Design, synthesis and biological investigation of certain pyrazole-3-carboxylic acid derivatives as novel carriers for nitric oxide

Article abstract of DOI:10.1016/j.bmc.2009.04.037

Some novel pyrazole-NO hybrid molecules 5a-e, 6, 8 and 10 were prepared through binding of the pyrazole-3-carboxylic acid derivatives with nitric oxide donor moiety like oxime or nitrate ester. The prepared compounds were evaluated for nitric oxide release, antibacterial and anti-inflammatory activities. The organic nitrate 10 exhibited the highest percentage of NO release using Griess diazotization method. Some of the prepared compounds exhibited remarkable antibacterial activity against Escherichia coli C-600, Pseudomonas aeruginosa, Bacillus subitilis and Staphylococcus aureus NCTC 6571 compared to ciprofloxacin. Most of the tested compounds showed significant anti-inflammatory activity compared to indomethacine using carrageenan induced paw edema method. In general, structural modification of compound 2 either to nitrate ester or oxime hybrids showed better anti-inflammatory with less ulcerogenic liability than their corresponding starting intermediates.

Full text of DOI:10.1016/j.bmc.2009.04.037

Bioorganic & Medicinal Chemistry 17 (2009) 3829–3837
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological investigation of certain pyrazole-3-carboxylic
acid derivatives as novel carriers for nitric oxide
a
El-Shimaa M. N. Abdel-Hafez a, Gamal El-Din A. A. Abuo-Rahma a, , Mohamed Abdel-Aziz ,
*
Mohamed F. Radwan 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:
Some novel pyrazole-NO hybrid molecules 5a–e, 6, 8 and 10 were prepared through binding of the pyr-
azole-3-carboxylic acid derivatives with nitric oxide donor moiety like oxime or nitrate ester. The pre-
pared compounds were evaluated for nitric oxide release, antibacterial and anti-inflammatory
activities. The organic nitrate 10 exhibited the highest percentage of NO release using Griess diazotiza-
tion method. Some of the prepared compounds exhibited remarkable antibacterial activity against Esch-
erichia coli C-600, Pseudomonas aeruginosa, Bacillus subitilis and Staphylococcus aureus NCTC 6571
compared to ciprofloxacin. Most of the tested compounds showed significant anti-inflammatory activity
compared to indomethacine using carrageenan induced paw edema method. In general, structural mod-
ification of compound 2 either to nitrate ester or oxime hybrids showed better anti-inflammatory with
less ulcerogenic liability than their corresponding starting intermediates.
Received 29 December 2008
Revised 15 April 2009
Accepted 17 April 2009
Available online 23 April 2009
Dedicated to Professor Jochen Lehmann on
the occasion of his 65th birthday
Keywords:
Nitric oxide donors
Anti-inflammatory
Antibacterial
Ó 2009 Elsevier Ltd. All rights reserved.
Pyrazole-3-carboxylic acid
Oximes
Nitrate esters
Ulcerogenicity
Histopathological investigation
1. Introduction
trite are mediated through liberation of NO after metabolic
bioactivation.18
Pyrazole derivatives are well established in the literatures as
important biologically effective heterocyclic compounds. These
derivatives are the subject of many research studies due to their
widespread potential pharmacological activities such as anti-
inflammatory,1 antipyretic,2 antimicrobial,3 antiviral,4 antitumour,5
anticonvulsant,6 antihistaminic7 and antidepressant8 activities. The
widely prescribed anti-inflammatory pyrazole derivatives, cele-
coxib9 and deracoxib10 are selective COX-2 inhibitors with reduced
ulcerogenic side effects.
Nitricoxide (NO)is a magicmoleculeendogenously discoveredas
endothelium derived relaxing factor.11 Besides its vasodilatation ef-
fect,12 NO is involved in many physiological and pathophysiological
processes13 like inhibition of platelets aggregation14,15 and immune
defense against viruses,16 bacteria16 and cancerous cells.17 Now, it is
clear that the vasodilatory effects of the old known organic nitrate
and nitrite esters, glyceryltrinitrate, isosorbid dinitrate and amyl ni-
Recent strategies devoted to minimize the ulcerogenic side ef-
fects of non-steroidal anti-inflammatory drugs (NSAIDs) by bind-
ing them to NO donating moieties.19 In such hybrids, NO
supports several endogenous GIT defense mechanisms against ul-
cer formation including increase in mucous and bicarbonate
secretions, increase in mucosal blood flow and inhibition of pro-
inflammatory caspase process.20 A series of NO/aspirin (e.g., NCX-
4016)19 acquired anti-inflammatory effects devoid of acute gas-
trotoxicity and showed potent antiaggregatory effects, principally
as a consequence of their NO donor ability.
Based on the above mentioned research results, the objective of
this study aimed to synthesize some novel pyrazole-3-carboxylic
amides and esters as a type of prodrugs in order to improve the ex-
pected ulcerogenic liability. In addition some novel pyrazole-NO
hybrids molecules were prepared by binding the pyrazole-3-car-
boxylic derivatives with NO donor moiety like oxime or nitrate es-
ter, for the purpose of synergism and/or decreasing the expected
ulcerogenic side effects. Also, a histopathological investigation for
ulcer formation liability was carried out. The synthesis of the target
compounds was illustrated in Schemes 1 and 2.
* Corresponding author. Tel.: +20 103069431; fax: +20 862369075.
E-mail address: gamalaburahma@yahoo.com (G.A.A. Abuo-Rahma).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.037

3830
E.-S. M. N. Abdel-Hafez et al. / Bioorg. Med. Chem. 17 (2009) 3829–3837
O
O
O
O
O
O
N
Ph
Ph
O
COOH
Ph
Ph
Cl
Ph
Ph
Ph
Ph
R
Ph-NH-N=CH-Ph
Amine
SOCl2
N
N
O
N
N
O
N
Ph
Ph
Ph
1
2
3
4a-g
O
NOH
Ph
R
NH2OH.HCl
4a-e
Base, > 4hr
N
Ph
N
Ph
5a-e
O
O
NOH
R
NH
C
NH2OH.HCl
4f
CH3
Pyridine, 4 hr
N
N
Ph
Ph
6
4 or 5
R
g
e
a
Cl
b
f
c
d
CH3
O
H
N
CH3
COOH
OCH3
N
N
O
S
H
N
HN
O
N
NH
NH
NH
NH
NH
Scheme 1. Synthesis of the pyrazole-3-carboxylic acid derivatives 4a–g, 5a–e, and 6.
O
O
O
O
ONO2
Br
Ph
Ph
O
Ph
Ph
Br
HO
O
AgNO3
CH3CN
3
N
N
Pyridine, 3 hr
N
N
Ph
Ph
7
8
O
O
N
O
ONO2
O
Cl
N
N
N
N
Ph
Ph
ClCH2COCl
K2CO3
Ph
Ph
O
4g
AgNO3
CH3CN
O
N
N
N
Ph
Ph
9
10
O
O
OH
Ph
Ph
OH
Pyridine, 3 hr
HO
O
3
N
N
Ph
11
Scheme 2. Synthesis of the pyrazole-3-carboxylic acid derivatives 7–10.
phenylfurane-2,3-dione22 1 with 1-benzylidine-2-phenylhydraz-
one without any solvent. Heating at reflux of the pyrazole-3-car-
boxylic acid 2 with thionyl chloride on a steam bath afforded the
corresponding acid chloride 3.23 Reaction of the acid chloride 3
with the appropriate nucleophiles afforded the corresponding
amides 4a–g in 50–78% yield. Condensation of the amides 4a–e
2. Results and discussion
2.1. Chemistry
The key intermediate, pyrazole-3-carboxylic acid 2 was pre-
pared as reported21 by heating the freshly prepared 4-benzoyl-5-

E.-S. M. N. Abdel-Hafez et al. / Bioorg. Med. Chem. 17 (2009) 3829–3837
3831
Table 2
with hydroxylamine hydrochloride in absolute ethanol for (4–
30 h) using triethylamine or pyridine as a base afforded the corre-
sponding oximes 5a–e in 60–82% yield. The oxime 6 was prepared
by the same procedure with a shorter reaction time (less than 4 h).
The IR spectra of the prepared oximes 5a–e and 6 revealed the
disappearance of the respective carbonyl stretching absorption and
appearance of the characteristic oximic OH stretching as broad
medium band ranging from 2500 to 3200 cmꢀ1. The 1H NMR spec-
tra of the prepared oximes revealed the characteristic proton of
–C@N–OH as a broad signal exchangeable with D2O in the range
of 8.5–12.3 ppm. It is noteworthy to focus on the change in the
chemical shift of the methyl group of the oxime 6, it is upfield
shifted from 2.5 to 2.3 ppm due to the change of the adjacent
C@O to the less electronegative –C@N–OH.
The mass spectrum of some of the oximes showed the disap-
pearance of M+ and appearance of M+ÅꢀOH peak due to the rapid
fragmentation of M+ to the more stable fragment of M+ÅꢀOH. On
the other hand, the oxime 5e as a representative example revealed
the molecular ion peak at m/z = 615 in addition to the M+ÅꢀOH
fragment at m/z = 598, that underwent loss of the amide moiety
giving a fragment at m/z = 348. This last fragment will subse-
quently fragmented either by cleavage of the pyrazole ring afford-
ing a fragment at m/z = 180 or complete loss of the pyrazole ring
giving a fragment for the remaining at m/z = 103. Subsequent loss
of (C@N) from the last fragment gives a fragment at m/z = 77
(100%).
Minimal inhibitory concentration of the tested compounds 2, 4a–g, 5a–e and 6–11
Compound
Minimal inhibitory concentration (MIC) (lg/mL)
B. subitilis
S. aureus
E. coli
P. aeruginosa
Ciprofloxacin
2
0.61
52.40
ꢀve
ꢀve
ꢀve
ꢀve
64.00
ꢀve
ꢀve
ꢀve
90.36
ꢀve
ꢀve
50.50
ꢀve
ꢀve
ꢀve
ꢀve
ꢀve
ꢀve
ꢀve
1.20
30.80
1.92
25.09
4.30
22.90
25.33
44.34
32.40
84.53
10.32
7.99
10.76
ꢀve
16.10
ꢀve
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
6
33.80
43.80
34.80
47.03
1.75
16.14
4.50
30.90
ꢀve
39.90
1.92
2.18
5.82
2.87
14.09
28.64
2.88
2.80
30.86
1.62
24.04
15.28
1.37
63.97
7.95
43.86
29.50
54.67
90.36
47.50
21.68
11.19
2.45
9.70
13.60
23.34
27.22
6.04
11.31
61.52
93.33
1.29
7
8
9
10
11
3.88
ꢀve means no antibacterial activity.
Found concentration of NO
Theoretical concentration of NO
% of NO release ¼
ꢁ 100
The titled ester 7 was prepared by heating at reflux of the acid
chloride 3 with bromoethanol. Heating at reflux the ester 7 with
AgNO3 in acetonitril afforded the corresponding nitrate ester 8.
The 1H NMR spectrum of the nitrate ester 8 showed the –CH2CH2–
pattern as a singlet at 4.4 ppm, where the two methylenes are con-
sidered magnetically equivalent.
Results revealed that all of the tested compounds 5a–e, 6, 8 and 10
release NO with relatively similar percentage. The nitrate ester 10
exhibited the highest percentage of NO release (Table 1).
2.3. Screening of the antibacterial activity
Treatment of the amide 4g with chloroacetyl chloride in K2CO3
at 0 °C gave the corresponding acetylated derivative 9 in low yield.
The objective nitrate ester 10 was prepared by heating at reflux of
the intermediate 9 with AgNO3 in acetonitril. The downfield shift
of the absorption of the –CH2 group at 5.2 ppm was the basis for
the identification of the nitrate ester 10. The hydroxyethyl ester
11 of compound 2 was prepared for comparison with its corre-
sponding nitrate ester 8 by heating at reflux of the acid chloride
3 with ethylene glycol.
The synthesized compounds 2, 4a–g, 5a–e, 6–11 were screened
for their antibacterial activity against Escherichia coli C-600, Pseu-
domonas aeruginosa (as examples for Gram-negative bacteria),
Bacillus subitilis and Staphylococcus aureus NCTC 6571 (as examples
for Gram-positive bacteria) using the agar cup diffusion method.25
The MICs (lg/mL) of the tested compounds in comparison to cipro-
floxacin are listed in Table 2.
Data in Table 2 revealed that the esters 7 and 11 exhibited the
highest antibacterial activity against P. aeruginosa with MIC value
of 6.04 and 1.29 lg/mL, respectively that is more active than cipro-
2.2. Nitric oxide release
floxacin, while the amide 4c, 4g the oximes 5c, 5d, 6 and 9 showed
the highest activity with MIC values of 4.50, 1.92, 2.88, 2.80, 1.62,
1.37 lg/mL, respectively against E. coli. All of the tested com-
pounds revealed no antibacterial activity against S. aureus except
the amide 4d and the oximes 5a and 5d which showed a markedly
weak activity. Compound 4f, 4g, 5b and 10 are the most active
derivative against B. subitilis strains with MIC of 1.75, 2.18, 2.87,
The percentage of nitric oxide release from the prepared NO
donating hybrids 5a–e, 6, 8 and 10 was determined. The spontane-
ous oxidation of NO into nitrite was the basis for the colorimetric
measurement using Griess diazotization reaction method.24 The
experiment was carried out in phosphate buffer of pH 7.4 in the
presence of L-cysteine using sodium nitrite solution (1–50 nmol/
mL) as a standard to establish the sodium nitrite standard curve
according to GRAPH PAD INSTANT program. The formed intense purple
colour was measured at kmax 540 nm after 25 min. The percentage
of NO release is calculated from the following equation:
2.45 lg/mL, respectively. Some of the nitric oxide donating hybrids
exhibited better antibacterial activity than their corresponding
intermediates that may be attributed to the synergistic antibacte-
rial effect of NO.16
Table 1
2.4. Screening of anti-inflammatory activity
Percentage of NO release of compounds 5a–e, 6, 8 and 10
Compound
Percentage of NO release
The synthesized compounds 2, 4a–g, 5a–e, 6–8, 10 and 11 were
evaluated for their in vivo anti-inflammatory activity using the car-
rageenan-induced paw edema method described by Winter et al.26
Rat paw edema was induced by subcutaneous injection of 0.1 mL
of 1% suspension of carrageenan in saline into the left paw. The
right paw was injected with an equal volume of saline and served
as a control. After 3 h, the average percentage increase in foot ede-
ma thickness measurement reaches maximum and served as a
5a
5b
5c
5d
5e
6
7.80
10.06
8.00
9.30
7.40
11.58
7.10
13.84
8
10

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E.-S. M. N. Abdel-Hafez et al. / Bioorg. Med. Chem. 17 (2009) 3829–3837
Table 3
Percentage of Edema inhibition SEM after administration of indomethacin, 2, 4a–g, 5a–e, 6–8, 10 and 11
Compound
Dose (
l
mol/kg)
Percentage of edema inhibition
3 h
1 h
2 h
4 h
5 h
Indomethacin
2
27.94
27.17
20.92
22.29
21.12
20.52
16.66
20.60
22.72
20.28
21.16
20.47
19.89
16.24
19.98
21.04
21.86
18.53
24.24
40.00 0.33**
32.72 0.57**
45.45 0.33**
42.54 0.17**
39.99 0.44**
23.63 0.58*
18.18 0.29**
29.09 1**
36.60 0.31**
35.00 0.88**
39.99 0.33**
29.09 0.33**
42.54 0.67**
25.00 0.76*
18.18 0.33**
45.45 0.66**
41.67 0.88**
23.33 0.33**
19.99 0.29**
25.00 0.5**
44.99 0.33**
26.67 0.29**
33.30 1.00**
28.38 0.67**
34.99 0.58**
29.99 0.44**
51.51 0.66**
63.17 0.33**
36.50 0.88**
34.99 0.58**
28.33 0.33**
28.33 0.88**
26.98 0.58*
24.99 0.33**
44.99 0.58**
28.57 0.88**
47.61 0.33**
36.50 0.58**
36.50 0.58**
47.62 0.29**
31.74 0.58**
33.30 0.57**
30.16 0.67**
47.62 0.88**
42.99 0.67**
66.43 0.00**
66.17 0.35**
51.47 0.33**
36.50 1**
69.86 0.33**
56.62 0.88**
55.88 0.33**
51.17 0.57**
52.94 0.33**
46.38 0.33*
41.18 0.88**
61.76 0.58**
66.67 0.66**
60.87 0.58**
82.60 0.33**
56.52 1**
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
6
30.16 0.33**
42.99 1**
41.18 0.20*
26.98 0.58**
47.62 0.88**
51.47 0.88**
55.88 0.33**
45.59 0.67**
51.47 0.33**
61.76 0.33**
51.47 0.33**
51.47 0.67**
41.18 1.00*
58.82 0.60**
45.59 0.88**
68.60 0.33**
34.99 0.72**
23.63 0.45**
12.72 0.67**
30.90 0.33**
45.45 0.29**
16.36 0.33**
16.36 0.88**
25.45 0.44**
29.09 0.44**
45.45 0.50**
19.66 0.57**
81.16 0.67**
56.52 0.58**
62.32 0.58**
52.17 0.67**
71.01 0.58**
68.12 0.58**
71.74 0.33**
7
8
10
11
Significantly different from control at *p < 0.05, **p < 0.01.
control. The treated groups received the appropriate dose (100 mg/
kg) of the tested compounds orally half an hour prior to carra-
geenan injection. The measurement was carried out at 0, 1, 2, 3,
4 and 5 h after administration of the tested compounds, indometh-
acine and vehicle. The obtained results were subjected to statistical
analysis by student’s t-test using the ‘ANOVA T program’. The differ-
ence in the results was considered significant when the values of P
are less than 0.05 or P less than 0.01. The percentage of edema inhi-
bition standard error of the mean was calculated28 and listed in
Table 3. The following equations is used for calculation of percent-
age edema inhibition.
according to the length of the lesions. Severity factor is as follows,
0 = no lesions; 1 = petechiae; 2 = erosions < 1 mm; 3 = erosions of
1–2 mm; 4 = erosions of 2–4 mm and 5 = erosions > 4 mm. The
partial scores were then summed to obtain the ulcer index for
the examined animal. The UI for each group was taken as the mean
lesion score for all rats in those groups. The preventive index (PI) of
the tested compounds was calculated from the following equation
according to Hano et al.28
UI of ulcered group ꢀ UI of treated group
Preventive indexðPIÞ ¼
UI of ulcered group
ꢁ 100
ðVR ꢀ VLÞ control ꢀ ðVR ꢀ VLÞ treated
% Edema inhibition ¼
ðVR ꢀ VLÞ control
The results listed in Table 4 revealed that all of the tested com-
pounds exhibited marked low UI in comparison to the reference
(indomethacin) that indicates their gastroprotective effect. Unex-
pectedly, the pyrazole-3-carboxylic acid 2 exhibited lower UI. Most
of the synthesized pyrazole-NO hybrid compounds containing
ꢁ 100
where VR is the average right paw thickness; VL is the average left
paw thickness.
Results revealed that all of the tested compounds showed a var-
iant significant anti-inflammatory activity at the administered
dose (100 mg/kg) compared to indomethacin as a reference. It is
obvious that most of the tested amides 4a–g revealed relatively
higher activities than their starting, pyrazole-3-carboxylic acid 2.
The results also revealed that in some cases, the presence of the
NO donating moiety enhances the anti-inflammatory activity in
comparison with their corresponding starting derivatives like in
compounds 5d and 5e. It is obvious that, there is no direct
correlation between enhancement of the anti-inflammatory activ-
ity and the presence of NO donating moiety. Also, it is clear that
maximal anti-inflammatory activity was obtained after 4–5 h of
administration.
Table 4
Ulcer index of indomethacin, carboxymethylcellulose, 2, 4a–g, 5a–e, 6–8, 10 and 11
Compound
UI
Indomethacin
Carboxymethylcellulose
Control
2
69.5
2.6
0.3
8.0
20.3
4.0
33.3
17.0
9.3
11.0
23.0
7.5
3.0
8.5
8.5
7.6
4.0
11.0
7.5
5.5
9.0
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
6
2.5. Assessment of gastric mucosal ulcerogenicity
In order to investigate the ulcerogenic liability of the tested
compounds 2, 4a–g, 5a–e and 6–10, the rats used in testing the
anti-inflammatory activity were sacrificed, their stomach were re-
moved, opened and examined for the presence of visible lesions.
Gastric mucosal lesions were expressed in terms of the ulcer index
(UI) according to the method of Till et al.27 which depends on the
calculation of lesion index by using of a 0–5 scoring system based
on the severity of each lesion. The severity factor was defined
7
8
10
11

E.-S. M. N. Abdel-Hafez et al. / Bioorg. Med. Chem. 17 (2009) 3829–3837
3833
tive ion mode with leucine enkephalin as an internal lock mass
standard. Elemental microanalyses were performed on a Perkin El-
mer 2400 CHN Elemental analyzer. All chemicals and solvents
were obtained commercially and were of the highest pure form.
The reactions follow up and checking the purity of the compounds
were made by TLC (Kieselgel 60 F254 precoated plates, E. Merck,
Dermastadt, Germany), the spots were detected by exposure to
UV lamp at kmax 254 nm. Absorbance was measured by ‘Spectronic
Genesys’ spectrophotometer connected to an IBM computer loaded
with the Winspec application software (Miton Roy, USA). Bright
field microscopy was used for investigation and capturing images
for the histology and immunoperoxidase staining. Slides were pho-
tographed using Olympus digital camera. The furandione 1 was
80
70
60
50
PI 40
30
20
10
0
4a-5a 4b-5b 4c-5c 4d-5d 4e-5e 4f-6
7-8 4g-10 11-8
prepared as reported.22 Also, the pyrazole-3-carboxylic acid21
2
and the corresponding acid chloride23 3 were prepared as reported.
Figure 1. The preventive index of the tested NO donating hybrids calculated by
comparison with their starting derivatives.
4.1.1. General procedure for synthesis of 4-benzoyl-1,5-
diphenylpyrazole-1H-3-carboxamide derivatives 4a–g
A mixture of the pyrazole-3-carboxylic acid chloride 3 (0.386 g,
1 mmol) and the respective amine (1 mmol) was heated at reflux in
toluene for 4–5 h. The solvent was evaporated under reduced pres-
sure and the obtained crude product was recrystallized from abso-
lute ethanol.
either oxime or nitrate moiety have higher percentage of preven-
tion index. This may be attributed to the release of NO in vivo
which possesses gastroprotective activity (Fig. 1).
2.6. Histopathological investigation
The histological slides were prepared according to the reported
procedures.29 The slides obtained from the control group showed
no lesions and characterized by continuous mucosal layer
(Fig. 2a). The slides of the intermediates 4a (Fig. 2b) and 4g
(Fig. 2d) showed greater irregular mucosa with ulcers of approxi-
mately higher number and severity with UI values of 23 and
20.3, respectively. On the contrary, the slides of the oxime 5a
(Fig. 2c) and the nitrate ester 10 (Fig. 2e) exhibited ulcers of low
number and severity with UI of 7.5 and 5.5, respectively. This
may be attributed to the ability of NO donating hybrids to release
NO with well known gastroprotective effect.
4.1.1.1. N-(4-Chlorophenyl)-4-benzoyl-1,5-diphenyl-1H-3-pyra-
zolecarboxamide 4a. Compound 4a was prepared according to
the general procedure above in 73% yield; mp, 177–178 °C; IR (
m
cmꢀ1): 3365 (NH), 1664 (C@O, benzoyl), 1655 (C@O, amide), and
1595 (C@N); 1H NMR (200 MHz, CDCl3): d 9.00 (s, 1H, NH), 7.1–
7.86 (m, 19H, Ar-H). FAB-MS: m/z 478 (M+1).
4.1.1.2. N-(4-Methylphenyl)-4-benzoyl-1,5-diphenyl-1H-3-pyra-
zolecarboxamide 4b. Compound 4b was prepared according to the
general procedure above in 50% yield; mp, 183–185 °C; IR (m
cmꢀ1):
3434 (NH), 1715 (C@O, benzoyl), 1635 (C@O, amide), and 1596
(C@N); 1H NMR (60 MHz, CDCl3): d 9.5 (s, 1H, NH), 7.3–8.5 (m,
19H, Ar-H) and 2.4 (s, 3H, CH3). Anal. Calcd for. C30H23N3O2ꢂ0.5H2O:
C, 77.23; H, 5.19; N, 9.01. Found: C, 77.22; H, 5.19; N, 8.71.
3. Conclusion
A group of pyrazole-3-carboxylic acid derivatives in addition to
their corresponding NO hybrid molecules were prepared. Some of
the prepared compounds exhibited good antibacterial activity
compared to ciprofloxacin. Most of the synthesized compounds
exhibited significant anti-inflammatory activity using indometha-
cin as a reference. The NO hybrid molecules either oxime or nitrate
ester showed pronounced gastroprotective activity better than
their corresponding starting intermediate amides or esters. This
may be attributed to the release of NO, the oxime 5c and the ni-
trate ester 10 showed the highest PI (74%, 76.08%, respectively).
Histopathological examination of some amide intermediates and
their corresponding oximes indicates that the NO donating moiety
either oxime or nitrate reduced greatly the incidence of gastric
ulceration.
4.1.1.3. N-(4-Methoxyphenyl)-4-benzoyl-1,5-diphenyl-1H-3-py-
razolecarboxamide 4c. Compound 4c was prepared according to
the general procedure above in 78% yield; mp, 179–181 °C; IR (
m
cmꢀ1): 3381 (NH), 2986 (C–H str.), 1680 (C@O, benzoyl), 1669
(C@O, amide), and 1593 (C@N); 1H NMR (200 MHz, CDCl3): d
8.88 (s, 1H, NH), 6.81–7.87 (m, 19H, Ar-H), 3.77 (s, 3H, –OCH3).
Anal. Calcd for C30H23N3O3: C, 76.09; H, 4.90; N, 8.87. Found: C,
75.90; H, 5.03; N, 8.84.
4.1.1.4. 4-[(4-Benzoyl-1,5-diphenyl-1H-3-pyrazolyl)carbonyl]
aminobenzoic acid 4d. Compound 4d was prepared according
to the general procedure above in 60% yield; mp, 286–288 °C; IR
(m
cmꢀ1): 2700–3364 (br, OH and NH), 1690 (br, C@O, carboxylic
and C@O of benzoyl), 1661 (C@O, amide), and 1594 (C@N); 1H
NMR (60 MHz, CDCl3): d 10.00 (br s, COOH), 9.30 (br s, 1H, NH)
and 7.5–8.8 (m, 19H, Ar-H). FAB-MS: m/z 488 (M+1). Anal. Calcd
for C30H21N3O4: C, 73.91; H, 4.34; N, 8.62. Found: C, 73.40; H,
4.51; N, 8.59.
4. Experimental
4.1. Chemistry
Melting points were recorded on Stuart electrothermal melting
point apparatus and were uncorrected. IR spectra were recorded as
KBr disks on a Brukar Vector 22 IR spectrometer and also on Perkin
Elmer 298 spectrometer. 1H NMR spectra were run on GEMINI-200
NMR spectrometer (200 MHz) and on MERCURY-300BB NMR spec-
trometer (300 MHz) and also on Varian Em-360L NMR spectrome-
ter (60 MHz) using tetramethylsilane (TMS) as internal standard.
Chemical shifts are expressed in d ppm. Mass spectra were ob-
tained on HP mass spectrometer which was recorded in the posi-
4.1.1.5. 4-Benzoyl-1,5-diphenyl-N{4-[(pyrimidin-2-yl-amino)sul-
fonyl]phenyl}-1H-pyrazole-3-carboxamide 4e. Compound 4e was
prepared according to the general procedure above in 63.0% yield;
mp, 255–256 °C; 1H NMR (60 MHz, CDCl3): d 11.00 (br s, NH), 9.35
(br s, 1H, NH), 7.60–8.65 (m, 19H, Ar-H). Anal. Calcd for
C33H24N6O4S: C, 65.99; H, 4.03; N, 13.99. Found: C, 66.31; H, 4.13;
N, 13.61.

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E.-S. M. N. Abdel-Hafez et al. / Bioorg. Med. Chem. 17 (2009) 3829–3837
Figure 2. (a) Photomicrograph of the mucosa of fundic stomach of control. (b) Photomicrograph of the mucosa of fundic stomach of compound 4a. (c) Photomicrograph of the
mucosa of fundic stomach of compound 5a. (d) Photomicrograph of the mucosa of fundic stomach of compound 4g. (e) Photomicrograph of the mucosa of fundic stomach of
compound 10.
4.1.1.6. N-(4-Acetylphenyl)-4-benzoyl-1,5-diphenyl-1H-3-pyra-
zolecarboxamide 4f. Compound 4f was prepared according to the
mide 4g, mp, >300 °C; yield = 63%; IR (m
cmꢀ1): 3423 (NH), 1657
(C@O, benzoyl), 1629 (C@O, amide), and 1597 (C@N); 1H NMR
(200 MHz, DMSO-d6): d 7.18–7.70 (m, 15H, Ar-H), 2.80–3.92 (m,
9H, piperazine 8H + NH). Anal. Calcd for C27H24N4O2: C, 74.29; H,
5.54; N, 12.84. Found: C, 74.32; H, 5.54; N, 12.92.
general procedure above in 65% yield; mp, 189–190 °C; IR (
m
cmꢀ1): 3368 (NH), 1680 (C@O, acetyl), 1659 (br s, C@O, benzoyl)
and (C@O, amide), and 1595 (C@N); 1H NMR (60 MHz, CDCl3): d
9.75 (s, 1H, NH), 7.4–8.3 (m, 19H, Ar-H) and 2.5 (s, 3H, CH3). Anal.
Calcd for C31H23N3O3: C, 76.69; H, 4.77; N, 8.65. Found: C, 76.47; H,
4.52; N, 8.53.
4.1.2. General procedure for synthesis of the oximes 5a–e
A solution of hydroxylamine hydrochloride (0.347 g, 5 mmol)
and triethylamine (0.505 g, 5 mmol) or pyridine (0.395 g, 5 mmol)
in 15 mL absolute ethanol was added dropwise with stirring to the
respective amide 4a–e (1 mmol). The mixture was heated at reflux
for 4–30 h and the solvent was evaporated under vacuum. To the
cooled residue, 10 mL water was added, cooled in an ice bath
and stirred until the oxime crystallized. The precipitated product
4.1.1.7. (4-Benzoyl-1,5-diphenyl-1H-pyrazol-3-yl)(piperazin-1-
yl)methanone 4g. Compound 4g was prepared by a modification
of the general procedure above where a solution of the acid chlo-
ride 3 in toluene was added in a dropwise manner to a solution
of piperazine in toluene in an equimolar ratio to give the monoa-

E.-S. M. N. Abdel-Hafez et al. / Bioorg. Med. Chem. 17 (2009) 3829–3837
3835
was filtered off and washed with little water, then dried. The crude
product was recrystallized from the appropriate solvent to afford
the oximes 5a–e in yields ranging from 60% to 82%.
the time of the reaction is 3 h using TEA as a base and crystalliza-
tion from methanol; mp = 236–238 ꢀC; yield = 52%; IR (KBr,
m
cmꢀ1): 3225–3370 (br, OH, oxime), 3368 (NH), 1670 (C@O, amide),
and 1595 (C@N). 1H NMR (60 MHz, CDCl3). d 9.7 (s, 1H, NH,
exchangeable), 7.8–8.5 (m, 19H, Ar-H), 2.3 (s, 3H, CH3). M.S (m/z)
(%): 500 (19.1%) (M+Å), 351 (100%), 150 (1.5%), 105 (28.9%), 103
(10.3%) and 77 (43.5%). Anal. Calcd for C31H24N4O3ꢂ0.5H2O: C,
73.07; H, 4.95; N, 11.00. Found: C, 73.33; H, 4.99; N, 11.13.
4.1.2.1. N-(4-Chlorophenyl)-4-[hydroxyimino(phenyl)methyl]-
1,5-diphenyl-1H-3-pyrazolecarboxamide 5a. Compound 5a was
prepared using the general procedure above in 81% yield; mp,
116–118 °C (ethanol); time of the reaction is about 18 h using
TEA as a base; IR (m
cmꢀ1): 3100–3600 (br, OH, oxime and NH),
1676 (C@O, amide) and 1595 (C@N). 1H NMR (60 MHz, DMSO-
d6): d 12.3 (br s, 1H, oximic OH, exchangeable with D2O), 9.5 (br
s, 1H, NH, exchangeable with D2O) and 7.8–8.6 (m, 19H, Ar-H).
MS: 478 (8.2%) (MÅ2+ꢀOH) 476 (MÅ+ꢀOH) (30.7%), 350 (34.8%),
272 (10.4%), 180 (45.8%), 129 (12.4%), 127 (35.1%), 105 (33.4%),
and 77 (100%). Anal. Calcd for: C29H21ClN4O2ꢂH2O: C, 68.17; H,
4.54; N, 10.96. Found: C, 68.50; H, 4.58; N, 10.59.
4.1.4. 2-Bromoethyl 4-benzoyl-1,5-diphenyl-1H-pyrazole-3-car-
boxylate 7
A mixture of the acid chloride 3 (0.386 g, 1 mmol) and 2-bromo-
ethanol (0.248 g, 2 mmol) was heated at reflux in 10 mL dry tolu-
ene with a catalytic amount of pyridine for 3 h. After cooling the
solution was acidified by adding 0.5 mol HCl, a crude solid was pre-
cipitated, filtered off and dried. The obtained crude product was
purified by column chromatography using silica gel and dichloro-
methane as an eluent system to afford 0.20 g, (% yield = 49) of col-
ourless needles of, mp = 142–144 °C; 1H NMR (200 MHz, CDCl3). d
7.16–7.88 (m, 15H, Ar-H), 4.4 (t, 2H, CH2), 3.2 (t, 2H, CH2). Anal.
Calcd for C25H19BrN2O3: C, 63.17; H, 4.03; N, 5.89. Found: C,
63.41; H, 4.29; N, 5.81.
4.1.2.2. N-(4-Methylphenyl)-4-[hydroxyimino(phenyl)methyl]-
1,5-diphenyl-1H-3-pyrazolecarboxamide 5b. Compound 5b was
prepared using the general procedure above in 64% yield; mp,
187–189 °C (ethanol); time of reaction is about 18 h using TEA as
a base; IR (
m
cmꢀ1): 3100–3700 (br, OH, oxime and NH), 1682
(C@O, amide), and 1595 (C@N). 1H NMR (60 MHz, DMSO-d6): d
9.9 (s, 1H, OH, exchangeable), 9.8 (s, 1H, NH), 7.1–8.9 (m, 19H,
Ar-H) and 2.3 (s, 3H, CH3); MS: 473.3 (100%) (M+Å+H+), 455.3
(32%) (M+ÅꢀOH), 351 (7%). Anal. Calcd for C30H24N4O2ꢂ0.4H2O: C,
75.11; H, 5.21; N, 11.68. Found: C, 75.25; H, 5.38; N, 11.21.
4.1.5. 2-(Nitrooxy)ethyl 4-benzoyl-1,5-diphenyl-1H-pyrazole-3-
carboxylate 8
A mixture of the ester 7 (0.474 g, 1 mmol), and AgNO3 (0.676 g,
4 mmol) was heated at reflux in 15 mL acetonitrile overnight at
80 °C. The inorganic solid was filtered and discarded. The filtrate
was evaporated till dryness, the residue was dissolved in 15 mL
dichloromethane and washed with water (3 ꢁ 10 mL), brine
(2 ꢁ 10 mL) and finally with water (2 ꢁ 10 mL). The organic layer
was dried over anhydrous sodium sulfate, evaporated under vac-
uum and the obtained residue was recrystallized from absolute
ethanol to give 0.10 g, (42%) of white needles, mp = 150–151 °C;
1H NMR (200 MHz, CDCl3). d 7.2–7.9 (m, 15H, Ar-H), 4.4 (s, 4H, –
CH2CH2–). MS (m/z) (%): 457 (20.8%) (M+Å), 367 (1.62%), 352
(24.0%), 351 (72.7%), 291 (6.02%), 219 (4.7%), 180 (27%), 105
(68%) and 77 (100%). Anal. Calcd for C25H19N3O6: C, 65.64; H,
4.19; N, 9.19. Found: C, 66.24; H, 4.38; N, 8.84.
4.1.2.3. N-(4-Methoxyphenyl)-4-[hydroxyimino(phenyl)meth-
yl]-1,5-diphenyl-1H-3-pyrazolecarboxamide 5c. Compound 5c
was prepared using the general procedure above in 63% yield;
mp, 125–127 °C (ethanol); time of reaction is 4 h using pyridine
as a base; IR (m
cmꢀ1): 2625–3500 (br, OH, oxime and NH), 1676
(C@O, amide), and 1598 (C@N). 1H NMR (200 MHz, CDCl3) d 8.8
(s, 1H, OH), 8.6 (s, NH) and 6.7–7.5 (m, 19H, Ar-H). MS: 488
(23.3%) (M+Å), 366 (40.6%), 350 (39.6%), 272 (11.2%), 123 (100%)
and 77 (79%). Anal. Calcd for C30H24N4O3ꢂ1.6H2O: C, 69.65; H,
5.30; N, 10.83. Found: C, 69.69; H, 5.24; N, 11.07.
4.1.2.4. 4-[(4-[Hydroxyimino(phenyl)methyl]-1,5-diphenyl-1H-
3-pyrazolylcarbonyl)- amino]benzoic acid 5d. Compound 5d was
prepared using the general procedure above in 60% yield; mp, 258–
260 °C, (ethanol); time of reaction is 10 h using pyridine as a base; IR
4.1.6. 1-4-[(4-Benzoyl-1,5-diphenyl-1H-3-pyrazolyl)
carbonyl]piperazino}-2-chloro-1-ethanone 9
(m
cmꢀ1): 2500–3500 (br, OH oxime, OH carboxylic and NH), 1686
(C@O, acid), 1650 (C@O, amide) and 1600 (C@N). 1H NMR
(60 MHz, CDCl3) d 10.3 (s, OH oxime), 10.2 (s, NH) and 7.4–8.6 (m,
19H, Ar-H); MS: 485 (12.2%) (M+ÅꢀOH), 366 (23.8%), 348 (11.7%),
137 (44.2%) and 77 (100%). Anal. Calcd for C30H22N4O4ꢂH2O: C,
69.22; H, 4.65; N, 10.76. Found: C, 69.41; H, 4.58; N, 10.51.
To an ice cooled stirred solution of the amide 4g (1.833 g,
0.0042 mol) in dichloromethane. a solution of potassium carbonate
(0.868 g, 0.0063 mol) in 100 mL water was added. Chloroacetyl
chloride (0.519 g, 0.0046 mol) in 20 mL dichloromethane was
added to this solution, chloroacetyl chloride (0.519 g, 0.0046 mol)
in 20 mL dichloromethane was added in a dropwise manner while
stirring over 30 min. The mixture was stirred for 2 h at 0 °C and
24 h at room temperature. The organic layer was separated and
the aqueous layer was extracted with (2 ꢁ 30 mL) dichlorometh-
ane, the combined organic layer was washed with distilled water
(3 ꢁ 20 mL). The organic layer was dried over anhydrous sodium
sulfate and evaporated under vacuum. The obtained crude product
was recrystallized from toluene to give 0.330 g of white crystals
(20% yield); mp = 259–260 °C; 1H NMR (200 MHz, CDCl3) d 7.2–
7.8 (m, 15H, Ar-H) and 2.5–4.0 (m, 10H, piperazine 8H + O@C–
CH2). FAB-MS: m/z 513 (M+1). Anal. Calcd for C29H25ClN4O3: C,
67.90; H, 4.91; N, 10.92. Found: C, 67.40; H, 4.74; N, 10.63.
4.1.2.5. N-4-[(2-Pyrimidinylamino)sulfonyl]phenyl-4-[hydrox-
yimino(phenyl)methyl]-1,5-diphenyl-1H-3-pyrazolecarbox-
amide 5e
dure above in 63% yield; mp, 192–193 °C (CH2Cl2/n-hexane); time
of reaction about 30 h using pyridine as a base; IR (
cmꢀ1): 2900–
. Compound 5e was prepared using the general proce-
m
3500 (OH, oxime), 3300 (NH), 1670 (C@O, amide) and 1590 (C@N).
1H NMR (60 MHz, CDCl3). d 12.2 (NH–SO2), 8.5 (OH, oxime), 8.4
(NH–C@O) 7.1–8.2 (m, 22H, Ar-H); MS: 598 (9.4%) (M+ÅꢀOH), 535
(12.9%), 348 (60.6%), 185 (28.0%), 103 (10.3%) and 77 (100%). Anal.
Calcd for C33H25N7O4Sꢂ0.5H2O: C, 63.45; H, 4.20; N, 15.70. Found:
C, 63.74; H, 4.64; N, 15.68.
4.1.7. 2-[4-(4-benzoyl-1,5-diphenyl-1H-pyrazole-3-carbonyl)
piperazin-1-yl]-2-oxoethyl nitrate 10
4.1.3. N-[4-(1-Hydroxyiminoethyl)phenyl]-4-benzoyl-1,5-diph
enyl-1H-3-pyrazole- carboxamide 6
The nitrate ester 10 was prepared from the amide 9 (0.30 g,
0.5 mmol) using the previously described procedure for synthesis
The oxime 6 was prepared from the intermediate 4f by the pre-
viously described general procedure for synthesis of oximes 5a–e;

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E.-S. M. N. Abdel-Hafez et al. / Bioorg. Med. Chem. 17 (2009) 3829–3837
of the nitrate ester 8 to give the compound 10; mp = 229–230 °C;
yield (0.095 g, 30%). 1H NMR (200 MHz, DMSO-d6) d 7.1–7.7 (m,
15H, Ar-H), 5.4 (s, 2H, CH2–ONO2) and 2.4–4 (m, 8H, piperazine).
Anal. Calcd for C29H25N5O6: C, 64.56; H, 4.67; N, 12.98; Found: C,
64.62; H, 4.43; N, 13.12.
bated for 24 h at 37 °C, and then the inhibition zone diameter
was measured in mm. The minimal inhibitory concentration
(MIC) in
lg/mL was determined in lg/mL. The MIC was defined
to be the intercept of the curve of logarithm concentrations versus
diameter of the inhibition zones.
4.1.8. 4-Benzoyl-1,5-diphenyl-1H-pyrazole-3-carboxylic acid 2-
hydroxyethyl ester 11
4.4. Screening of the anti-inflammatory activity
The ester 11 was prepared by reaction between equimolar mix-
ture of the acid chloride 3 and ethylene glycol in a similar proce-
dure to the ester 7. The crude oily product was purified using
silica gel column chromatography with dichloromethane/metha-
nol in 9:1 ratio as eluent in (0.59 g, 55% yield) as oily product solid-
ified upon standing.
4.4.1. Carrageenan induced paw edema method
The anti-inflammatory activity of compounds 2, 4a–g, 5a–e and
6–10 was screened according to the method described by Winter
et al.26 where a pedal inflammation in rat paws induced by subpla-
nar injection of carrageenan suspension (1% w/v) into the right
hind of the rats. Male albino rats 140–150 g were randomly di-
vided into groups which consisted of 5 rats each. All the animals
were left for three days in the laboratory for acclimatization before
the day of experiment, and on the last day they were given water
only. The thickness of rat paw was measured by a digital plethys-
mometer before and 30 min after carrageenan injection to detect
the inflammation induced by carrageenan. Reference group was ta-
ken indomethacin orally at a dose 100 mg/mL. Control group re-
1H NMR (60 MHz, CDCl3). d 3.85–4.20 (m, 4H, –CH2CH2–); 4.80
(br s, 1H, OH), 8.0–8.4 (m, 15H, Ar-H).
4.2. Evaluation of nitric oxide release
A solution of the appropriate compound (20
l
L of 1 ꢁ 10ꢀ4 mol
in 100 mL DMSO) was added to 2 mL of 1:1 V/V mixture of 50 mM
phosphate buffer (pH 7.4) and methanol containing (5 ꢁ 10ꢀ4 M)
ceived
a
vehicle (carboxymethylcellulose), while tested
L
-cysteine. After 1 h at 37C°, 1 mL of the reaction mixture was trea-
compounds 2, 4a–g, 5a–f and 6–10 at dose of 100 mg/kg were ta-
ken orally to different groups of rats. The difference between the
thicknesses of the two paws was taken as a measure of edema.
The measurement was carried out at 0, 1, 2, 3, 4 and 5 h after
administration of the tested compounds, reference and vehicle.
ted with 250 lL of Griess reagent. After 25 min, the absorbance
was measured at 540 nm. Sodium nitrite standard solutions (10–
50 nmol/mL) were used to construct the calibration curve.24,30
The Griess reagent was prepared as follows: [sulfanilamide (4 g),
N-naphthylethylenediamine dihydrochloride (0.2 g), 85% phospho-
ric acid (10 mL) in distilled water (final volume: 100 mL)].24,30
4.5. Investigation of the gastric protective activity
4.3. Investigation of the antibacterial activity
After measuring the anti-inflammatory activity the rats were
killed by an overdose of ether. All experiments were performed
at the same time of the day to avoid variations due to diurnal
rhythms of putative regulators of gastric functions. The stomachs
were removed and opened along the greater curvature. The stom-
achs were washed with ice-cold saline and scored for macroscopic
gross mucosal lesions.31
Compounds 4a, 4c–f, 5a, 5c–e, 6, 7 and 8 were evaluated for
their in vitro antibacterial activity using agar cup diffusion meth-
od.25 Ciprofloxacin was used as a reference.
4.3.1. Materials and methods
The selected compounds were evaluated for antibacterial activ-
ity against E. coli C-600, Klebsila pneumonia (as Gram negative), B.
subitilis and S. aureus NCTC 6571 (as Gram positive) using the agar
cup diffusion method.25
4.6. Histopathological investigation
The histological slides were prepared according to the reported
procedures29 for examination of ulcers under light microscope.
4.3.2. Preparation of the media
All strains were cultured on sterile nutrient agar medium which
was supplied by oxoid, and prepared according to the instructions
of the manufactures. The media were molten on a steam bath inoc-
ulated with few drops of the culture of the specific microorganism
and poured into sterile Petri dishes to form a layer of about 3–
4 mm thickness. The layer was allowed to cool and harden, the un-
der side of each plate was marked into approximately equal six
sectors. With the aid of Wassrman tube, a single cup of about
10 mm diameter was cut in the centre of each sector to produce
a total of six cups per dish. Five of these cups were devoted for
the testing of the desired compounds, while the last one was left
as a control guide for the solvent.
4.6.1. Procedures
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.
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