Synthesis and pharmacological evaluation of new pyrazolyl benzenesulfonamides linked to polysubstituted pyrazoles and thiazolidinones as anti-inflammatory and analgesic agents

Article abstract of DOI:10.1007/s00706-015-1549-x

New compounds comprising the pyrazolyl benzenesulfonamide scaffold linked to polysubstituted pyrazoles and thiazolidinones were synthesized and evaluated for their anti-inflammatory and analgesic activities. The results revealed that most of the compounds

Full text of DOI:10.1007/s00706-015-1549-x

Monatsh Chem
DOI 10.1007/s00706-015-1549-x
ORIGINAL PAPER
Synthesis and pharmacological evaluation of new pyrazolyl
benzenesulfonamides linked to polysubstituted pyrazoles
and thiazolidinones as anti-inflammatory and analgesic agents
Hayam M. A. Ashour1 Ibrahim M. El-Ashmawy2,3 Aida E. Bayad2
•
•
Received: 19 June 2015 / Accepted: 30 July 2015
Ó Springer-Verlag Wien 2015
Abstract New compounds comprising the pyrazolyl
Keywords Pyrazoles Á Thiazolidinones Á
benzenesulfonamide scaffold linked to polysubstituted
pyrazoles and thiazolidinones were synthesized and eval-
uated for their anti-inflammatory and analgesic activities.
The results revealed that most of the compounds displayed
distinctive anti-inflammatory and analgesic activity. Two
thiazolidinone derivatives emerged with the highest anti-
inflammatory activity in this study, whereas two pyrazole
derivatives displayed high analgesic activity with a fast
onset of action compared to the reference drug. Moreover,
the active compounds showed minimal potential for gastric
injury in addition to a good safety margin (ALD50[0.25 g/
kg). In vitro COX-1/COX-2 inhibition study revealed that
the most active compounds showed relatively more selec-
tivity toward COX-2 than COX-1. Among the test
compounds, a thiazolidinone derivative possessed the
lowest IC50 value against both COX-1 and COX-2.
Graphical abstract
Anti-inflammatory Á Analgesic Á COX-1/COX-2 Á
Bioorganic synthesis
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most
widely prescribed agents in the treatment of various inflam-
matory diseases such as arthritis, rheumatism as well as mild to
moderate pain and fever [1]. The pharmacological activity of
NSAIDs is closely related to their ability to inhibit both iso-
forms of the cyclooxygenase enzyme (COX), COX-1 and
COX-2 which catalyzes the bioconversion of arachidonic acid
to inflammatory prostaglandins (PGs). The constitutive COX-1
isoform is mainly responsible for the synthesis of PGs that exert
cytoprotective effect in the gastrointestinal (GI) tract and con-
trol renal function in the kidneys, whereas the inducible COX-2
isoform is selectively activated by pro-inflammatory stimuli
and facilitates the release of PGs involved in the inflammatory
process [2]. Consequently, their long-term clinical use is
associated with several side effects such as gastrointestinal
lesions, bleeding, and nephrotoxicity [3]. These observations
provided an acceptable rationale for the development of
selective COX-2 inhibitors that should retain the therapeutic
potency of classical NSAIDs with less GI adverse effects [4].
However, recent concerns regarding these drugs and their
association with significant cardiovascular side effects led to
reconsideration of their appropriate use [5]. Therefore, devel-
opment of novel compounds having anti-inflammatory activity
with an improved safety profile is still a necessity.
& Hayam M. A. Ashour
hayamashour@ymail.com
1
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Alexandria University, Alexandria 21521, Egypt
2
Pharmacology Department, Faculty of Veterinary Medicine,
Alexandria University, Alexandria, Egypt
Since the discovery of antipyrine, the first pyrazolone
derivative used in the management of pain, inflammation,
and fever, great attention has been focused on pyrazole
derivatives as potent anti-inflammatory, analgesic, and
3
Department of Veterinary Medicine, Faculty of Agricultural
and Veterinary Medicine, Qassim University, P.O.Box 1482,
Buraydah, Al-Qassim, Saudi Arabia
123

H. M. A. Ashour et al.
antipyretic agents [6–10]. As a result, a large number of
pyrazoles have been obtained and some have gained appli-
cation on the clinical level such as celecoxib (Fig. 1), a
potent and GI-safe anti-inflammatory and analgesic agent. It
is considered as a typical model of the diaryl heterocycle
template that is known to selectively inhibit the COX-2
enzyme [11].
particular, potential anti-inflammatory activity and remark-
able gastrointestinal safety were displayed by the lead
structures A [17], B [18], and C [19] (Fig. 1).
Encouraged by these results, it was attempted in the
present study to synthesize and evaluate the anti-inflam-
matory and analgesic activities of some new structure
hybrids comprising basically the pyrazolyl benzenesul-
fonamide skeleton linked either to polysubstituted
pyrazoles at position 4 through a carbonyl bridge (struc-
tures D–G, Fig. 1), or to substituted thiazolidinones
through a carbonyl hydrazinylidene moiety (structure H,
Fig. 1). The substitution pattern of the pyrazole ring
includes various functionalities such as the carbonyl,
amino, cyano, and carbethoxy groups in addition to the
antipyrine moiety that is documented to be an important
integral counterpart in many analgesic and anti-inflamma-
tory agents [6, 20]. Moreover, the incorporated
thiazolidinone ring was substituted with a ylidene group
which is suggested to be an additive anti-inflammatory
pharmacophoric element [12]. In addition to the targeted
anti-inflammatory and analgesic activities, the ulcerogenic,
acute toxicity profiles and inhibitory activities of COX-1
and COX-2 enzymes were also investigated.
On the other hand, the 4-thiazolidinone ring system is one
of the privileged structure fragments in modern medicinal
chemistry, owing to its broad pharmacological activities and
affinity for various biotargets. Among these biological
activities, the anti-inflammatory and analgesic activities of
thiazolidinones have been of particular interest recently [12–
14]. Moreover, some thiazolidinones have been considered
as effective lead anti-inflammatory COX-2 inhibitors [15,
16].
In the course of a research program devoted to the
development of new anti-inflammatory agents devoid of the
undesirable side effects associated with classical NSAIDs,
we have reported the synthesis and anti-inflammatory
activity of some pyrazolyl benzenesulfonamides substituted
with various functionalities and attached to different hete-
rocyclic ring systems through various linkages [17–19]. In
Fig. 1 Chemical structure of reported anti-inflammatory pyrazolyl benzenesulfonamides A–C and the newly synthesized compounds D–H
123

Synthesis and pharmacological evaluation of new pyrazolyl benzenesulfonamides linked to…
pyrazol-4-ylazo)pentane-2,4-dione, ethyl 2-(1,5-dimethyl-
Results and discussion
Chemistry
3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylazo)-3-oxobu-
tanoate, or diethyl 2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-ylazo)malonate gave rise to the
proposed pyrazole 6, pyrazolinone 7, and pyrazolidinone 8,
respectively.
The synthetic strategies adopted for the synthesis of the
intermediate and final compounds are depicted in
Schemes 1, 2, and 3. In Scheme 1, the amino ester 1 [21]
and the acid hydrazide 2 [22] were prepared as previously
described. Condensation of the acid hydrazide 2 with the
diketo compounds acetylacetone, ethyl acetoacetate, or
diethyl malonate in ethanol/glacial acetic acid mixture
yielded the corresponding pyrazole 3 [22], pyrazolinone 4,
and pyrazolidinone 5, respectively. Similarly, heating 2
In Scheme 2, heating
2
with ethoxymethylene-
malononitrile or ethyl (ethoxymethylene)cyanoacetate in
dry DMF resulted in the formation of 5-amino-4-substi-
tuted-1H-pyrazol-1-yl derivatives 9a, 9b in moderate
yields. On the other hand, refluxing
[bis(methylthio)methylene]malononitrile or
2
with
methyl
2-cyano-3,3-bis(methylthio)acrylate in dry DMF following
previously published reaction conditions [23] furnished the
with
3-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
Scheme 1
COOEt
NH2
N
N
H3C
H3C
O
N
N
NH2
N
O
N
An
N
N
N
NH2
SO2NH2
CH3
CH3
N
N
1
N
i
v
ii
O
NH2
SO2NH2
SO2NH2
N
H
3
6
O
O
O
O
N
An
N
NH2
N
N
N
N
N
N
CH3
CH3
iii
iv
N
N
vi
NH2
NH2
N
N
SO2NH2
2
`vii
O
O
SO2NH2
SO2NH2
O
O
N
An
N
4
7
N
HN
NH2
N
HN
NH2
O
O
N
N
N
N
CH3
N
H3C
N
An =
SO2NH2
8
SO2NH2
O
5
Reagents and Conditions. i)NH2NH2.xH2O 80% / reflux; ii) CH3COCH2COCH3 / glacial acetic acid /absolute ethanol
/ reflux; iii) CH3COCH2COOC2H5 / gl.acetic acid /abs.ethanol/ reflux;iv) CH2(COOC2H5)2 / glacial acetic acid /
abs. ethanol/ reflux; v) 3-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylazo)pentane-2,4-dione /
gl. acetic acid / abs. ethanol / reflux; vi) ethyl 2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylazo)-3-oxo
butanoate / gl. acetic acid / abs. ethanol/ reflux; vii) diethyl 2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-
4-ylazo)malonate / gl.acetic acid /abs. ethanol/ reflux
123

H. M. A. Ashour et al.
Scheme 2
NH2
NH2
O
O
O
R1
R
N
N
NHNH2
NH2
N
NH2
N
NH2
N
N
N
SCH3
N
N
N
i
ii
SO2NH2
9a, b
SO2NH2
SO2NH2
2
10a,b
iii
NH2
O
N
N
NH2
N
9a: R = CN, 9b: R = COOEt
N
10a: R1 = CN, 10b: R1 = COOMe
11a; R2 = H, 11b: R2 = Cl
R2
SO2NH2
11a,b
Reagents and Conditions: i) EtOCH=C(CN) or EtOCH=C(CN)COOEt / DMF / reflux;
2
ii) (SCH ) C=C(CN) or (SCH ) C=C(CN)COOMe / DMF / reflux;
3 2
2
3 2
iii) R2C H COCH CN/gl. acetic acid / abs. ethanol / reflux
6
4
2
proposed 5-amino-3-methylsulfanyl-4-substituted pyra-
zolyl derivatives 10a, 10b. Synthesis of the target 3-aryl-5-
aminopyrazolyl derivatives 11a, 11b was achieved by
refluxing 2 with phenacyl or 4-chlorophenacyl cyanides in
ethanol/glacial acetic acid mixture as described for the
synthesis of analogous compounds [22].
15 in moderate yields. 1H NMR spectra of compounds 13–
15 revealed signals for NH proton of the thiazolidinone
ring at 11.66–12.54 ppm, accounting for a lactam proton
[25]. The Z configuration of the exocyclic C=C bond in the
5-arylidene derivatives 14 and 15 was confirmed on the
basis of 1H NMR spectral analysis, since the methine
proton resonated, as expected, at higher chemical shift
values due to the deshielding effect of the adjacent C=O,
than it would do in E isomers, because of the lower
deshielding effect of the S atom [25].
In Scheme 3, stirring 2 with chloroacetyl chloride in dry
DMF at room temperature furnished the corresponding
chloroacetyl derivative 12, which upon cyclization with
ammonium thiocyanate in boiling ethanol produced thia-
zolidin-4-one 13 in excellent yield and purity. Finally,
thiazolidinone 13 was allowed to undergo Knoevenagel
condensation with aromatic aldehydes or 3-aryl-1-phenyl-
1H-pyrazole-4-carboxaldehydes [24] to give the target
arylidene derivatives 14 and 15, respectively. Due to poor
solubility of compound 13 in organic solvents, conven-
tional heating in glacial acetic acid in the presence of
anhydrous sodium acetate [25] or in ethanol in the presence
of piperidine [26] as previously described for synthesis of
analogous compounds failed to give the target compounds
14 and 15. However, carrying out the reaction in a mixture
of absolute ethanol/dry DMF (2:1) in the presence of
piperidine yielded the target arylidene derivatives 14 and
Anti-inflammatory (AI) activity
The AI activity of the synthesized compounds was evalu-
ated utilizing the formalin-induced paw edema bioassay
using diclofenac sodium (20 mg/kg) as a reference stan-
dard anti-inflammatory agent [27]. The data obtained are
presented in Table 1 and expressed as mean SE. Sta-
tistical differences of control and test groups were carried
out using the analysis of variance (ANOVA) followed by
‘Student–Newman–Keuls multiple comparison test’. They
were performed using computer package of the Statistical
Analysis System (SAS, 1987), SAS Incorporation Institute.
123

Synthesis and pharmacological evaluation of new pyrazolyl benzenesulfonamides linked to…
Cl
Scheme 3
O
O
HN
NH
NHNH2
NH2
O
i
N
N
NH2
N
N
SO2NH2
SO2NH2
2
12
ii
S
O
N
NH
HN
O
N
NH2
N
R
N N
iv
iii
SO2NH2
R
S
S
O
O
13
N
NH
N
NH
HN
HN
O
O
N
N
N
NH2
NH2
N
SO2NH2
SO2NH2
15a, b
14a, b
a: R = H, b: R = Cl
Reagents and Conditions: i) ClCOCH2Cl /dry DMF /r.t.; ii) NH4SCN /ethanol /reflux;
iii) C6H5CHO or 4-ClC6H4CHO / DMF/ abs.ethanol /piperidine / reflux;
iv) 3-aryl-1-phenyl-1H-pyrazole-4-carboxaldehyde / DMF / abs. ethanol / piperidine/ reflux
The difference in results was considered significant when
compounds showed distinctive pharmacokinetic profiles as
revealed from their potent and rapid onset of action which
was nearly equivalent to or higher than diclofenac sodium
at a dose of 20 mg/kg, po. An outstanding AI % was
recorded for the pyrazolyl derivatives 4 (52 %) and 5
(56 %) when compared with diclofenac sodium (36 %),
whereas the pyrazolyl derivatives 3, 8, 9a, 9b, 11b and the
thiazolidinones 13, 14a, 14b, 15a, 15b showed anti-in-
flammatory activity (32–40 %) nearly equivalent to the
reference drug. The other compounds showed moderate to
weak activity. After 2 h interval, the pyrazole 5 and the
thiazolidinones 14a, 14b, 15a displayed anti-inflammatory
activity (45–48 %) superior to diclofenac sodium (38 %),
whereas the pyrazoles 4, 7, 8, 9a, and 10a were nearly
effective in inhibiting the paw edema with percentage
activity of 35–38 % compared to the reference drug. After
p \ 0.05.
Formalin-induced paw edema bioassay
In this acute inflammatory model, each test compound was
dosed orally (20 mg/kg body weight) 1 h prior to induction
of inflammation by formalin injection. Diclofenac sodium
was utilized as a reference drug at a dose of 20 mg/kg, po.
The anti-inflammatory activity was then calculated 1–4 h
after induction of inflammation and presented in Table 1 as
the mean paw volume (cm3) and the percentage anti-in-
flammatory activity (AI %).
A comparative study of the anti-inflammatory activity of
the test compounds relative to the reference drug at dif-
ferent time intervals indicated the following: after 1 h, 13
123

H. M. A. Ashour et al.
Table 1 Anti-inflammatory activity (AI) of the synthesized compounds in formalin-induced rat paw edema bioassay (acute inflammatory
model)
Comp.a
Volume of edema/cm3b
ED50/mg/kg
0
1 h
2 h
4 h
Control
0.33 0.02
0.34 0.02
0.32 0.01
0.35 0.02
0.36 0.02
0.34 0.01
0.35 0.01
0.34 0.01
0.35 0.02
0.33 0.01
0.35 0.02
0.33 0.02
0.33 0.01
0.31 0.03
0.32 0.01
0.32 0.01
0.34 0.01
0.32 0.02
0.34 0.02
0.58 0.02
0.64 0.01
0.83 0.03
3
0.49 0.01c (40)
0.44 0.02c (52)
0.46 0.01c (56)
0.57 0.01c (16)
0.55 0.02c (16)
0.52 0.02c (32)
0.51 0.01c (32)
0.52 0.01c (32)
0.51 0.01c (28)
0.53 0.01c (28)
0.51 0.01c (28)
0.49 0.01c (36)
0.46 0.01c (40)
0.48 0.02c (36)
0.47 0.01c (40)
0.49 0.02c (40)
0.47 0.03c (40)
0.50 0.01c (36)
0.56 0.03c (29)
0.51 0.01c (38)
0.51 0.03c (48)
0.58 0.01c (29)
0.54 0.02c (35)
0.55 0.02c (35)
0.53 0.02c (38)
0.55 0.01c (35)
0.53 0.02c (35)
0.56 0.01c (32)
0.53 0.01c (35)
0.54 0.03c (32)
0.52 0.01c (32)
0.49 0.03c (45)
0.49 0.01c (45)
0.50 0.02c (48)
0.49 0.02c (45)
0.53 0.01c (38)
0.71 0.01c (26)d
0.62 0.01c (40)
0.64 0.02c (42)
0.66 0.02c (40)
0.57 0.01c (54)
0.57 0.01c (56)
0.60 0.02c (48)
0.62 0.01c (46)
0.61 0.02c (44)
0.65 0.01c (40)
0.58 0.02c (50)
0.61 0.01c (44)
0.63 0.03c (36)
0.58 0.01c (48)
0.54 0.03c (56)
0.62 0.01c (44)
0.55 0.01c (54)
0.55 0.02c (58)
4
5
6
7
17.12e
16.34
8
9a
9b
10a
10b
11a
11b
13
14a
14b
14.54
15a
15b
15.13
13.95
Diclofenac sodium
a
Dose levels, po: test compounds and diclofenac sodium (20 mg/kg b.wt.)
Values are expressed as mean SE (number of animals = 6 rats)
Significantly different compared to corresponding control, p B 0.05
Between parentheses (percentage anti-inflammatory activity, AI %)
ED50 is the effective dose calculated after 4 h
b
c
d
e
4 h, the pyrazoles 7, 8 and the thiazolidinones 14b, 15b
showed anti-inflammatory activity (54–56 %) nearly
equivalent to diclofenac sodium (58 %), whereas the
pyrazole 11a displayed anti-inflammatory activity (50 %)
slightly lower than the reference.
group by carbonyl (compounds 5 and 8) did not result in
a significant change in the overall activity. Moreover, the
presence of antipyrinylazo moiety in compounds 6–8
greatly enhanced the anti-inflammatory activity after 4 h
compared to the corresponding antipyrinylazo free ana-
logs. Concerning the 5-aminopyrazoles 9–11, it was
noticed that introduction of methylsulfanyl moiety
(compounds 10a, 10b) or removal of the cyano or ester
group and introduction of phenyl or 4-chlorophenyl
group at position 3 (compounds 11a, 11b) did not sig-
nificantly affect the overall activity in this series of
compounds.
The most active compounds 7, 8, 14b, and 15b were
further tested at 5, 10, 20, 40, and 50 mg/kg body weight to
determine their ED50 values after 4 h interval. The thia-
zolidinones 14b and 15b were found to be nearly
equipotent (ED50 = 14.54 and 15.13 mg/kg respectively,
Table 1) to diclofenac sodium (ED50 = 13.95 mg/kg),
whereas compounds 7 and 8 were found to be less potent
(ED50 = 16.34 and 17.12 mg/kg respectively, Table 1).
A deep insight into the structures of the tested com-
pounds revealed that they represent two different series,
namely the pyrazole series (compounds 3–11) and the
thiazolidinone series (compounds 13–15). Among the
pyrazole derivatives 3–8, it was found that replacing one
methyl group (compounds 3 and 6) by carbonyl func-
tionality (compounds 4 and 7) led to a marked increase
in activity, whereas replacement of the second methyl
On the other hand, within the thiazolidinones 13–15,
derivatization of the methylene group to a substituted yli-
dene moiety (compounds 14 and 15) led to a noticeable
enhancement in the overall activity. Moreover, the ylidene
derivatives containing 4-chlorophenyl group (14b and 15b)
were more active than their unsubstituted phenyl analogs
(14a and 15a) after 4 h intervals.
Collectively, compounds comprising the antipyrine
moiety (7, 8) and the thiazolidinones substituted with
123

Synthesis and pharmacological evaluation of new pyrazolyl benzenesulfonamides linked to…
Table 2 Analgesic activity of the synthesized compounds using the rat tail withdrawal technique
Comp.a
Reaction time/sb
0
1 h
2 h
4 h
Control
2.86 0.20
2.38 0.20
2.75 0.20
2.39 0.10
2.38 0.20
3.03 0.10
2.58 0.10
2.68 0.20
2.33 0.10
2.65 0. 10
2.63 0.20
2.31 0.20
2.66 0.20
2.83 0.20
2.34 0.20
2.58 0.20
2.35 0.20
2.79 0.10
2.79 0.20
2.90 0.10
2.97 0.20
3.01 0.20
3
2.82 0.10 (18)d
3.20 0.10c (16)
3.15 0.20c (31)
2.7 0.20 (13)
3.53 0.20 (16)
3.02 0.20c (17)
3.63 0.20c (35)
3.00 0.20c (28)
3.32 0.20 (25)
3.23 0.10 (22)
3.10 0.10c (34)
3.78 0.20c (42)
3.05 0.10 (07)
3.04 0.20c (30)
3.06 0.30c (18)
3.02 0.20c (28)
3.08 0.20 (10)
3.25 0.20c (16)
3.06 0.20c (32)
3.74 0.20c (36)
3.68 0.20c (53)
2.92 0.20c (22)
3.88 0.30c (28)
3.65 0.20c (41)
3.88 0.40c (44)
3.45 0.20c (48)
3.65 0.30c (38)
3.54 0.20c (34)
3.52 0.20c (52)
3.99 0.30c (50)
3.32 0.20c (17)
3.27 0.20c (40)
3.45 0.20c (33)
3.25 0.20c (38)
3.19 0.20c (14)
3.99 0.10c (43)
3.38 0.20c (42)
4.15 0.20c (50)
3.89 0.10c (62)
3.18 0.20c (33)
4.25 0.20c (40)
3.85 0.10c (49)
4.25 0.20c (58)
3.69 0.30c (58)
3.88 0.20c (46)
3.74 0.20c (42)
3.78 0.20c (63)
4.25 0.30c (59)
3.63 0.20c (28)
3.85 0.20c (64)
3.70 0.20c (43)
3.85 0.20c (63)
3.74 0.20c (34)
4.51 0.30c (61)
4
5
6
7
8
9a
9b
10a
10b
11a
11b
13
14a
14b
15a
15b
Diclofenac sodium
a
Dose levels, po: test compounds (20 mg/kg b.wt.), diclofenac sodium (20 mg/kg b.wt.)
Values are expressed as mean SE (number of animals = 6 rats)
Significantly different compared to corresponding control, p B 0.05
Between parentheses (percentage analgesic activity)
b
c
d
chlorobenzylidene group (14b, 15b) displayed anti-in-
flammatory activity equivalent to diclofenac sodium after
4 h.
diclofenac sodium (16 %), whereas the pyrazoles 3, 4, 6, 7,
8 were nearly equipotent to it. After 2 h, the pyrazoles 5,
9b, 11a, 11b exhibited analgesic activity (48–53 %) higher
than the reference drug (43 %), while the pyrazoles 8, 9a
and the thiazolidinone 14a showed similar activity
(40–44 %), and the pyrazole 10a and the thiazolidinone
15a displayed slightly lower activity (38 %). After 4 h, the
pyrazoles 5, 9a, 9b, 11a, 11b and the thiazolidinones 14a,
15a were found to be nearly equipotent (58–64 %) to the
reference drug.
Analgesic activity
The analgesic activity of the synthesized compounds was
evaluated using the rat tail withdrawal technique in
response to immersion in water at 55 °C using diclofenac
sodium as a reference drug (20 mg/kg, po) [28]. The
analgesic activity was measured at 1–4 h time intervals
after pain induction. The results were recorded as the
average values of six administrations and the percentage
increase of the reaction time in comparison with the basal
values. The results are presented in Table 2 and expressed
as mean SE. Statistical differences of control and test
groups were carried out as described in the
‘‘Experimental’’.
Based on the results in Table 2, the following prelimi-
nary structure–activity relationships could be tentatively
deduced. Among the pyrazoles 3–8, replacing the two
methyl groups by carbonyl functionality (compounds 5 and
8) markedly improved the anti-inflammatory activity at all
time intervals. Unexpectedly, pyrazoles containing the
antipyrinylazo moiety (compounds 6–8) were less active
than their corresponding antipyrinylazo free analogs
(compounds 3–5). Concerning the 5-aminopyrazoles 9–11,
it was noticed that introduction of methylsulfanyl moiety
(compounds 10a, 10b) markedly decreased the activity at
all time intervals, whereas removal of the cyano or ester
group and introduction of phenyl or 4-chlorophenyl group
A comparative study of the analgesic activity of the test
compounds relative to the reference drug at different time
intervals revealed the following: after 1 h, the pyrazoles 5,
9a, 9b, 10a, 10b, 11a, 11b and the thiazolidinones 14a, 15a
exhibited fast analgesic activity (22–42 %) superior to
123

H. M. A. Ashour et al.
at position 3 (compounds 11a, 11b) improved the overall
activity.
Table 3 In vitro COX-2 selectivity of the synthesized compounds
Comp
IC50/lMa
COX-1/COX-2b
On the other hand, among the thiazolidinones 13–15,
derivatization of the methylene group to a substituted yli-
dene moiety led to a marked improvement in the overall
activity. Moreover, the ylidene derivatives containing
unsubstituted phenyl group (14a and 15a) were more active
than their 4-chlorophenyl analogs (14b and 15b). Thus,
compound 14a emerged as the most potent derivative
among the thiazolidinones 13–15. Collectively, the pyra-
zoles 5, 9a, 9b, 11a, 11b and the thiazolidinones 14a, 15a
were the most active analgesic agents in this study with a
fast onset of action compared to the reference drug.
Comparing the experimental results from both anti-in-
flammatory and analgesic screening, it could be concluded
that the parameters that modulate one activity were dif-
ferent from those which modulate the other one. This could
explain the observation that highly active compounds in
anti-inflammatory screening, e.g., compounds 14b and
15b, were not potent in the analgesic screening and vice
versa.
COX-1
COX-2
5
11.59
16.0
25.86
12.32
26.66
22.5
20.3
5.6
2.24
5.35
8.23
3.60
6.90
6.33
7.30
1.52
1.06
5.0
5.17
2.99
3.14
3.42
3.86
3.55
2.78
3.68
4.24
3.64
3.17
0.27
407
7
8
9a
9b
11a
11b
14a
14b
4.5
15a
18.2
28.5
0.06
28.5
15b
8.98
0.22
0.07
Diclofenac sod
Celecoxib
a
Values are means of three determinations acquired using an ovine
COX-1/COX-2 assay kit (catalog no. 760111, Cayman Chemicals,
MI, USA) and the deviation from the mean is \10 % of the mean
value
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50
)
Ulcerogenic activity
activity is assayed colorimetrically by monitoring the
appearance of oxidized
N,N,N0,N0-tetramethyl-p-
The tested compounds 5, 7, 8, 9a, 9b, 11a, 11b, 14a, 14b,
15a, and 15b that exhibited promising anti-inflammatory or
analgesic profiles in the pre-mentioned animal models were
further evaluated for their ulcerogenic potential in rats [29].
Gross observation of the isolated rat stomachs showed
weak ulcerogenic potential of the test compounds at an oral
dose of 250 mg/kg, when administered twice at 2 h interval
in fasted rats compared with diclofenac sodium which
caused serious gastric ulcers under the same experimental
conditions.
phenylenediamine (TMPD), which is produced during the
reduction of PGG2 to PGH2, at 590 nm. The efficacies of
the tested compounds were determined as the concentration
causing 50 % enzyme inhibition (IC50) and recorded in
Table 3. The data revealed that the pyrazoles 5, 7, 8, 9a,
9b, 11a, 11b and the thiazolidinones 14a, 14b, 15a, and
15b showed relatively more selectivity toward COX-2 than
COX-1. Among the tested compounds, the thiazolidinone
derivative 14b demonstrated the highest activity as it
showed IC50 value of 4.5 lM for COX-1 and 1.06 lM for
COX-2.
Acute toxicity
The same selected compounds, 5, 7, 8, 9a, 9b, 11a, 11b,
14a, 14b, 15a, and 15b, were further evaluated for their
approximate acute lethal dose ALD50 in male rats follow-
ing a reported literature method [30]. The results indicated
the tested compounds were non-toxic and showed a high
safety margin when screened at graded doses (0.1–0.25 g/
kg, po), where their ALD50 values were found to be
[0.25 g/kg.
Conclusion
The present study reports the synthesis and investigation of
the anti-inflammatory and analgesic activities of new
pyrazolyl benzenesulfonamides linked to polysubstituted
pyrazole and thiazolidinone ring systems. The obtained
results revealed that the thiazolidinones 14b and 15b were
the most potent anti-inflammatory agents in this study,
whereas the pyrazoles 11a and 11b were the most active
analgesic agents with a fast onset of action compared to the
reference drug. Additionally, the active compounds
revealed lower ulcerogenic potential than the reference
drug diclofenac sodium and were well tolerated by exper-
imental animals with a high safety margin (ALD50
In vitro COX-1/COX-2 inhibition assay
The compounds which showed significant in vivo activity
were further evaluated for their ability to inhibit COX-1
and COX-2 isoenzymes by in vitro colorimetric COX
(ovine) inhibitor assay method [31], which utilizes the
peroxidase component of cyclooxygenase. The peroxidase
123

Synthesis and pharmacological evaluation of new pyrazolyl benzenesulfonamides linked to…
reaction mixture was left overnight and the separated solid
[0.25 g/kg). In vitro COX-1/COX-2 inhibition study
revealed that the most active compounds showed relatively
more selectivity toward COX-2 than COX-1 which might
account for their low ulcerogenic potential. Among the test
compounds, compound 14b demonstrated the highest
activity with IC50 value of 4.5 lM for COX-1 and 1.06 lM
for COX-2.
product was filtered, washed with ethanol, dried, and
recrystallized from dimethyl formamide/ethanol as white
crystals (82 %). M.p.: 297 °C (Ref. [22] 296–297 °C).
General procedure for the synthesis of 4-[5-amino-4-
substituted-1H-pyrazol-1-yl]benzenesulfonamides
3–8
Finally, these compounds represent a new structure
scaffold that could be further optimized for future devel-
opment of more potent anti-inflammatory and/or analgesic
agents with better GIT tolerance.
A mixture of 0.59 g acid hydrazide 2 (2 mmol) and the
selected diketo derivative (2 mmol) in 20 cm3 of glacial
acetic acid/absolute ethanol mixture (2:1) was heated under
reflux for 8–12 h. The reaction mixture was allowed to
attain room temperature and the separated solid pro-
duct was filtered, dried, and crystallized from the proper
solvent.
Experimental
All reagents and solvents were purchased from commercial
suppliers and dried and purified when necessary by stan-
dard techniques. Melting points were determined in open
glass capillaries using Stuart capillary melting point
apparatus (Stuart scientific Stone, Staffordshire, UK).
Infrared (IR) spectra were recorded on Perkin Elmer 1430
infrared spectrophotometer (Perkin Elmer, Beaconsfield,
UK) and measured using KBr cell. NMR spectra were
scanned on Varian Mercury VX-300 or VX-400 using
tetramethylsilane (TMS) as internal standard and DMSO-
d6 as solvent (chemical shifts are given in d/ppm). Mass
spectra were run on a Finnigan mass spectrometer model
SSQ/7000 (70 eV) or on a gas chromatograph/mass spec-
trometer Schimadzu GCMS-QP 2010 Plus (70 eV).
Microanalyses were performed at the regional Center for
Mycology and Biotechnology, El-azhar University, and the
found values were within 0.3 % of the theoretical values.
Follow-up of the reactions and checking the purity of the
compounds were made by thin layer chromatography
(TLC) on silica gel-precoated aluminum sheets (Type 60
GF254, Merck, Germany) and the spots were detected by
exposure to UV lamp at k = 254 nm for a few seconds.
4-[5-Amino-4-[(3,5-dimethyl-1H-pyrazol-1-yl)carbonyl]-
1H-pyrazol-1-yl]benzenesulfonamide (3)
White crystals (0.6 g, 84 %); m.p.: 244 °C (Ref. [22]
244–245 °C).
4-[5-Amino-4-[(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
yl)carbonyl]-1H-pyrazol-1-yl]benzenesulfonamide
(4, C14H14N6O4S)
Grayish white crystals (0.46 g, 64 %); m.p: [300 °C
(dimethyl formamide/ethanol 4:1); 1H NMR (300 MHz,
DMSO-d6): d = 2.16 (s, 3H, CH3), 4.0 (s, 2H, CH2, keto
tautomer), 6.62 (s, 2H, pyrazole NH2, D2O exchangeable),
7.49 (s, 2H, SO2NH2, D2O exchangeable), 7.80, 7.96 (2d,
J = 8.3 Hz, each 2H, benzenesulfonamide C3,5–H and
C2,6–H), 8.09 (s, 1H, pyrazole C3–H), 9.82 (s, 1H, OH, enol
tautomer) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 14.93 (CH3), 59.60 (CH2), 95.59 (pyrazole C4),
124.24, 127.49, 140.90, 141.43 (benzenesulfonamide-C),
142.43, 142.98 (pyrazole and pyrazolinone C3), 150.54
(pyrazole C5), 162.82, 164.69 (C=O) ppm; IR (KBr):
vꢀ = 3406, 3325, 3188 (OH, NH), 3071, 2985 (CH), 1670,
1650 (C=O), 1619, 1589 (C=N), 1544, 1495, 1438 (C=C),
1326, 1163 (SO2) cm-1; MS (70 eV): m/z (%) = 362 (M?,
78.0), 125 (100.0).
Ethyl 5-amino-1-(4-sulfamoylphenyl)-1H-pyrazole-4-car-
boxylate (2)
A mixture of 16.92 g ethyl ethoxymethylenecyanoacetate
(100 mmol), 22.37 g sulfanilamide hydrazine hydrochlo-
ride (100 mmol), and 8.2 g anhydrous sodium acetate
(100 mmol) in 50 cm3 ethanol was heated under reflux for
3 h. The reaction mixture was left overnight and the
separated solid product was filtered, washed with ethanol,
dried, and recrystallized from ethanol. White crystals
(70 %); m.p.: 231–232 °C (Ref. [21] 230 °C).
4-[5-Amino-4-[(3,5-dioxopyrazolidin-1-yl)carbonyl]-1H-
pyrazol-1-yl]benzenesulfonamide (5, C13H12N6O5S)
Grayish white crystals (0.5 g, 69 %); m.p: 259–260 °C
(dimethyl formamide/ethanol 2:1); 1H NMR (300 MHz,
DMSO-d6): d = 4.28 (s, 2H, CH2), 6.51 (s, 2H, pyrazole
NH2, D2O exchangeable), 7.45 (s, 2H, SO2NH2, D2O
exchangeable), 7.78 (d, J = 7.7 Hz, 2H, benzenesulfon-
amide C3,5–H), 7.92–7.95 (m, 3H, benzenesulfonamide
4-[5-Amino-4-(hydrazinocarbonyl)-1H-pyrazol-1-yl]ben-
zenesulfonamide (3)
A suspension of 6.2 g 2 (20 mmol) in 30 cm3 hydrazine
hydrate (80 %) was heated under reflux for 8 h. The
C
2,6–H and pyrazole C3–H), 9.17 (s, 1H, NH, D2O
exchangeable) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 20.65 (CH2), 95.88 (pyrazole C4), 123.23, 127.11,
139.14, 140.69 (benzenesulfonamide-C), 142.29 (pyrazole
123

H. M. A. Ashour et al.
C3), 150.12 (pyrazole C5), 163.50, 169.06, 174.90 (C=O)
ppm; IR (KBr): vꢀ = 3387, 3343, 3221 (NH), 3018 (CH),
1692 (C=O), 1623 (C=N), 1543, 1507, 1434 (C=C), 1327,
1164 (SO2) cm-1; MS (70 eV): m/z (%) = 364 (M?, 76.0),
247 (100.0).
1165 (SO2) cm-1; MS (70 eV): m/z (%) = 576 (M?, 84.0),
125 (100.0).
4-[5-Amino-4-[[4-[(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihy-
dro-1H-pyrazol-4-yl)azo]-3,5-dioxopyrazolidin-1-yl]car-
bonyl]-1H-pyrazol-1-yl]benzenesulfonamide
4-[5-Amino-4-[[4-[(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihy-
dro-1H-pyrazol-4-yl)azo]-3,5-dimethyl-1H-pyrazol-1-yl]-
carbonyl]-1H-pyrazol-1-yl]benzenesulfonamide
(8, C24H22N10O6S)
Orange crystals (0.71 g, 62 %); m.p.: 236–238 °C
(dimethyl formamide/ethanol 2:1); 1H NMR (400 MHz,
DMSO-d6): d = 2.42 (s, 1H, pyrazolidinone C4–H), 2.58
(s, 3H, dimethylpyrazolinone C–CH3), 3.19 (s, 3H,
dimethylpyrazolinone N–CH3), 7.15 (s, 2H, pyrazole
NH2, D2O exchangeable), 7.39–7.58 (m, 7H, phenyl-H
and SO2NH2), 7.79, 7.99 (2d, J = 8.4 Hz, each 2H,
benzenesulfonamide C3,5–H and C2,6–H), 8.32 (s, 1H,
pyrazole C3–H), 9.17 (s, 1H, NH, D2O exchangeable) ppm;
13C NMR (100 MHz, DMSO-d6): d = 31.33 (C–CH3),
36.46 (N–CH3), 84.24, 96.27, 109.46 (pyrazolidinone C4,
aminopyrazole C4, and dimethylpyrazolinone C4), 123.31,
123.94, 124.38, 127.45, 127.98, 129.72, 140.47, 141.13
(benzenesulfonamide-C and phenyl-C), 142.57 (aminopy-
razole C3), 151.35, 153.16 (aminopyrazole C5 and
dimethylpyrazolinone C5), 162.84, 163.16, 164.45, 165.50
(C=O) ppm; IR (KBr): vꢀ = 3376, 3333, 3246 (NH), 3016
(CH), 1692, 1646 (C=O), 1623 (C=N), 1547, 1516, 1435
(C=C), 1326, 1161 (SO2) cm-1; MS (70 eV): m/z
(%) = 578 (M?, 73.0), 125 (100.0).
(6, C26H26N10O4S)
Orange crystals (0.85, 74 %); m.p: 276–277 °C (dioxane);
1H NMR (300 MHz, DMSO-d6): d = 2.48, 2.62 (2 s, each
3H, pyrazole CH3), 2.80 (s, 3H, pyrazolinone C–CH3), 3.35
(s, 3H, pyrazolinone N–CH3), 6.92 (s, 2H, pyrazole NH2,
D2O exchangeable), 7.18–7.59 (m, 7H, phenyl-H and
SO2NH2), 7.80, 7.97 (2d, J = 8.0 Hz, each 2H, benzene-
sulfonamide C3,5–H and C2,6–H), 8.53 (s, 1H, pyrazole C3–
H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 11.25,
12.50, 31.25 (C–CH3), 36.25 (N–CH3), 96.32, 96.36,
109.70 (dimethylpyrazole C4, aminopyrazole C4, and
dimethylpyrazolinone C4), 123.64, 123.78, 124.33,
127.48, 127.56, 129.71, 140.0, 140.94 (benzenesulfon-
amide-C and phenyl-C), 142.82, 142.86 (aminopyrazole C3
and dimethylpyrazole C3), 150.0, 152.50, 153.75
(aminopyrazole
C5,
dimethylpyrazole
C5,
and
dimethylpyrazolinone C5), 162.70, 162.79 (C=O) ppm;
IR (KBr): vꢀ = 3325, 3289, 3175 (NH), 3073, 2975, 2931
(CH), 1669, 1641 (C=O), 1591 (C=N), 1537, 1493, 1428
(C=C), 1352, 1158 (SO2) cm-1; MS (70 eV): m/z
(%) = 574 (M?, 76.0), 247 (100.0).
General procedure for the synthesis of 4-[5-amino-4-
[(5-amino-4-substituted-1H-pyrazol-1-yl)carbonyl]-
1H-pyrazol-1-yl]benzenesulfonamides 9a, 9b
4-[5-Amino-4-[[4-[(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)azo]-3-methyl-5-oxo-4,5-dihydro-
1H-pyrazol-1-yl]carbonyl]-1H-pyrazol-1-yl]benzenesul-
fonamide (7, C25H24N10O5S)
A mixture of 0.59 g 2 (2 mmol) and ethoxymethylene
malononitrile or ethyl ethoxymethylenecyanoacetate
(2 mmol) in 10 cm3 dry DMF was heated under reflux for
4 h. The reaction mixture was concentrated under reduced
pressure and poured into ice-cold water. The obtained
precipitate was filtered, washed with ethanol, dried, and
crystallized from the proper solvent.
Reddish crystals (0.83 g, 72.3 %); m.p.: 266–267 °C
(dimethyl formamide/ethanol 4:1); 1H NMR (300 MHz,
DMSO-d6): d = 2.26 (s, 3H, methylpyrazolinone CH3),
2.44 (s, 1H, methylpyrazolinone C4–H), 2.54 (s, 3H,
dimethylpyrazolinone C–CH3), 3.18 (s, 3H, dimethylpyra-
zolinone N–CH3), 7.15 (s, 2H, pyrazole NH2, D2O
exchangeable), 7.36–7.57 (m, 7H, phenyl-H and SO2NH2),
7.79, 7.97 (2d, J = 8.4 Hz, each 2H, benzenesulfonamide
4-[5-Amino-4-[(5-amino-4-cyano-1H-pyrazol-1-yl)car-
bonyl]-1H-pyrazol-1-yl]benzenesulfonamide
(9a, C14H12N8O3S)
C
3,5–H and C2,6–H), 8.30 (s, 1H, pyrazole C3–H) ppm; 13
C
Grayish white crystals (0.5 g, 68 %); m.p.: [300 °C
(dimethyl formamide/water 2:1); 1H NMR (300 MHz,
DMSO-d6): d = 5.89 (s, 2H, NH2, D2O exchangeable),
6.70 (s, 2H, NH2, D2O exchangeable), 7.49 (s, 2H,
SO2NH2, D2O exchangeable), 8.0, 8.31 (2d, J = 9.2 Hz,
each 2H, benzenesulfonamide C3,5–H and C2,6–H), 8.48,
8.58 (2 s, each 1H, pyrazole C3–H) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 93.81, 94.12 (pyrazole C4),
112.30 (CN), 124.82, 127.95, 140.66, 143.13 (benzenesul-
fonamide-C), 143.74, 142.98 (pyrazole C3), 153.11, 154.36
(pyrazole C5), 163.33 (C=O) ppm; IR (KBr): vꢀ = 3337,
NMR (75 MHz, DMSO-d6): d = 12.14, 31.33 (C–CH3),
36.46 (N–CH3), 83.28 (methylpyrazolinone C4), 96.30,
109.63 (aminopyrazole C4 and dimethylpyrazolinone C4),
123.62, 123.82, 124.29, 127.64, 127.92, 129.78, 140.22,
140.89 (benzenesulfonamide-C and phenyl-C), 142.76,
142.88 (aminopyrazole C3 and methylpyrazolinone C3),
150.65, 153.84 (aminopyrazole C5 and dimethylpyrazoli-
none C5), 162.50, 162.70, 162.79 (C=O) ppm; IR (KBr):
vꢀ = 3337, 3325 (NH), 3075, 2975, 2900 (CH), 1695, 1631
(C=O), 1613, 1588 (C=N), 1546, 1525, 1498 (C=C), 1323,
123

Synthesis and pharmacological evaluation of new pyrazolyl benzenesulfonamides linked to…
3255 (NH), 3045 (CH), 2202 (CN), 1697 (C=O), 1632
(C=N), 1597, 1574, 1528, 1432 (C=C), 1335, 1163 (SO2)
cm-1; MS (70 eV): m/z (%) = 372 (M?, 100.0).
1362, 1154 (SO2) cm-1; MS (70 eV) m/z (%) = 418 (M?,
30.5), 416 (100.0).
Methyl 5-amino-1-[[5-amino-1-(4-sulfamoylphenyl)-1H-
pyrazol-4-yl]carbonyl]-3-methylsulfanyl-1H-pyrazole-4-
carboxylate (10b, C16H17N7O5S2)
Ethyl 5-amino-1-[[5-amino-1-(4-sulfamoylphenyl)-1H-
pyrazol-4-yl]carbonyl]-1H-pyrazole -4-carboxylate
(9b, C16H17N7O5S)
White crystals (0.63 g, 70 %); m.p.: 291–292 °C; 1H NMR
(300 MHz, DMSO-d6): d = 2.49, 3.74 (2 s, each 3H,
SCH3 and OCH3), 7.25 (s, 2H, NH2, D2O exchangeable),
7.52 (s, 2H, SO2NH2, D2O exchangeable), 7.57 (s, 2H,
NH2, D2O exchangeable), 7.79, 7.98 (2d, J = 8.7 Hz, each
2H, benzenesulfonamide C2,6 and C3,5–H), 8.52 (s, 1H,
pyrazole C3–H) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 13.11 (SCH3), 51.24 (OCH3), 91.83, 96.20 (pyrazole
C4), 124.32, 127.81, 140.43, 143.29 (benzenesulfonamide-
C), 143.50, 153.29 (pyrazole C3), 153.35, 155.16 (pyrazole
C5), 163.51, 163.77 (C=O) ppm; IR (KBr): vꢀ = 3411,
3343, 3297, 3208 (NH), 3096, 2960 (CH), 1679 (C=O),
1617 (C=N), 1515, 1436, 1400 (C=C), 1335, 1158 (SO2)
cm-1; MS (70 eV) m/z (%) = 451 (M?–CH3O, 0.88), 265
(100).
Beige crystals (0.53 g, 64 %); m.p.: 258–259 °C (dimethyl
formamide/ethanol 2:1); H NMR (300 MHz, DMSO-d6):
1
d = 1.27 (t, J = 6.9 Hz, 3H, CH3), 4.23 (q, J = 6.9 Hz,
2H, CH2), 7.29, 7.45 (2 s, each 2H, 2 NH2, D2O
exchangeable), 7.54 (s, 2H, SO2NH2, D2O exchangeable),
7.79 (d, J = 8.9 Hz, 2H, benzenesulfonamide C2,6–H),
7.85 (s, 1H, pyrazole C3–H), 7.98 (d, J = 8.9 Hz, 2H,
benzenesulfonamide C3,5–H), 8.50 (s, 1H, pyrazole C3–H)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 14.89 (CH3),
59.72 (CH2), 93.81, 96.17 (pyrazole C4), 124.43, 127.53,
140.32, 143.15 (benzenesulfonamide-C), 143.40, 143.68
(pyrazole C3), 153.37, 154.05 (pyrazole C5), 163.45,
164.45 (C=O) ppm; IR (KBr): vꢀ = 3378, 3334 (NH),
3056 (CH), 1688, 1667 (C=O), 1625 (C=N), 1527, 1486
(C=C), 1331, 1161 (SO2) cm-1; MS (70 eV): m/z
(%) = 374 (M?–C2H5O, 1.3), 266 (100 %).
General procedure for the synthesis of 4-[5-amino-4-
[(5-amino-3-aryl-1H-pyrazol-1-yl)carbonyl]-1H-
pyrazol-1-yl]benzenesulfonamides 11a, 11b
General procedure for the synthesis of 4-[5-amino-4-
[(5-amino-3-methylsulfanyl-4-substituted-1H-
pyrazol-1-yl)carbonyl]-1H-pyrazol-1-
To a suspension of 0.59 g 2 (2 mmol) in 20 cm3 ethanol/
acetic acid mixture (4:1), the selected phenacyl cyanide
(2 mmol) was added. The reaction mixture was heated
under reflux for 12 h during which a crystalline precipitate
separated out. The reaction mixture was cooled, filtered,
washed with ethanol, and recrystallized from dimethyl
formamide/ethanol (2:1).
yl]benzenesulfonamides 10a, 10b
A
mixture
of
0.59 g
2
(2 mmol)
or
and
[bis(methylthio)methylene]malononitrile
methyl
2-cyano-3,3-bis(methylthio)acrylate (2 mmol) in 10 cm3
dry DMF was heated under reflux for 4 h. The reaction
mixture was evaporated under reduced pressure and the
remaining residue was triturated with ethanol. The sepa-
rated solid product was filtered, washed with ethanol, dried,
and crystallized from dimethyl formamide/ethanol (3:1).
4-[5-Amino-4-[(5-amino-3-phenyl-1H-pyrazol-1-yl)car-
bonyl]-1H-pyrazol-1-yl]benzenesulfonamide
(11a, C19H17N7O3S)
1
White fine crystals (0.55 g, 65 %); m.p.: 258–259 °C; H
NMR (300 MHz, DMSO-d6): d = 5.87 (s, 1H, pyrazole
C4–H), 6.78, 7.21 (2 s, each 2H, 2 NH2, D2O exchange-
able), 7.38–7.47 (m, 3H, phenyl C3,4,5–H), 7.50 (s, 2H,
SO2NH2, D2O exchangeable), 7.82 (d, J = 8.4 Hz, 2H,
benzenesulfonamide C3,5–H), 7.87 (d, J = 7.8 Hz, 2H,
phenyl C2,6–H), 8.0 (d, J = 8.4 Hz, 2H, benzenesulfon-
amide C2,6–H), 8.74 (s, 1H, pyrazole C3–H) ppm; 13C
NMR (75 MHz, DMSO-d6): d = 84.23, 94.10 (pyrazole
C4), 123.83, 127.06, 127.58, 128.84, 129.35, 133.36,
140.09, 142.76 (benzenesulfonamide-C and phenyl-C),
143.89, 152.12 (pyrazole C3), 152.76, 152.98 (pyrazole
C5), 164.19 (C=O) ppm; IR (KBr): vꢀ = 3461, 3440, 3335,
3224 (NH), 3060 (CH), 1649 (C=O), 1614 (C=N), 1531,
1494, 1436 (C=C), 1323, 1159 (SO2) cm-1; MS (70 eV) m/
z (%) = 423 (M?, 65.5), 366 (100).
4-[5-Amino-4-[(5-amino-4-cyano-3-methylsulfanyl-1H-
pyrazol-1-yl)carbonyl]-1H-pyrazol-1-yl]benzenesulfon-
amide (10a, C15H14N8O3S2)
White crystals (0.58 g, 70 %); m.p.: 283–284 °C; 1H NMR
(300 MHz, DMSO-d6): d = 2.61 (s, 3H, SCH3), 7.23 (s,
2H, NH2, D2O exchangeable), 7.50 (s, 2H, SO2NH2, D2O
exchangeable), 7.78, 7.96 (2d, J = 8.7 Hz, each 2H,
benzenesulfonamide C3,5 and C2,6–H), 8.07 (s, 2H, NH2,
D2O exchangeable), 8.46 (s, 1H, pyrazole C3–H) ppm; 13
C
NMR (75 MHz, DMSO-d6): d = 13.20 (CH3), 72.44,
95.99 (pyrazole C4), 113.50 (CN), 124.41, 127.55,
140.35, 143.38 (benzenesulfonamide-C), 143.47, 153.13
(pyrazole C3), 153.39, 156.65 (pyrazole C5), 163.36 (C=O)
ppm; IR (KBr): vꢀ = 3439, 3311 (NH), 2922 (CH), 2203
(CN), 1659 (C=O), 1615 (C=N), 1529, 1502, 1467 (C=C),
123

H. M. A. Ashour et al.
4-[5-Amino-4-[[5-amino-3-(4-chlorophenyl)-1H-pyrazol-
1-yl]carbonyl]-1H-pyrazol-1-yl]benzenesulfonamide
(11b, C19H16ClN7O3S)
2H, SO2NH2, D2O exchangeable), 7.78, 7.95 (2d,
J = 7.8 Hz, each 2H, benzenesulfonamide C3,5–H and
C2,6–H), 8.12 (s, 1H, pyrazole C3–H), 10.26, 11.66 (2 s,
Beige crystals (0.55 g, 60 %); m.p.: 263–264 °C; 1H NMR
(300 MHz, DMSO-d6): d = 5.88 (s, 1H, pyrazole C4–H),
6.84, 7.24 (2 s, each 2H, 2 NH2, D2O exchangeable), 7.51
(d, J = 7.7 Hz, 2H, chlorophenyl C3, 5–H), 7.54 (s, 2H,
SO2NH2, D2O exchangeable), 7.83 (d, J = 7.7 Hz, 2H,
benzenesulfonamide C3,5–H), 7.90 (d, J = 7.7 Hz, 2H,
chlorophenyl C2,6–H), 8.01 (d, J = 7.7 Hz, 2H, benzene-
sulfonamide C2,6–H), 8.74 (s, 1H, pyrazole C3–H) ppm;
13C NMR (75 MHz, DMSO-d6): d = 84.59, 96.13 (pyra-
zole C4), 123.83, 126.97, 127.09, 127.60, 128.77, 131.25,
140.09, 142.76 (benzenesulfonamide-C and chlorophenyl-
C), 143.81, 152.12 (pyrazole C3), 152.78, 152.93 (pyrazole
C5), 164.19 (C=O) ppm; IR (KBr): vꢀ = 3386, 3357, 3308,
3273 (NH), 3062 (CH), 1651 (C=O), 1617 (C=N), 1574,
1529, 1495 (C=C), 1332, 1162 (SO2) cm-1; MS (70 eV)
m/z (%) = 459 ([M ? 2]?, 27.6), 457 (M?, 70.8), 425
(100.0).
each 1H, 2 NH, D2O exchangeable) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 44.42 (thiazolidinone C5),
105.32 (pyrazole C4), 132.50, 132.75, 136.43, 148.50
(benzenesulfonamide-C), 149.96 (pyrazole C3), 151.65
(pyrazole C5), 159.58 (thiazolidinone C2), 166.60, 171.77
(C=O) ppm; IR (KBr): vꢀ = 3453, 3356, 3275, 3161 (NH),
3009 (CH), 1702 (C=O), 1626 (C=N), 1538, 1474, 1429
(C=C), 1327, 1160 (SO2) cm-1; MS (70 eV) m/z
(%) = 395 (M?, 70.3), 266 (100.0).
General procedure for the synthesis of 4-[5-amino-4-
[[(2E)-2-[(5Z)-5-substituted-4-oxo-1,3-thiazolidin-2-
ylidene]hydrazinyl]carbonyl]-1H-pyrazol-1-
yl]benzenesulfonamides 14 and 15
A mixture of 0.79 g thiazolidinone 13 (2 mmol), the
selected aryl aldehyde (2.2 mmol), and piperidine
(2 mmol) in 10 cm3 of dry DMF/absolute EtOH mixture
(4:1) was heated under reflux for 12 h. The reaction mix-
ture was allowed to attain room temperature and the
separated product was filtered, washed with ethanol, dried,
and crystallized from dimethyl formamide/ethanol (2:1).
4-[5-Amino-4-[[2-(2-chloroacetyl)hydrazinyl]carbonyl]-
1H-pyrazol-1-yl]benzenesulfonamide
(12, C12H13ClN6O4S)
To a stirred ice-cooled solution of 2.96 g 2 (10 mmol) in
10 cm3 dry DMF, 1.25 g chloroacetyl chloride (0.88 cm3,
11 mmol) was added dropwise. The reaction mixture was
left stirred at room temperature overnight and then poured
onto crushed ice. The formed precipitate was filtered, dried,
and crystallized from ethanol. White crystals (2.9 g, 78 %);
m.p.: 144–145 °C; 1H NMR (300 MHz, DMSO-d6):
d = 4.18 (s, 2H, CH2), 6.62 (s, 2H, NH2, D2O exchange-
able), 7.49 (s, 2H, SO2NH2, D2O exchangeable), 7.78, 7.95
(2d, J = 8.9 Hz, each 2H, benzenesulfonamide C3,5–H and
4-[5-Amino-4-[[(2E)-2-((5Z)-5-benzylidene-4-oxo-1,3-thi-
azolidin-2-ylidene)hydrazinyl]carbonyl]-1H-pyrazol-1-
yl]benzenesulfonamide (14a, C20H17N7O4S2)
1
Yellow solid (0.5 g, 52 %); m.p.: 291–292 °C; H NMR
(300 MHz, DMSO-d6): d = 6.68 (s, 2H, NH2, D2O
exchangeable), 7.42–7.56 (m, 5H, phenyl C3,4,5–H and
SO2NH2), 7.61 (d, J = 7.5 Hz, 2H, phenyl C2,6–H), 7.65 (s,
1H, = CH), 7.80, 7.96 (2d, J = 8.7 Hz, each 2H, benzene-
sulfonamide C3,5–H and C2,6–H), 8.09 (s, 1H, pyrazole C3–
H), 10.58, 12.31 (2 s, each 1H, 2 NH, D2O exchangeable)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 104.45 (pyra-
zole C4), 119.54 (thiazolidinone C5), 126.23, 127.40,
129.13, 131.65, 132.43, 136.38, 137.16, 148.29 (phenyl-C
and benzenesulfonamide-C), 149.22 (pyrazole C3), 149.43
(=CH), 151.27 (pyrazole C5), 158.14 (thiazolidinone C2),
166.45, 171.26 (C=O) ppm; IR (KBr): vꢀ = 3410, 3328,
3248 (NH), 3010, 2969 (CH), 1686 (C=O), 1645 (C=N),
1599, 1540 (C=C), 1332, 1158 (SO2) cm-1; MS (70 eV) m/
z (%) = 483 (M?, 94.2), 413 (100.0).
C2,6–H), 8.03 (s, 1H, pyrazole C3–H), 9.97, 10.20 (2 s,
each 1H, 2 NH, D2O exchangeable) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 45.7 (CH2), 94.62 (pyrazole
C4), 123.78, 127.63, 140.94, 141.62 (benzenesulfonamide-
C), 142.55 (pyrazole C3), 150.75 (pyrazole C5), 162.80,
163.45 (C=O) ppm; IR (KBr): vꢀ = 3358, 3209 (NH), 3020
(CH), 1692 (C=O), 1623 (C=N), 1575, 1535, 1500, 1431
(C=C), 1328, 1163 (SO2) cm-1
.
4-[5-Amino-4-[[(2Z)-2-(4-oxo-1,3-thiazolidin-2-yli-
dene)hydrazinyl]carbonyl]-1H-pyrazol-1-yl]benzenesul-
fonamide (13, C13H13N7O4S2)
A mixture of 3.73 g 12 (10 mmol) and 1.52 g ammonium
thiocyanate (20 mmol) in 20 cm3 ethanol (95 %) was
refluxed for 2 h. The reaction mixture was concentrated
and diluted with ice-cold water. The obtained precipitate
was filtered, dried, and crystallized from dimethyl for-
mamide/water (3:1). White crystals (3.36 g, 85 %); m.p.:
251–252 °C; 1H NMR (300 MHz, DMSO-d6): d = 3.94 (s,
2H, CH2), 6.63 (s, 2H, NH2, D2O exchangeable), 7.46 (s,
4-[5-Amino-4-[[(2E)-2-[(5Z)-5-(4-chlorobenzylidene)-4-
oxo-1,3-thiazolidin-2-ylidene]hydrazinyl]carbonyl]-1H-
pyrazol-1-yl]benzenesulfonamide
(14b, C20H16ClN7O4S2)
1
Yellow solid (0.5 g, 50 %); m.p: 298–299 °C; H NMR
(300 MHz, DMSO-d6): d = 6.69 (s, 2H, NH2, D2O
exchangeable), 7.48–7.57 (m, 6H, chlorophenyl-H and
SO2NH2), 7.76 (s, 1H, =CH), 8.15, 8.42 (2d, J = 9.0 Hz,
123

Synthesis and pharmacological evaluation of new pyrazolyl benzenesulfonamides linked to…
each 2H, benzene sulfonamide C3,5–H and C2,6–H), 8.49 (s,
1H, pyrazole C3–H), 10.53, 12.19 (2 s, each 1H, 2 NH,
D2O exchangeable) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 104.84 (pyrazole C4), 118.13 (thiazolidinone C5),
128.80, 129.43, 132.43, 133.15, 135.33, 136.12, 137.46,
147.84 (chlorophenyl-C and benzenesulfonamide-C),
148.64 (pyrazole C3), 149.22 (=CH), 150.69 (pyrazole
C5), 158.28 (thiazolidinone C2), 166.15, 171.23 (C=O)
ppm; IR (KBr): vꢀ = 3347, 3216 (NH), 3097, 2927 (CH),
1695 (C=O), 1619 (C=N), 1596, 1546, 1495 (C=C), 1334,
1160 (SO2) cm-1; MS (70 eV) m/z (%) = 519 ([M ? 2]?,
31.5), 517 (M?, 91.2), 413 (203.0).
Anti-inflammatory (AI) activity
Formalin-induced paw edema bioassay
Male albino rats weighing 180–200 g were used throughout
the assay. They were kept in the animal house under standard
conditions of light and temperature with free access to food
and water. The animals were randomly divided into groups
each of six rats. One group of six rats was kept as a control
and another group received the standard drug diclofenac Na
(at a dose of 20 mg/kg body weight po). A solution of for-
malin (2 %, 0.1 cm3) was injected into the subplantar region
of the left hind paw under light ether anesthesia 1 h after oral
administration (po) of the test compound (at a dose level of
20 mg/kg body weight). The paw volume (cm3) was mea-
sured by means of water plethysmometer and remeasured
again 1, 2, and 4 h after administration of formalin. The
edema was expressed as an increase in the volume of paw,
and the percentage of edema inhibition for each rat and each
group was obtained as follows:
4-[5-Amino-4-[[(2E)-2-[(5Z)-5-[(1,3-diphenyl-1H-pyrazol-
4-yl)methylene]-4-oxo-1,3-thiazolidin-2-ylidene]hy-
drazinyl]carbonyl]-1H-pyrazol-1-yl]benzenesulfonamide
(15a, C29H23N9O4S2)
1
Yellow solid (0.7 g, 56 %); m.p.: 301–302 °C; H NMR
(300 MHz, DMSO-d6): d = 6.68 (s, 2H, NH2, D2O
exchangeable), 7.40–7.62 (m, 11H, N-phenyl–H, phenyl
% Inhibition ¼ ðVt À V0ÞcontrolÀ ðVt À V0Þtested compound
ðVt À V0Þcontrol 100;
=
C
3,4,5–H, SO2NH2 and =CH), 7.66 (d, J = 8.6 Hz, 2H,
benzenesulfonamide C3,5–H), 7.96 (d, J = 8.7 Hz, 2H,
phenyl C2,6–H), 7.99 (d, J = 8.6 Hz, 2H, benzenesulfon-
amide C2,6–H), 8.10 (s, 1H, pyrazole C3–H), 8.73 (s, 1H,
pyrazole C5–H), 10.70, 12.54 (2 s, each 1H, 2 NH, D2O
exchangeable) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 94.65, 115.76 (pyrazole C4), 119.75 (thiazolidinone
C5), 120.48, 123.32, 123.62, 124.13, 127.78, 128.16,
129.66, 128.58, 130.45, 130.59, 130.64, 134.42, 139.85,
141.64, 142.79, 150.16, 152.18, 152.48 (Ar–C), 162.76,
172.69 (C=O) ppm; IR (KBr): vꢀ = 3442, 3339, 3230 (NH),
3099, 3059, 2948 (CH), 1690, 1660 (C=O), 1616 (C=N),
1545, 1501, 1435 (C=C), 1336, 1163 (SO2) cm-1; MS
(70 eV) m/z (%) = 625 (M?, 45), 88 (100.0).
where Vt is the volume of edema at a specific time interval
and V0 is the volume of edema at zero time interval.
Determination of effective dose (ED50)
50
The selected compounds were further tested at 5, 10, 20,
40, and 50 mg/kg body weight and the ED50 was deter-
mined by measuring the inhibition of edema volume 4 h
after formalin injection.
Analgesic activity
Analgesic activity was determined using the tail with-
drawal response to immersion of rat tail in water at 55 °C
according to the procedure described by Janssen et al. [28].
Male albino rats weighing 120–150 g were used through-
out this assay. They were kept in the animal house under
standard conditions of light and temperature with free
access to food and water. The animals were randomly
divided into groups each of six rats. One group of six rats
was kept as a control and another group received the
standard drug diclofenac sodium (at a dose of 20 mg/kg
body weight po). The tested compounds were adminis-
trated orally at a dose of 20 mg/kg. The recorded values
were the average of six administrations SE and the per-
centage increase of the reaction time (after 1–4 h time
intervals) was calculated in comparison with the basal
values according to the following equation:
4-[5-Amino-4-[[(2E)-2-[(5Z)-5-[[3-(4-chlorophenyl)-1-
phenyl-1H-pyrazol-4-yl]methylene]-4-oxo-1,3-thiazolidin-
2-ylidene]hydrazinyl]carbonyl]-1H-pyrazol-1-yl]benzene-
sulfonamide (15b, C29H22ClN9O4S2)
1
Yellow solid (0.66 g, 50 %); m.p.: 296–297 °C; H NMR
(400 MHz, DMSO-d6): d = 6.68 (s, 2H, NH2, D2O
exchangeable), 7.39–7.98 (m, 16H, N-phenyl–H, chloro-
phenyl–H, SO2NH2, = CH and benzenesulfonamide-H),
8.11 (s, 1H, pyrazole C3–H), 8.72 (s, 1H, pyrazole C5–H),
10.53, 12.40 (2 s, each 1H, 2 NH, D2O exchangeable) ppm;
13C NMR (75 MHz, DMSO-d6): d = 96.55, 116.28 (pyra-
zole C4), 119.72 (thiazolidinone C5), 119.92, 123.46,
123.52, 127.47, 127.90, 128.45, 129.24, 129.50, 130.08,
130.74, 130.79, 134.27, 139.49, 141.02, 142.76, 150.76,
152.31, 152.42 (Ar–C), 162.78, 172.9 (C=O) ppm; IR
(KBr): vꢀ = 3393, 3240 (NH), 3050, 2922 (CH), 1696
(C=O), 1613 (C=N), 1528, 1504, 1433 (C=C), 1331, 1158
(SO2) cm-1; MS (70 eV) m/z (%) = 661 ([M ? 2]?, 16),
659 (M?, 49.4), 88 (100.0).
% increase of the reaction time
¼ ðTt=T0Þtested compoundÀ 1 Â 100;
123

H. M. A. Ashour et al.
where Tt is the reaction time at a specific time interval and
T0 is the reaction time at zero time interval.
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123