Discovery of novel urea-diarylpyrazole hybrids as dual COX-2/sEH inhibitors with improved anti-inflammatory activity and highly reduced cardiovascular risks

Article abstract of DOI:10.1016/j.ejmech.2020.112662

Herein we describe our efforts to develop novel anti-inflammatory/analgesic agents devoid of known cardiovascular drawbacks. In doing so, two 1,5-diarylpyrazole series of urea linked (9a-f) and amide linked (11a-f) compounds were synthesized and evaluated

Full text of DOI:10.1016/j.ejmech.2020.112662

Journal Pre-proof
Discovery of novel urea-diarylpyrazole hybrids as dual COX-2/sEH inhibitors with
improved anti-inflammatory activity and highly reduced cardiovascular risks
Ahmed H. Abdelazeem, Asmaa G. Safi El-Din, Maha M. Abdel-Fattah, Noha H. Amin,
Samir M. El-Moghazy, Mohammed T. El-Saadi
PII:
S0223-5234(20)30634-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112662
EJMECH 112662
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 June 2020
Revised Date: 10 July 2020
Accepted Date: 11 July 2020
Please cite this article as: A.H. Abdelazeem, A.G. Safi El-Din, M.M. Abdel-Fattah, N.H. Amin, S.M. El-
Moghazy, M.T. El-Saadi, Discovery Of Novel Urea-Diarylpyrazole Hybrids As Dual COX-2/sEH Inhibitors
With Improved Anti-Inflammatory Activity And Highly Reduced Cardiovascular Risks, European Journal
of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112662.
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Discovery Of Novel Urea-Diarylpyrazole Hybrids As Dual COX-
2/sEH Inhibitors With Improved Anti-Inflammatory Activity And
Highly Reduced Cardiovascular Risks
Ahmed H. Abdelazeem1,*; Asmaa G. Safi El-Din1; Maha M. Abdel-Fattah2; Noha H.
Amin1; Samir M. El-Moghazy3*; Mohammed T. El-Saadi1, 4
1Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt;
2Department of pharmacology and Toxicology, Faculty of pharmacy, Beni-Suef University, Beni-Suef 62514,
Egypt; 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo 11562,
Egypt; 4Department of Medicinal Chemistry, Faculty of Pharmacy, Sinai University, Qantara, Egypt.
*To whom correspondence should be addressed:
Ahmed H. Abdelazeem, Ph.D. Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-
Suef University, Beni-Suef 62514, Egypt.
E-mail address: ahmed.abdelazeem@pharm.bsu.edu.eg
Samir M. El-Moghazy, Ph.D. Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Cairo University, Kasr-El-Eini Street, 11562 Cairo, Egypt.
E-mail address: samirelmoghazy@gmail.com
1

Graphical Abstract:
ABSTRACT
:
Herein we describe our efforts to develop novel anti-inflammatory/analgesic agents devoid of known
cardiovascular drawbacks. In doing so, two 1,5-diarylpyrazole series of urea linked (9a-f) and amide
linked (11a-f) compounds were synthesized and evaluated in vitro as dual COX-2/sEH inhibitors
using recombinant enzyme assays. The in vivo anti-inflammatory and analgesic activities were then
examined using reported animal models. Compounds 9b and 9c showed the highest inhibitory
activities against both COX-2 and sEH (IC50 of COX-2 = 1.85 and 1.24 µM; sEH = 0.55 and 0.40
nM, respectively), besides showing the best activity as anti-inflammatory agents. Interestingly, the
cardiovascular profile of the two compounds 9b and 9c was evaluated through measuring some
biochemical parameters such as prostacyclin (PGI2), lactate dehydrogenase (LDH), troponin-1 (Tn-1),
tumor necrosis factor- α (TNF-α), creatine kinase-M (CK-M) and reduced glutathione (GSH) in
addition to a histo-pathological study to investigate the changes in the heart muscle. The results
confirmed that compounds 9b and 9c have a more favorable cardio-profile than celecoxib with much
less cardiovascular risks associated with the common selective COX-2 inhibitors. Finally, the current
work provided a promising approach that can be optimized to serve as a lead project to overcome the
cardiovascular toxicity related to the traditional selective COX-2 inhibitors.
Keywords: 1,5-Diarylpyrazole; Dual COX-2/sEH; sEH inhibitor; Cardiotoxicity, Anti-inflammatory.
2

1. Introduction:
For many years, the arachidonic acid (AA) cascade mediators have been focused on the therapy of
several medical conditions including inflammation, pain, cancer, asthma and cardiovascular disorders
[1–8]. It is well established that the primer step of the AA cascade is its release from the cellular
membrane under the effect of the phospholipase A2 (PLA2) [4,5,9]. In consecutive steps, AA is bio-
converted by several specific oxygenases to pro-inflammatory, pro-resolving in addition to anti-
inflammatory mediators [10,11]. The inhibition of these specific oxygenases blocks their output
mediators release, hence they can be utilized in many kinds of pharmaceutical researches and
conditions [12]. For instance, cyclooxygenase inhibitors (COX pathway), are broadly used in the
treatment of inflammatory diseases, exemplified by celecoxib, rofecoxib and valdecoxib [5,9,13]. On
the other hand, zileuton, the drug of choice for allergy treatment, acts through the inhibition of
lipoxygenase (LOX pathway; the second pathway of arachidonic acid cascade metabolism) as shown
in Fig. 1 [9,13,14].
H2NO2S
H2NO2S
H3CO2S
N
N
CF3
O
O
N
O
H3C
Rofecoxib
Celecoxib
Validoxib
Cl
OH
O
H2N
S
O
N OH
N
Zileuton
Licofelone
Fig. 1. Some known inhibitors of COX-2 and 5-LOX enzymes
Traditional non-steroidal anti-inflammatory drugs (NSAIDs) were considered the first pivot of
inflammation treatment due to its ability to put down prostaglandins (PGs), the prominent prostanoids
associated with several biological and pathological functions [15]. Unfortunately, their long-term use
3

was well-known to be accompanied by severe gastrointestinal toxicity as a consequence of their
COX-1 inhibition, the enzyme responsible for generation of the gastro-protective prostaglandin E2
(PGE2) [16]. Accordingly, improvement of selective COX-2 inhibitors was considered as a promising
approach that circumvents NSAIDs-gastrointestinal injury adverse effects. However, the appearance
of a novel side adverse effect, cardio-toxicity, lead to the rebound of some selective COX-2 inhibitors
from clinical fields [17]. The incidence of thrombotic cardiovascular disorders caused by COX-2
inhibitors were primarily due to the imbalance in the ratio of the thromboxane A2 (TX A2;
vasoconstrictor/prothrombotic agent via action thromboxane-A synthase) to prostacyclin (PGI2;
vasodilator/anti-aggregative agents synthesized via the action of COX-2) levels [18,19]. This serious
episode had a resounding effect which proved the need for developing new selective COX-2
inhibitors with a better cardiovascular profile. Recently, great attention has been devoted to the third
arachidonic acid metabolic pathway (cytochrome P450) which involves release of
vasoconstrictive/pro-inflammatory 20-hydroxyeicosatetranoic acid (20-HETE) and epoxy-
eicosatrienoic acids (EETs) [20,21] as shown in Fig. 2.
Fig. 2. The arachidonic acid metabolism pathways
4

EETs are known as effective contributors to modulate numerous homeostatic biological process
including: inflammation [22,23], pain [24,25], vascular reactivity [26], blood pressure [27] as well as
cardiovascular protective features [28,29]. It was reported that the cardioprotective activity of EETs
are exhibited by several mechanisms including vasodilation of coronary vessels via stimulation of
endothelial calcium-activated potassium channel (Kca) [30]. Moreover, EETs showed pro-
angiogenic properties that is associated with cardioprotection in chronic phases through the EET-
peroxisome proliferator activated receptor γ (PPARγ) pathway [31]. However, EETs are metabolized
into pro-inflammatory dihydroxy eicosatrienoic acids (DHETs) by the action of soluble epoxide
hydrolase enzyme (sEH), Fig. 2 [32].
Accordingly, the inhibition of sEH has been proposed as a novel therapeutic approach for treatment
of numerous biological disorders including pain, inflammation, hypertension and cardiovascular
disorders depending on its ability for preserving the advantageous effects of EETs through enhancing
their levels in the plasma and biological tissues [33–37]. Despite the various developed sEH
inhibitors (e.g. AUDA, CDU, AUCB and TPAU), no sEH inhibitor has been presented to the market
yet. Only some hopeful sEH inhibitor candidates are subjected now to clinical trials such as
GSK2256294A in Phase-I and AR9281 in Phase-II against chronic obstructive pulmonary disease
(COPD) and hypertension[38–40], as shown in Fig. 3.
Fig. 3. Some reported sEH inhibitors with high potency in nanomolar range
5

In particular, hybridization of inhibitors of COX-2 and sEH enzymes has been proposed for the
development of a harmonious target as an anti-inflammatory agent by poly-pharmacology technique.
The amalgamation of selective COX-2 inhibitor and sEH inhibitor has been proved to act as a dual
synergistic effect that caused reduction of both inflammation and cardiovascular risks [41]. This
hypothesis was illustrated by several mechanisms including ability of COX-2/sEH combination to
decrease PGE2 without any change in PGI2/TXA2 ratio due to up-regulation of PGI2 synthases, the
enzyme responsible for producing PGI2 [41,42]. Subsequently, COX-2/sEHI combination will allow
to reduce COX-2 dose and concomitant dose-dependent cardiovascular adverse effects [43].
Based on the aforementioned studies, it was conceptualized that the development of novel dual
inhibitors of COX-2 and sEH would be a promising approach to resuscitate COX-2 inhibitors with an
overall successful outcome of both anti-inflammatory treatment for ulcer patients and profound safety
margins against cardiovascular risks.
Rational design:
One of the most important drug design approaches used to reduce drug toxicity and improve
biological activity is multi-target-directed ligand (MTDL) technique carried out by the covalent
conjugation of various active moieties (pharmacophores) having different mechanisms of actions, yet
with precise pharmacophoric selection for each individual target. Based on this concept, the design of
our newly suggested COX-2/sEH dual inhibitors depends on finding two pharmacophores to hit the
both targets concurrently with maintaining good pharmacodynamic and pharmacokinetic profiles.
Celecoxib- and SC-558-based pharmacophore was placed as the first part in our strategy for targeting
COX-2 enzyme since it contains all the required COX-2 pharmacophoric moieties including Y-
shaped structure (diaryl-heterocycles) with a sulfonamide group at the p-position of one of the two
phenyl rings. Moreover, the presence of adjacent five-membered heterocyclic core, a pyrazole ring,
which has been critical for high activity.
6

Exploring the sEH pharmacophoric moieties to build the second part in our newly suggested dual
ligands was extracted from the study of critical interactions and the positioning of known selective
sEH inhibitors inside the catalytic domain of its 3D protein structure. Consequently, this investigation
revealed that the substituted urea or amide moieties were found to be essential chemical entities
involved in these interactions and important for the whole activity [40]. This was clear in several
reported sEH inhibitors such as AR9281, AUDA and GSK2256294. Additionally, some other
substituents including trifluoromethyl aniline, cyclohexyl, adamantyl, piperidine and long chain dual
amine/fatty acid have been pointed as secondary pharmacophoric moieties that significantly
contribute in the sEH potency [40]. Taking these bullets into our consideration, we have combined
the structural features of both COX-2 and sEH pharmacophores into one hybrid scaffold as depicted
in Fig. 4.
7

Fig. 4. The design strategy of our newly designed dual COX-2/sEH scaffold
2. Results and discussion:
2.1. Chemistry:
In the present study, the novel pyrazole-based derivatives were synthesized as represented in Scheme
1. The β-diketone 2 was prepared by condensation of acetophenone 1 and diethyl oxalate (Claisen
Condensation) as reported [44]. The (4-aminosulfonyl) phenyl hydrazine hydrochloride (4) was
obtained by diazotization of sulfanilamide 3, followed by reduction of the diazonium salt with tin (II)
8

chloride [45]. The regio-selective cyclization of β-diketone 2 with hydrochloride salt of phenyl
hydrazine 4 in ethanol to afford the 1,5-diarylpyazole compound 5 according to Knorr pyrazole
synthesis method was adopted [46]. The hydrazide intermediate 6 was furnished by reacting 1, 5-
diarylpyrazol ester 5 with hydrazine hydrate under reflux as reported [47].
Scheme 1:
Scheme 1. The synthesis of compound 6. Reagents and reaction conditions: a) Diethyl oxalate,
ᴏ
EtO-Na+, EtOH, rt, overnight; b) i. NaNO2/ HCl, 0-5 C, ii. SnCl2/ HCl, overnight, refrigerator; c)
EtOH, reflux, 12 h; d) NH2NH2.H2O, EtOH, reflux at 80 ᴏC, 6 h (65%).
The acyl azide 7 was prepared by the reaction of hydrazide 6 with in situ generated nitrous acid, then
simultaneously was undergone Curtius rearrangement as outlined in Scheme 2. The Curtius
rearrangement allowed the intra-molecular transformation of acyl azide 7 into isocyanate intermediate
8 through reflux in toluene. Consecutively, the isocyanate was treated in situ with aliphatic primary,
secondary amine or amino acids in the presence of pyridine to obtain derivatives of urea. The
proposed chemical structure of these derivatives was confirmed by their spectral data, where 1H NMR
of compound 9a was confirmed by the appearance of two single peaks of two NH at δ 9.00 and 6.69
13
ppm, in addition to the characteristic peaks of cyclohexyl at range δ 1.17-3.55 ppm. C NMR
9

spectrum showed signals at δ 22.99, 23.77, 33.17, and 48.17 ppm assigned to the characteristic
cyclohexyl carbons.
Scheme 2:
Scheme 2. The synthesis of compounds 9a-9f. Reagents and reaction conditions: a)
ᴏ
NaNO2/ HCl, 0-5 C; b) Toluene, reflux at 110-115 C, 0.5h; c) appropriate amine,
pyridine, reflux, 24 h (51-63%).
Regarding the amide derivatives 11a-f, they were synthesized through the formation of the
corresponding acyl chlorides. The acid of pyrazole 10 was synthesized through ester saponification
by heating with KOH in methanol [48] followed by treating with thionyl chloride under reflux in the
presence of drops of dimethylformamide for 3h. A simple condensation reaction was undergone
between the reactive intermediates acyl chlorides and different amines to afford the target 1, 5-
disubstituted pyrazol-3-carboxamide 11a-f as described in Scheme 3. The postulated compounds
10

were confirmed by their IR, NMR and mass spectral data. Evidence for the formation of compounds
1
11a-f was supported by H NMR spectrum and revealed the characteristic peaks of the attached
1
amine at their specific signals. H NMR of compound 11c showed the characteristic peaks of
triflouromethyl aniline at δ 8.01-7.24 ppm in addition of another peak at δ 10.16 ppm attributed to the
13
amide NH group where it disappeared upon deuteration. Also, C NMR revealed the peak of amide
at 160.53 ppm.
Scheme 3:
Scheme 3. The synthesis of compounds 11a-11f.Reagents and reaction conditions: a)
ᴏ
KOH, MeOH, reflux at 65 C, 12 h; b) i. SOCl2, DMF, reflux, 3h ii) appropriate amine,
TEA, CH2Cl2, rt, overnight (45-85%).
2.2. Pharmacology:
2.2.1. In vitro COXs/sEH assays:
Evaluation of all synthesized compounds for their in vitro inhibition of human COX-1/COX-2
enzymes using enzyme immunoassay (EIA) kits and human sEH enzyme using a cell-based assay kit
was carried out by measuring IC50 (The concentration causing 50% enzyme inhibition). Moreover, in
case of in vitro COX-1/COX-2 assay, the selectivity of these compounds is another efficacy index,
where it could be estimated as COX-1 IC50/COX-2 IC50. The obtained results of both IC50 and
selectivity were listed as shown in Table 1. The tested compounds results showed good COX-2
inhibitory activity (IC50 = 1.24-4.12 μM) and selectivity index (SI) values between 2.85 and 7.03
11

compared to celecoxib (IC50 = 0.95 μM; SI = 6.44) as selective COX-2 inhibitor reference. On the
other hand, all the compounds showed moderate inhibitory activity against sEH with IC50 range 0.98–
4.00 nM comparing to AUDA (IC50 = 0.49 nM) as a sEH inhibitor reference except two compounds
9b and 9c which elicited good sEH inhibition with IC50 of 0.55 and 0.40 nM respectively. Obviously,
the results revealed that the compounds possessing a urea linker (9a-f) were more potent as selective
COX-2 inhibitors (IC50 = 1.24-2.89 μM range) and sEH inhibitors (IC50 = 0.40-4.01 nM)
corresponding to amide linker as shown in compounds 11a-f (COX-2 IC50 = 2.05-4.12 μM range;
sEH IC50 = 1.15-2.38 nM). Moreover, compounds 9a-c showed the highest selectivity index among
the tested compounds towards COX-2 (in range of 5.23-7.03) and the highest potency against sEH
with IC50 of 0.98-0.44 nM, in order of 9c > 9b > 9a. Obviously, 4-(1-phenyl-3-(3-(4-
(trifluoromethyl)phenyl) ureido)-1H-pyrazol-5-yl)benzenesulfonamide (PTPUP) 9c, which possessed
a p-triflouromethyl anilinyl moiety directly attached to urea linker, was the most potent dual COX-
2/sEH inhibitor with (COX-2, IC50 = 1.24 μM; SI = 7.03; sEH, IC50 = 0.40 nM).
Table 1: In vitro COX-1\COX-2 and sEH inhibition results of the newly synthesized compounds 9a-
f, 11a-f, celecoxib and AUDA as a reference drug.
COX Inhibition (IC50 µM)
Compound
code
Selectivity
Indexa
sEH inhibition
(
IC50 nM)b
COX-1
10.62
11.85
8.72
COX-2
2.03
1.85
1.24
2.89
2.18
1.99
2.48
2.15
9a
9b
9c
5.23
6.41
7.03
3.49
5.01
4.96
4.12
4 .18
0.98±0.013
0.55±0.009
0.40±0.011
1.74±0.025
1.57±0.015
4.01±0.012
2.38±0.041
1.15±0.029
10.1
9d
9e
10.92
9.87
9f
10.22
8.99
11a
11b
12

9.12
11.74
10.54
12.75
6.12
2.05
4.12
3.04
4
4.45
2.85
3.47
3.19
6.44
1.21±0.011
1.61±0.037
3.31±0.037
1.84±0.022
261.14±15.1
11c
11d
11e
11f
0.95
Celecoxib
----
----
----
AUDA
0.49±0.009
aSelectivity index was calculated by dividing COX-1 IC50 by COX-2 IC50)
bThe values are the mean ± SEM (n = 3)
The reference drugs and the most active compounds results were written in Bold
2.2.2. In vivo Evaluation:
2.2.2.1. Analgesic activity:
Acetic acid-induced writhing method [49] was applied to evaluate the analgesic activity of all the
tested compounds. The efficacy of the targeted compounds was determined through calculating the
reduction in the number of acetic acid-induced writhing episodes and hence detecting their potency.
The detected results were outlined in Table 2. The results exposed that all the compounds exhibited
moderate analgesic activity with % inhibition in the number of writhing range between 22.38-
65.67%, if compared to the reference drug, celecoxib with 13.43%. Interestingly enough, compound
9c, the most potent in vitro dual COX-2/sEH inhibitor, elicited also the highest analgesic activity
among the rest of other compounds (% inhibition = 65.67 %; Potency = 4.88).
Table 2: Results of analgesic activity of the new compounds 9a-f and 11a-f using acetic acid-induced
writhing assay in mice compared to celecoxib.
Compound
code
No. of writhes in 5–15 min
after treatment a
(Mean±SE)
Potencyb
% Inhibition
23.50±0.2
25.25±0.7
11.50±0.6
20.75±0.3
29.85
24.62
65.67
38.05
2.23
1.83
4.88
2.83
9a
9b
9c
9d
13

23.00±0.3
20.25±0.7
23.00±0.4
12.50±0.4
19.25±0.4
24.50±0.6
22.25±0.6
26.00±0.6
29.00±0.6
33.50±0.8
31.34
39.55
31.34
62.68
42.53
26.86
33.58
22.38
13.43
--
2.33
2.94
2.33
4.66
3.16
2
9e
9f
11a
11b
11c
11d
2.5
1.66
1
11e
11f
Celecoxib
Control
--
aValues are given as mean ± SE
bPotency are calculated according to equation of relative potency % = % of
inhibition of tested compound / % of inhibition of control X 100
Celecoxib and the most active compounds results were written in Bold
2.2.2.2. Anti-inflammatory activity:
The anti-inflammatory activity of the tested compound was determined using carrageenan-induced rat
paw edema method [50]. The efficacy of the compounds was expressed as the decrease in edema paw
volume and calculated % edema inhibition (EI %) after 1, 3 and 5 h compared to standard celecoxib
drug, Table 3. The results elicited that all the compounds showed wide range in edema inhibition
percentages after the three-time intervals 1, 3 and 5 hours. After 1 hour, all compounds showed weak
to moderate EI% in range 28.18-78.73% comparing with celecoxib 81.17%. Meanwhile, after 3
hours, EI% of all compounds was increased to reach to the range of 57.18-95.82% in contrast to
celecoxib whose EI% slightly increased to become 82.71%. Finally, after 5 hours, the increase in EI%
was continued to be recorded in the range of 69.87-98.15% in comparison with celecoxib which
showed EI% of 88.30%. Using 5 hours results as a criterion for the comparison, all compounds
showed lower anti-inflammatory activity than celecoxib except compounds 9a-c that showed the
highest EI% among all compounds in addition to celecoxib (EI% = 98.15-88.76% for 9a-c; EI% =
88.30% for celecoxib). The aforementioned results concluded that compound 9c elicited the highest
14

anti-inflammatory activity among all the rest of the compounds with EI % = 98.15 in comparison
with celecoxib EI % = 88.30 at the same time interval.
Table 3: Results of anti-inflammatory activity of synthesized compounds 9a-f and 11a-f using
carrageenan-induced rat paw edema model in rats compared to celecoxib.
Change in paw volume in (mm)
after drug digestion(±SEM)
Anti-inflammatory activity
(Edema inhibition %)
Compound
code
1h
3h
5h
1h
3h
5h
3.48±0.16 3.17±0.01
0.08±6.18 0.31±1.08
2.19±0.07
0.11±1.21
9a
9b
78.73
62.22
81.35
92.02
88.76
93.79
0.21±5.88 0.27±0.71
0.16±10.81 0.60±6.84
5.44±0.14 5.52±0.10
0.29±9.40 0.28±4.68
0.36±11.28 0.27±7.28
0.36±9.48 0.51±.3.99
0.97±6.03 0.64±4.28
0.18±10.68 0.23±6.18
6.73±0.31 7.28±0.35
0.51±11.75 0.21±5.76
3.08±0.15 2.94±0.08
0.24±16.36 17.00±0.28
0.03±0.36
0.35±4.92
4.40±0.3
9c
9d
64.06
33.92
66.75
42.54
31.05
42.05
63.14
34.72
58.86
28.18
81.17
--
95.82
59.76
67.53
72.47
57.18
76.53
74.82
63.65
57.18
66.12
82.71
--
98.15
74.74
77.41
74.74
74.74
84.19
86.76
69.87
72.59
69.87
88.3
9e
0.17±4.92
0.22±4.92
0.760±3.08
0.4202.58
0.09±5.87
5.34±0.16
0.19±5.87
2.28±0.14
19.48±0.26
9f
11a
11b
11c
11d
11e
11f
Celecoxib
Control
--
The edema inhibition % is calculated according to the following equation:
EI(%) = (1-Wt/Wc) x 100 where Wt symbolized the main increase in the tested compound
groups paw thickness and Wc symbolized the mean increase in control group paw
thicknesses.
The results analyzed by ANOVA (n = 4), P < 0.05 were all significant from the control.
Celecoxib and the most active compounds were written in Bold
2.2.2.3. Cardiovascular Evaluation:
The possible cardiovascular risks of the most active compounds 9b and 9c was evaluated using the
celecoxib induced cardio-toxicity in rats as reported [51,52]. The response of the heart towards the
tested compounds was expressed as the change in biochemical parameters obtained from both sera
15

including lactate dehydrogenase (LDH), troponin-I (Tn-I), tumor necrosis factor-α (TNF-α), creatine
kinase-MB (CK-MB), prostacyclin (PGI2) and tissues including glutathione (GSH). Also, the study of
changes of histo-pathological parameters of the evaluated compounds compared to celecoxib as a
reference standard was performed. All the obtained results were shown in Table 4 and Fig. 4. The
results revealed that celecoxib administration induced significant increase in the diagnostic
biomarkers of myocardial damage [53–55] including Tn-I, LDH and CK-MB levels, as compared to
normal control. Whereas, both 9b and 9c exhibited a significant lowering in these biochemical levels
when compared to celecoxib treatment levels proofing their protective cardiovascular activity and
much less risks. Moreover, serum TNF-α, a key player in the inflammatory response and cardiac
depression [56], significantly increased after celecoxib treatment if compared to the control group,
unlike groups that administered the newly synthesized compounds 9b and 9c that showed remarkably
decrease in TNF-α concentration if compared to the celecoxib induced cardio-toxicity group.
Table 4: Results of serum TNF-α, Tn-I, LDH and CK-MB measurements in 9b, 9c and celecoxib.
TNF-α
Troponine-I
LDH
CK-MB
Groups
(pg/ml)
(pg/ml)
IU/L
IU/L
Normal
control
78.67±3.75
74±4.61
1536±100
16±2.3
748.67±43.15a
322±11.9b
340±11.56a
150±11.23b
108.67±2.60b
2091.3±99.9a
1585±74.2b
95.33±4a
22±4b
Celecoxib
9b
9c
265.67±7.3ab
1473.3±28.57b 17±2.3b
Data analyzed by one-way ANOVA followed by Bonferroni test to enable comparing
a
groups pairwise, (n = 6), Significantly different from normal control group at p <
0.05. bSignificantly different from celecoxib group at p < 0.05.
Tissue GSH, the important antioxidant in cardiac tissues, remarkably decreased in the celecoxib
treated group confirming the previous studies which reported that celecoxib in chronic administration
elevated the oxidative stress markers as lipid peroxidation and reduced GSH level through increasing
16

the oxidative damage of the kidney [57]. On the contrary, compounds 9b and 9c restored heart GSH
level, where they significantly increased GSH levels compared to celecoxib group as shown in Fig. 4.
Meanwhile, PGI2 plays an important role in cardiovascular health, [58] especially its ratio to
thromboxane A2 in blood, as the most potent endogenous inhibitor of platelet aggregation, where the
decrease in PGI2 levels indicates cardiomyopathy. In this regard, it was found that celecoxib and 9b
significantly decreased PGI2 levels, if compared to normal control, while compound 9c was not
significantly different from the normal control. Furthermore, each of the tested compounds
significantly increased PGI2 level if compared to celecoxib, as shown in Fig. 5.
Fig. 5. Cardic glutathione (GSH) content and serum prostacyclin (PGI2) level in different
groups. Data analyzed by one-way ANOVA followed by Bonferroni test to compare groups
b
pairwise, (n = 6). aSignificantly different from normal control group at p < 0.05. Significantly
different from celecoxib group at p < 0.05.
Interestingly, histo-pathological studies revealed that the heart section obtained from rats exposed to
celecoxib showed severe hemorrhage and edema between muscle bundles (B). While the heart
sections obtained from rats exposed to 9b (C) showed mild to moderate congestion and edema in
myocardium blood vessels and in coronary artery. Also, the heart sections obtained from rats exposed
17

to 9c (D) showed mild decongestant and mild edema on cardiac blood vessels and showed more or
less normal muscle bundles, as shown in Fig. 6.
Fig. 6. Routine hematoxylin and eosin (H&E) stained sections of the left ventricle in animals’ groups
where (A) Control group; (B) Celecoxib administered group; (C) 9b administered group; (d) 9c
administered group. (Routine hematoxylin and eosin (H&E) stained; 200x). Figure (A) shows normal
myocardium. Figure (B) shows sever hemorrhage and edema between muscle bundles. Figures (C)
and (D) show mild to moderate congestion and edema in the myocardium.
2.3. Molecular Docking:
The current study was accomplished to recognize the activity of the newly synthesized urea
disubstituted-1,5-diarylpyrazole derivatives and know some structural perceptions into their binding
manners and probable interactions with sEH enzyme. Accordingly, some active derivatives (9a, 9c,
11a and 11c) in addition to some other inactive ones (9e, 9f and 11e), for comparison, were docked
into the active sites of sEH using LigandFit embedded in the Discovery Studio software (San Diego,
18

USA). The 3D crystal structure of sEH (PDB ID: 1VJ5) in complex with CIU781, respectively, were
used for this docking simulation study [59,60]. The amino acid residues within a distance of 8 Å
around the sEH co-crystallized ligands in the active pocket were isolated and the potential
interactions and ligands orientation were inspected. The overlay of the top docking poses into sEH
protein binding pocket was presented in Fig. 7, where the poses 9a, 9c, 11a, 11c and AUDA showed
good shape complementarity with the active site of sEH enzyme.
Fig. 7. Overlay of the top docked poses 9a, 9c, 11a, 11c and AUDA as a reference ligand into the
sEH binding pocket (PDB code: 1VJ5); sEH protein is represented as: (A) Solid surface colored
according to atom charges; (B) Secondary structure displayed in a flat ribbon style.
It was reported that the catalytic pocket of sEH consists of three main amino acids; Asp333, Tyr381,
Tyr465, which are responsible for the activity of the enzyme [59–61]. Therefore, it always noticed
that a network of hydrogen bonds formed between the urea or amide moieties in the potent sEH
inhibitors and these important amino acids inside the hydrolase catalytic pocket. In doing so, the
inspection of the docking results of the active derivatives 9a, 9c and 11c showed that they all fit
precisely inside the active site engaging some interactions including hydrogen bonds and π-π
stacking. In general, it was found that the top docked poses adopted a common binding pattern and
orientation where the phenyl-bearing sulfonamide moiety in the three compounds aligned towards
His523, Trp524, Val415 and Leu416 amino acids residues while the phenyl-bearing trifluoromethyl
19

group was extended towards Trp334, Met337, Ilu373 and Phe379 residues forming some
hydrophobic interactions, Fig. 8(A-C). Their noticeable potency against sEH enzyme may greatly due
to the obvious similarity between the binding patterns of these derivatives. It was conceptualized that
the number and type of these interactions could explain the variation between these ligands in their
potency. Compound 9c was the most active derivative (IC50 = 0.4 nM) where it involved in the
highest number and types of interactions. It formed five different H-bonds; one between the carbonyl
oxygen of urea moiety and Try381, two between NH of urea and Asp333, one between the NH of the
pyrazole ring and Try464 and the fifth between the NH2 of the sulfonamide group and Leu416
residue. In addition, several π-π stacking between pyrazole ring and imidazole of His523 residue and
two others between phenyl-bearing sulfonamide and the indole heterocycle of Trp524 amino acid,
Fig. 8(A). The decrease in the activity of 9a (IC50 = 0.98 nM) compared with 9c could be explained
by the absence of some interactions such as H-bond with Leau416 and π-π stacking with Trp524
residue, Fig. 8(B). However, the amide derivatives 11c showed the least activity (IC50 = 1.2 nM)
between the most active ones due to the sharp reduction in the number of interactions despite of
adopting a similar orientation. The replacement of urea moiety with an amide one resulted in the
absence of an important H-bond with Asp333 in the catalytic active site of sEH enzyme, Fig. 8(C).
20

Fig. 8. (A) Docking and binding pattern of compound 9c (green) into catalytic active pocket of sEH
enzyme (PDB code: 1VJ5); (B) Docking and binding pattern of compound 9a (orange) into catalytic
active pocket of sEH enzyme; (C) Docking and binding pattern of compound 11c (violet) into
catalytic active pocket of sEH enzyme; (D) The superimposition of 9a and 9c docked poses within
the catalytic active pocket of sEH enzyme. The poses were rendered as ball and stick model. π- π
Interactions were represented as orange solid line. Hydrogen bonds were represented as dashed
orange lines. All hydrogens were removed for the purposes of clarity.
Moreover, the analysis of the docking results of the inactive compounds 9f and 11e into the sEH
active pocket specified that these two ligands shared a completely opposing orientation and
dispositioning compared with the previously docked active derivatives (9a, 9c, 11a and 11c), Fig. 9
(A and B). It was found that the phenyl-bearing sulfonamide core was located near Trp334, Met337,
Ilu373 and Phe379 residues causing some bulkiness reflected in the sharp decrease in activity as in
compound 9f which showed the least potency (IC50 = 4.01 nM). While, the phenyl-bearing
21

trifluoromethyl directed towards His523, Trp524, Val415 and Leu416 amino acids residues, Fig.
9(A). Moreover, amide derivative 11e adopted a similar orientation to that of 9f with a slight increase
in activity (IC50 = 3.31 nM) noticed with increasing the carbon side chain length, Fig. 9(B). The
docking study was compatible with the in vitro assay results, proving that hybridization between the
urea or amide sEH pharmacophoric moiety and the sulfonamide-bearing diarylpyrazole COX-2 core
are good leads for further optimization.
Fig. 9. (A) Docking and binding pattern of compound 9f (magenta red) into the catalytic active
pocket of sEH enzyme (PDB code: 1VJ5); (B) Docking and binding pattern of compound 11e (blue)
catalytic active pocket of sEH enzyme. The poses were rendered as ball and stick model. All
hydrogens were removed for the purposes of clarity.
2.4. Physicochemical parameters:
According to the great effect of physicochemical properties of any compound on its bioavailability
and its possible druggability, computational prediction of the physicochemical properties of the
synthesized compounds has been performed on the basis of Lipinski's rule of five (RO5) [62]. The
chemo-informatic properties of the synthesized compounds were predicted using Molinspiration tool
[63,64] and outlined in Table 5. All synthesized compounds exhibited good oral bioavailability
because all of them showed no or only one violation from Lipinski's rule except two compounds 9e
and 11f. Moreover, calculated absorption (%ABS) complied the ability of the compounds to elicit
promising pharmacokinetic properties, where %ABS is ranging from 55-72%.
22

Table 5. Calculated absorption, polar surface area and Lipinski parameters of the target compounds
9a-f and 11a-f.
nON nOHNH miLogP MW n violations
Compound %ABSa tPSAb Nrotbc
≤10d
≤5e
≤5
≤500
≤1
67.9
67.9
67.9
119.12
119.12
119.12
5
5
8
8
4
4
4
3
5
5
3
3
3
2
4
4
3.56
4.58
4.25
2.92
4.98
1.45
3.37
4.38
4.05
2.72
4.78
1.25
439.54
491.62
501.49
497.58
541.67
443.49
424.53
476.6
0
0
1
0
3
1
0
0
0
0
3
1
9a
9b
6
8
9c
61.86 136.63
55.04 156.41
55.04 156.41
72.06 107.09
72.06 107.09
72.06 107.09
66.01 124.61
59.19 144.39
59.19 144.39
7
10
10
10
7
9d
15
8
9e
9f
5
11a
11b
11c
11d
11e
11f
5
7
6
7
486.48
482.56
526.66
7
9
15
8
9
9
428.47
b
c
a% ABS: Calculated absorption; tPSA: Topological polar surface area; nrotb: Number of
d
rotatable bonds; nON≤10: Number of hydrogen bond acceptors less than or equal to 10 and
enOHNH≤5: Number of hydrogen bond donors less than or equal to 5.
3. Conclusion:
Novel series of 1,5- diarylpyrazole series of urea linked (9a-f) and amide linked (11a-f) compounds
were designed, synthesized and evaluated as dual inhibitors of both COX-2/sEH enzymes in an
attempt to preserve the preferred pharmacological activity of the selective COX-2 inhibitors with
avoiding their serious associated cardiovascular side effects through sEH inhibition. The in vivo and in
vitro biological results revealed that compound 9c, which possessed p-triflouromethyl anilinyl moiety
directly attached to a urea linker, was the most potent dual COX-2/sEH inhibitor (COX-2 IC50 = 1.24
μM; SI = 7.03; sEH IC50 = 0.40 nM) and showed the highest in vivo activities as analgesic (65.67%
writhing inhibition) and anti-inflammatory (98.15% edema inhibition) agent, followed by compound
9b, which elicited good COX-2/sEH inhibition (COX-2 IC50 = 1.85 μM; SI = 6.41; sEH IC50 = 0.55
23

nM) and high anti-inflammatory activity (93.79% Edema inhibition). Regarding the cardiovascular
system evaluation, the results were consistent with both in vivo and in vitro inhibitory activities values
that affirmed the good activities of compound 9c affording a perfect cardio-protection and less
cardiovascular liabilities, in contrast to the selective COX-2-induced cardiotoxicity. This was proved
via decreasing the cardiac diagnostic biomarkers of myocardial damage including Tn-I, LDH and CK-
MB level, consort with increasing in the antioxidant biomarker GSH, and the anti-platelets aggregation
agent PGI2 levels. The aforementioned results revealed that compound 9c, named 4-(1-phenyl-3-(3-(4-
(trifluoromethyl)phenyl)ureido)-1H-pyrazol-5-yl)benzenesulfonamide (PTPUP), could be considered
as a promising candidate with potent dual COX-2/sEH inhibitory activities to be further developed as
a safe anti-inflammatory/analgesic lead attending with significant less cardiovascular risks than current
selective COX-2 drugs.
4. Experimental protocols:
4.1. Chemistry:
All chemical reagents and solvents were procured from commercial suppliers and were utilized with
no further purification. Melting points were uncorrected and were implemented by open capillary
tube methods using IA 9100MK-Digital melting point apparatus. Elemental analysis was performed
at the micro-analytical center at the regional center for mycology and biotechnology, Al-Azhar
University. Infrared spectra were reported on Bruker FT-IR spectrophotometer Vector 22 and
recorded in wave number (cm-1) using KBr discs at the micro-analytical center, Faculty of Science,
Cairo University. 1H NMR and 13C NMR spectra were executed with Bruker APX400 spectrometer at
400 MHz and 101 MHz, respectively in the specified solvent at the Faculty of Pharmacy, Beni-Suef
University. The chemical shifts were outlined on the δ scale and J values were recorded in Hz. Mass
spectra were reported on Finnegan MAT, SSQ 7000, Mass spectrometer, at 70 eV (EI) at the micro-
analytical center, Faculty of Science, Cairo University. Thin layer chromatography (TLC) was carried
out using Macherey-Nagel Alugram Sil G/UV254 silica gel plats and Petroleum ether-ethyl acetate
(6:4) as the eluting system.
24

1-phenyl-5-(4-sulfamoylphenyl)-1H-pyrazole-3-carbonyl azide 7. A solution of NaNO2 (0.87 g, 1.0
mmol) in cold water (3 mL) was added to a cold solution (0-5 ᴏC) of hydrazide 6 (0.357 g, 1.0 mmol)
in acetic acid (6 mL), 1N HCl (3 ml), and water (25 mL). The reaction mixture was allowed to be
stirred at 0-5 °C for 15 min followed by extraction with ethyl acetate (30 mL) producing yellow
syrup. The formed yellow syrup was washed with cold 3% NaHCO3, H2O, dried over anhydrous
Na2SO4 and used in the next step without purification.
General procedure A for synthesis of compounds (9a-9f): The acyl azide 7 (0.78 g, 2.14 mmol)
was dissolved in anhydrous toluene (5 mL) and the mixture was exposed to heating under reflux for 1
hour under N2. After completion of the reaction, the reaction mixture was allowed to cool to room
temperature followed by addition of the appropriate amine (2 mmol) in the presence of pyridine. The
mixture was heated under reflux for 24 hours subsequently pouring onto ice/H2O containing few
drops of conc HCl. The compound was extracted with ethyl acetate, drying over anhydrous
magnesium sulfate followed by purification of the resulted residue by column chromatography using
petroleum ether to ethyl acetate gradient to elute the final compounds 9a-9f in good yields.
4-(3-(3-cyclohexylureido)-1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide 9a. General procedure A,
as white solid (54%); m.p. 215°C. IR (cm-1): 3415 (NH), 3100 (CH aromatic), 2929 (CH aliphatic),
1
1660 (NHCONH), 1510 (C=N), 1339, 1161 (SO2NH2). H NMR (400 MHz, DMSO-d6): δ 9.00 (s,
1H, NH exchangeable with D2O), 7.77-7.79 (d, J = 8 Hz, 2H, Ar-H), 7.41-7.43 (m, 5H, 3 H of Ar-H
and 2H of SO2NH2 exchangeable with D2O ), 7.35-7.37 (m, 2H, Ar-H), 7.27-7.29 (m, 2H, Ar-H),
6.69 (s, 1H, NH exchangeable with D2O), 6.59 (s, 1H, Pyrazolyl-H), 3.55 (m, 1H, -NHCHCH2-),
1.81-1.83 (m, 2H, -CHCH(H)CH2-), 1.61-1.70 (m, 2H, -CHCH(H)CH2-), 1.51-1.55 (m, 2H, -
(CH2)2CH2(CH2)2-), 1.17-1.35 (m, 4H, -CH2CH2CH2CH-). 13C NMR (101MHz, DMSO-d6): δ 160.5,
148.2, 145.2, 144.9, 143.9, 142.0, 131.9, 129.5, 129.0, 126.1, 125.5, 108.8, 48.2, 32.8, 25.6, 25.4. MS
(EI): m
/
z 439 (M+). Anal. Calcd. For C22H25N5O3S: C, 60.12; H, 5.73; N, 15.93. Found: C, 59.95; H,
5.88; N, 16.12.
25

4-(3-(3-adamantylureido)-1-phenyl-1H-pyrazol-5-yl) benzenesulfonamide 9b. General procedure A,
white solid (53%); m.p. 265°C. IR (cm-1): 3387 (NH), 3278 (CH aromatic), 2904 (CH aliphatic),
1678 (NHCONH), 1339, 1161 (SO2NH2), 1508 (C=N).1H NMR (400 MHz, DMSO-d6): δ 8.88 (s,
1H, NH exchangeable with D2O), 7.77-7.79 (d, J = 8 Hz, 2H, Ar-H), 7.69-7.72 (s broad, 1H, NH
exchangeable with D2O), 7.41-7.43 (m, 5H, 3 H of Ar-H and 2H of SO2NH2 exchangeable with D2O
), 7.33-7.36 (m, 2H, Ar-H), 7.27-7.29 (m, 2H, Ar-H), 6.58 (s, 1H, Pyrazolyl-H), 2.02-2.05 (m, 3H, -
(CH2)2CH(CH2)2-), 1.90-1.89 (m, 6H, -NHCCH2-), 1.65-1.59 (m, 6H, -CHCH2CH-). 13C NMR
(101MHz, DMSO-d6): δ 153.5, 143.4, 142.4, 142.2, 130.3, 129.3, 128.9, 127.2, 127.1, 124.6, 119.6,
99.9, 50.4, 42.1, 36.5, 29.4. MS (EI): m/
z 491 (M+). Anal. Calcd. For C26H29N5O3S: C, 63.52; H,
5.95; N, 14.25. Found: C, 63.81; H, 6.08; N, 14.37.
4-(1-phenyl-3-(3-(4-(trifluoromethyl)phenyl)ureido)-1H-pyrazol-5-yl)benzenesulfonamide 9c.General
procedure A, yellow solid (63%); m.p. 278°C. IR (cm-1): 3371 (NH), 3167 (CH aromatic), 1689
1
(NHCONH), 1330, 1161 (SO2NH2), 1508. (C=N). H NMR (400 MHz, DMSO-d6): δ 10.13 (s, 1H,
NH exchangeable with D2O), 9.77 (s, 1H, NH exchangeable with D2O), 7.88-7.90 (d, J = 8 Hz, 2H,
Ar-H), 7.59-7.61 (d, J = 8 Hz, 2H, Ar-H), 7.51-7.54 (m, 3H, Ar-H), 7.43-7.44 (m, 4H, 2H of Ar-H
and 2H of SO2NH2 exchangeable with D2O), 7.34-7.36 (m, 2H, Ar-H), 7.18 (s, 1H, Pyrazolyl-H),
13
7.16-7.17 (m, 2H, Ar-H) . C NMR (101MHz, DMSO-d6): δ 159.9, 144.3, 144.1, 144.0, 130.8,
129.9, 129.5, 129.3, 129.3, 127.4, 126.6, 126.4, 126.4, 126.2, 125.9, 120.7, 109.1. MS (EI): m/z 501
(M+). Anal. Calcd. For C23H18F3N5O3S: C, 55.09; H, 3.62; N, 13.97. Found: C, 55.24; H, 3.81; N,
14.15.
Ethyl 1-((1-phenyl-5-(4-sulfamoylphenyl)-1H-pyrazol-3-yl)carbamoyl)piperidine-4-carboxylate 9d.
General procedure A, buff solid (57%); m.p. 189 °C. IR (cm-1): 3414 (NH), 3100 (CH aromatic),
2930 (CH aliphatic), 1720 (COOCH2CH3), 1630 (NHCONH), 1508 (C=N), 1338, 1161 (SO2NH2).
1H NMR (400 MHz, DMSO-d6): δ 9.42 (s, 1H, NH exchangeable with D2O), 7.77-7.79 (d, J = 8 Hz,
2H, Ar-H), 7.41-7.43 (m, 5H, 3 H of Ar-H and 2H of SO2NH2 exchangeable with D2O ), 7.35-7.37
(m, 2H, Ar-H), 7.27-7.29 (m, 2H, Ar-H), 6.59 (s, 1H, Pyrazolyl-H), 4.03-4.09 (m, 4H, 2H of CH2CH3
26

and 2H of -NCH(H)CH2-), 2.93 (m, 1H, -NCH(H)CH2-), 2.60 (m, 1H, -NCH(H)CH2-), 2.25 (m, 1H, -
CH2CHCO-), 1.77-1.92 (m, 2H, -CHCH2CH2-), 1.51-1.56 (m, 2H,-CHCH2CH2-), 1.17-1.20 (t, J = 6
Hz, 3H, -CH2CH3-). 13C NMR (101MHz, DMSO-d6): δ 174.4, 161.7, 148.3, 144.0, 143.7, 142.0,
129.5, 129.3, 129.3, 129.2, 127.2, 126.0, 110.4, 60.5, 48.2, 29.1, 28.2, 14.6. MS (EI): m
Anal. Calcd. For C16H15N5O3S: C, 58.69; H, 5.71; N, 13.69. Found: C, 58.90; H, 5.73; N, 13.84.
11-(3-(1-phenyl-5-(4-sulfamoylphenyl)-1H-pyrazol-3-yl)ureido)undecanoic acid 9e. General
/
z 497 (M+).
procedure A, white solid (63%); m.p. 245 °C. IR (cm-1): 3400-2500 (COOH), 3035 (CH aromatic),
2970 (CH aliphatic), 1697 (C=O), 1630 (C=O), 1504 (C=N), 1334, 1161 (SO2NH2). 1H NMR (400
MHz, DMSO-d6): δ 9.08 (s, 1H, NH exchangeable with D2O), 7.77-7.79 (d, J = 8 Hz, 2H, Ar-H),
7.41-7.43 (m, 5H, 3 H of Ar-H and 2H of SO2NH2 exchangeable with D2O ), 7.36-7.38 (m, 2H, Ar-
H), 7.27-7.29 (m, 2H, Ar-H), 6.66 (s, 1H, NH exchangeable with D2O), 6.59 (s, 1H, Pyrazolyl-H),
3.09-3.14 (m, 2H,-NHCH2CH2-), 2.16-2.19 (m, 2H,-CH2CH2COOH), 1.44-1.50 (m, 4H, 2H of -
CH2CH2CH2COOH and 2H of –NHCH2CH2CH2-), 1.26-1.28 (m, 12H, -CH2CH2CH2-). 13C NMR
(101MHz, DMSO-d6): δ 175.1, 163.2, 154.9, 150.8, 143.6, 142.4, 142.3, 130.3, 129.3, 128.9, 127.0,
124.7, 99.9, 34.2, 30.1, 29.4, 29.3, 29.2, 29.0, 26.8, 25.0. MS (EI): m/
z 541 (M+). Anal. Calcd. For
C27H35N5O5S: C, 59.87; H, 6.51; N, 12.93. Found: C, 60.04; H, 6.61; N, 13.19.
4-(3-(1-phenyl-5-(4-sulfamoylphenyl)-1H-pyrazol-3-yl)ureido)butanoic acid 9f. General procedure A,
white solid (51%); m.p. 245 °C. IR (cm-1): 3500-2800 (COOH), 3367 (NH), 3070 (CH aromatic),
2929 (CH aliphatic), 1693 (COOH), 1589 (NHCONH), 1512 (C=N), 1334, 1161 (SO2NH2). 1H NMR
(400 MHz, DMSO-d6): δ 10.12 (s, 1H, COOH exchangeable with D2O), 9.75 (s, 1H, NH
exchangeable with D2O), 7.88-7.90 (d, J = 8 Hz, 2H, Ar-H), 7.59-7.61 (d, J = 8 Hz, 2H, Ar-H), 7.50-
7.52 (m, 2H, Ar-H), 7.43-7.44 (m, 3H, 1H of Ar-H and 2H of SO2NH2 exchangeable with D2O),
7.34-7.36 (m, 2H, Ar-H), 7.18 (s, 1H, Pyrazolyl-H), 7.16 (s, 1H, NH exchangeable with D2O), 3.17
(m, 2H, -NHCH2CH2), 2.27 (m, 2H, -CH2CH2COOH), 1.24 (m, 2H, -CH2CH2CH2-). 13C NMR
(101MHz, DMSO-d6): δ 208.0, 154.4, 154.2, 149.5, 143.6, 142.6, 142.3, 130.2, 129.3, 129.3, 127.1,
27

125.1, 100.5, 61.0, 28.5, 14.9. MS (EI): m/
z 443 (M+). Anal. Calcd. For C20H21N5O5S: C, 54.17; H,
4.77; N, 15.79. Found: C, 54.37; H, 4.89; N, 16.01.
General procedure B for preparation of compounds 11a-11f: A solution of ester 5 (0.37g, 1
mmol) in methanol was treated with KOH (0.11 g, 2 mmol) and heated under reflux for 12 hours. The
mixture was acidified with 1N HCl after cooling to room temperature. The solvent was then
evaporated to give buff solid that recrystallized from methanol to furnish the desired compound
(0.33g, 97% yield) as reported [48]. To a suspension of acid (0.342 g, 1mmol) in CH2Cl2, 2 mL of
SOCl2 was added and heated under reflux for 3 hours. Evaporation the solvent was carried out under
vacuum to give a residue of the corresponding acyl chloride that utilized in the following step without
purification. To a solution of acyl chloride in CH2Cl2, few drops of trimethylamine were added
followed by appropriate amine (1 mmol) and was left under stirring at room temperature overnight.
The solvent was evaporated, washing the residue with water, extraction with ethyl acetate followed
by drying on anhydrous MgSO4. Purification of the obtained residue was carried out by column
chromatography technique using hexane and ethyl acetate (8:2) to afford the final compounds 11a-
11f in good yields.
to afford the titled compound.
N-cyclohexyl-1-phenyl-5-(4-sulfamoylphenyl)-1H-pyrazole-3-carboxamide 11a. General procedure B,
white solid (85%); m.p. 240 °C. IR (cm-1): 3390 (NH), 3132 (CH aromatic), 2931 (CH aliphatic),
1643 (CONH), 1323, 1161 (SO2NH2), 1550 (C=N). 1H NMR (400 MHz, DMSO-d6): δ 8.06-8.08 (m,
1H, Ar-H), 7.86-7.88 (d, J = 8 Hz, 2H, Ar-H), 7.51-7.57 (m, 4H, 2H of Ar-H and 2H of SO2NH2
exchangeable with D2O), 7.41 (m, 3H, 2H of Ar-H and 1H of NH exchangeable with D2O), 7.30-7.31
(m, 2H, Ar-H), 7.04 (s, 1H, Pyrazolyl-H), 3.78 (m, 1H, -NHCHCH2-), 1.72-1.82 (m, 4H, -
NHCH(CH2)2CH2-), 1.61-1.63 (m, 2H, -(CH2)2CH2(CH2)2-), 1.29-1.39 (m, 4H, -CHCH2CH2CH2-).
13C NMR (101MHz, DMSO-d6): δ 160.6, 148.5, 144.9, 143.9, 142.1, 129.6, 129.4, 129.30 129.2,
28