European Journal of Medicinal Chemistry 171 (2019) 25e37

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis and biological evaluation of pyridazinone derivatives as

selective COX-2 inhibitors and potential anti-inflammatory agents
Eman M. Ahmed a, Asmaa E. Kassab a, *, Afaf A. El-Malah a, b, Marwa S.A. Hassan a
a
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, 33 Kasr El-Aini Street, Cairo, 11562, Egypt
b
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy and Drug Technology, Heliopolis University, Cairo, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: A series of pyridazinone derivatives, bearing an aryl or pyridyl moiety linked through an ethenyl spacer
Received 3 December 2018 to position-6 was designed and synthesized. The newly synthesized compounds were screened for
Received in revised form preferential inhibition of COX-2 over COX-1 isoforms. Compounds 2c, 2d, 2e, 2f, 3a, 3b, 3c, 3d and 3e are
19 February 2019
highly potent COX-2 inhibitors with IC50 values in nano-molar range. Moreover, they showed clear
Accepted 14 March 2019
Available online 19 March 2019
preferential COX-2 over COX-1 inhibition with selective indices (SIs) ranging from 4 to 38. Of particular
interest, compounds 2d, 2f, 3c and 3d exhibited the most prominent COX-2 inhibitory activity with IC50
values range of 15.56e19.77 nM. They showed SIs of 24, 38, 35 and 24, respectively which were 1.4e2.2
Keywords:
Pyridazinone
fold higher than celecoxib (SI 17). These four compounds were further investigated in vivo for anti-
Synthesis inflammatory activity using the carrageenan induced rat paw edema method and ulcerogenic liability.
COX-2 inhibitors Compounds 2f, 3c and 3d demonstrated superior anti-inflammatory activity relative to both indo-
Anti-inflammatory methacin and celecoxib. None of these compounds showed gastric ulcerogenic effect. On the other hand,
compound 2d was found equipotent to celecoxib at the second hour of oral administration. At the fourth
hour, it exhibited more potent anti-inflammatory activity than celecoxib, becoming equipotent to
indomethacin. It showed mild hyperemia in vivo compared to indomethacin and celecoxib. The molec-
ular docking study of compounds 2d, 2f, 3c and 3d into COX-2 active site revealed a similar binding mode
to celecoxib, explaining their remarkable COX-2 inhibitory activity. Taken together, these results indi-
cated that these derivatives are good leads for potential COX-2 inhibitors to be used as potent and safe
anti-inflammatory agents.
© 2019 Elsevier Masson SAS. All rights reserved.

1. Introduction effects are due to inhibition of COX-l enzyme. This isoform is

Inflammation is a part of the body defensive mechanism to
different triggers, such as pathogens, chemicals or physical tissue
injury [1]. An acute inflammatory response in the body is not that
dangerous, it may produce edema and cellular influx due to
changes in vascular permeability and local hemodynamics, how-
ever, the chronic inflammatory response produces diseases like
asthma, rheumatoid arthritis and cancer [2,3]. Long-term use of
nonsteroidal anti-inflammatory drugs (NSAIDs) like indomethacin,
ibuprofen and naproxen has been associated with gastrointestinal
ulceration, bleeding and nephrotoxicity [4]. These undesirable side
* Corresponding author. Pharmaceutical Organic Chemistry Department, Faculty
of Pharmacy, Cairo University, Cairo, 11562, Egypt.
E-mail addresses: asmaa.kassab@pharma.cu.edu.eg, eman.ahmed@pharma.cu.
edu.eg (A.E. Kassab).
involved in the physiological production of prostaglandins (PGs)
that are responsible for maintaining gastric and renal integrity. On
the other hand, inhibition of COX-2 isozyme accounts for the
therapeutic benefits of NSAIDs, since COX-2 induces inflammatory
conditions and is involved in the production of prostaglandins
mediating pain [5].
Several highly selective COX-2 inhibitors (coxibs) have been
reported and marketed [6], including celecoxib [7] and etoricoxib
[8] (Fig. 1). Even though these drugs produce less ulceration than
conventional NSAIDs, coxibs still produce significant gastric and
cardiovascular side effects due to the decrease in the protective
prostacyclin (PGI2) production [9,10].
Therefore, there is an ongoing need for the discovery of new
generations of selective COX-2 inhibitors with an improved gastric
safety profile and minimal side effects. Luong et al. designed and
synthesized derivative I (Fig. 2) by replacing carboxylic group
present in zomepirac (non-selective NSAID) with the pyridazinone

https://doi.org/10.1016/j.ejmech.2019.03.036
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.
26 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37
Fig. 1. Some famous COXIBs.
Fig. 2. Zompirac and its pyridazinone derivative I.
moiety. This modification highly improved the selectivity ratio of
more than 1500 in favor of COX-2 enzyme [11,12]. Literature suc-
cessfully used non-classic isosteres of the sulfonyl aryl group, such
as pyridazinone (RS-57067 and Syntex compound) (Fig. 3) to make
potent COX-2 inhibitors [13,14]. In addition, the presence of het-
erocyclic ring at position-6 of the pyridazinone ring has been re-
ported to improve the anti-inflammatory activity with nil or very
low ulcerogenicity, including for example, compounds II and III
(Fig. 3) [15,16]. Furthermore, pyridazinone having an aryl group
linked to position-6 through an ethenyl spacer is reported as a
promising scaffold for the synthesis of COX-2 selective inhibitors.
Some of these compounds are substituted at the amidic pyr-
idazinone N (2), such as for example, compound IV (Fig. 3) [17].
Inspired by all these findings and in the same direction, we have
constructed pyridazinone A and B scaffolds as promising candi-
dates for selective COX-2 inhibition (Fig. 3). Our strategy aims to
designing a variety of modifications in the pyridazinone core, tar-
geting exploring the impact of such on the desired selectivity,
ulcerogenicity and anti-inflammatory activity and hoping to iden-
tify efficient COX-2 inhibitor, potent and safe anti-inflammatory
lead. In our design strategy, we have utilized either the 4,5-
dihydropyridazinone or pyridazinone core connected at position-
6 with m-tolyl, o-methoxyphenyl or pyridyl moiety via an ethenyl
spacer. We aimed to keep the amidic pyridazinone N (2) non-
substituted or to incorporate a phenyl or 4-fluorophenyl group to
the same. These synthesized compounds were initially tested for
their in vitro COX-2 and COX-1 inhibition followed by the in vivo
anti-inflammatory activity and the gastric ulcerogenic effect for the
pyridazinones with the highest selectivity on COX-2 isoform.
2. Results and discussions
2.1. Chemistry
The synthesis of the target compounds is illustrated in Scheme 1.
Oxohexenoic acid derivatives 1a-c represent the key intermediates
for the synthesis of 4,5-dihydropyridazinone derivatives 2a-f.
Compounds 1a-c were synthesized via condensation of levulinic
acid with o-methoxybenzaldehyde, m-tolualdyhyde or 3-
pyridinecarboxaldhyde in the presence of morpholine or piper-
dine and glacial acetic acid using dry benzene as solvent for 6e14 h
adopting reported procedures [18,19]. Thereafter, the obtained keto
acids 1a-c were cyclized with hydrazine hydrate or its phenyl de-
rivatives in ethanol for 3e30 h to afford the desired 4,5 dihy-
dropyridazinone derivatives 2a-f, according to the reported
methods [14,17,20]. In case 4-fluorophenyl hydrazine HCl salt was
used, triethylamine was employed to liberate the free base from its
salt. On the other hand, pyridazinones 3a-e were obtained through
dehydrogenation of the corresponding 4,5-dihydropyridazinones
2a-e, under mild conditions, using anhydrous CuCl2 in dry aceto-
nitrile via halogenation and spontaneous HCl elimination. Litera-
ture pointed that the dehydrogenation of the dihydro derivatives
might be achieved by heating this reaction mixture, under reflux
for 30 min [21]. In the present study, compounds 3a-e were ob-
tained by stirring a mixture of compounds 2a-e with 2 equivalents
of anhydrous CuCl2 using dry acetonitrile as solvent at 60
C for
3e5 h.
The structures of the novel compound 1b, 4,5-dihydropyridazin-
3(2H)-ones 2a-f and pyridazin-3(2H)-ones 3a-e were structurally
elucidated based on microanalyses and spectral data. IR spectrum
for 1b showed characteristic broad band at the range of
3051e2542 cm1 for the OH stretching, a band at 1708 cm1 for the
carboxylic C]O stretching and a band at 1658 cm1 for the ketonic
C]O stretching. 1H NMR of 1b proved the presence of a singlet
peak, which corresponds to methyl protons at 2.34 ppm, in addition
to two characteristic triplets of two methylene protons at 2.51 ppm
and 2.94 ppm. The olefinic and aromatic protons were observed in
the range of 6.89e7.59 ppm. Further evidence involved the pres-
ence of the exchangeable singlet signal corresponding to OH proton
at 12.15 ppm. Furthermore, 13C NMR of 1b revealed the presence of
ketonic and acidic carbonyl carbons at 174.2 and 198.8 ppm,
respectively. The IR spectra of the 4,5-dihydropyridazinone 2a, d, f
showed the disappearance of the band corresponding to carboxylic
C]O stretching in the range of 1708e1701 cm1, the appearance of
absorption bands in the range of 3209e3186 cm1, indicating the
presence of NH groups. Moreover, 1H NMR spectra for these com-
pounds revealed the presence of the 2 triplets corresponding to 2
CH2 groups in the region 2.39e2.80 ppm and exchangeable singlet
signals for NH protons in the region 10.85e10.93 ppm. The 13C NMR
spectra of compounds 2a, d, f exhibited amidic carbonyl signal at
167.6 ppm. 1H NMR of N-substituted 4,5-pyridazinones 2b, c, e
showed the 2 triplets corresponding to 2 CH2 groups in the region
2.66e2.98 ppm and additional aromatic protons signals, along with
the disappearance of the OH exchangeable singlet. The 13C NMR
spectra of compounds 2b, c, e displayed amidic carbonyl signal at
166.0e166.1 ppm. 1H NMR spectra for compounds 3a-e proved the
dehydrogenation of 4,5-dihydropyridazin-3(2H)-ones 2a-e by the
disappearance of the 2 triplets corresponding to 2 CH2 groups in
the region 2.39e2.98 ppm. The 13C NMR spectra of compounds 3a-e
showed disappearance of 2 CH2 groups in the region
20.5e27.7 ppm.
2.2. In vitro COX-1 and COX-2 inhibition assays
All the newly synthesized pyridazinones 2a-f and 3a-e were
screened for in vitro COX-1/COX-2 inhibition assays, using the COX-
1(human) Inhibitor Screening Assay Kit and COX-2 (human) In-
hibitor Screening Assay Kit (Cayman Chemical Company, Ann Ar-
bor, MI, USA). The half-maximal inhibitor concentrations IC50
values were determined, being the means of three determinations
acquired, and the selectivity index (SI) values were calculated as
IC50 (COX-1)/IC50 (COX-2) (Table 1).
 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37
Fig. 3. Pyridazinone-containing compounds with anti-inflammatory activities and the target compounds (A and B).
IC50 values for the screened compounds were determined and
compared to indomethacin and celecoxib, as reference drugs. It is
evident from the in vitro assay that many of the synthesized de-
rivatives demonstrated marked potency and selectivity against
COX-2 isoform. Compounds 2c, 2d, 2e, 2f, 3a, 3b, 3c, 3d and 3e are
highly potent COX-2 inhibitors, having IC50 values in nano-molar
range. Moreover, they showed clear preferential COX-2 over COX-
1 inhibition with SIs of 11, 24, 4, 38, 5, 5, 35, 24 and 6, respec-
tively, which were greater than that of standard drug indomethacin
(SI 3). Particularly interesting were compounds 2d, 2f, 3c and 3d,
they exhibited the most prominent COX-2 inhibitory activity (IC50
values: 15.56e19.77 nM). They showed SIs of 24, 38, 35 and 24,
respectively, 1.4e2.2 fold higher than celecoxib, with SI 17 (Fig. 4).
Structural activity relationship analysis revealed that the substitu-
tion pattern on positions 6 and 2 of the newly synthesized pyr-
idazinones was a crucial element for the COX-2 inhibition and
selectivity. It was noticed that presence of m-tolyl group on
position-6 of either 4,5-dihydropyridazinone 2d or pyridazinone
3d highly enhanced COX-2 selectivity more than the two reference
drugs, indomethacin and celecoxib. Incorporation of the hetero-
cyclic 3-pyridyl moiety on position-6 of 4,5-dihydropyridazinone 2f
greatly improved the COX-2 selectivity and afforded the most
prominent compound (2.2 fold more selective than celecoxib). On
the other hand, 2-methoxyphenyl substituent at same position,
either of 4,5-dihydropyridazinone 2a or pyridazinone 3a, resulted
in a marked decrease in COX-2 inhibition and selectivity.
Regarding position-2, it is worth noting that keeping the amidic
nitrogen unsubstituted in pyridazinone derivatives resulted in
marked COX-2-selective inhibition (2d, 2f and 3d). Grafting a
27
phenyl group in compounds 2b and 3b caused a dramatic decrease
in both the inhibitory activity and selectivity against COX-2. Pyr-
idazinone 3b exhibited higher COX-2 selectivity than 4,5-
dihydropyridazinone 2b. This observation was consistent with the
previously published work reporting that substitution of amidic
nitrogen of 4,5-dihydropyridazinone ring with a phenyl moiety
decreased the anti-inflammatory activity [22]. Incorporating 4-
fluorophenyl moiety at position-2 and m-tolyl group at position-6
(2e and 3e) showed moderate inhibitory activity and selectivity
against COX-2 isoform. The presence of 4-fluorophenyl moiety at
position-2 and 2-methoxyphenyl substituent at position-6 of the
pyridazinone ring (2c and 3c) was tolerated for both COX-2 inhib-
itory activity and selectivity. Compound 3c showed highly
improved affinity and remarkable selectivity for COX-2 enzyme
activity, superior to that of standard drugs, indomethacin and cel-
ecoxib. Compounds 2d, 2f, 3c and 3d with the highest COX-2 SIs
were selected for further pharmacological evaluation of in vivo
anti-inflammatory activity and gastrointestinal safety profile.
2.3. 3. In vivo anti-inflammatory activity: carrageenan -induced rat
paw edema test
Based on the previous in vitro test results; four compounds (2d,
2f, 3c and 3d) were selected to be tested for their in vivo anti-
inflammatory activity, using carrageenan-induced rat paw edema
bioassay method reported by Winter et al. [23]. The carrageenan-
induced rat paw edema method is highly reproducible and is the
most traditional model to evaluate anti-inflammatory drugs. The
anti-inflammatory properties were compared to celecoxib and
28 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37

Scheme 1. The synthetic path and reagents for the preparation of the target compounds 1e3. Reagents and conditions: a) Base/gl. acetic acid/dry benzene/6e14 h, b) Ethanol/
3e30 h, c) Anhyd. CuCl2/dry acetonitrile/60
C/3e5 h.

Table 1 indomethacin (in a dose of 10 mg/kg) as two reference standards.

In vitro COX-1/COX-2 inhibition results and selectivity index (SI) . The four tested compounds showed potent anti-inflammatory ac-
tivities. Three compounds; namely 2f, 3c and 3d, exhibited the
most prominent and consistent anti-inflammatory activity, which
was superior, compared to both indomethacin and celecoxib. They
showed rapid onset of action and sustained duration until the
fourth hour after the administration of the compounds. Compound
2d was equipotent to celecoxib at the second hour of oral admin-
istration, while at the fourth hour; it exhibited more potent anti-
inflammatory activity than celecoxib, becoming equipotent to
Compound R R1 COX-1 COX-2 c
SI indomethacin (Table 2 and Fig. 5). According to these findings, we
a b
IC50 IC50 concluded that pyridazinone scaffold with non-substituted amidic
2a 2-CH3OC6H4 H 0.27 ± 0.01 103.17 ± 4.00 2.65 nitrogen and connected at position-6 via an ethenyl spacer with m-
2b 2-CH3OC6H4 C6H5 0.39 ± 0.01 134.58 ± 5.22 2.91 tolyl or pyridyl moiety is a satisfactory lead to design highly effi-
2c 2-CH3OC6H4 4-FC6H4 0.54 ± 0.02 48.79 ± 1.89 11.25 cient COX-2 inhibitors, as potent anti-inflammatory agents.
2d 3-CH3C6H4 H 0.45 ± 0.01 18.35 ± 0.71 24.52
2e 3-CH3C6H4 4-FC6H4 0.21 ± 0.01 52.34 ± 2.03 4.09
2f 3-Pyridyl H 0.75 ± 0.02 19.32 ± 0.74 38.92 2.4. Gastric ulcerogenic activity
3a 2-CH3OC6H4 H 0.53 ± 0.01 98.03 ± 3.80 5.48
3b 2-CH3OC6H4 C6H5 0.46 ± 0.01 89.80 ± 3.48 5.14 Gastrointestinal erosions and ulcers are two of the most com-
3c 2-CH3OC6H4 4-FC6H4 0.70 ± 0.02 19.77 ± 0.76 35.65
3d 3-CH3C6H4 H 0.37 ± 0.01 15.56 ± 0.60 24.33
mon side-effects associated with the chronic administration of
3e 3-CH3C6H4 4-FC6H4 0.24 ± 0.01 36.54 ± 1.41 6.59 NSAIDs. Therefore, we were interested in determining the ulcero-
Celecoxib 0.30 ± 0.01 17.79 ± 0.69 17.18 genic potential of the most potent compounds, 2d, 2f, 3c and 3d,
Indomethacin 0.22 ± 0.01 67.72 ± 2.62 3.28 when administered orally. Macroscopic observation of rat intestinal
The bold text indicates the compounds that are more selective for COX-2 than mucosa for the presence of lesions following oral administration of
celecoxib and indomethacin. 10 mg/kg of tested compounds as well as reference compounds
a
IC50 is the concentration (mM) needed to cause 50% inhibition of COX-1 enzy- (celecoxib and indomethacin) was used to gauge the ulcerogenic
matic activity. All values are expressed as a mean of three replicates ± SD.
b
IC50 is the concentration (nM) needed to cause 50% inhibition of COX-2 enzy-
effects of the these compounds. Observation of the isolated rat
matic activity. All values are expressed as a mean of three replicates ± SD. stomachs showed no ulceration in gastric region for compounds 2f,
c
Selectivity index ¼ (COX-1 IC50(nM)/COX-2 IC50(nM)). 3c and 3d, while compound 2d showed mild hyperemia without
 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37 29

Fig. 4. Graphical representation of IC50 (COX-1 and COX-2) values for the screened compounds in comparison with indomethacin and celecoxib.

Table 2
Results of in vivo anti-inflammatory activity of compounds 2d, 2f, 3c, 3d, celecoxib and indomethacin (10 mg/kg) in male albino rats (n ¼ 6).

Zero 1h 2h 3h 4h

Paw diameter (mm) Paw diameter (mm) % Edema Paw diameter (mm) % Edema Paw diameter (mm) % Edema Paw diameter (mm) % Edema

Control 3.46 ± 0.08 4.51 ± 0.09* 30.3 4.72 ± 0.08* 36.4 4.79 ± 0.1* 38.4 4.88 ± 0.1* 41.0
2d 3.51 ± 0.09 3.99 ± 0.06* 13.6 3.92 ± 0.05* 11.6 3.84 ± 0.03* 10.2 3.80 ± 0.04* 8.2
2f 3.53 ± 0.06 3.92 ± 0.07* 11.0 3.86 ± 0.06* 9.3 3.81 ± 0.06* 7.9 3.78 ± 0.06* 7.0
3c 3.59 ± 0.07 3.97 ± 0.05* 10.5 3.93 ± 0.04* 9.4 3.88 ± 0.01* 8.0 3.85 ± 0.05* 7.2
3d 3.55 ± 0.03 3.96 ± 0.08* 11.5 3.86 ± 0.03* 8.7 3.84 ± 0.03* 8.1 3.82 ± 0.07* 7.6
Celecoxib 3.48 ± 0.09 3.93 ± 0.09* 12.9 3.88 ± 0.07* 11.4 3.83 ± 0.06* 10.0 3.78 ± 0.04* 8.6
Indomethacin 3.49 ± 0.06 3.92 ± 0.08* 12.3 3.85 ± 0.06* 10.3 3.81 ± 0.03* 9.4 3.73 ± 0.04* 8.0

* Significantly different from zero time at p < 0.05.

Data are expressed as mean ± SE. Statistical analysis of the data was done using one way Anova followed by Duncan's test for multiple group comparisons. Probability levels of
P < 0.05 were considered statistical significant.

Fig. 5. Graphical representation of in vivo anti-inflammatory activities of the selected compounds in comparison with indomethacin and celecoxib in carragneenan-induced rat paw
edema bioassay.
30 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37

Table 3 19.32 and 15.56 nM, respectively) with almost the same binding
Gastric ulcerative effect of tested compounds 2d, 2f, 3c and 3d compared to cele- poses (Fig. 7). However, Compound 3c (IC50 ¼ 19.77 nM) interacted
coxib and indomethacin in male albino rats (n ¼ 6).
with the same amino acids, in a different way. The N1 of pyridazine
Groups Score ring interacted as H-bond acceptor with the His 90. Moreover,
No. of gastric ulcers Severity lesions compound 3c interacted with its methoxy group as H-bond
acceptor, with the key amino acid Arg 513 (Fig. 8). An overlay of
Control (1 mL saline) 0 0
2d 0.2 ± 0.01 0.4 ± 0.01 compounds 2d, 2f and 3d with the celecoxib showed a perfect
2f 0 0 superimposition of the pyridazinone nucleus with N1-phenyl ring
3c 0 0 carrying the sulfonamide group in celecoxib and 3-methyl phenyl
3d 0 0
or pyridyl ring with the orthogonal 4-methylphenyl group in cel-
Celecoxib 2.7 ± 0.1* 6.2 ± 0.2*
Indomethacin 8.3 ± 0.4* 12.4 ± 0.7* ecoxib (Figs. 9e12).
An overlay of the docking poses of compounds 2d, 2f and 3d
Data are expressed as mean ± SE.
* Statistically significant from control group at p < 0.01.
showed that they are almost superimposable on each other, which
indicate that the COX-2 active site is capable of accommodating all
of the pyridazinone nucleus, ethenyl spacer and 3-methylphenyl or
gross ulceration (Table 3). The results showed that compounds 2d, pyridyl ring, with almost the same binding pattern (Fig. 12). The
2f, 3c and 3d possessed selective COX-2 inhibitory profile in vitro, pyridazin-4(5H)-one ring was within the vicinity of COX-2 second-
potent anti-inflammatory activity in vivo and they did not exert ary pocket, surrounded by amino acids like His90, Ser353, Tyr355,
gastric toxicity. Arg513 and Val523. The pyridazinone carbonyl group interacted as
H-bond acceptor with the amino acids His 90 and Arg 513, which
2.5. Molecular docking of compounds 2d, 2f, 3c and 3d in the active suggests favorable binding interactions allowing this ring to enter
site of COX-2 the mouth of the COX-2 active site. The 3-methylphenyl or pyridyl
ring was oriented toward the apex of the COX-2 active site in a re-
To support in vitro and in vivo anti-inflammatory profile exerted gion comprised of amino acids like Phe381, Leu384, Tyr385, Trp387,
by the most active compounds 2d, 2f, 3c and 3d, we also carried out Met522, Gly526 and Ala527 (Figs. 9e12). The results obtained in
molecular docking studies. Interestingly, the test compounds these docking studies suggested that the synthesized compounds
exerted favorable interactions with COX-2 enzyme active site, with 2d, 2f, 3c and 3d were fit on the active site of COX-2 enzyme and
almost the same binding pattern as celecoxib. To that end, we used they were able to form the same binding interactions, as indicated
cyclooxygenase-2 enzyme (COX-2) (PDB entry 1CX2) for molecular by its docking pattern, compared to that of celecoxib. Overall, these
docking of compounds 2d, 2f, 3c, 3d and celecoxib [24]. Celecoxib interactions and binding patterns into COX-2 active site rationalized
showed interactions via sulfonamide group, with two key amino the remarkable COX-2 inhibitory activity of these compounds.
acids His 90 and Arg 513 (Fig. 6). The pyridazinone carbonyl group
interacted as H-bond acceptor with the amino acids His 90 and Arg
513, in the case of compounds 2d, 2f and 3d (COX-2 IC50 ¼ 18.35,

Fig. 6. Docking and binding pattern of celecoxib into COX-2 active site (PDB 1CX2) in 2D. Numbers indicate H-Bond distances.
 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37
Fig. 7. Docking and binding pattern of 2d (upper panel), 2f (middle panel) and 3d
(lower panel) into COX-2 active site (PDB 1CX2) in 2D. Numbers indicate H-Bond
distances.
3. Conclusion
The present study describes the synthesis of a series of pyr-
idazinone derivatives 2a-f and 3a-e linked at postion-6 to an aryl or
31
pyridyl moiety, through two carbons spacer. The synthesized
compounds were evaluated as COX-2 inhibiting anti-inflammatory
drug candidates, using indomethacin and celecoxib, as reference
drugs. All the synthesized pyridazinones were highly potent COX-2
inhibitors, having IC50 values in nano-molar range. Moreover, they
showed clear preferential COX-2 over COX-1 inhibition. Com-
pounds 2d, 2f, 3c and 3d were of particular interest, as they
exhibited the most prominent COX-2 inhibitory activity (IC50
values: 15.56e19.77 nM). They showed SIs of 24, 38, 35 and 24,
respectively, which were 1.4e2.2 fold higher than celecoxib (SI 17).
These four compounds were further investigated in vivo for anti-
inflammatory activity and ulcerogenic liability. Compounds 2f, 3c
and 3d demonstrated superior anti-inflammatory activity, relative
to both indomethacin and celecoxib. None of these compounds
showed gastric ulcerogenic effect. Compound 2d was equipotent to
celecoxib at the second hour of oral administration, while at the
fourth hour, it exhibited more potent anti-inflammatory activity
than celecoxib, becoming equipotent to indomethacin. It showed
mild hyperemia in vivo, compared to indomethacin and celecoxib.
Compounds 2d, 2f, 3c and 3d were docked into the binding site
pocket of COX-2 and showed perfect fitting within the pocket, with
substantial interactions with the key amino acid residues His 90
and Arg 513. Accordingly, these active compounds represent
promising leads to pursue as potential COX-2 inhibitors, to be used
as potent and safe anti-inflammatory agents.
Further studies are needed as continuation of this work to
explore the effects of type and position of substituents of pyr-
idazinone ring on anti-inflammatory activity profile. Taking into
consideration that the phenyl moiety at position 2 wasn't a favor-
able substituent, even certain studies may be required to explore
the effect of different substitution pattern of phenyl group at
postion-2 of pyridazinone ring on in vitro COX-2 inhibitory, in vivo
anti-inflammatory activity and gastric safety. Regarding postion-6,
an important contribution is to synthesize novel pyridazin-3(2H)-
ones with different heterocyclic moieties at postion-6 and study
their effect on COX-2 inhibitory, anti-inflammatory activity and
gastric safety profile. Compound 2f may be used as a starting point
for further derivatives' synthesis owing to its COX-2 selectivity and
gastric safety. Further optimizations for compound 2d are still
required to improve its GI safety. As a continuation of this work, we
will study the effect of replacing the carbonyl of pyridazinone de-
rivatives with other isosteric groups such as thione on the binding
to COX-2 enzyme and in vitro COX-2 inhibition. Last but not least,
progressive development of the attractive pyridazinone scaffold is
still needed for the design and synthesis of selective COX-2 in-
hibitors, useful and safe anti-inflammatory agents as the pyr-
idazinone skeleton still offers various options to medicinal
chemists for further structural exploration.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were obtained on a Griffin apparatus and were
uncorrected. Microanalyses for C, H and N were carried out at the
Regional Center for Mycology and Biotechnology, Faculty of Phar-
macy, Al-Azhar University. IR spectra were recorded on Shimadzu
IR 435 spectrophotometer (Shimadzu Corp., Kyoto, Japan) Faculty of
Pharmacy, Cairo University, Cairo, Egypt and values were repre-
sented in cm1. 1H NMR spectra were carried out on Bruker
400 MHz (Bruker Corp., Billerica, MA, USA) spectrophotometer,
Faculty of Pharmacy, Cairo University, Cairo, Egypt. Tetramethylsi-
lane (TMS) was used as an internal standard and chemical shifts
were recorded in ppm on d scale and coupling constants (J) were
32 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37
Fig. 8. Docking and binding pattern of N-substituted pyridazine 3c into COX-2 active site (PDB 1CX2) in 2D. Numbers indicate H-Bond distances.
given in Hz. 13C NMR spectra were carried out on Bruker 100 MHz
spectrophotometer, Faculty of Pharmacy, Cairo University, Cairo,
Egypt. Progress of the reactions was monitored by TLC using pre-
coated aluminum sheet silica gel MERCK 60F 254. The spots were
visualized using UV lamp. The used solvent system was benzene:
methanol: chloroform: TEA [9: 3: 1.5: 0.1]. The preparation of
compounds 1a and 1c were performed according to reported pro-
cedures [18,19].
4.1.2. 4-Oxo-6-(m-tolyl)hex-5-enoic acid (1b)
A mixture of levulenic acid (5.8 g, 0.05 mol), m-tolualdhyde (6 g,
0.05 mol), morpholine (2 mL) and glacial acetic acid (6 mL) in dry
benzene (40 mL) was heated under reflux using Dean-Stark water
separator until the theoretical amount of water had been collected
(6 h). After cooling, the separated solid was filtered, washed with
ethanol (5 mL) and crystallized from ethanol to give compound 1b.
Yield 46%, m.p. 144e145
C, IR (KBr, cm1): 3051-2542 (OH),
1708 (acidic C]O), 1658 (ketonic C]O).1H NMR (DMSO‑d6) ppm:
d 2.34 (s, 3H, CH3), 2.51 (t, 2H, CH2, J ¼ 8 Hz), 2.94 (t, 2H, CH2,
J ¼ 8 Hz), 6.89 (d, 1H, CH, J ¼ 16 Hz), 7.24e7.55 (m, 4H, AreH), 7.59
(d, 1H, CH, J ¼ 16 Hz), 12.15 (brs, 1H, OH, D2O exchangeable).13C
NMR (DMSO‑d6) ppm: 21.3 (CH3), 28.2 (CH2), 35.3 (CH2), 126.1,
126.5, 129.2, 129.3, 131.6, 134.8, 138.6, 142.6 (ArCs þ2CH), 174.2 (C]
O), 198.8 (C]O). Anal. Calcd. for C13H14O3 (218.25): C, 71.54, H, 6.47.
Found: C, 71.69, H, 6.31.
4.1.3. 6-[2-Substitutedethenyl]-4,5-dihydropyridazin-3(2H)-ones
(2a, d, f)
A solution of an appropriate hex-5-enoic acid 1a-c (0.01 mol) in
ethanol (25 mL) containing hydrazine hydrate 99% (1 g, 0.02 mol)
was heated under reflux for 3 h. The mixture was concentrated to
half its volume, cooled, diluted with water and the separated solid
was filtered, dried and crystallized from ethanol to yield com-
pounds 2a, d. In case of 2f, the mixture was evaporated; residue
was triturated with ethanol (5 mL), filtered and crystallized from
ethanol.
4.1.3.1. 6-(2-(2-Methoxyphenyl)ethenyl)-4,5-dihydropyridazin-
3(2H)-one (2a). Yield 45%, m.p. 146e147
C, IR (KBr, cm1): 3209
(NH), 1678 (C]O), 1585 (C]N).1H NMR (DMSO‑d6) ppm: 2.39 (t,
2H, CH2, J ¼ 8 Hz), 2.75 (t, 2H, CH2, J ¼ 8 Hz), 3.85 (s, 3H, OCH3),
6.86e7.63 (m, 6H, 2CHþ4Ar-H), 10.85 (s, 1H, NH, D2O exchangea-
ble).13C NMR (DMSO‑d6) ppm: 20.6 (CH2), 26.3 (CH2), 55.9 (OCH3),
111.9, 121.2, 124.8, 127.3, 127.4, 128.2, 130.3, 151.3, 157.2
(ArCsþ2CH þ C]N), 167.6 (C]O). Anal. Calcd. for C13H14N2O2
(230.26): C, 67.81, H, 6.13, N, 12.17. Found: C, 68.09, H, 6.27, N, 12.40.
4.1.3.2. 6-(2-(3-Methylphenyl)ethenyl)-4,5-dihydropyridazin-3(2H)-
one (2d). Yield 51%, m.p. 96e97
C, IR (KBr, cm1): 3209 (NH), 1670
(C]O), 1593 (C]N).1H NMR (DMSO‑d6) ppm: d 2.32 (s, 3H, CH3),
2.39 (t, 2H, CH2, J ¼ 8 Hz), 2.78 (t, 2H, CH2, J ¼ 8 Hz), 6.88 (d, 1H, CH,
J ¼ 16 Hz), 7.01 (d, 1H, CH, J ¼ 16 Hz), 7.13e7.41 (m, 4H, AreH), 10.87
(s, 1H, NH, D2O exchangeable).13C NMR (DMSO‑d6) ppm: 20.5 (CH2),
21.4 (CH3), 26.3 (CH2), 124.6, 126.6, 127.8, 129.1, 129.6, 133.9, 136.5,
138.4, 151.1(ArCsþ2CH þ C]N), 167.6 (C]O). Anal. Calcd. for
C13H14N2O (214.26): C, 72.87, H, 6.59, N, 13.07. Found: C, 72.70, H,
6.75, N, 13.21.
4.1.3.3. 6-(2-(Pyridin-3-yl)ethenyl)-4,5-dihydropyridazin-3(2H)-one
(2f). Yield 30%, m.p. 212e213
C, IR (KBr, cm1): 3186 (NH), 1678
(C]O), 1570 (C]N). 1H NMR (DMSO‑d6) ppm: 2.41 (t, 2H, CH2,
J ¼ 8 Hz), 2.80 (t, 2H, CH2 J ¼ 8 Hz), 7.02 (d, 1H, CH, J ¼ 16 Hz), 7.09
 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37
Fig. 9. An overlay of the docked pose of compound 2d (green) with celecoxib (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
(d, 1H, CH, J ¼ 16 Hz), 7.38e7.42 (m, 1H, ArH), 8.05e8.07 (m, 1H,
ArH), 8.30e8.49 (m, 1H, ArH), 8.60e8.76 (m, 1H, ArH), 10.93 (s, 1H,
NH, D2O exchangeable). 13C NMR (DMSO‑d6) ppm: 20.5 (CH2), 26.2
(CH2), 124.3, 128.7, 130.3, 132.3, 133.6, 149.1, 149.6, 150.8 (ArCsþ
2CH þ C]N), 167.6 (C]O). Anal. Calcd. for C11H11N3O (201.22): C,
65.66, H, 5.51, N, 20.88. Found: C, 65.80, H, 5.73, N, 21.09.
4.1.4. 2-[4-Fluorophenyl]-6-[2-Substitutedethenyl]-4,5-
dihydropyridazin-3(2H)-ones (2c and e)
A mixture of the appropriate hex-5-enoic acid 1a, b (0.001 mol),
4-fluorophenyl hydrazine hydrochloride (0.16 g, 0.001 mol) and
triethylamine (0.1 g, 0.001 mol) in absolute ethanol (20e30 mL)
was heated under reflux for 30 h. The reaction mixture was
concentrated to one-third of its volume, diluted with water and left
at room temperature, when a solid separated out. The crude
product was filtered off, washed with ethanol (5 mL); the product
was dried and crystallized from ethanol to yield compounds (2c
and e).
4.1.4.1. 2-(4-Fluorophenyl)-6-(2-(2-methoxyphenyl)ethenyl)-4,5-
dihydropyridazin-3(2H)-one (2c). Yield 50%, m.p. 143e144
C, IR
(KBr, cm1): 1685 (C]O), 1597 (C]N). 1H NMR (DMSO‑d6) ppm:
2.66 (t, 2H, CH2, J ¼ 8 Hz), 2.95 (t, 2H, CH2, J ¼ 8 Hz), 3.86 (s, 3H,
OCH3), 6.95e7.67 (m, 10H, 2CH þ8Ar-H). 13C NMR (DMSO‑d6) ppm:
21.0 (CH2), 27.5 (CH2), 56.0 (OCH3), 112.0, 115.4, 115.6, 121.2, 124.5,
126.5, 127.7, 127.8, 130.1, 130.7, 138.0, 154.0, 157.4 (ArCsþ2CH þ C]
N), 166.1(C]O). Anal. Calcd. for C19H17FN2O2 (324.35): C, 70.36, H,
5.28, N, 8.64. Found: C, 70.58, H, 5.43, N, 8.50.
33
4.1.4.2. 2-(4-Fluorophenyl)-6-(2-(3-Methylphenyl)ethenyl)-4,5-
dihydropyridazin-3(2H)-one (2e). Yield 67%, m.p. 147e148
C, IR
(KBr, cm1): 1670 (C]O), 1585 (C]N).1H NMR (DMSO‑d6) ppm:
2.32 (s, 3H, CH3), 2.67 (t, 2H, CH2, J ¼ 8 Hz), 2.98 (t, 2H, CH2,
J ¼ 8 Hz), 6.95e7.52 (m, 10H, 2CH þ8Ar-H).13C NMR (DMSO‑d6)
ppm: 20.9(CH2), 21.3 (CH3), 27.5 (CH2), 115.3, 124.9, 125.9, 127.6,
127.7, 128.0, 129.2, 130.0, 135.7, 136.2, 138.0, 138.4, 153.7
(ArCsþ2CH þ C]N), 166.1(C]O). Anal. Calcd. for C19H17FN2O
(308.35): C, 74.01, H, 5.56, N, 9.08. Found: 74.29, H, 5.70, N, 9.23.
4.1.5. 6-(2-(2-Methoxyphenyl)ethenyl)-2-phenyl-4,5-
dihydropyridazin-3(2H)-one (2b)
Required amount of phenyl hydrazine (0.23 g, 0.002 mol) was
added to a stirred solution of compound 1a (0.49 g, 0.002 mol) in
ethanol (40 mL). The reaction mixture was heated under reflux for
24 h under continuous stirring. The reaction mixture was concen-
trated to half the volume, cooled and the solid was filtered, washed
with ethanol and crystallized from ethanol to yield compound 2b.
Yield 47%, m.p. 118e119
C, IR (KBr, cm1): 1678 (C]O), 1597
(C]N).1H NMR (DMSO‑d6) ppm: d 2.67 (t, 2H, CH2 J ¼ 8 Hz), 2.95 (t,
2H, CH2 J ¼ 8 Hz), 3.87 (s, 3H, OCH3), 6.61e7.46 (m, 11H, 2CHþ9Ar-
H). 13C NMR (DMSO‑d6) ppm: 21.0 (CH2), 27.7 (CH2), 56.0 (OCH3),
112.0, 121.2, 124.6, 125.7, 126.6, 126.8, 127.8, 128.7, 129.2, 130.0,
130.6, 153.9, 157.4 (ArCsþ2CH þ C]N), 166.0 (C]O). Anal. Calcd. for
C19H18N2O2(Mwt.: 306.36): C, 74.49, H, 5.92, N, 9.14. Found: C,
74.31, H, 6.08, N, 9.31.
34 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37
Fig. 10. An overlay of the docked pose of compound 2f (cyan) with celecoxib (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
4.1.6. 6-[2-Substitutedethenyl]pyridazin-3(2H)-ones (3a,d) and 2-
substituted-6-[2-substitutedethenyl]pyridazin-3(2H)-ones (3b,c,e)
To a solution of an appropriate 4,5-dihydropyridazine derivative
2a-e (0.001 mol) in dry acetonitrile (25 mL), anhydrous CuCl2
(0.26 g, 0.002 mol) was added and the mixture was stirred at 60
C
for 3e5 h. After cooling, the separated solid was filtered, washed
with acetonitrile and crystallized from ethanol to give compounds
3a-e.
4.1.6.1. 6-(2-(2-Methoxyphenyl)ethenyl)pyridazin-3(2H)-one (3a).
Yield 90%, m.p. 191e192
C, 3 h, IR (KBr, cm1): 3217 (NH), 1643 (C]
O).1H NMR (DMSO‑d6) ppm:: 3.80 (s, 3H, OCH3), 6.77e7.81 (m, 8H,
2CHþ6Ar-H), 12.94 (s, 1H, NH, D2O exchangeable).13C NMR
(DMSO‑d6) ppm: 56.2 (OCH3), 112.0, 121.2, 124.7, 124.9, 127.3, 127.7,
130.1, 130.3, 131.5, 144.4, 157.3(ArCsþ2CH), 160.8 (C]O). Anal.
Calcd. for C13H12N2O2 (228.25): C, 68.41, H, 5.30, N, 12.27. Found: C,
68.35, H, 5.43, N, 12.43.
4.1.6.2. 6-(2-(2-Methoxyphenyl)ethenyl)-2-phenylpyridazin-3(2H)-
one (3b). Yield 42%, m.p 222e223
C, 5 h, IR (KBr, cm1): 1627 (C]
O). 1H NMR (DMSO‑d6) ppm: d 3.86 (s, 3H, OCH3), 6.96e8.01 (m,
13H, 2CHþ11Ar-H).13C NMR (DMSO‑d6) ppm: 56.0 (OCH3), 112.0,
121.2, 124.6, 124.7, 126.2, 128.0, 128.5, 128.7, 129.1, 130.6, 130.8,
130.9, 142.0, 145.0, 157.5 (ArCsþ2CH), 159.1(C]O). Anal. Calcd for
C19H16N2O2 (304.34): C, 74.98, H, 5.30, N, 9.20. Found: C, 75.20, H,
5.47, N, 9.38.
4.1.6.3. 2-(4-Fluorophenyl)-6-(2-(2-methoxyphenyl)ethenyl)pyr-
idazin-3(2H)-one (3c). Yield 35%, m.p. 230e231
C, 5 h, IR (KBr,
cm1): 1624 (C]O).1H NMR (DMSO‑d6) ppm: 3.86 (s, 3H, OCH3),
6.96e8.01 (m, 12H, 2CHþ10Ar-H). 13C NMR (DMSO‑d6) ppm: 56.0
(OCH3), 112.0, 115.8, 116.0, 121.2, 124.6, 128.0, 128.4, 128.5, 128.8,
130.6, 130.9, 131.0, 138.3, 145.0, 157.5 (ArCsþ2CH), 159.1(C]O).
Anal. Calcd. for C19H15FN2O2 (322.33): C, 70.80, H, 4.69, N, 8.69.
Found: C, 71.13, H, 4.78, N, 8.90.
4.1.6.4. 6-(2-(3-Methylphenyl)ethenyl)pyridazin-3(2H)-one (3d).
Yield 76%, m.p. 246e247
C, 3 h, IR (KBr, cm1): 3213 (NH), 1660
(C]O). 1H NMR (DMSO‑d6) ppm: d 2.32 (s, 3H, CH3), 6.92 (d, 1H,
AreH, J ¼ 8 Hz), 7.03 (d, 1H, CH, J ¼ 16 Hz), 7.12e7.43 (m, 5H, 4Ar-
H þ CH), 7.92 (d, 1H, AreH, J ¼ 8 Hz), 13.02 (s, 1H, NH, D2O
exchangeable).13C NMR (DMSO‑d6) ppm: 21.5 (CH3), 124.3, 124.7,
127.9, 129.2, 129.7, 130.2, 131.1, 132.7, 136.3, 138.3, 144.2
(ArCsþ2CH), 160.7 (C]O). Anal. Calcd. for C13H12N2O (212.25): C,
73.56, H, 5.70, N, 13.20. Found: C, 73.80, H, 5.79, N, 13.04.
4.1.6.5. 2-(4-Fluorophenyl)-6-(2-(3-methylphenyl)ethenyl)pyridazin-
3(2H)-one (3e). Yield 40%, m.p. 214e215
C, 5 h, IR (KBr, cm1):
1627 (C]O).1H NMR (DMSO‑d6) ppm: 2.33 (s, 3H, CH3), 7.09e7.67
(m, 11H, 2CH þ9Ar-H), 8.05 (d, 1H, AreH, J ¼ 8 Hz).13C NMR
(DMSO‑d6) ppm: 21.3 (CH3), 115.8, 116.0, 123.8, 124.8, 128.0, 128.4,
128.5, 129.2, 129.9, 130.8, 130.9, 134.0, 136.2, 138.4, 144.8
(ArCsþ2CH), 159.1(C]O). Anal. Calcd. for C19H15FN2O (306.33): C,
74.50, H, 4.94, N, 9.14. Found: C, 74.68, H, 4.73, N, 9.28.
 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37
Fig. 11. An overlay of the docked pose of compound 3d (purple) with celecoxib (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
4.2. In vitro COX-1 and COX-2 inhibitory assay
All the newly synthesized compounds were screened for their
ability to inhibit COX-1 and COX-2 enzymes using ten folds serial
dilutions (1, 0.1, 0.01, 0.001 mg/mL). This was carried out using the
COX 1(human) Inhibitor Screening Assay Kit and COX 2 (human)
Inhibitor Screening Assay Kit (supplied by Cayman chemicals
(catalog no. 560131), Ann Arbor, MI, USA). The preparation of re-
agents and testing procedures were according to the instructions
recommended by the supplier. COX catalyzes the first step in the
biosynthesis of arachidonic acid to PGH2. The PGF2a produced from
PGH2 by reduction with stannous chloride is measured by enzyme-
linked immunosorbent assay (ELISA).
In brief, the compounds were dissolved in dimethylsulfoxide
(DMSO). The enzyme COX-1 and COX-2 (10 mL), heme (10 mL) and
samples (20 mL) were added to the supplied reaction buffer solution
(160 mL, 0.1 M TriseHCl, pH 8 containing 5 mM ethylenediamine
tetra acetate (EDTA) and 2 mM phenol) and pre-incubated for
10 min in a water bath (37
C). After that, COX reactions were
initiated by the addition of arachidonic acid (10 mL, final concen-
tration in reaction mixture 100 mM). After 2 min, The COX reactions
were stopped by the addition of saturated stannous chloride (30 mL)
followed by incubation for 5 min at room temperature. The PGF2a
formed in the samples by COX reactions was quantified by ELISA.
Following transfer to a 96-well plate, the plate was incubated with
samples for 18 h at room temperature. After incubation, the plate
was washed to remove any unbound reagent and then Ellman's
reagent (200 mL), which contains substrate to acetyl cholinesterase,
35
was added and incubated at room temperature for 60e90 min until
the absorbance of Bo well is in the range 0.3e0.8 A U. at 410 nm. The
plate was then read by an ELISA plate reader.
The IC50 of inhibition of COX-1 and COX-2 was calculated by the
comparison of the sample treated incubations to control in-
cubations. Celecoxib and indomethacin were used as reference
standard drugs in the study.
4.3. In vivo anti-inflammatory assay
Adult male albino rats of Sprague Dawley strain weighing
130e150 g were kept in the animal house unit of the Pharmacology
Dept., National Research Center (Dokki, Giza, Egypt) for at least one
week prior to the experiments under standard conditions of light
and temperature. All animals were accessed to standard laboratory
diet consisting of vitamin mixture (1%), mineral mixture (4%), corn
oil (10%), sucrose (20%), cellulose (0.2%), casein 95% pure (10.5%)
and starch (54.3%). All test compounds were dispensed in 10%
Tween-80 solution in distilled water. Compounds 2d, 2f, 3c and 3d
were evaluated for their in vivo anti-inflammatory activity applying
the carrageenan-induced rat paw edema screening protocol as an
acute inflammation model [23]. The rats were marked and divided
into 7 experimental groups of six rats each. The first group received
1 mL saline and served as untreated control. The second to fifth
groups received 10 mg/kg of tested compounds 2d, 2f, 3c and 3d,
respectively. The sixth and seventh group received 10 mg/kg of the
reference drugs celecoxib and indomethacin, respectively and
served as positive control group. After 1 h of oral administration of
36 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37
Fig. 12. An overlay of the docked poses of compounds 2d (green), 2f (cyan) and 3d (purple) with celecoxib (red). (For interpretation of the references to colour in this figure legend,
the reader is referred to the Web version of this article.)
test compounds (10 mg/kg), a sub-plantar injection of 0.1 mL of 1%
carrageenan solution to the right hind paw of each animal was
paw diameter after carrageenan  paw diameter before carrageenan
% edema ¼ x 100
paw diameter before carrageenan
performed. Rat paw volumes were measured immediately after
injection of carrageenan and 1, 2, 3, and 4 h later. The right hind
paw edema was measured by caliber and the % edema were
calculated (Table 2).
Animals’ treatment protocol was approved by the Faculty of
Pharmacy, Cairo University Animal Rights Committee (OC1989). In
all tests, adequate considerations were adopted to reduce pain or
discomfort of animals.
4.4. Gastric ulcerogenic activity
Compounds 2d, 2f, 3c and 3d were also evaluated for acute
gastric ulcerogenic effect in adult male albino rat. Rats were starved
for 18 h prior and were divided into seven groups of six rats each
and tested compounds, references (celecoxib and indomethacin) or
saline as control were administered orally at a dose of 10 mg/kg
body weight. Four hour after the treatment the animals were
sacrificed and their stomachs were removed and examined
macroscopically using a magnifying lens. A longitudinal incision
along the greater curvature was made with fine scissor. The pres-
ence of a single or multiple lesions, erosion, ulcer or perforation
was evaluated [25]. The number of ulcers and the occurrence of
hyperemia were noted. The gastric lesions were stretched out and
scored from 0 (no lesion) to 5 (3 or more marked ulcers), according
to the method of Clementi et al. [26] (Table 3).
4.5. Molecular docking of compounds 2d, 2f, 3c and 3d in the active
site of COX-2
The molecular modeling studies of the selected compounds 2d,
2f, 3c and 3d was performed using Molecular Operating Environ-
ment (MOE, 10.2008) software. The X-ray crystallographic structure
of Cyclooxygenase-2 enzyme (COX-2) (PDB entry 1CX2) was
downloaded from the protein data bank website (http://www.rcsb.
org).
The protein structure was prepared by deleting the repeating
chains and water molecules. Hydrogen atoms were added to the
system using Protonate 3D application and the partial charges were
 E.M. Ahmed et al. / European Journal of Medicinal Chemistry 171 (2019) 25e37
calculated followed by isolation of the determined pocket and the
back bone was hidden. Triangle Matcher as method of displace-
ment and London dG as the main scoring function were used for
docking. The database of active compounds and celecoxib was
prepared by adding hydrogen (Protonate 3D option), calculating
partial charges and minimizing energy by Merck Molecular force
field (MMFF94) until an RMSD gradient of 0.05 kcal mol1A
1
followed by docking into the active site using the MOE Dock tool
(Figs. 6e12).
Acknowledgments
The authors are grateful to Dr. Amany Ameen Sleem, Professor of
Pharmacology, Pharmacology Department, National Research
Center, Dokki, Giza, Egypt for carrying out the in vivo anti-
inflammatory assay and gastric ulcerogenic activity. The authors
thank Dr. Esam Rashwan, Head of the confirmatory diagnostic unit
VACSERA-EGYPT, for carrying out the in vitro COX-1 and COX-2
inhibitory assay. The authors would like to acknowledge Dr. Amr
Sayed Motawi, Lecturer of Pharmaceutical Organic Chemistry,
Pharmaceutical Organic Chemistry Department, Faculty of Phar-
macy, Cairo University for helping in Molecular Docking.
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