Design, synthesis, analgesic, anti-inflammatory activity of novel pyrazolones possessing aminosulfonyl pharmacophore as inhibitors of COX-2/5-LOX enzymes: Histopathological and docking studies

Article abstract of DOI:10.1016/j.bioorg.2018.03.011

A series of newly synthesized 4-aryl-hydrazonopyrazolones were designed and their structures were confirmed by spectral and elemental analyses. All synthesized compounds were evaluated for their in vitro COXs, 5-LOX inhibition, in vivo analgesic and anti-

Full text of DOI:10.1016/j.bioorg.2018.03.011

Accepted Manuscript
Design, synthesis, analgesic, anti-inflammatory activity of novel pyrazolones
possessing aminosulfonyl pharmacophore as inhibitors of COX-2/5-LOX en-
zymes: Histopathological and docking studies
Mohamed A. Abdelgawad, Madlen B. Labib, Waleed A.M. Ali, Gehan Kamel,
Amany A. Azouz, EL-Shaymaa EL-Nahass
PII:
S0045-2068(17)30927-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.03.011
YBIOO 2301
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 December 2017
27 January 2018
7 March 2018
Please cite this article as: M.A. Abdelgawad, M.B. Labib, W.A.M. Ali, G. Kamel, A.A. Azouz, E-S. EL-Nahass,
Design, synthesis, analgesic, anti-inflammatory activity of novel pyrazolones possessing aminosulfonyl
pharmacophore as inhibitors of COX-2/5-LOX enzymes: Histopathological and docking studies, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.03.011
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Graphical abstract
Design, synthesis, analgesic, anti-inflammatory activity of novel pyrazolones
possessing aminosulfonyl pharmacophore as inhibitors of COX-2/5-LOX enzymes:
Histopathological and docking studies
Mohamed A. Abdelgawad1,2*, Madlen B. Labib1, Waleed A.M. Ali3, Gehan Kamel4,
Amany A. Azouz5and EL-Shaymaa EL-Nahass6
O
SO2NH2
N
N
N
H
N
COOH
5f
COX-2 IC50 = 0.72 µM
5-LOX IC50 = 0.52 µM
COX-2 SI = 5.29
ED50 = 0.044 (2h), 0.064 (4h)mmol/Kg
Ulcer Index = 2.33
1

Design, synthesis, analgesic, anti-inflammatory activity of novel pyrazolones
possessing aminosulfonyl pharmacophore as inhibitors of COX-2/5-LOX enzymes:
Histopathological and docking studies
Mohamed A. Abdelgawad1,2*, Madlen B. Labib1, Waleed A.M. Ali3, Gehan Kamel4,
Amany A. Azouz5 and EL-Shaymaa EL-Nahass6
1Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef
University, Beni-Suef 62514, Egypt;
2Department of Pharmaceutical Chemistry, College of Pharmacy, Aljouf University,
Sakaka, Aljouf-2014, Saudi Arabia;
3 Biochemistry Department, Cairo General Hospital, Egypt;
4 Department of Pharmacology, Faculty of Veterinary, Cairo University, Cairo, Egypt;
5 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-Suef
University, Beni-Suef, Egypt;
6Department of Pathology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-
Suef, 62511, Egypt.
* Author to whom correspondence should be addressed;
Email mohamedabdelwahab976@yahoo.com;
Official email; mohamed.abdelgawad@pharm.bsu.edu.eg, mhmdgwd@ju.edu.sa
Tel.:0020238482600, mobile +966595435214
Address: Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-
Suef University, Beni-Suef 62514, Egypt. Pharmaceutical chemistry Department,
College of Pharmacy, Aljouf University, Aljouf, KSA.
2

Abstract
A series of newly synthesized 4-aryl-hydrazonopyrazolones were designed and
their structures were confirmed by spectral and elemental analyses. All synthesized
compounds were evaluated for their in vitro COXs, 5-LOX inhibition, in vivo analgesic
and anti-inflammatory activities. Compounds 5d, 5f and 5i were found to be the most
potent COX-2/5-LOX inhibitors with superior COX-2 selectivity index values (SI = 5.29
- 5.69) to reference standard celecoxib (SI = 3.52). Four compounds; 5b, 5c, 5d and 5f
showed excellent anti-inflammatory activity (% edema inhibition = 72.72 - 54.54%) and
perfect ED50 values (ED50 = 0.044 - 0.104 mmol/Kg) relative to celecoxib (ED50 = 0.032
mmol/Kg). To explore the most active compounds, ulcerogenic effect on stomach in
comparison with indomethacin and celecoxib in addition to histopathological
investigations were performed. Compound 5f showed better gastric profile (UI = 2.33)
than celecoxib (UI = 3.00). Also, 5f caused 50% increase in thermal pain threshold close
to reference drug indomethacin (53.13%). Docking study of all the target compounds into
COX-2 and 5-LOX active sites was performed to rational their anti-inflammatory
activities.
Key words: pyrazolone, analgesic, anti-inflammatory, COX-2, 5-LOX,
3

1. Introduction
The inflammatory mediators, prostaglandins (PGs), leukotrienes (LTs) and
thromboxanes (TXs) are responsible for inflammation, other pathological and
physiological processes. [1] They are generated from arachidonic acid (AA), a poly
saturated fatty acid released from membrane phospholipids metabolism by the action of
cyclooxygenase (COX-1, -2, -3) and lipoxygenase (5-LOX, 8-, 12-, 15-) enzymes. [2-4]
COX enzymes are responsible for the production of PGs and TXs. COX-1 is a
constitutive enzyme found in the stomach, platelets and kidneys as a “house-keeper”
enzyme and involved in gastric protection, platelet aggregation and normal kidney
functions. COX-2 is an inducible enzyme found in macrophages, fibroblasts and
leukocytes and stimulated in response to pro-inflammatory mediators. COX-3, the third
cyclooxygenase is latterly discovered and present in central nervous system. [5-8]
Classical non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and
indomethacin exert their therapeutic action via suppressing PGs bio-synthesis through
non-selective inhibition of COX-1 and COX-2 enzymes resulting in serious adverse
effects like gastric pain, bleeding, ulcer and kidney complications. [9-10]
COX-2 selective NSAIDs (Coxibs) illustrated by celecoxib (Celebrex®),
valdecoxib (Bextra®) and rofecoxib (Vioxx®) have no effect on gastric mucosal
prostaglandin. But recent studies have shown the risk of some highly selective COX-2
inhibitors to increase the incidence of myocardial infarction leading to cardiac arrest due
to alteration in the COX-1/COX-2 biochemical pathway. [3, 11-12]
5-LOX is a human non-heme enzyme responsible for the production of LTs
involved in the inflammatory process. Zileuton, licofelone and meclofenamate sodium
(Meclomen®) are examples of orally active 5-LOX inhibitors. [13-14]
The incident of NSAIDs side effects is thought to be due to inhibition of one
enzyme pathway (COX) over the other (LOX) pathway leading to shift in AA
metabolism [15], hence the development of new anti-inflammatory (AI) agents targeting
both metabolic pathways of AA (COX-2 and 5-LOX) inhibition is a worthy rational
approach to obtain effective and safe NSAIDs. [14, 16-17]
Pyrazolone ring system is a core structure in numerous drugs displaying analgesic
and AI activities such as aminoantipytine, propyphenazone and famorofazone [18-20]
4

Several research studies reported the enhanced biological activities of
heterocyclic compounds incorporating hydrazono pharmacophore as analgesic and AI
agents with improved gastric profile due to its dual COX/5-LOX inhibitory activities.
[18, 21-23]
Furthermore, structure activity relationship studies of selective COX-2 inhibitors
demonstrated the importance of aminosulfonyl (SO2NH2) pharmacophore for COX-2
selectivity. [24-26]
On the basis of these findings and in continuation of our previous work [27-32] to
develop effective AI agents devoid from adverse effects, we describe the design,
synthesis, analgesic and AI activities of novel 4-aryl-hydrazonopyrazolone derivatives
incorporating both sulfonamoyl and hydrazone pharmacophores as COX-2 and 5-LOX
inhibitors. (Fig. 1) Ulcerogenic liability and histopathological screening were performed
in order to identify the non-ulcerogenic AI active compounds. Docking studies were also
performed to understand the possible binding modes of the synthesized compounds into
both COX-2 and 5-LOX active sites in order to explain their AI activities.
2. Results and Discussion
2.1. Chemical Synthesis
The synthetic pathways adopted for starting materials 2a-e and target compounds 3,
4, 5a-i are illustrated in Scheme 1, 2.
Hydrazones 2a-d were prepared via coupling the diazonium salt of different
primary aromatic amines with the active methylene group of ethyl acetoacetate. [33]
3-Oxo-2-[(4-sulfamoylphenyl)-hydrazono]-butyric acid ethyl ester (2e) was
prepared through diazotization of sulfanilamide 1e and coupling the formed diazonium
salt with ethyl acetoacetate in presence of sodium acetate. IR spectrum of compound 2e
demonstrated two absorption bands at 1690 and 1676 cm-1 corresponding to two C=O
1
groups while H NMR spectrum showed a D2O exchangeable peak at δ 11.60 ppm
indicating NH proton. Also, the presence of a singlet CH3, a triplet CH3 and a quartet
CH2 peaks at δ 2.47, 1.28 and 4.29 ppm corresponding to acetyl (COCH3) and ethoxy
(OCH2CH3) protons respectively confirmed the structure. These spectral data
demonstrated the existence of compounds 2a-f in hydrazone form (i) rather than azo form
(ii). (Scheme 1)
5

[Please insert Scheme 1 about here]
Cyclo-condensation ethanolic solution of 2e with an equimolar amount of hydrazine
1
hydrate afforded pyrazolone 3 in 74% yield. H NMR spectrum of 3 revealed the
disappearance of the signals due to ethoxy protons of the parent ester 2e. The presence of
additional D2O exchangeable singlet signal at δ 13.22 ppm corresponding to pyrazolone
NH proton, confirmed the reaction. In addition, the mass spectra of 3 displayed molecular
ion peak at m/z 281(92.94%).
Heating pyrazolone 3 with acetyl chloride afforded the N-acetylpyrazolone 4 in a
good yield of 63%. IR spectrum of 4 displayed an additional absorption band at 1749 cm-
1
due to the carbonyl group of N-acetyl moiety. The absence of pyrazolone NH peak of
the precursor 3 and presence of a peak of an additional signal of three protons integration
1
due to acetyl moiety at δ 2.45 ppm in H NMR spectrum of 4 confirmed the reaction.
13
Also, C NMR spectrum of compound 4 revealed the presence of two peaks at δ 24.20
and δ 171.61 ppm corresponding to COCH3 and COCH3, respectively.
Different substituted phenylhydrazine hydrochlorides were heated with hydrazones
2a-e under reflux in absolute ethanol to give the target compounds 5a-i in excellent yield
(68-85%). The reaction proceeds via addition of the more nucleophilic NH2 hydrazine
group to the reactive acetyl carbonyl group (COCH3) followed by intra-molecular
cyclization through nucleophilic substitution of the good leaving ethoxy group and loss of
an ethanol molecule. (Scheme 2)
The structure of diarylpyrazolones 5a–i was investigated by elemental and spectral
analyses. The IR spectra of compounds 5a-i indicated the presence of two absorption
bands at 3460-3313 cm-1 and 3279-3247 cm-1 corresponding to NH2 and NH groups in
addition to an absorption band at 1672-1650 cm-1 indicating C=O group at C-5 of
pyrazolone ring. Carboxy compounds, 5b, 5c, 5d, 5f, 5g, 5h displayed two additional
absorption bands at 3440-3416 cm-1 and 1701-1680 cm-1 due to carboxylic OH and C=O
groups, respectively. 1H NMR spectra of 5a-i showed the presence of a singlet signal at δ
2.29-2.34 ppm corresponding to methyl protons at C-3 of pyrazolone ring, in addition to
two exchangeable singlet signals at δ 7.22-7.42 corresponding to NH2 protons of
aminosulfonyl moiety, and at δ 13.03-13.66 for NH protons. Carboxy compounds 5b, 5c,
5d, 5f, 5g and 5h displayed carboxyl protons at δ 13.03-15.05 ppm. Also, 13C
NMR
6

spectra of 5a-i exhibited the methyl carbon at δ 12.14-12.30 ppm and carbonyl carbons at
13
δ 154.71-156.99 ppm. In Addition, C NMR spectra of carboxy compounds 5b, 5c, 5d,
5g, 5h displayed carboxy carbons at δ 167.17-168.67 ppm which confirmed the structure.
[Please insert Scheme 2 about here]
2.2. Biological activity
2.2.1. Analgesic activity (Hot plate latency test)
Analgesic activity of compounds 3, 4 and diarylpyrazolones 5a-i was evaluated
applying hot plate latency test [34]. Oral administration of tested compounds produced a
significant delay in the latency time relative to basal values except for compounds 3, 4,
5e and 5i which displayed weak delay in latency time and didn’t reach statistical
significance (Table 1). Compound 5f caused the highest percentage increase in pain
threshold (50%) compared to reference drug indomethacin (PIP = 53.13%). Mice treated
with compounds 5a, 5b and 5c showed moderate analgesic activity on thermal pain (PIP
= 28.13, 31.25, 21.88 % sequentially). Three compounds 5d, 5g and 5h were less potent
in order of 5h (PIP = 18.75%) > 5d = 5g (PIP = 15.63%)
[Please insert Table 1 about here]
2.2.2. In vitro cyclooxygenase (COX-1/2) and lipoxygenase (5-LOX) inhibition assays
(IC50 values, μM) were measured to evaluate the potency of prepared compounds
3, 4, 5a-i, indomethacin and celecoxib to inhibit COX-1/2 enzymes as the concentration
causing 50% enzyme inhibition using a colorimetric EIA kit [35-36]. Also, COX-2 SI
(COX-2 selectivity index) values were calculated as [IC50 (COX-1) / IC50 (COX-2)] and
compared to standard drugs: a non-selective COX inhibitor; indomethacin and a selective
COX-2 inhibitor; celecoxib. (Table 2)
All the tested compounds inhibited COX-1 enzyme at higher concentrations (IC50
= 1.32 - 4.66 μM) than indomethacin (IC50 = 1.14 μM). Compounds 3, 5b, 5d, 5f and 5i
showed COX-1 inhibitory activity at a higher dose range (IC50 = 3.76 - 4.66 μM) than
celecoxib (IC50 = 3.14 μM), while compounds 4, 5a, 5c, 5e, 5g and 5h were close in
potency to celecoxib with IC50 range of (1.32 - 2.68 μM). Accordingly, all target
compounds are considered as weak COX-1 enzyme inhibitors and expected to have a safe
gastric profile.
7

Alternatively, all tested compounds inhibited COX-2 enzyme at lower dose range
of (IC50 = 0.66 - 2.04 μM) than indomethacin (IC50 = 7.24 μM). Three compounds; 5d
(IC50 = 0.77 μM), 5f (IC50 = 0.72 μM) and 5i (IC50 = 0.66 μM) were more potent than
celecoxib (IC50 = 0.89 μM) while compounds 3, 4, 5b, 5c, 5h showed moderate COX-2
inhibitory activity (IC50 = 1.00 – 1.42 μM). Three compounds (5a, 5e, 5g) were weak
COX-2 inhibitors (IC50 = 1.59 - 2.04 μM).
Regarding COX-2 selectivity index values, all target compounds were more
selective to COX-2 enzyme than COX-1 (SI = 1.32 - 5.69) except 5g (SI = 0.82).
Compounds 5d, 5f and 5i (SI = 5.29- 5.69) exhibited superior SI values than celecoxib
(SI = 3.52) while compounds 3, 5b showed selectivity indices of (3.41 and 3.47)
respectively close to that of celecoxib. The rest of compounds displayed SI values (SI =
1.32 - 2.17) in order of 5c>5e>5a>5h>4. Presence of two aminosulfonyl (SO2NH2)
moieties; the most important pharmacophore for COX-2 selectivity in pyrazolone 5i may
explain its maximum COX-2 selectivity (SI = 5.96).
Concerning data acquired from in vitro lipoxygenase (5-LOX) inhibition assay
demonstrated that three compounds (5d, 5f and 5i) showed higher 5-LOX inhibitory
activity (IC50 = 0.53, 0.52 and 0.57 µM in sequent) than standard drug, Zileuton (IC50 =
0.77 µM). Six compounds (3, 4, 5b, 5c, 5e, and 5h) displayed moderate activities (IC50 =
0.84 – 1.17 µM) close to that of Zileuton. Two compounds; 5a (IC50 = 1.42 µM) and 5g
(IC50 = 1.59 µM) exhibited the least 5-LOX inhibitory activity. (Table 2)
Collectively, three derivatives (5d, 5f, and 5i) of the designed target compounds
were found to have superior inhibitory activity against both COX-2 and 5-LOX with
excellent COX-2 SI values and can be considered as promising safe AI agents via dual
COX-2/5-LOX inhibition.
[Please insert Table 2 about here]
2.2.3. In vivo anti-inflammatory activity
2.2.3.1. Carrageenan-induced rat paw edema assay)
AI activities of all synthesized compounds were evaluated employing carrageen-
induced rat foot paw edema model using celecoxib as a reference drug [37-38]. Increase
in paw thickness, edema inhibition percentage (EIP) and ED50 values displayed by the
8

prepared pyrazolones and celecoxib at 2h and 4h after carrageenan injection are listed in
Table 3.
Tested compounds 3, 4, 5a-i showed a wide range of EIP in the range of 18.18 to
72.72% after both time intervals used. The benzoic acid derivatives 5b and 5f that
possess carboxyl moiety at position 2 displayed the highest AI activity (EIP = 72.72%; 2h
and 67.67%; 4h). This could be due to the possibility of intra-molecular hydrogen bond
formation which enhances membrane permeability and absorption. (Fig. 2) Compounds
5a, 5c, 5d and 5h showed good AI activity (EIP = 58.18 - 49.09%) in order of 5c = 5h
>5a = 5d after 2h and 5d >5c >5h >5a after 4h. Five derivatives 3, 4, 5e, 5g, 5i displayed
lower AI activity in sequence of 3 = 5i >4 = 5e >5g at two different time intervals.
[Please insert Fig. 2 about here]
Regarding ED50 values, compound 5f was the most potent AI agent (ED50 = 0.044
(2h) and 0.064 (4h) mmol/Kg) relative to celecoxib (ED50 = 0.032 mmol/Kg).
Compounds 5b, 5c and 5d exhibited promising degree of AI activity (ED50 = 0.079 –
0.084 mmol/Kg; 2h and 0.072 - 0.089 mmol/Kg; 4h). Compounds 3, 4, 5a, 5e, 5g, 5h and
5i showed moderate AI activity with ED50 values in the range of (0.104 – 0.588
mmol/Kg) and compounds’ potency were in order of 5h >5a>5i>3>5g>4>5e.
[Please insert Table 3 about here]
Ulcerogenic liability
Test compounds 5b, 5c, 5d and 5f that showed potent in vivo AI activity (ED50 =
0.044 – 0.104 mmol/kg) were further evaluated for their ulcerogenic liability and
compared to indomethacin; an ulcerogenic drug and celecoxib; a safe drug [39]. Ulcer
index values were calculated and tabulated (Table 4). The data obtained showed that
celecoxib and tested compounds caused a much smaller number of ulcers with ulcer
index (UI = 2.33 – 9.67) when compared to indomethacin (UI = 21.33). Compound 5f
was less ulcerogenic (UI = 2.33) than celecoxib (UI = 3.00) while the other analog, 5b
(UI = 4.67) showed a close ulcerogenic effect to celecoxib. Compounds 5c and 5d caused
higher gastric ulceration effect (UI = 9.67, 6.67 in sequent).
[Please insert Table 4 about here]
Although 5b, 5c, 5d and 5f hold an acidic center; carboxyl group and was
expected to have a local ulcerogenic effect, they were much safer than indomethacin.
9

Moreover, the most preferable pyrazolone 5f with the carboxyl group at position 2 of
phenyl ring was safer than celecoxib. This may be due to the formation of intra-molecular
hydrogen bond between the carboxyl group and pyrazolone N-2 forming a pseudo-ring
that withdraws topical irritant effect of the carboxyl group on the epithelium (Fig. 2).
[Please insert Fig. 2 about here]
2.2.3.3. Histopathological study
Examining histopathological lesions produced after drug intake was performed to
explore the ulcerogenic effect of compounds 5b, 5c, 5d and 5f on stomach in comparison
to celecoxib or indomethacin (Table 5). In control negative group, more or less normal
histological structure of glandular gastric mucosa, and submucosa. The non-glandular
portion showed normal histological structure (Fig. 3 (i), A-I, A-II). Administrations of
indomethacin led to severe degenerative changes and necrosis in both glandular and non-
glandular portions of the stomach. The glandular stomach showed variable degrees of
erosive and ulcerative changes accompanied by submucosal leukocytic infiltration,
edema, and congestion. The muscular layer suffered from severe degenerative changes,
hyalinosis, and massive leukocytic infiltration. The non-glandular stomach showed focal
erosive gastritis and hyperkeratosis (Fig. 3 (i), B-I— B-IV). The celecoxib treated rats
showed moderate pathological lesions in the glandular stomach in form of degenerative
changes of mucosal lining, submucosal congestion and mild leukocytic infiltration.
Additionally, the mucosal lining of the non-glandular stomach portion was more or less
normal and mild congestion could be detected (Fig. 3 (i), C-I, C-II).
[Please insert Table 5 about here]
Treatment with compound 5b caused mild to moderate degeneration of mucosal
lining of the glandular portion of the stomach. The degenerative changes were mainly in
form of hydropic degeneration which consequently led to microvesicle formation in
certain areas and presence of mild congestion and edema in the submucosal blood
vessels. In addition, the muscular layer showed mild hyalinosis. More or less normal
histological structure of the non-glandular stomach with moderate hyperkeratosis in small
focal areas was also detected (Fig. 3 (ii), 5b-I, 5b-II).
Compound 5c intake caused severe degenerative changes and sloughing of
glandular portion of stomach in certain areas accompanied with massive leukocytic
10

infiltration while the muscular layer showed moderate hyalinosis. Also, moderate
degenerative changes of mucosa and submucosa were detected in other areas. Besides,
the presence of focal areas of hyperkeratosis in the non-glandular stomach was also
observed (Fig. 3 (ii), 5c-I, 5c-II).
Scanning stomach of rats treated with compound 5d, showed presence of
multifocal areas of mucous degeneration in the lining epithelium associated with some
necrotic changes in another area. The submucosa was suffering from moderate
congestion and leukocytic infiltration while the muscular layer suffered from mild to
moderate degenerative changes and hyalinosis. Also, mild hyperkeratosisof the non-
glandular stomach was detected (Fig. 3 (ii), 5d-I, 5d-II).
Stomach specimens isolated from rats treated with 5f showed more or less normal
mucosal lining of glandular stomach accompanied with minimal congestion, edema and
leukocytic infiltration of the submucosa and very mild degenerative changes of the
musculosa. The normal histological structure of the non-glandular stomach was also
observed (Fig. 3 (ii), 5f-I, 5f-II).
[Please insert Fig. 3 about here]
In summary, the preferable pyrazolone 5f induced minimal pathological lesions of
both glandular and non-glandular stomach and can be considered as a safe AI agent while
the use of 5b caused mild lesions. Alternatively, compound 5c caused severe
degenerative and necrotic changes in the glandular stomach associated with degenerative
changes and hyperkeratosis in the non-glandular stomach relative to the less severe
lesions detected in glandular and non-glandular stomach by the use of 5d. On the other
hand, severe ulcerative changes, congestion, and leukocytic infiltration could be detected
with indomethacin treatment.
2.3. Molecular docking study
In order to investigate the possible binding conformations of all synthesized
compounds with either COX-2 (PDB code 3LN1) or 5-LOX (PDB code 3V99) receptors,
molecular docking study was carried out using MOE (version 2008.10) modeling
software [41-42]. Docking scores, amino acid residues forming Hydrogen bonding (H-
bonds) interactions and their lengths were summarized in Table 6.
[Please insert Table 6 about here]
11

Regarding COX-2 enzyme, Celecoxib aminosulfonyl moiety (NH2 and SO2
groups) made two H-bonding interactions with Gln178 and Arg499 amino acids in a
distance of 3.20 and 2.95 Ao in sequent, with an energy score equals to -12.96 Kcal/Mol.
All the compounds subjected to molecular docking fitted well to COX-2 active
site inside the pocket and showed good binding energy scores ranged from -11.22 to -
16.35 Kcal/Mol. Also, aminosulfonyl moiety of all compounds showed H-bond
interactions with Gln178, Arg499, Ser516, Tyr341, Tyr371, Arg106 and Phe504 amino
acids of the active site which proved the importance of SO2NH2 as a pharmacophoric
moiety in COX-2 selectivity. The most active AI compounds 5b, 5c, 5d and 5f made one
to four H-bonds with distance of (2.32 – 3.17 Ao) showing excellent docking scores (-
14.18 to -16.35 Kcal/Mol).
For 5-LOX enzyme, AA made one H-bond with His367 amino acid with distance
of 2.77 Ao and energy score of -9.00 Kcal/Mol. The synthesized compounds made one or
two H-bond interactions with distance in range of 1.53 to 3.03 Ao exhibiting energy
scores (-6.46 — -8.29 Kcal/Mol). The proposed binding modes of compound 5f inside
COX-2 and 5-LOX receptor active sites are displayed in Fig. 4 (I and II).
[Please insert Fig. 4 about here]
3. Conclusion
In this work, we represent the design and synthesis of novel 4-aryl-
hydrazonopyrazolones 3, 4, 5a-i as analgesic and anti-inflammatory agents. All target
compounds were screened for their in vitro COX-1, COX-2 and 5-LOX inhibitory
activity in addition to their in vivo analgesic and anti-inflammatory activities. Most s
compounds were found to be good inhibitors of both COX-2 (IC50 = 0.66 -2.04 μM,
reference celecoxib IC50 = 0.89 μM) and 5-LOX (IC50 = 0.52 - 1.59 μM, reference
zileuton IC50 = 0.77 μM). In vivo biological testing showed that compounds 5b, 5c, 5d
and 5f [ED50 = 0.044 – 0.084 mmol/Kg (2h) and 0.064 – 1.104 mmol/Kg (4h)] were the
most potent AI agents and were further screened for their ulcerogenic liability and
histopathological studies to give an evidence for the most preferable AI agent. pyrazolone
5f (UI = 2.33, references: celecoxib UI = 3.00 and indomethacin UI = 21.33) caused
minimal pathological lesions on both glandular and non-glandular stomach. Also, 5f
showed 50% delay in latency time to thermal pain induced by hot plate close to reference
12

drug indomethacin (53.13%). Docking studies showed the importance of aminosulfonyl
moiety in celecoxib and prepared compounds in hydrogen bond formation with the COX-
2 active site.
4. Experimental
4.1. Chemistry
General Information: Melting points were determined on a Thomas-Hoover capillary
apparatus and are uncorrected. Infrared (IR) spectra were recorded as films on KBr desks
1
using a Nicolet 550 Series II Magna FT-IR spectrometer (Middleton, WI). H NMR and
13C NMR spectra were measured on a Bruker Avance III 400 MHz (Bruker BioSpin AG,
1
Fa¨llanden, Switzerland) for H and 100 MHz for 13C with BBFO Smart Probe and
Bruker 400 AEON Nitrogen-Free Magnet, Faculty of Pharmacy, Beni-Suef University,
Egypt, in DMSO-d6 with TMS as the internal standard, where J (coupling constant)
values are estimated in Hertz (Hz) and chemical shifts were recorded in ppm on δ scale.
Mass spectra (MS) were recorded on a Hewlett Packard 5988 spectrometer (Palo Alto,
CA). Microanalyses for C, H and N were carried out on Perkin-Elmer 2400 analyzer
(Perkin-Elmer, Norwalk, CT) at the Micro analytical unit of Cairo University, Egypt, and
all compounds were within ± 0.4% of the theoretical values.
General Procedure for synthesis of 3-oxo-2-aryl-hydrazonobutyric acid ethyl ester
(2a-e): To an ice cooled solution of various substituted aromatic amines 1a-e (0.01 mol)
in hydrochloric acid (2.5 mL) and distilled water (5 mL), a solution of sodium nitrite (0.9
g, 0.013 mol) in distilled water (5 mL) was added portion-wise. The resulting diazonium
salt solution was then added to a cold solution of ethyl acetoacetate (1.3 g, 0.01 mol) in
aqueous ethanol (50%, 10 mL) containing sodium acetate (0.9 g, 0.011 mol). The
reaction mixture was stirred in ice bath for 2 h and then filtered, dried and crystallized
from ethanol.
3-Oxo-2-[(4-sulfamoylphenyl)-hydrazono]-butyric acid ethyl ester (2e). Yellow solid;
o
94% yield; mp 239-241 C; IR (KBr, υ cm-1): 3344, 3253 (NH2), 3232(NH), 3080 (C-H
aromatic), 2964 (C-H aliphatic), 1690 (COOCH2CH3), 1676 (COCH3), 1551 (C=N),
1
1343, 1155 (SO2); H NMR (DMSO-d6, δ ppm): 1.28 (t, J = 7.2 Hz, 3H, CH2CH3), 2.47
(s, 3H, COCH3), 4.29 (q, J = 7.2 Hz, 2H, CH2CH3), 7.30 (s, 2H, NH2, exchangeable with
D2O), 7.55 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.81 (d, J = 8.4 Hz, 2H,
13

aminosulfonylphenyl H-2, H-6), 11.61 (s, 1H, NH, exchangeable with D2O); MS (m/z,
%): 313 (M+., 46.26). Anal. Calcd. for C12H15N3O5S: C, 46.00; H, 4.83; N, 13.41;. Found:
C, 46.35; H, 4.92; N, 13.54.
Procedure for synthesis of 4-[(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-ylidene)-
hydrazin-1-yl]-benzenesulfonamide (3): A mixture of 3-oxo-2-[(4-sulfamoylphenyl)-
hydrazono]-butyric acid ethyl ester (2e, 7.8 g, 0.025 mol) and hydrazine hydrate 99% (0.8
g, 0.025 mol) in absolute ethanol (30 mL) was heated under reflux for 4 h. After cooling
to room temperature, the separated solid was filtered off. The obtained crude product was
crystallized from aqueous ethanol to afford 3 as orange solid; 74% yield; mp 135-137 oC;
IR (KBr, υ cm-1): 3460, 3340 (NH2), 3311, 3214(2NH), 3090 (C-H aromatic), 2927 (C-H
1
aliphatic), 1671 (C=O), 1551 (C=N), 1337, 1150 (SO2); H NMR (DMSO-d6, δ ppm):
2.16 (s, 3H, CH3), 7.35 (s, 2H, NH2, exchangeable with D2O), 7.67 (d, J = 8.4 Hz, 2H,
aminosulfonylphenyl H-3, H-5), 7.84 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-2, H-6),
11.65 (s, 1H, NH, exchangeable with D2O), 13.22 (s, 1H, NH, exchangeable with D2O);
13C NMR (DMSO-d6, δ ppm): 12.07 (CH3), 115.99 (CH, aminosulfonylphenyl C-3, C-5),
127.83 (CH, aminosulfonylphenyl C-2, C-6), 130.23 (C, aminosulfonylphenyl C-1),
140.10 (C, aminosulfonylphenyl C-4), 144.54 (C, pyrazole C-4), 147.59 (C, pyrazole C-
3), 160.35 (C, C=O); MS (m/z, %): 281 (M+., 92.94). Anal. Calcd. for C10H11N5O3S: C,
42.70; H, 3.94; N, 24.90. Found: C, 42.45; H, 4.12; N, 24.81.
Procedure for synthesis of 4-[(1-Acetyl-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-
ylidene)-hydrazin-1-yl]-benzenesulfonamide (4): A well-stirred mixture of 4-[(3-methyl-
5-oxo-4,5-dihydro-1H-pyrazol-4-ylidene)-hydrazin-1-yl]-benzenesulfonamide (3, 0.807
g, 0.0025 mol) and glacial acetic acid (15 mL) was heated under reflux for 6 h. After
cooling, the formed solid was filtered off, dried and crystallized from n-butanol to afford
o
4 as yellow solid; 63% yield; mp 245-247 C; IR (KBr, υ cm-1): 3422, 3329 (NH2),
3235(NH), 3118 (C-H aromatic), 2927 (C-H aliphatic), 1749, 1672 (2C=O), 1549 (C=N),
1346, 1157 (SO2); 1H NMR (DMSO-d6, δ ppm): 2.25 (s, 3H, CH3), 2.45 (s, 3H, COCH3),
7.39 (s, 2H, NH2, exchangeable with D2O), 7.78 (d, J = 7.6 Hz, 2H, aminosulfonylphenyl
H-3, H-5), 7.87 (d, J = 7.6 Hz, 2H, aminosulfonylphenyl H-2, H-6), 13.03 (s, 1H, NH,
13
exchangeable with D2O); C NMR (DMSO-d6, δ ppm): 12.81 (CH3), 24.20 (COCH3),
116.88 (CH, aminosulfonylphenyl C-3, C-5), 127.84 (CH, aminosulfonylphenyl C-2, C-
14

6), 128.31(C, aminosulfonylphenyl C-1), 141.06 (C, aminosulfonylphenyl C-4), 151.28
(C, pyrazole C-4), 157.71 (C, pyrazole C-3), 166.59 (C, C=O), 171.61 (C, COCH3);
Anal. Calcd. for C12H13N5O4S: C, 44.58; H, 4.05; N, 21.66. Found: C, 44.49; H, 4.02; N,
21.62.
General procedure for preparation of 1,4-diarylpyrazolones (5a-i): A mixture of the
appropriate hydrazone 2a-e (0.035 mol), anhydrous sodium acetate (0.5 g) and the proper
phenyl hydrazine hydrochloride (0.035 mol) in absolute ethanol (25 mL) was heated
under reflux for 10 h. The separated solid was filtered off, dried and crystallized from
aqueous ethanol to afford 5a-i in excellent yield (68 – 85%).
4-[3-methyl-5-oxo-4-(2-phenylhydrazin-1-ylidene)-4,5-dihydro-1H-pyrazol-1-yl]benzene-
o
1-sulfonamide (5a). Orange solid; 81% yield; mp 163-165 C; IR (KBr, υ cm-1): 3426,
3348 (NH2), 3258 (NH), 3070 (C-H aromatic), 2924 (C-H aliphatic), 1665 (C=O), 1592
(C=N), 1339, 1164 (SO2);1H NMR (DMSO-d6, δ ppm): 2.34 (s, 3H, CH3), 7.27-7.29 (m,
1H, phenyl H-4), 7.36 (s, 2H, NH2, exchangeable with D2O), 7.46-7.48 (m, 2H, phenyl
H-2, H-6), 7.65 (d, J = 7.2 Hz, 2H, phenyl H-3, H-5), 7.91 (d, J = 8.4 Hz, 2H,
aminosulfonylphenyl H-3, H-5), 8.13 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-2, H-6),
13.23 (s, 1H, NH, exchangeable with D2O); MS (m/z, %): 357 (M+., 1.87). Anal. Calcd.
for C16H15N5O3S: C, 53.77; H, 4.23; N, 19.60. Found: C, 53.63; H, 4.19; N, 19.73
2-{2-[3-methyl-5-oxo-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazol-4-ylidene]hydrazin-
o
1-yl}benzoic acid (5b). Orange red solid; 88% yield; mp 183-185 C; IR (KBr, υ cm-1):
3422-3313 (OH and NH2), 3247(NH), 3066 (C-H aromatic), 2925 (C-H aliphatic), 1701,
1664 (2C=O), 1588 (C=N), 1329, 1156 (SO2); 1H NMR (DMSO-d6, δ ppm): 2.33 (s, 3H,
CH3), 7.26-7.28 (m, 1H, benzoic acid H-3), 7.39 (s, 2H, NH2, exchangeable with D2O),
7.66-7.68 (m, 1H, benzoic acid H-5), 7.90 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-3,
H-5), 7.99-8.02 (m, 2H, benzoic acid H-4, H-6), 8.13 (d, J = 8.8 Hz, 2H,
aminosulfonylphenyl H-2, H-6), 13.66 (s, 1H, NH, exchangeable with D2O), 15.08 (s,
1H, COOH, exchangeable with D2O); 13C NMR (DMSO-d6, δ ppm): 12.30 (CH3), 115.22
(CH, benzoic acid C-3), 117.38 (CH, benzoic acid C-5), 119.12 (C, benzoic acid C-1),
125.03 (CH, aminosulfonylphenyl C-3, C-5), 127.37 (CH, aminosulfonylphenyl C-2, C-
6), 129.27 (C, aminosulfonylphenyl C-1), 131.91 (CH, benzoic acid C-6), 134.28 (CH,
benzoic acid C-4), 139.87 (C, aminosulfonylphenyl C-4), 140.98 (C, benzoic acid C-2),
15

144.21 (C, pyrazole C-4), 150.19 (C, pyrazole C-3), 156.54 (C, C=O), 168.67 (C,
COOH); MS (m/z, %): 401 (M+., 10.52). Anal. Calcd. for C17H15N5O5S: C, 50.87; H,
3.77; N, 17.45. Found: C, 50.63; H, 3.69; N, 17.43
3-{2-[3-methyl-5-oxo-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazol-4-ylidene]hydrazin-
1-yl}benzoic acid (5c). Orange solid; 68% yield; mp 190-192 oC; IR (KBr, υ cm-1): 3416-
3353 (OH and NH2), 3263(NH), 3097 (C-H aromatic), 2981 (C-H aliphatic), 1693, 1672
(2C=O), 1554 (C=N), 1337, 1159 (SO2); 1H NMR (DMSO-d6, δ ppm): 2.29 (s, 3H, CH3),
7.37 (s, 2H, NH2, exchangeable with D2O), 7.67-7.69 (m, 3H, benzoic acid H-4, H-5, H-
6), 7.92-7.97 (m, 4H, aminosulfonylphenyl H-2, H-3, H-5, H-6), 8.08 (s, 1H, benzoic
acid H-2), 13.05 (s, 2H, NH and COOH, exchangeable with D2O); 13C NMR (DMSO-d6,
δ ppm): 12.14 (CH3), 115.82 (CH, benzoic acid C-2), 116.41 (CH, benzoic acid C-6),
117.56 (CH, benzoic acid C-4), 121.46 (CH, aminosulfonylphenyl C-3, C-5), 127.41
(CH, aminosulfonylphenyl C-2, C-6), 127.78 (C, benzoic acid C-1), 129.31 (C,
aminosulfonylphenyl C-1), 131.40 (CH, benzoic acid C-5), 140.21 (C,
aminosulfonylphenyl C-4), 140.63 (C, benzoic acid C-4), 145.29 (C, pyrazole C-4),
150.10 (C, pyrazole C-3), 156.99 (C, C=O), 167.17 (C, COOH); MS (m/z, %): 401 (M+.,
20.84). Anal. Calcd. for C17H15N5O5S: C, 50.87; H, 3.77; N, 17.45. Found: C, 51.03; H,
3.79; N, 17.48
4-{2-[3-methyl-5-oxo-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-pyrazol-4-ylidene]hydrazin-
o
1-yl}benzoic acid (5d). Orange red solid; 77% yield; mp 198-200 C; IR (KBr, υ cm-1):
3425-3354 (OH and NH2), 3263(NH), 3100 (C-H aromatic), 2925 (C-H aliphatic), 1693,
1672 (2C=O), 1604 (C=N), 1337, 1160 (SO2); 1H NMR (DMSO-d6, δ ppm): 2.33 (s, 3H,
CH3), 7.37 (s, 2H, NH2, exchangeable with D2O), 7.72 (d, J = 8.4 Hz, 2H, benzoic acid
H-3, H-5), 7.92 (d, J = 8.4 Hz, 2H, benzoic acid H-2, H-6), 8.00 (d, J = 8 Hz, 2H,
aminosulfonylphenyl H-3, H-5), 8.11 (d, J = 8 Hz, 2H, aminosulfonylphenyl H-2, H-6),
13
13.09 (s, 2H, NH and COOH, exchangeable with D2O); C NMR (DMSO-d6, δ ppm):
12.18 (CH3), 116.53 (CH, benzoic acid C-3, C-5), 117.59 (CH, aminosulfonylphenyl C-3,
C-5), 127.42 (CH, aminosulfonylphenyl C-2, C-6), 127.80 (C, benzoic acid C-1), 129.28
(C, aminosulfonylphenyl C-1), 131.42 (CH, benzoic acid C-2, C-6), 140.13 (C,
aminosulfonylphenyl C-4), 140.71 (C, benzoic acid C-4), 145.93 (C, pyrazole C-4),
150.23 (C, pyrazole C-3), 156.97 (C, C=O), 167.23 (C, COOH); MS (m/z, %): 401 (M+.,
16

5.16). Anal. Calcd. for C17H15N5O5S: C, 50.87; H, 3.77; N, 17.45. Found: C, 50.78; H,
3.84; N, 17.57
4-{2-[3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-ylidene]hydrazin-1-
yl}benzene-1-sulfonamide (5e). Dark yellow solid; 68% yield; mp 184-186 oC; IR (KBr, υ
cm-1): 3440, 3320 (NH2), 3247(NH), 3070 (C-H aromatic), 2922 (C-H aliphatic), 1655
(C=O), 1601 (C=N), 1329, 1155 (SO2); 1H NMR (DMSO-d6, δ ppm): 2.32 (s, 3H, CH3),
7.24 (t, J = 7.6 Hz, 1H, phenyl H-4), 7.39 (s, 2H, NH2, exchangeable with D2O), 7.47 (t, J
= 7.6 Hz, 2H, phenyl H-3, H-5), 7.78 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-3, H-5),
7.87 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-2, H-6), 7.92 (d, J = 8 Hz, 2H, phenyl H-
2, H-6), 13.24 (s, 1H, NH, exchangeable with D2O); 13C NMR (DMSO-d6, δ ppm): 12.14
(CH3), 116.62 (CH, aminosulfonylphenyl C-3, C-5), 118.21 (CH, phenyl C-2, C-6),
125.47 (CH, phenyl C-4), 127.81 (CH, aminosulfonylphenyl C-2, C-6), 129.87 (CH,
phenyl C-3, C-5), 129.87 (C, aminosulfonylphenyl C-1), 138.23 (C, phenyl C-1), 140.72
(C, aminosulfonylphenyl C-4), 144.48 (C, pyrazole C-4), 149.26 (C, pyrazole C-3),
154.71 (C, C=O); MS (m/z, %): 357 (M+., 9.34). Anal. Calcd. for C16H15N5O3S: C, 53.77;
H, 4.23; N, 19.60. Found: C, 53.64; H, 4.19; N, 19.63.
2-[3-methyl-5-oxo-4-[2-(4-sulfamoylphenyl)hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-
1-yl]benzoic acid (5f). Orange solid, 80% yield; mp 185-187 oC; IR (KBr, υ cm-1): 3460-
3330 (OH and NH2), 3279(NH), 3081 (C-H aromatic), 2923 (C-H aliphatic), 1692, 1650
(2C=O), 1601 (C=N), 1338, 1153 (SO2); 1H NMR (DMSO-d6, δ ppm): 2.30 (s, 3H, CH3),
7.32 (s, 2H, NH2, exchangeable with D2O), 7.49 (d, J = 8 Hz, 2H, aminosulfonylphenyl
H-3, H-5), 7.56 (t, J = 7.6 Hz, 1H, benzoic acid H-5), 7.68-7.71 (m, 3H, benzoic acid H-
3, H-4, H-6), 7.81 (d, J = 8 Hz, 2H, aminosulfonylphenyl H-2, H-6), 13.22 (s, 2H, NH
and COOH, exchangeable with D2O); MS (m/z, %): 401 (M+., 1.48). Anal. Calcd. for
C17H15N5O5S: C, 50.87; H, 3.77; N, 17.45. Found: C, 50.80; H, 3.56; N, 17.19
3-[3-methyl-5-oxo-4-[2-(4-sulfamoylphenyl)hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-
o
1-yl]benzoic acid (5g). Orange red solid; 74% yield; mp 180-182 C; IR (KBr, υ cm-1):
3435-3350 (OH and NH2), 3253(NH), 3090 (C-H aromatic), 2925 (C-H aliphatic), 1680,
1671 (2C=O), 1594 (C=N), 1337, 1159 (SO2); 1H NMR (DMSO-d6, δ ppm): 2.29 (s, 3H,
CH3), 6.86 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.22 (s, 2H, NH2,
exchangeable with D2O), 7.51-7.57 (m, 2H, benzoic acid H-3, H-4), 7.76 (d, J = 7.2 Hz,
17

1H, benzoic acid H-6), 7.90 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-2, H-6), 8.21 (s,
1H, benzoic acid H-2), 13.06 (s, 2H, NH and COOH exchangeable with D2O); 13C NMR
(DMSO-d6, δ ppm): 12.29 (CH3), 120.04 (CH, aminosulfonylphenyl C-3, C-5), 121.32
(CH, benzoic acid C-2), 123.93 (CH, benzoic acid C-4), 124.46 (CH, benzoic acid C-6),
126.95 (CH, aminosulfonylphenyl C-2, C-6), 129.87 (C, aminosulfonylphenyl C-1),
130.55 (CH, benzoic acid C-5), 131.91 (CH, benzoic acid C-1), 136.18 (C, benzoic acid
C-3), 144.43 (C, pyrazole C-4), 150.82 (C, aminosulfonylphenyl C-4), 151.21 (C,
pyrazole C-3), 156.34 (C, C=O), 168.05 (C, COOH); MS (m/z, %): 401 (M+., 1.34).
Anal. Calcd. for C17H15N5O5S: C, 50.87; H, 3.77; N, 17.45. Found: C, 50.92; H, 3.62; N,
17.23
4-[3-methyl-5-oxo-4-[2-(4-sulfamoylphenyl)hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-
o
1-yl]benzoic acid (5h). Orange red solid; 71% yield; mp 190-192 C; IR (KBr, υ cm-1):
3430-3340 (OH and NH2), 3253(NH), 3060 (C-H aromatic), 2923 (C-H aliphatic), 1680,
1666 (2C=O), 1592 (C=N), 1332, 1154 (SO2); 1H NMR (DMSO-d6, δ ppm): 2.31 (s, 3H,
CH3), 7.42 (s, 2H, NH2, exchangeable with D2O), 7.77 (d, J = 8.4 Hz, 2H,
aminosulfonylphenyl H-3, H-5), 7.88 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-2, H-6),
8.02 (d, J = 8.8 Hz, 2H, benzoic acid H-3, H-5), 8.06 (d, J = 8.8 Hz, 2H, benzoic acid H-
2, H-6), 13.03 (s, 2H, NH and COOH, exchangeable with D2O); 13C NMR (DMSO-d6, δ
ppm): 12.24 (CH3), 116.96 (CH, aminosulfonylphenyl C-3, C-5), 117.19 (CH, benzoic
acid C-3, C-5), 126.92 (C, benzoic acid C-1), 127.75 (CH, aminosulfonylphenyl C-2, C-
6), 129.20 (C, aminosulfonylphenyl C-1), 131.11 (CH, benzoic acid C-2, C-6), 140.88
(CH, benzoic acid C-4), 141.81 (C, aminosulfonylphenyl C-4), 144.82 (C, pyrazole C-4),
150.18 (C, pyrazole C-3), 156.54 (C, C=O), 167.24 (C, COOH); MS (m/z, %): 401 (M+.,
17.01). Anal. Calcd. for C17H15N5O5S: C, 50.87; H, 3.77; N, 17.45. Found: C, 50.79; H,
3.71; N, 17.52
4-[3-methyl-5-oxo-4-[2-(4-sulfamoylphenyl)hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-
1-yl]benzene-1-sulfonamide (5i). Dark orange solid; 83% yield; mp 215-217 oC; IR (KBr,
υ cm-1): 3425-3340 (2NH2), 3260(NH), 3098 (C-H aromatic), 2925 (C-H aliphatic), 1670
(C=O), 1590 (C=N), 1331, 1157 (2SO2); 1H NMR (DMSO-d6, δ ppm): 2.34 (s, 3H, CH3),
7.40 (s, 2H, NH2, exchangeable with D2O), 7.43 (s, 2H, NH2, exchangeable with D2O),
7.79 (d, J = 8.4 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.88 (d, J = 8.4 Hz, 2H,
18

aminosulfonylphenyl H-2, H-6), 7.93 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-2’, H-
6’), 8.11 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-3’, H-5’), 13.21 (s, 1H, NH,
exchangeable with D2O); 13C NMR (DMSO-d6, δ ppm): 12.21 (CH3), 116.79 (CH,
aminosulfonylphenyl C-3’, C-5’), 117.56 (CH, aminosulfonylphenyl C-3, C-5), 127.47
(CH, aminosulfonylphenyl C-2, C-6), 127.81 (CH, aminosulfonylphenyl, C-2’, C-6’),
129.47 (C, aminosulfonylphenyl C-1’), 140.28 (C, aminosulfonylphenyl C-1), 140.64 (C,
aminosulfonylphenyl C-4), 140.97 (C, aminosulfonylphenyl C-4’), 144.43 (C, pyrazole
C-4), 150.22 (C, pyrazole C-3), 156.99 (C, C=O); MS (m/z, %): 436 (M+., 10.67). Anal.
Calcd. for C16H16N6O5S2: C, 44.03; H, 3.69; N, 19.25. Found: C, 44.14; H, 3.62; N, 19.40
4.2. Biological activity
Animals: Adult male Swiss albino mice (20–25 g) and Wistar albino rats (100 - 150 g)
were used all through the study to examine analgesic activity, AI activity and ulcerogenic
liability. The animals (five per cage) were housed under controlled laboratory conditions
o
(12/12 h light/dark cycle, humidity 60 ± 10 % and temperature 23 ± 2 C), and were
allowed standard chow and water ad libitum. All experimental procedures were carried
out in strict compliance with the Institutional Animal Ethics Committee regulations of
Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt.
4.2.1. Analgesic activity (hot plate latency test)
Hot plate latency (seconds) was evaluated in animals receiving normal saline or
test agents (50 mg/kg) at 2 h after administration [34]. Celecoxib and indomethacin (10
mg/kg) were used as reference standards. Temperature was adjusted at 50 ± 1oC and test
compounds were administered using an oral gavage 1 h before starting the experimental
protocol. A cut-off latency of 30 seconds was used to prevent heat-induced tissue
damage. Time taken for each mouse to jump out or lick the paw was recorded as latency
time and pain inhibition percentage (PIP) was calculated (Table 1).
4.2.2. In vitro cyclooxygenase (COX-1/2) and lipoxygenase (5-LOX) inhibition assays
Enzyme immune assay (EIA) kits; i) catalog no. 560131, Cayman Chemical, Ann
Arbor, MI, USA and ii) catalogue no 760709, Cayman Chemical, Ann Arbor, MI, USA,
were used to evaluate the ability of test compounds to inhibit ovine COX-1/2 and 5-LOX
enzymes, respectively according to the manufacturer’s procedure [35-36]. For COX-1/2
assay, incubation of the enzymes with different concentrations of test compounds for a
19

o
period of 5 min at 25 C was carried out before addition of arachidonic acid and the
colorimetric substrate; absorbance was measured at 590 nm using plate reader (Table 2).
Considering 5-LOX assay, stock solutions were freshly prepared before use and a
buffer solution (0.1 M Tris HCl, PH, 7.4) was used. 10 µL of different compounds were
dissolved in the least amount of DMSO and diluted with the stock solution to prepare
(0.001, 0.1, 1, 5, 10 µM) concentrations in a final volume of 210 µL, Table 2.
4.2.3. In vivo anti-inflammatory activity
4.2.3.1. Carrageenan-induced rat paw edema assay: The AI activity of test compounds
was determined in vivo applying carrageenan-induced rat paw edema model [37-38].
After oral administration of test compounds and celecoxib, induction of paw edema was
performed by subcutaneous injection of a 1% saline solution of carrageenan into the right
hind paw of each rat. Paw thickness of each rat was measured using plethysmometer and
Percentage Inhibition in edema thickness (EIP, %) was determined after 2 h and 4 h of
carrageenan injection. ED50 values for each tested compound were also calculated using
three different doses (Table 3).
4.2.3.2. Ulcerogenic liability
The ulcerogenic effect of the most active compounds 5b, 5c, 5d, 5f and celecoxib
was evaluated and compared to that of indomethacin. 21 rats were used in this study,
divided into 7 groups and fasted for 18 h before drug administration. The control group
received the vehicle (2.5 % Tween 80). Other groups received test compounds, celecoxib,
or indomethacin at a dose of 50 mg/kg, then the animals were fed after 2 h. Rats were
given the required dose orally for three successive days. After 2 h of the last dose, rats
were sacrificed; the stomach of each rat was removed then opened along the greater
curvature and rinsed with saline. In order to examine the stomach, it was stretched by
pins on a corkboard. The gastric mucosa was carefully inspected for the occurrence of
ulcers with the aid of an illuminated magnifying lens (l0x), the ulcer index was then
calculated according to the previously described method [39]. Lesions were counted and
measured along the greater diameter using the transparent ruler. Every five hemorrhagic
spots were considered equivalent to 1 mm of an ulcer. The ulcer index (mm) was
calculated from the sum of the total length of ulcers and hemorrhagic spots in each
stomach (Table 4).
20

4.2.3.3. Histopathological study
Tissue specimens were collected from rat stomach (glandular and non-glandular
portions). They were fixed in 10% buffered formalin for 72 h followed by routine
histological processing and paraffin embedding according to Bancroft and Gamble
(2008). Five-micron tissue sections were stained with routine Hematoxylin and Eosin
stain [40]. (Table 5)
4.3. Molecular docking study
Molecular Operating Environment, Montreal, QC, Canada (MOE version
2008.10) was chosen as the computer software in this docking study. Crystal structure of
celecoxib in complex with COX-2 active side (PDB code 3LN1) and AA bound to 5-
LOX (PDB code 3V99) were downloaded from protein data bank at Research
Collaboration for Structural Bioinformatics (RCSB) [41-42].
Docking of the co-crystallized ligands was performed to determine the binding
energy score, amino acid interactions and relative mean square deviation (rmsd) in order
to confirm the validity of the docking steps. 3D structures of synthesized compounds to
be docked were built by MOE and docking steps were accomplished using London DG
force, structures were protonated using the 3D protonation application and energy was
minimized using the MMFF94x force field energy to produce the lowest energy
conformer in order to obtain refined results. For each docked compound, one pose was
selected based on superposition with the original ligand, docking scores and the number
of binding interactions with amino acid residues forming H-bonds (Table 6).
Acknowledgments
The authors would like to thank all members of Pharmaceutical Organic Chemistry,
Faculty of Pharmacy, Beni-Suef University for their great support and encouragement
during the whole work.
Conflicts of Interest
The authors have no conflict of interest to declare.
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26

Tables, Figures and Schemes captions
Table 1: Analgesic activity of test compounds using a hotplate latency test
Table 2: In vitro COX-1, COX-2 and 5- LOX enzyme inhibition, and COX-2 selectivity
index (SI) data for 3, 4, 5a-i, reference drugs indomethacin, celecoxib and zileuton.
Table 3: In vivo anti-inflammatory activities for pyrazolones 3, 4, 5a-i and celecoxib
against Carrageenan-induced rat paw edema.
Table 4: Ulcerogenic liability of compounds 5b, 5c, 5d, 5f, indomethacin, and celecoxib.
Table 5: Scoring of different pathological lesions for groups treated with compounds 5b,
5c, 5d, 5f, indomethacin, and celecoxib.
Table 6: The docking scores and binding interactions of 3, 4, 5a-i inside COX-2 and 5-
LOX active sites.
Fig. 1: Structure of aminoantipyrine, CBS 1108, celecoxib, and general structure of
targeted pyrazolones (A) and (B).
Fig. 2: Intra-molecular hydrogen bonds in 5band 5f
Fig. 3: Histological structure of glandular stomach (first row) and non-glandular stomach
(second row) in: (i) negative control (A-I, A-II), indomethacin (B-I—B-IV), celecoxib
(C-I, C-II) and (ii) compounds 5b (I, II), 5c (I, II), 5d (I, II), 5f (I, II). L: leukocytic
infiltration; H: hyalinosis; Ed: edema; C: congestion; K: hyperkeratosis
Fig. 4: I) The proposed binding modes of the most active pyrazolone 5f inside COX-2
receptor active site. IA: 2D interaction of 5f with Ser516, Gln178 and Phe504 amino
acids residues forming H-bonds, IB: 3D interaction of 5f (yellow) with ligand (red).
II) The proposed binding modes of the most active pyrazolone 5f inside 5-LOX receptor
active site (PDB code 3V99). IIA: 2D interaction of 5f with His367 and Ser171 amino
acids residues forming H-bonds, IIB: 3D interaction of 5f (red) with ligand (yellow).
Scheme 1: Synthesis of starting materials 2a-e
Scheme 2: Synthesis of target pyrazolones 3, 4, 5a-i.
27

Table 1: Analgesic activity of test compounds using the hotplate latency test
Compound no Latency time
(sec) a
Compound no Latency time
(sec) a
PIP
9.38
6.25
PIP
3
5f
3.5 ± 0.16
4.8 ± 0.26***
50
4
5g
3.4 ± 0.19
3.6 ± 0.20*
15.63
18.75
9.38
93.75
53.13
----
5a
5h
4.1 ± 0.22*** 28.13
4.2 ± 0.29*** 31.25
3.9 ± 0.23*** 21.88
3.8 ± 0.23*
5b
5c
5d
5e
5i
3.5 ± 0.16
Celecoxibb
6.2 ± 0.27***
Indomethacinb
3.7 ± 0.21*
15.63
4.9 ± 0.25***
control
3.4 ± 0.19
6.25
3.2 ± 0.16
a The results are expressed as means ± SD (n = 5)
*Significantly different from normal control group at p < 0.05
*** Significantly different from normal control group at p < 0.001
b values are determined using10mg/Kg dose
Dose = 50mg/Kg
28

Table 2: In vitro COX-1, COX-2, 5- LOX enzyme inhibition, and COX-2 selectivity
index (SI) data for 3, 4, 5a-i and reference drugs indomethacin, celecoxib and zileuton.
Compound
COX-1
IC50 (µM)a
4.48
COX-2
IC50 (µM)a
1.31
5-LOX
IC50 (µM)a
1.03
SIb
no
3.41
1.32
1.36
3.47
2.17
5.29
1.68
5.29
0.82
1.33
5.69
3.52
0.15
NDc
3
1.32
1.00
1.06
4
2.63
1.93
1.42
5a
4.66
1.34
1.17
5b
2.54
1.17
0.89
5c
4.08
0.77
0.53
5d
2.68
1.59
1.09
5e
3.81
0.72
0.52
5f
5g
1.69
2.04
1.59
1.89
1.42
0.84
5h
3.76
0.66
0.57
5i
3.14
0.89
NDc
Celecoxib
Indomethacin
Zileuton
1.14
NDc
7.24
NDc
NDc
0.77
a In vitro test compound concentration that produce 50% inhibition of COX-1, COX-2
and 5-LOX enzyme, the result (IC50, μM) is the mean of two values obtained using an
ovine COX-1/COX-2 assay Kits (Cayman Chemicals Inc., Ann Arbor, MI, USA). the
deviation from the mean is <10% of the mean value.
b The in vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
c ND: Not determined.
29