH2 of pyr-
azoline), 3.94 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 6.04 (q, 1H,
J ¼ 6.90 Hz, CH), 6.26 (dd, 1H, J ¼ 12.60 and 8.20 Hz, CH of pyrazo-
line), 6.9 (d, 1H, J ¼ 8.60 Hz, Ar-H), 7.10–7.40 (m, 5H, Ar-H). MS: m/z
(%): 469 (32) [M þ 2], 467 (37) [Mþ], 422 (16), 377 (59), 205 (100),
200 (80), 163 (57), 77 (24). Anal. calcd for C20H19Cl2N3O6: C, 51.30;
H, 4.09; N, 8.97. Found: C, 51.89; H, 4.15; N, 8.72.
1503 (CfC). 1H NMR (60 MHz, DMSO-d6)
d (ppm): 1.95 (s, 3H, CH3),
3.40–4.00 (m, 2H, CH2 of pyrazoline), 4.20 (s, 6H, 2 OCH3), 4.35 (s,
6H, 2 OCH3), 4.80 (s, 2H, CH2), 6.00–6.30 (m, 1H, CH of pyrazoline),
6.80–9.00 (m, 10H, Ar-H). MS: m/z (%): 532 (8) [Mþ], 437 (18), 418
(40), 341 (48), 79 (100). Anal. calcd for C29H32N4O6: C, 65.40; H,
6.06; N, 10.52. Found: C, 65.31; H, 5.89; N, 10.71.
6.1.6. General procedure for the preparation of compounds 12a and
12b [44]
6.2. NO release assay [46]
To a stirred solution of p-amino acetophenone (0.68 g, 5 mmol),
bromoacetyl derivatives 8a and 8b (5 mmol) in 80 mL acetone, and
anhydrous K2CO3 (0.25 g, 1.80 mmol) were added. The mixture was
heated at reflux for about 13–15 h on a water bath, the solvent was
evaporated under reduced pressure and the resulting solid was
poured into distilled water and then filtered off, washed with
distilled water and recrystallized from methanol.
A solution of the appropriate compound (20 mL) in dime-
thylsulfoxide (DMSO) was added to 2 mL of 1:1 v/v mixture of
50 mM phosphate buffer (pH 7.4) with MeOH, containing
5 ꢃ10ꢁ4 M of
L-cysteine. The final concentration of drug was
10ꢁ4 M. After 1 h at 37 ꢀC, 1 mL of the reaction mixture was treated
with 250 mL 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
6.1.6.1. 1-[2-(4-Acetylphenylamino)acetyl]-5-(2-furyl)-4,5-dihydro-3-
(3,4-dimethoxyphenyl)-1H-pyrazole (12a). White powder (1.56 g,
70.0% yield), m.p. 184–185 ꢀC. IR (KBr) nmax (cmꢁ1): 3369 (NH),
3058–3001 (Ar-CH), 2937–2840 (Aliph-CH), 1662 (C]O), 1601
room temperature, the absorbance was measured at
l
¼ 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.
(C]N), 1520 (CfC). 1H NMR (60 MHz, CDCl3)
d (ppm): 2.70 (s, 3H,
CH3), 3.70–4.00 (m, 2H, CH2 of pyrazoline), 4.30 (s, 3H, OCH3), 4.35
(s, 3H, OCH3), 4.70 (s, 2H, CH2), 6.20 (dd, 1H, J ¼ 12.00 and 6.00 Hz,
CH of pyrazoline), 6.90 (s, 1H, Ar-H), 7.20 (d, 2H, J ¼ 9.00 Hz, Ar-H),
7.40–8.10 (m, 5H, Ar-H), 8.50 (d, 2H, J ¼ 9.00 Hz, Ar-H). MS: m/z (%):
447 (45) [Mþ], 383 (66), 272 (100), 148 (72), 120 (37), 77 (20).
The results were expressed as amount of NO released relative to
a theoretical maximum release of 1 mol NO/mol of test compound.
6.3. Pharmacology
6.1.6.2. 1-[2-(4-Acetylphenylamino)acetyl]-4,5-dihydro-5-(2,4-dimethoxy-
phenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrazole (12b). White powder
(1.84 g, 71.30% yield), m.p. 107–108 ꢀC. IR (KBr) nmax (cmꢁ1): 3388
(NH), 3062–2999 (Ar-CH), 2958–2836 (Aliph-CH), 1658 (C]O), 1598
6.3.1. Anti-inflammatory activity
The experiments were performed on adult male albino rats,
weighing 140–200 g and 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 7a–d, 8a–d,
9a–d, 10a–c, 11a–c, 13a and 13b and reference drug (indo-
methacin) in carboxy methyl cellulose (CMC) solution (0.5% w/v
in water) was administered orally in a dose level of 100 mg/kg.
Control animals were treated similarly 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. [31]. 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.
(C]N), 1515 (CfC). 1H NMR (60 MHz, CDCl3)
d (ppm): 2.70 (s, 3H,
CH3), 3.05–4.00 (m, 2H, CH2 of pyrazoline), 4.10 (s, 6H, 2 OCH3), 4.25
(s, 6H, 2 OCH3), 4.70 (s, 2H, CH2), 6.20 (dd, 1H, J ¼ 12.00 and 6.00 Hz,
CH of pyrazoline), 6.80–8.00 (m, 10H, Ar-H). MS: m/z (%): 517 (25)
[Mþ], 462 (35), 341 (100), 309 (44), 178 (33), 77 (10).
6.1.7. General procedure for the preparation of compounds 13a and
13b [45]
To a solution of 12a and 12b (5 mmol) in 20 mL absolute ethanol,
hydroxylamine hydrochloride (0.35 g, 5 mmol) was added; the
mixture was heated at reflux for 3 h and then left to cool. The
obtained precipitate was filtered off, washed with dilute ammonia
solution and then with distilled water, dried and recrystallized from
aqueous ethanol.
6.1.7.1. 5-(2-Furyl)-4,5-dihydro-1-[2-(4-(1-hydroxyiminoethyl)phenyl-
amino)acetyl]-3-(3,4-dimethoxyphenyl)-1H-pyrazole (13a). Pale brown
crystals (1.69 g, 73.0% yield), m.p. 225–226 ꢀC. IR (KBr) nmax (cmꢁ1):
3491 (OH), 3392 (NH), 3080–3004 (Ar-CH), 2964–2838 (Aliph-CH),
1660 (C]O), 1606 (C]N), 1521 (CfC). 1H NMR (300 MHz, CDCl3)
The anti-inflammatory activity of the tested compounds and
indomethacin was calculated as the percentage decrease in edema
thickness induced by carrageenan.
d
(ppm): 2.20 (s, 3H, CH3), 3.47 (dd, 1H, J ¼ 17.40 and 4.80 Hz, CH2 of
pyrazoline), 3.60 (dd, 1H, J ¼ 17.40 and 11.40 Hz, CH2 of pyrazoline),
3.95 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 4.36 (s, 2H, CH2), 5.71 (dd, 1H,
J ¼ 11.50 and 4.95 Hz, CH of pyrazoline), 6.32–6.36 (m, 2H, Ar-H), 6.55
(d, 2H, J ¼ 8.70 Hz, Ar-H), 6.93 (d, 1H, J ¼ 8.10 Hz, Ar-H), 7.23–7.31 (m,
2H, Ar-H), 7.45 (s, 1H, Ar-H), 7.50 (d, 2H, J ¼ 8.70 Hz, Ar-H). MS: m/z (%):
462 (64) [Mþ], 348 (23), 272 (100), 163 (83), 77 (26). Anal. calcd
6.3.2. Ulcerogenic toxicity
Adult male albino rats weighing 140–200 g were divided into
different groups consisting of four animals in each group. Animals
were deprived of food but not water 24 h before the experiment.
Ulcerogenic activity was evaluated after oral administration of
a suspension of the tested compounds 7a–d, 8a–d, 9a–d, 10a–c,

3076
M.E. Shoman et al. / European Journal of Medicinal Chemistry 44 (2009) 3068–3076
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