Novel anti-inflammatory agents based on pyridazinone scaffold; design, synthesis and in vivo activity

Article abstract of DOI:10.1016/j.bmc.2008.04.007

Herein, we report the design, synthesis, and pharmacological properties of a series of arylethenylpyridazinones 3a-h and arylethylpyridazinone derivatives 3k-i from the corresponding aryloxohexenoic 1a-e and aryloxohexanoic acids 2a,d, respectively. The synthesized compounds were tested for their anti-inflammatory activity in carrageenan-induced rat paw edema model. Compound 3j demonstrated the greatest in vivo activity with ED₅₀ equal to 17 μmol compared with celecoxib with no ulceration on the gastric mucosa. Docking study of the synthesized compounds into the active site of COX-2 revealed a similar binding mode to RS-57067, a COX-2 inhibitor.

Full text of DOI:10.1016/j.bmc.2008.04.007

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5547–5556
Novel anti-inflammatory agents based on pyridazinone
scaffold; design, synthesis and in vivo activity
Khaled Abouzida,* and Salma A. Bekhitb
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo 11566, Egypt
bDepartment of Pharmacology, Faculty of Veterinary Medicine, University of Alexandria, Edfina, Beheira, Egypt
Received 11 December 2007; revised 2 April 2008; accepted 4 April 2008
Available online 9 April 2008
Abstract—Herein, we report the design, synthesis, and pharmacological properties of a series of arylethenylpyridazinones 3a–h and
arylethylpyridazinone derivatives 3k–i from the corresponding aryloxohexenoic 1a–e and aryloxohexanoic acids 2a,d, respectively.
The synthesized compounds were tested for their anti-inflammatory activity in carrageenan-induced rat paw edema model. Com-
pound 3j demonstrated the greatest in vivo activity with ED50 equal to 17 lmol compared with celecoxib with no ulceration on
the gastric mucosa. Docking study of the synthesized compounds into the active site of COX-2 revealed a similar binding mode
to RS-57067, a COX-2 inhibitor.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
panied the rapid development of this field and numerous
reviews have been published.2–4 The three-dimensional
crystal structures of both COX-1 and COX-2 have been
solved.5 The two enzymes are highly homologous, with
63% identity and 77% similarity. The COX-1 active site
contains an isoleucine residue (Ile523) which is replaced
by a valine residue in COX-2 (Val523). This single ami-
no acid difference allows access to a polar side pocket in
COX-2, and binding in this pocket, along with a differ-
ence in binding kinetics, largely accounts for the selectiv-
ity observed with COX-2 inhibitors.6 Selective
cyclooxygenase-2 (COX-2) inhibitors represent a new
generation of anti-inflammatory drugs as they demon-
strated less gastrointestinal side effects than the classical
NSAIDs, which also inhibit the cytoprotective action of
COX-1 in the GI tract.7–9 During the last decade, several
selective inhibitors (coxibs) have been reported10 and
many of them have now reached the market such as
rofecoxib,11 celecoxib,12 valdecoxib,13 and etoricoxib14
(Fig. 1). Later on, many publications emerged in this
field aiming at finding new lead in this area.15,16 Clinical
experience with marketed COX-2 inhibitors has con-
firmed the utility of these agents in the treatment of
inflammatory pain and symptoms of osteoarthritis and
rheumatoid arthritis with an improved gastrointestinal
safety profile relative to NSAID comparators. More-
over, withdrawal of rofecoxib from the market recently
by its originators due to adverse cardiovascular effects
puts a big question mark on the safety profile of other
COXIBs in the long-term therapy.17 Consequently, there
Non-steroid anti-inflammatory drugs (NSAIDs) which
inhibit the activity of cyclooxygenases (COXs) are
widely used medication for the treatment of pain and
inflammation. The common dose limiting toxicity of
the classical NSAIDs is the increased risk of gastrointes-
tinal ulceration and hemorrhage.1a Cyclooxygenase cat-
alyzes the conversion of arachidonic acid into
prostaglandin H2 as the first committed step of prosta-
glandin biosynthesis. This enzyme has been known as
the target of non-steroidal anti-inflammatory drugs
(NSAIDs). In 1991 it was recognized that this enzyme
existed in two isoforms, designated COX-1, a constitu-
tive enzyme, and COX-2, an enzyme induced in response
to a variety of pro-inflammatory stimuli.1b,2 This recog-
nition led to the hypothesis that the anti-inflammatory
and analgesic effects of NSAIDs were due to the inhibi-
tion of COX-2, while many of the undesirable side ef-
fects of these drugs, particularly gastric irritation, were
due to non-selective cyclooxygenase inhibition. The
identification of selective inhibitors of COX-2 (coxibs)
followed by clinical trials has largely validated this
hypothesis. An extensive body of literature has accom-
Keywords: Pyridazinone; Anti-inflammatory; Selective COX-2 inhib-
itors; Docking study.
*
Corresponding author. Tel.: +202 2508 0848; fax: +202 2508 0728;
e-mail: abouzid@yahoo.com
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.007

5548
K. Abouzid, S. A. Bekhit / Bioorg. Med. Chem. 16 (2008) 5547–5556
a
H3C
O
O
CF3
N
N
H3CO2S
H2NO2S
Celecoxib
Rofecoxib
H3CO2S
CH3
N
N
O
CH3
N
H2NO2S
Cl
Valdecoxib
Etoricoxib
O
b
O
O
NH
Cl
CH3
N
N
NH
O
H3C
O
N
RS-57067
Syntex compound
F
F
R1
R2
O
N
H
N
N
N
Ra
N
H
O
O
Benzylpyridazinone derivative
Pyridazinone scaffold
Figure 1. (a) Some famous COXIBs and (b) pyridazinone lead compounds and our pyridazinone scaffold.
is a current need to develop safer anti-inflammatory
agents based on alternative templates.
and after their exact mechanism became established,
molecular design of selective inhibitors became straight-
forwardly accessible.23–26
Later on, a number of new indications have been
explored clinically with celecoxib and/or rofecoxib.
The role of selective COX-2 inhibitors in the prevention
and treatment of a variety of cancers has been explored
extensively as COX-2 is expressed at high levels in
numerous cancer tissues and this expression has corre-
lated with lower survival rates in patients.18
Taking the reports about the potential cardiovascular
dangers posed by COXIBs into cognizance, and the cost
and time involved in the discovery of a new drug, our
complementary strategy is concerned with the modifica-
tion of structures of some conventional NSAIDs. This
could be verified through conversion of the carboxylic
group of NSAIDs into cyclic amide functions aiming
to enhance the selectivity of these compounds towards
COX-2 enzyme taking advantage of the larger COX-2
active site and thus impart their selectivity. This effort
has been aided greatly by the availability of structural
information on both COX enzymes.27
Since it has been reported that pyridazinone nucleus was
found to be an excellent template for many selective
COX-2 inhibitors such as the syntex compound and
RS-57067 and benzylpyridazinone derivatives19–22
have been described. In the same direction, we planned
to design new pyridazinone scaffolds as potential
anti-inflammatory agents which could act as selective
COX-2 inhibitors (Fig. 1).
Based on the literature data in the anti-inflammatory
area, pyridazinone skeleton was considered to be an
excellent template for novel selective COX-2 inhibitors.
In this respect, we describe a new template for COX-2
inhibitors based on pyridazinone system.22 In these de-
signed compounds we utilized certain feature from
non-selective anti-inflammatory drugs such as biphenyl
fragment in flurbiprufen and fenbufen, and ethylene di-
2. Rational and design
After identification of theX-ray three-dimensional crystal
structure of both human COX-1 and COX-2 isozymes

K. Abouzid, S. A. Bekhit / Bioorg. Med. Chem. 16 (2008) 5547–5556
5549
oxy or methylene dioxy fragment in the previously re-
ported NSAIDs.28
duced with Pd/C to the corresponding oxohexanoic
acids. Thereafter, the obtained keto acids 1a–e were cyc-
lized with hydrazine hydrate or its methyl or phenyl
derivative in acetic acid to afford the dihydropyridazi-
none derivatives. On the other hand, oxohexanoic acid
derivatives were cyclized more smoothly with hydrazine
hydrate or methylhydrazine to provide the correspond-
ing dihydropyridazinones (3i–k) in refluxing ethanol
for 2–3 h. On the other hand, pyridazinones 4a and
4b were obtained in moderate yield from the corre-
sponding 4,5-dihydropyridazinones (3c and 3i) under
mild conditions with anhydrous CuCl2 in acetonitrile
via halogenation and spontaneous HCl elimination un-
der the previously described reaction condition.29
In addition, we investigated further attempts to refine
the basic pyridazinone framework designed previously22
by connecting pyridazinone ring at C(6) with an aryl
moiety via ethyl or ethenyl chain instead of a methylene
spacer. Also, by placing various substituents on the phe-
nyl ring or on the amidic pyridazinone N(2). Also, par-
tially reduced pyridazinone ring could replace the
pyridazinone ring without affecting their binding energy
to the active site of COX-2.
Besides, we employed protein docking study to discover
structurally novel inhibitors of cyclooxygenase-2 (COX-
2) by screening a selected number of pyridazinone
derivatives, thus providing an alternative to extensive
screening. Also, structural analysis of various anti-
inflammatory agents and studying their docking mode
in the flexible active site of COX-2 isozyme revealed that
certain structural modification of pyridazinone skeleton
could be afforded. From the results of virtual screening
for many pyridazinone derivatives, a computational
model for COX-2 inhibition was constructed and used
to decide on candidate inhibitors to be synthesized (see
Scheme 1).
Compound
R1
R2
R
3a
3b
3c
3d
3e
3f
3g
3h
3i
Ph
Ph
H
F
H
H
H
H
H
CH3
CH3
CH3
Ph
H
OPh
–OCH2CH2O–
Ph
–OCH2CH2O–
–OCH2O–
Ph
Ph
H
H
H
3j
–OCH2CH2O–
Ph
H
CH3
H
3k
4a
4b
H
3. Chemistry
OPh
Ph
H
H
H
We report the synthesis of two series of pyridazinone
target compounds from the corresponding keto acids
as depicted in the following scheme. Aryloxohexenoic
acids represent a key intermediate for the synthesis of
the target compounds which was synthesized via con-
densation of the appropriately substituted araldehyde
with levulinic acid according to the method previously
reported.28 Moreover, aryl oxohexenoic acids were re-
4. Molecular modeling
A variety of COX-2-ligand crystal structures have
been solved over the last years.23 These X-ray struc-
R1
R1
R2
a
COOH
COOH
1a,d
R2
O
O
1a-e
2a,d
1a; R1=Ph, R2= H
2a; R1=Ph, R2= H
2d; R1R2= -OCH2CH2O-
1b; R1=Ph, R2=F
b
1c; R1=OPh, R2= H
b
1d; R1R2= -OCH2CH2O-
R1
R2
R1
R2
N
N
3a-h
3c
3i-k
3i
N
R
O
N
O
R
c
c
R1
R2
R1
R2
N
N
N
H
O
N
H
O
4a
4b
Scheme 1. Reagents and condition: (a) H2 Pd/C, ethanol; (b) NH2NHR/ethanol or acetic acid, reflux; and (c) CuCl2/acetonitrile.

5550
K. Abouzid, S. A. Bekhit / Bioorg. Med. Chem. 16 (2008) 5547–5556
tures provide important information about the rele-
vant interaction possibilities at the COX-2 binding
pocket. The first crystal structure of human cyclooxy-
genase-2, in the presence of a selective inhibitor, is
similar to that of cyclooxygenase-1. The structure of
the non-steroidal anti-inflammatory drug (NSAID)
binding site is also well conserved, although there
are differences in its overall size and shape which
may be exploited for the further development of selec-
tive COX-2 inhibitors. A second COX-2 structure with
a different bound inhibitor displays a new, open con-
formation at the bottom of the NSAID binding site,
without significant changes in other regions of the
COX-2 structure. These two COX-2 structures provide
evidence for the flexible nature of cyclooxygenase,
revealing details about how substrate and inhibitor
may gain access to the cyclooxygenase active site from
within the membrane.23
The predicted docked energy of the best scoring member
of the largest cluster obtained for each ligand and the
experimental ED50 values are given in Table 1.
Figure 2 shows straight line correlation between the
ED50 and docking energy, this confirms the validity of
docking study.
From the binding study we could conclude that most of
the ligands exhibit interactions with the amino acids;
TRP 328, MET 522, VAL 523, SER 530, ALA 527,
and SER 353. It was noticed that the ligands have
moved a little away from ARG 513, TYR 355, and
ARG 120 (Figs. 3–9). In the active site of COX-2, the
amide NH of pyridazinone forms hydrogen bond with
SER 530 in addition to extensive van der Waals interac-
tions with many amino acids in contact. On the other
hand, both compounds 3i and 3j showed the highest
binding affinity to the COX2 active site as both com-
pounds possessed the least docking energy compared
with the other compounds and at the same time pro-
duced the best activity in terms of ED50%. While com-
pound 3k with a flexible ethyl chain and N-methyl
group exhibits little increase in docking energy and les-
ser ED50% compared with its desmethyl derivative,
compound 3h which has N-phenyl group displayed the
highest energy at the binding site. This coincides with
the biological data which showed the least activity.
The docking study was performed using Autodock
software version 4 according to the reported proce-
dure.30 The COX-2 structure was obtained from the
protein databank (PDB entry: 1CVU). The structures
were minimized by Merck Molecular force field
(MMFF) to a gradient of 4.5 · 10ꢀ5 using Spartan’06.
The optimized geometries were exported as Sybyl
mol2 file which were exported to Autodock tools.
Gasteiger charges were calculated for each molecule
and the torsional degrees of freedom were set to the
maximum number of rotatable bonds. Water mole-
cules were deleted from the file and hydrogens were
added, then Kollman charges were calculated. No fur-
ther geometry optimization was performed. Non-polar
hydrogens were merged with the parent atoms for
both the ligands and the receptor.
5. Biological screening
5.1. Anti-inflammatory activity
Carrageenan-induced rat paw edema test: Male albino
rats weighing 120–150 g (Medical Research Institute,
Alexandria University) were used throughout the work.
They were kept in the animal house under standard con-
ditions of light and temperature with free access to food
and water. The animals were randomly divided into
groups of six rats each. The paw edema was induced
by subplantar injection of 50 lL of 2% carrageenan
solution in saline (0.9%). Indomethacin and the test
compounds were dissolved in DMSO and injected sub-
cutaneously in different dose levels of 5, 10, 15, 20 and
25 lmol/kg body weight, 1 h prior to carrageenan injec-
tion. DMSO was injected to the control group. The vol-
ume of paw edema (mL) was determined by means of
water plethysmometer immediately after injection of
carrageenan and 4 h later. ED50 was calculated for the
test compounds and reference drugs through dose re-
sponse curves by measuring the inhibition of edema vol-
ume 4 h after the carrageenan injection.31 These data
were calculated using Microsoft excel software version
2002 and presented in Table 2.
Structure preparation for the ligands and the receptor
was done in Autodock tools. Each docking run was
composed of 30 runs of the Lamarckian genetic algo-
rithm (LGA), the number of top individuals to survive
to next generation was set to two, and the clustering
RMS tolerance was set to 3 A, all the other docking
parameters were kept unmodified from their default
values.
˚
Table 1. Predicted docked energy of the best scoring member of the
largest cluster obtained for each ligand and experimental ED50 values
Compound
Docked energy
ED50
RS-57067
ꢀ10.18
ꢀ9.05
3a
3b
3c
3d
3e
3f
24.6
N/T
28.7
27.0
32.4
N/T
34.3
38.6
18.7
17.0
22.3
N/T
N/T
ꢀ10.2
ꢀ10.67
ꢀ9.74
ꢀ10.44
ꢀ9.74
The percentage protection against inflammation was cal-
culated as follows: Vc ꢀ Vd/Vc · 100, where Vc is the in-
crease in paw volume in the absence of the test
compound (control) and Vd is the increase of paw
volume after injection of the test compound. Data were
3g
3h
3i
ꢀ8.71
ꢀ8.04
ꢀ10.95
ꢀ10.72
ꢀ10.09
ꢀ10.18
ꢀ10.92
3j
3k
4a
4b
expressed as means SEM. Significant differences be-
tween the control and the treated groups were obtained

K. Abouzid, S. A. Bekhit / Bioorg. Med. Chem. 16 (2008) 5547–5556
5551
40
38
36
34
32
30
28
26
24
22
20
18
16
-11.5
-11.0
-10.5
-10.0
-9.5
-9.0
-8.5
-8.0
-7.5
Docked energy
Figure 2. Correlation coefficient between the docked energy and the ED50 = ꢀ0.71 (significant at P = 0.05).
Figure 5. Docking of compound 3j (spacefill) in the active site of
COX-2.
Figure 3. Large binding site of COX-2 appears as a green volume.
using Student’s t-test and P values. The differences in re-
sults were considered significant when P < 0.001. The
anti-inflammatory activity of the test compounds
relative to that of indomethacin was also determined
(Table 3).
5.2. Ulcerogenic effects
Most of the synthesized compounds were evaluated for
their ulcerogenic potential in rats.32 Indomethacin was
used as reference standard. Male albino rats (100–120
g) were fasted for 12 h prior to the administration of
the compounds. Water was given ad libitum. The ani-
mals were divided into groups, each of six animals.
The control group received 1% gum acacia orally. Other
groups received indomethacin or the test compounds or-
ally in two equal doses at 0 and 12 h for three successive
days at a dose of 30 lM/kg per day. Animals were sac-
rificed by diethyl ether 6 h after the last dose and the
stomach was removed. An opening at the greater curva-
Figure 4. RS-57067 (spacefill) bound in the active site of COX-2.

5552
K. Abouzid, S. A. Bekhit / Bioorg. Med. Chem. 16 (2008) 5547–5556
nyl moieties through two carbon spacers. The synthe-
sized compounds exhibited anti-inflammatory activity
and superior gastrointestinal safety profile. The results
of biological screening also revealed that compounds
3i and 3j in which ethyl spacer between the dihydropy-
ridazinone ring and the aryl moiety exhibits the highest
activity compared to the ethenyl analogs.
In addition, two potent and probable selective COX-2
inhibitors 3i and 3j have been identified among the test
compounds and showed excellent efficacy in animal mod-
els of inflammation. Moreover, since it was reported that
the safety profile of ulcerogenic test could be considered as
a measure for COX-2 selectivity. Accordingly, these com-
pounds could be speculated as selective COX-2 inhibi-
tors.33 This hypothesis is substantiated by the molecular
docking study which demonstrated that docking energy
of these compounds are comparable to that of the lead
compound RS-57067, which is previously reported as
selective COX-2. Also, their binding mode to the active
site of COX-2 with amino acids in the vicinity is very close
to that of the selective COX-2 inhibitors.
Figure 6. Compound 3j (ball and stick) docked in the active site of
COX-2 showing H bonding between amide H and ser-530 as dotted
green line.
7. Experimental
Melting points were determined with a Stuart Scientific
apparatus and are uncorrected. IR spectra were re-
corded on a Perkin-Elmer FT-IR series using KBr
1
cell. H NMR and 13C NMR were measured in d scale
on Varian-200 MHz, Gemini-200-software spectrometer
and Brucker 300 spectrometers. All the spectra were ob-
¨
tained in solutions in DMSO-d6 or CDCl3 and referred
to TMS. The electron impact (EI) mass spectra were re-
corded on Finnigan Mat SSQ 7000 (70 eV) mass spec-
trometer. Analytical thin layer chromatography (TLC)
on silica gel plates containing UV indicator was em-
ployed routinely to follow the course of reactions and
to check the purity of products. All reagents and sol-
vents were purified and dried by standard techniques.
Elemental microanalyses were performed at Microana-
lytical Center, Cairo University, and were within 4%
unless otherwise stated. All chemicals were purchased
from Sigma–Aldrich, Germany, and all the solvents
were of analytical grades.
Figure 7. Compound 3i (ball and stick) docked in the active site of
COX-2.
ture was made and the stomach was cleaned by washing
with cold saline and inspected with a 3· magnifying lens
for any evidence of hyperemia, hemorrhage, definite
hemorrhagic erosion, or ulcer. An arbitrary scale was
used to calculate the ulcer index which indicates the
severity of the stomach lesions.32 (Table 2). The % ulcer-
ation for each group was calculated as follows:
7.1. General procedure for preparation of (E)-6-(2-
arylvinyl)-4,5-dihydropyridazin-3(2H)-ones (3a–d)
To a solution of (E)-6-aryl-4-oxohex-5-enoic acids (1a–
d) (0.071 mmol) in acetic acid (5 mL) hydrazine hydrate
80% (0.71 mmol) was slowly added and the reaction
mixture was stirred under reflux for 4 h. The solvent
was removed under reduced pressure. The residue was
triturated with 10 mL distilled water, filtered and recrys-
tallized from the appropriate solvent.
Number of animals bearing ulcer in a group
% Ulceration ¼
Total number of animals in the same group
ꢁ100
7.1.1. (E)-6-[2-(Biphenyl-4-yl)vinyl]-4,5-dihydropyridazin-
3(2H)-one (3a). Compound 3a was prepared from
1a and recrystallized from ethanol as white needles.
Yield: 71%; mp: 216–217 ꢁC, H NMR (DMSO-d6)
(300 MHz) d: 2.41 (t, 2H, J = 7.85 Hz, CH2, dihydropy-
ridazinone), 2.81 (t, 2H, J = 7.85 Hz, CH2, dihydropy-
6. Conclusion
1
The present study describes the synthesis of a series of
pyridazinone derivatives linked at C(6) to aryl or biphe-

K. Abouzid, S. A. Bekhit / Bioorg. Med. Chem. 16 (2008) 5547–5556
5553
Figure 8. Compound 3c (ball and stick) docked in the active site of COX-2.
Figure 9. Overlaid docked pyridazinone structures showing that they all bind with the same orientation.
Table 2. Anti-inflammatory (ED50, lmol/kg)a and ulcerogenic activity
of 6-substituted-4,5-dihydropyridazin-3(2H)-ones (3a–k) and reference
ArH), 7.47 (t, 2H, J = 7.6 Hz, ArH), 7.69–7.71 (m, 6H,
ArH), 10.9 (s, 1H, NH), 13C NMR (20, 25, 126, 127,
128, 129, 132, 136, 140, 152, 170). IR (KBr) cmꢀ1
3250, 1686. MS m/z 276 (M+, 43%). Anal. Calcd for
C18H14N2 O: C, 78.24; H, 5.84; N, 10.14. Found: C,
78.16; H, 5.76; N, 10.21.
drugs
Compound
ED50 (lmol/kg)
% Ulceration
Indomethacin
Celecoxib
9.7 0.022
16.7 0.024
24.6 0.018
N/T
100
0
3a
3b
3c
3d
3e
3f
10
7.1.2. (E)-6-[2-(2-Fluorobiphenyl-4-yl)vinyl]-4,5-dihydro-
pyridazin-3(2H)-one (3b). Compound 3b was prepared
from 1b and recrystallized from ethanol as a white
N/T
0.0
0.0
20
28.8 0.033
27.0 0.020
32.4 0.026
N/T
1
solid. Yield: 80%; mp: 188–189 ꢁC, H NMR (CDCl3)
N/T
10
(200 MHz) d: 2.54 (t, 2H, J =8.0 Hz, CH2, dihydropyrid-
azinone), 2.84 (t, 2H, J = 8.1 Hz, CH2, dihydropyridaz-
inone), 6.87 (d, 1H, J = 16.4 Hz, –CH@), 7.23 (d, 1H,
J = 16.6 Hz, –CH@), 7.36 (m, 8H, ArH), 9.0 (s, 1H,
NH). IR (KBr) cmꢀ1 : 3220, 1680. MS m/z 294 (M+,
87.26%). Anal. Calcd for C18H15FN2 O: C, 73.45; H,
5.14; N, 9.52. Found: C, 73.37; H, 4.97; N, 9.35.
3g
3h
3i
34.3 0.028
38.6 0.031
18.7 0.025
17.0 0.027
22.3 0.019
10
10
3j
3k
0.0
0.0
N/T means not tested.
a All data are significantly different from control (P < 0.001).
7.1.3. (E)-6-(4-Phenoxystyryl)-4,5-dihydropyridazin-3
(2H)-one (3c). Compound 3c was prepared from 1c and
ridazinone), 6.93 (d, 1H, J = 16.4 Hz, –CH@), 7.08 (d,
1H, J = 16.6 Hz, –CH@), 7.37 (t, 1H, J = 7.6 Hz,

5554
K. Abouzid, S. A. Bekhit / Bioorg. Med. Chem. 16 (2008) 5547–5556
Table 3. Effect of compounds 3i and 3j on carrageenan-induced rat
paw edema (mL), % protection and activity relative to indomethacin
CH2O–), 6.74–7.0 (m, 5H, 2-CH@ and 3 ArH). IR
(KBr) cmꢀ1: 1661, 1578. MS m/z 272 (M+, 100%). Anal.
Calcd for C15H16N2O3: C, 66.16; H, 5.92; N, 10.29, C,
66.05; H, 5.88; N, 10.16.
Test compound Increase in paw % Protection Activity
edema
relative to
indomethacin
(mL) SEMa,b
7.1.7. (E)-6-(2-[Benzo[d][1,3]dioxol-5-yl)vinyl]-2-methyl-
4,5-dihydropyridazin-3(2H)-one (3g). Compound 3g was
prepared from 1e, N-methylhydrazine and refluxed for
4 h, recrystallized from aqueous ethanol as a white solid.
Control
Indomethacin
Celecoxib
0.98 0.027
0.25 0.024
0.22 0.016
0.41 0.015
0.38 0.012
0.0
74.4
0.0
100
77.5
58.16
61.22
104.23
78.17
82.28
3i
3j
1
Yield: 87 %; mp: 195–197 ꢁC, H NMR (DMSO-d6)
a SEM denotes the standard error of the mean.
b All data are significantly different from control (P < 0.001).
(300 MHz) d: 2.41 (t, 2H, J = 7.85 Hz, CH2, dihydropy-
ridazinone), 2.77 (t, 2H, J = 7.85 Hz, CH2, dihydropy-
ridazinone), 3.24 (s, 3H, CH3), 6.04 (s, 2H, –OCH 2
O–), 6.70 (d, 1H, J = 16.4 Hz, –CH@), 6.92 (d, 1H,
J = 8.2 Hz, ArH), 7.01 (d, 1H, J = 16.4 Hz, –CH@),
7.05–707 (dd, 1H, J = 2, 8.4 Hz, ArH), 7.29 (d, 1H,
J = 1.55 Hz, ArH), 13 C NMR (20, 26, 35, 100, 105,
recrystallized from ethanol as a beige solid. Yield: 76%;
mp: 173–175 ꢁC, H NMR (CDCl3) (300 MHz) d: 2.55
1
(t, 2H, J = 8.0 Hz, CH2, dihydropyridazinone), 2.81 (t,
2H, J = 8.1 Hz, CH2, dihydropyridazinone), 6.80 (d,
1H, J = 16.0 Hz, –CH@), 7.04 (d, 1H, J = 16.0 Hz,
–CH@), 7.11–7.45 (m, 9H, ArH), 8.67 (s, 1H, NH). IR
(KBr) cmꢀ1: 3210, 1675. MS m/z 292 (M+, 57.2%). Anal.
Calcd for C18H16N2O2: C, 74.47; H, 4.86; N, 9.65.
Found: C, 74.41; H, 4.77; N, 9.32.
108, 122, 123, 130, 134, 146, 152, 164). IR (KBr) cmꢀ1
:
1640. MS m/z 258 (M+, 48.1%). Anal. Calcd for
C14H14N2O3 : C, 65.11; H, 5.46; N, 10.85. Found: C,
65.08; H, 5.79; N, 10.68.
7.1.8. (E)-6-[2-(Biphenyl-4-yl)vinyl]-2-phenyl-4,5-dihydro-
pyridazin-3(2H)-one (3h). Compound 3h was prepared
from 1a, phenylhydrazine and refluxed for 4 h recrystal-
lized from aqueous ethanol as a white solid. Yield: 87 %;
7.1.4 . (E)-6-[2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)vi-
nyl]-4,5-dihydropyridazin-3(2H)-one (3d). Compound 3d
was prepared from 1d and recrystallized from ethanol as
off-white solid. Yield: 66%; mp: 179–181 ꢁC, H NMR
(DMSO-d6) (300 MHz) d: 2.36 (t, 2H, J = 7.85 Hz, CH2,
dihydropyridazinone), 2.73 (t, 2H, J = 7.85 Hz, CH2,
dihydropyridazinone), 4.25 (s, 4H, –OCH CH2 O–),
6.70 (d, 1H, J = 16.4 Hz, –CH@), 6.84 (d, 1H,
J = 8.2 Hz, ArH), 6.90 (d, 1H, J = 16.4 Hz, –CH@),
7.05–7.07 (dd 1H, J = 1.9, 8.4 Hz, ArH), 7.09 (d, 1H,
J = 2.2 Hz, ArH), 10.81 (s, 1H, NH), 13C NMR (20, 25,
64, 65, 115, 117, 120, 126, 130, 144, 145, 151, 167). IR
(KBr) cmꢀ1: 3440, 1670. MS m/z 258 (M+, 55.4%). Anal.
Calcd for C14H14N2O3 : C, 65.11; H, 5.46; N, 10.85.
Found: C, 65.34; H, 5.49; N, 10.67.
1
mp: 151–153 ꢁC, H NMR (CDCl3) (300 MHz) d: 2.76
1
(t, 2H, J = 7.6 Hz, CH2, dihydropyridazinone), 2.95 (t,
2H, J = 7.6 Hz, CH2, dihydropyridazinone), 6.91–7.62
(M, 16H, –CH@ and ArH). IR (KBr) cmꢀ1: 1635. MS
m/z 352 (M+, 44.4%). Anal. Calcd for C24H20N2O.C,
81.79; H, 5.72; N,7.95. Found: C, 81.66; H, 5.57; N, 7.69.
2
7.2. General procedure: 6-(2-arylethyl)-4,5-dihydropyri-
dazin-3(2H)-ones (3i–k)
To a solution of 6-aryl-4-oxohax-5-enoic acid (2a or 2d)
(0.071 mmol) in ethanol (10 mL), hydrazine hydrate or
N-methylhydrazine 80% (0.71 mmol) was added and the
reaction mixture was stirred under reflux for 3 h. The
reaction mixture was cooled and the precipitate formed
was filtered, washed with diethylether, and recrystallized
from the appropriate solvent.
7.1.5 . (E)-6-[2-(Biphenyl-4-yl)vinyl]-2-methyl-4,5-dihyd-
ropyridazin-3(2H)-one (3e). Compound 3e was prepared
from 1a, N-methylhydrazine and refluxed for 6 h, recrys-
tallized from ethanol as a faint yellow solid. Yield: 86%;
1
mp: 205–207 ꢁC, H NMR (DMSO-d ) (300 MHz) d:
7.2.1. 6-[2-(Biphenyl-4-yl)ethyl]-4,5-dihydropyridazin-3(2H)
-one (3i). Compound 3i was prepared from 1a and
hydrazine hydrate. Yield: 85 %; mp: 194–196 ꢁC H
6
2.45 (t, 2H, J = 8.2 Hz, CH2, dihydropyridazinone),
2.82 (t, 2H, J = 8.2 Hz, CH2, dihydropyridazinone),
3.27 (s, 3H, CH3), 6.91 (d, 1H, J = 16.4 Hz, –CH@),
7.06 (d, 1H, J = 16.6 Hz, –CH@), 7.32 (t, 1H,
J = 7.6 Hz, ArH), 7.44 (t, 2H, J = 7.6 Hz, ArH), 7.65
(m, 6H, ArH), 13C NMR (20, 27, 38, 126, 127, 128, 129,
132, 130, 138, 152, 167). IR (KBr) cmꢀ1: 1699. MS m/z
290 (M+, 77.1%). Anal. Calcd for C19H18N2O: C, 78.59;
H, 6.25; N, 9.65. Found: C, 78.43; H, 6.11; N, 9.34.
1
NMR (DMSO-d6) (300 MHz) d: 2.26 (t, 2H, J =
8.1 Hz, CH2), 2.46 (t, 2H, J = 7.9 Hz, CH2), 2.58 (t,
2H, J = 7.9 Hz, CH2, dihydropyridazinone), 2.86 (t,
2H, J = 8.2 Hz, CH2, dihydropyridazinone), 7.31–7.35
(m, 3H, ArH), 7.45 (t, 2H, J = 7.6 Hz, ArH), 7.57 (d,
2H, J = 8.2 Hz, ArH), 7.63 (d, 2H, J = 7.25 Hz, ArH),
10.46 (s, 1H, NH), 13C NMR (23, 27, 32, 38, 125, 126,
128, 138, 140, 141, 154, 168). IR (KBr) cmꢀ1: 3247,
1662. MS m/z 278 (M+, 17.1%), 167 (100%). Anal. Calcd
for C18H18N2 O: 77.67; H, 6.52; N, 10.06. Found: C,
77.38; H, 6.42; N, 9.86.
7.1.6. (E)-6-[2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)vinyl]-2-
methyl-4,5-dihydropyridazin-3(2H)-one (3f). Compound
3f was prepared from 1d, N-methylhydrazine and
refluxed for 7 h, recrystallized from aqueous ethanol as
a white solid. Yield: 90 %; mp: 176–178 ꢁC, H NMR
(CDCl3) (300 MHz) 2.49 (t, 2H, J = 8.1 Hz, CH2, dihyd-
ropyridazinone), 2.75 (t, 2H, J = 7.85 Hz, CH2, dihydro-
pyridazinone), 3.39 (s, 3H, CH3), 4.25 (s, 4H, –OCH2
1
7.2.2 . 6-[2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)ethyl]-
4,5-dihydropyridazin-3(2H)-one (3j). Compound 3j was
obtained from 2d and recrystallized from dioxan. Yield:
77%; mp: 139–141 ꢁC 1H NMR (DMSO-d6) (300 MHz)

K. Abouzid, S. A. Bekhit / Bioorg. Med. Chem. 16 (2008) 5547–5556
5555
d: 2.23 (t, 2H, J = 8.5 Hz, CH2), 2.41 (t, 2H, J = 7.9 Hz,
CH2), 2.47 (t, 2H, J = 7.25 Hz, CH2, dihydropyridazi-
none), 2.68 (t, 2H, J = 7.25 Hz, CH2, dihydropyridazi-
operation and for carrying out NMR spectra. I also
thank Dr. Azza Baraka, Department of Pharmacol-
ogy, Faculty of Medicine, University of Alexandria
for her indispensable support in the pharmacological
study.
none 4.18 (s, 4H, –OCH CH2O–), 6.64–6.66 (dd, 1H,
2
J = 2, 8.4, ArH), 6.70 (d, 1H, J = 2.2 Hz, ArH), 6.74
(d, 1H, J = 8.2 Hz, ArH), 10.5 (s, 1H, NH), 13C NMR
(20, 25, 64, 65, 115, 117, 120, 126, 130, 144, 145, 151,
167). IR (KBr) cmꢀ1 : 3442, 1649,1641,1598. MS m/z
260 (M+, 6.7%), 149 (100%). Anal. Calcd for
C14H16N2O3: C, 64.60; H, 6.20; N, 10.76. Found: C,
64.66; H, 6.15; N, 10.88.
References and notes
1. (a) Allison, M. C.; Howatson, A. G.; Torrance, C. J.; Lee,
F. D.; Russel, R. I. N. Engl. J. Med. 1992, 327, 749; (b)
Botting, R. M. J. Therm. Biol. 2006, 31, 208.
2. Vane, J. R.; Bakhle, Y. S.; Blotting, R. M. Annu. Rev.
Pharmacol. Toxicol. 1998, 38, 97.
3. Dannhardt, G.; Kiefer, W. Eur. J. Med. Chem. 2001, 36,
109.
4. Isakson, P. C. Prog. Exp. Tumor Res. 2003, 37, 25.
5. Garavito, R. M.; Mulichak, A. M. Annu. Rev. Biophys.
Biomol. Struct. 2003, 32, 183.
6. Kurumbail, R. G.; Stevens, A. M.; Gierse, J. K.;
McDonald, J. J.; Stegeman, R. A.; Pak, J. Y.; Gildehaus,
D.; Miyashiro, J. M.; Penning, T. D.; Seibert, K.; Isakson,
P. C.; Stallings, W. C. Nature 1996, 384, 644.
7. Bombardier, C. Am. J. Cardiol. 2002, 89 (Suppl. ), 3-D .
8. Ferreira, S. H.; Moncada, S.; Vane, J. R. Nature 1971,
231, 237.
7.2.3 . 6-(2-(Biphenyl-4-yl)ethyl)-2-methyl-4,5-dihydropy-
ridazin-3(2H)-one (3k). Compound 3k was prepared
from 1a and recrystallized from ethanol as white crys-
1
tals. Yield: 91%; mp: 145–146 ꢁC H NMR (DMSO-d
6) (300 MHz) d: 2.31 (t, 2H, J = 8.1 Hz, CH2), 2.49 (t,
4H, J = 7.9 Hz, CH2), 2.60 (t, 2H, J = 7.9 Hz, CH2),
2.86 (t, 2H, J = 8.2 Hz, CH2), 3.16 (s, 3H, CH3), 7.31–
7.36 (m, 3H, ArH), 7.48 (t, 2H, J = 7.9 Hz, ArH), 7.57
(d, 2H, J = 8.2 Hz, ArH), 7.63 (dd, 2H, J = 8.50,
0.95 Hz, ArH), 10.46 (s, 1H, NH), 13C NMR (24, 27,
32, 36, 39, 125, 126, 128,139,141, 142, 156, 168). IR
(KBr) cmꢀ1: 1660. MS 292 (M+, 33.2%), 167 (100%).
Anal. Calcd for C19H20N2 O: C, 78.05; H, 6.89; N,
9.58. Found: C, 77.75; H, 6.94; N, 9.79.
9. Murry, M. D.; Brater, D. C. Annu. Rev. Pharmacol.
Toxicol. 1993, 33, 435.
10. Dannhardt, G.; Kiefer, W. Eur. J. Med. Chem. 2001, 36,
109.
7.3. General procedure for dehydrogenation of 4,5-
dihydropyridazin-3(2H)-ones (3c and 3i)
11. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J.
S.; Collins, P. W.; Doctor, S.; Graneto, M. J.; Lee, L. F.;
Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,
D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn,
J. N.; Gregory, S. A.; Koboldt, C. M.; Perkins, W. E.;
Seibert, K.; Veenhuizen, A. M.; Zhang, Y. Y.; Isakson, P.
C. J. Med. Chem. 1997, 40, 1347.
12. Talley, J. J.; Brown, D. L.; Carter, J. S.; Graneto, M. J.;
Koboldt, C. M.; Masferrer, J. L.; Perkins, W. E.; Rogers,
R. S.; Shaffer, A. F.; Zhang, Y. Y.; Zweifel, B. S.; Seibert,
K. J. Med. Chem. 2000, 43, 775.
13. Li, J. J.; Norton, M. B.; Reinhard, E. J.; Anderson, G. D.;
Gregory, S. A.; Isakson, P. C.; Koboldt, C. M.; Masferrer,
J. L.; Perkins, W. E.; Seibert, K.; Zhang, Y.; Zweifel, B. S.;
Reitz, D. B. J. Med. Chem. 1996, 39, 1846.
14. Friesen, R. W.; Brideau, C.; Chan, C.-C.; Charleson, S.;
Deschenes, D.; Dube, D.; Ethier, D.; Fortin, T.; Gauthier,
J. Y.; Girard, Y.; Gordon, R.; Greig, G.; Riendeau, D.;
Savoie, C.; Wang, Z.; Wong, E.; Visco, D.; Xu, L.-J.;
Young, R. N. Bioorg. Med. Chem. Lett. 1998, 8, 2777.
15. Chen, Q.-A.; Rao, P. N. P.; Knaus, E. E. Bioorg. Med.
Chem. 2005, 13, 6425.
16. Anana, R.; Rao, P. N. P.; Chenb, Q.-A.; Knausb, E. E.
Bioorg. Med. Chem. 2006, 14, 5259.
17. Dogne, J. M.; Supuran, C. T.; Pratico, D. J. Med. Chem.
2005, 48, 2251.
18. Grossman, H. B. Drugs Today 2003, 39, 203.
19. Li, C. S.; Brideau, C.; Chan, C. C.; Savoie, C.; Claveau,
D.; Gordon, C. R.; Greig, G.; Gauthier, J. Y.; Lau, C. K.;
Riendeau, D.; Therien, M.; Wong, E.; Prasit, P. Bioorg.
Med. Chem. Lett. 2003, 13, 597.
20. Biancalani, C. N.; Vergelli, C. C.; Piaz, V. D.; Graziano,
A.; Biagini, P.; Ghelardini, C.; Galeotti, N.; Giovannoni,
M. P. J. Med. Chem. 2006, 49, 7826.
To a solution of 3c or 3i (0.01 mol) in absolute aceto-
nitrile (25 mL), anhydrous CuCl2 (2.68 g, 0.02 mol) was
added and the mixture was refluxed for 30 min. After few
minutes, the brown solution turned pale yellow. The prod-
uct that separated out on cooling was filtered, washed with
ether, and crystallized from a suitable solvent.
7.3.1. (E)-6-(4-Phenoxystyryl)pyridazin-3(2H)-one (4a).
Compound 4a was prepared from 3c and recrystallized
from acetone as a beige solid. Yield: 64%; mp: 188–
1
190 ꢁC, H NMR (CDCl3) (300 MHz) d: 4.89 (d, 1H,
J = 10.5 Hz, –CH@), 5.03 (d, 1H, J = 10.5 Hz, –CH@),
6.82–7.64 (m, 11H, pyridazinone and ArH), 10.4 (s,
1H, NH). IR (KBr) cmꢀ1 : 1716. MS m/z 290 (M+,
100%); Anal. Calcd for C 18H14N2O2 : C, 74.47; H,
4.86; N, 9.65. Found: C, 74.27; H, 4.65; N, 9.88.
7.3.2. 6-(2-(Biphenyl-4-yl)ethyl)pyridazin-3(2H)-one (4b).
Compound 4b was prepared from 3i and recrystallized
from aqueous ethanol as a white solid. Yield: 74%; mp:
1
180–182 ꢁC H NMR (CDCl3) (300 MHz) d: 2.96–3.03
(t, 4H, J = 6.1 Hz, 2CH2), 6.87 (d, 1H, J = 9.6 Hz, CH
pyridazinone), 7.08 (d, 1H, J = 9.6 Hz, CH pyridazi-
none), 7.31–7.58 (m, 9H, ArH), 11.20 (s, 1H, NH). IR
(KBr) cmꢀ1
:
1710. MS m/z 276 (M+, 3.75%), 167
(100%); Anal. Calcd for C18H16N2O: C, 78.24; H, 5.84;
N, 10.14. Found: C, 78.01; H, 5.55; N, 10.40.
Acknowledgments
21. Beswick, P.; Sharon, B.; Bountra, C.; Brown, T.;
Browning, K.; Campbell, I.; Chessell, I.; Clayton, N.;
Collins, S.; Corfield, J.; Guntrip, S.; Haslam, C.;
Lambeth, P.; Lucas, F.; Mathews, N.; Murkit, G.;
I thank professor Dr. Ernst Urban, Institute of Phar-
macy, Vienna University for his indispensable co-

5556
K. Abouzid, S. A. Bekhit / Bioorg. Med. Chem. 16 (2008) 5547–5556
Naylor, A.; Pegg, N.; Pickup, E.; Player, H.; Price, H.; 27. Trummlitz, G.; van Ryn, J. Curr. Opin. Drug Discovery
Stevens, A.; Stratton, S.; Wiseman, J. Bioorg. Med.
Chem. Lett. 2004, 14, 5445.
Dev. 2002, 5, 550.
28. Abouzid, K.; Frohberg, P.; Lehmann, J.; Decker, M.
Med. Chem. 2007, 3, 433.
´ ´ ´
29. Csende, F.; Szabo, Z.; Bernath, G.; Stajer, G. Synthesis
1995, 1995, 1240.
30. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.;
Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem.
1998, 19, 1639.
22. Chintakunta, V. K.; Akella, V.; Vedula, M. S.; Mamnoor,
P. K.; Mishra, P.; Casturi, S. R.; Vangoori, A.; Rajago-
palan, R. Eur. J. Med. Chem. 2002, 37, 339.
23. Christine, L.; Aaron, M.; Barnett, Jim; Chow, Joan;
Chakk, R.; Michelle, B. Inflamm. Nat. Struct. Biol. 1996,
3, 927.
24. Blobaum, A. L.; Marnett, L. J. J. Med. Chem. 2007, 50,
1425.
31. DiRosa, M.; Willoughby, D. A. J. Pharm. Pharmacol.
1971, 23, 297.
25. Habeeb, A. G.; Rao, P. N.; Knaus, E. E. J. Med. Chem.
2001, 44, 2921.
32. Abou Zeit-Har, M. S.; Verimer, T.; Long, J. P. Pharmazie
1982, 37, 593.
26. Soliva, R.; Almansa, C.; Kalko, S. G.; Luque, F. J.;
Orozco, M. J. Med. Chem. 2003, 46, 1372.
33. Naser, M. N. A.; Said, S. A. Arch. Pharm. Pharm. Med.
Chem. 2003, 336, 559.