Structure-based molecular design, synthesis, and in vivo anti-inflammatory activity of pyridazinone derivatives as nonclassic COX-2 inhibitors

Article abstract of DOI:10.1007/s00044-009-9218-4

A scaffold with bicyclic core carrying pyridazinone moiety, which exhibited potent in vivo anti-inflammatory activities, was introduced in this article. The design of these compounds was assisted by docking and superposition experiments on cyclooxygenase-

Full text of DOI:10.1007/s00044-009-9218-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:629–642
DOI 10.1007/s00044-009-9218-4
ORIGINAL RESEARCH
Structure-based molecular design, synthesis, and in vivo
anti-inflammatory activity of pyridazinone derivatives
as nonclassic COX-2 inhibitors
Khaled A. M. Abouzid Æ Nadia A. Khalil Æ
Eman M. Ahmed Æ Hekmat A. Abd El-Latif Æ
Moustafa E. El-Araby
Received: 28 January 2009 / Accepted: 17 April 2009 / Published online: 27 May 2009
¨
Ó Birkhauser Boston 2009
Abstract A scaffold with bicyclic core carrying pyridazinone moiety, which
exhibited potent in vivo anti-inflammatory activities, was introduced in this article.
The design of these compounds was assisted by docking and superposition experi-
ments on cyclooxygenase-2 enzyme. The activity of a chloro analogue was as high as
that of diclofenac in carrageenan-induced rat paw edema anti-inflammatory screening.
Keywords Quinoxaline Á Pyridazinone Á Anti-inflammatory Á Molecular design Á
Synthesis
Introduction
There is a great deal of renewed interest in cyclooxygenase-2 (COX-2), a key
inducible enzyme for the synthesis of prostaglandins, as a therapeutic target
(McKellar et al., 2008; Jankowski and Hunt, 2008). The introduction of COX-2
K. A. M. Abouzid (&)
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University,
Cairo 11566, Egypt
e-mail: abouzid@yahoo.com
N. A. Khalil Á E. M. Ahmed
Organic Chemistry Department, Faculty of Pharmacy, Cairo University,
Cairo 11562, Egypt
H. A. A. El-Latif
Pharmacology Department, Faculty of Pharmacy, Cairo University,
Cairo, Egypt
M. E. El-Araby
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University,
Helwan 11795, Egypt
e-mail: alamka@yahoo.com

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CF3
A
N
H3C
O
N
N
H2NO2S
H2NO2S
CH3
Celecoxib (Celebrex®)
Valdecoxib (Bextra®)
Cl
O
O
N
H3CO2S
H3CO2S
N
CH3
Rofecoxib (Vioxx®)
Etoricoxib (Arcoxia®)
B
O
O
H3C
N
N
CH3
N
H
O
O
N
Cl
N
H
O
O
N
N
H
O
1
2
3
R
O
F
S
N
N
O
N
N
H
SO2NH2
4
5a-c
Fig. 1 Representative of potent COX-2 inhibitors: a Market classic COX-2 inhibitors; b literature
pyridazinone derivatives 1–4 and our quinoxaline derivatives 5a–c
inhibitors (coxibs), such as celecoxib, rofecoxib, and valdecoxib (Fig. 1) in the
1990s was viewed as a milestone triumph over human suffering of pain and
inflammation with little or no damage to the gastric mucosa, a well-known side
effect of classic nonsteroidal anti-inflammatory drugs (NSAIDs) (Flower, 2003).
Unfortunately, rofecoxib (VioxxÒ) was withdrawn from the market in the fall of
2004 after clinical reports of its linkage to elevated risk of cardiac stroke (Abramson
and Greenberg, 2008). The only upside of the VioxxÒ crisis is that no concrete
evidence has been introduced to conclude that COX-2 inhibition itself is the sole
factor in such cardiac problems; otherwise, there would be widespread side effects
for all selective COX-2 inhibitors and, possibly, nonselective COX-1 and COX-2
inhibitors (classic NSAIDs). It was argued that these adverse actions of rofecoxib
may be mediated by a maleic anhydride metabolite rather than the inhibition of

Med Chem Res (2010) 19:629–642
631
prostacyclin, a prostaglandin involved in the prevention of cardiac stroke (Reddy
and Corey, 2005). Therefore, a new generation of COX-2 inhibitors that are
radically different from the first-generation coxibs are anticipated to avoid the
cardiac untoward effects. The second reason that spotlighted COX-2 was the
discovery of its increased expression in certain types of solid tumors, such as gastric
cancer (Yamac et al., 2008), pancreatic cancer (Tucker et al., 1999), esophageal
cancer (Li et al., 2001), and colorectal cancer (Arber, 2008). Although the exact
mechanism of COX-2 in carcinogenesis is not completely understood, it was
suggested that it is due to up-regulation of prostaglandin-induced angiogenesis
(Sinicrope and Gill, 2004).
The basic structural features of the classic COX-2 inhibitors included a central
trisubstituted monocyclic planar ring (usually five-membered heterocycles) attached
to two pendent benzene rings and a lipophilic group. One of the phenyl groups is
attached to sulfamyl (celecoxib and valdecoxib) or methylsulfonyl (rofecoxib and
etoricoxib). The sulfonated benzene moiety penetrates a narrow hydrophilic pocket,
which is not accessible in COX-1, and hence, it achieves the COX-2 selectivity
(Gierse et al., 1996). Recent reports successfully used nonclassic isosteres of the
sulfonylaryl group, such as pyridazinone (compounds 1–3) to make potent COX-2
inhibitors (Chintakunta et al., 2002; Li et al., 2003; Abouzid and Bekhit, 2008).
Other reports introduced an atypical central part, such as bicyclic rings
(Ranatunge et al., 2004; Beswick et al., 2004; Whitehead et al., 2007), Z-olifens
(Jashim Uddin et al., 2004), biphenyls (Chen et al., 2005), and other scaffolds
(Kalgutkar and Zhao, 2001). For instance, compound 4, a bicyclic derivative with
fused six-membered rings, was found to be a potent COX-2 inhibitor in vitro but it
lacked in vivo potency (Joo et al., 2003). In this report, we are used a combination
of these two approaches to design and synthesize novel COX-2 inhibitors 5
designed to keep the potency while moving away from the classic coxib structures
to reduce side effects.
Rationale and structure-based drug design
Our design is based on a two-point structural modification: 1) central bicyclic
quinoxaline scaffold carrying only one phenyl ring; and 2) pyridazinone moiety as a
replacement of sulfamylphenyl or sulfonylphenyl group. The benzene portion of the
fused quinoxaline ring was used to cover the area occupied by the CF3 group of
celecoxib. This was confirmed by structure-based molecular modeling studies,
which were performed using the SYBYL molecular modeling package. The COX-2
enzyme crystal structure coordinates were downloaded from the Protein Data Bank
(PDB entry: 6COX), which contains SC-558 as selective COX-2 inhibitor in its
active site (Kurumbail et al., 1996). SC-558 is a closely related congener to
celecoxib that differs only in having bioisosteric bromo group in replacement of the
para methyl on the pendent phenyl group.
The structure of 5a was constructed using the BUILD/EDIT option of SYBYL
using fragments, groups, and atoms from SYBYL substructure library. The structure
was given Gasteiger-Marsili charges. The built structure were subjected to geometry

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Med Chem Res (2010) 19:629–642
Fig. 2 a The pyridazine derivative 5a superposed with SC-558. b The potent COX-2 inhibitor 5b shown
inside the active site (expressed by Connolly surface)
optimization by running energy minimization using the following parameters:
˚
MAXIMIN2 minimizer, gradient energy change, 0.01 kcal/A mol; rms displace-
˚
˚
ment, 0.001 A; nonbonded cutoff, 8.000 A; dielectric function, distant dependent;
dielectric constant, 1.00; iteration: until convergence.
We performed the modeling studies in two ways. First, we tested the
superposition of the designed compound 5a with SC-558 (Fig. 2). Second, we
performed docking experiments of the new quinoxaline 5 structure models on
the active site of the COX-2 structure coordinates after removing SC-558. The
results showed very good superposition and binding opportunity for the designed
scaffold.
Chemistry
As shown in Scheme 1, the synthesis started with preparation of intermediate
benzylidenelevulenic acid 6 by condensation of levulenic acid with certain aromatic
aldehydes (Zaheer et al., 1956; Sircar et al., 1989). Cyclization of 6 with hydrazine
hydrate afforded the corresponding intermediate 4,5-dihydropyridazinones 7
according to the reported method (Sircar et al., 1989; Baddar et al., 1972).
Bromination of 7 followed by a second cyclization using o-phenylenediamine
afforded the partially saturated congeners 9, which were again treated with bromine
and acetic acid to furnish the target compounds 5a–c (Coudert et al., 1988).
Attempts for cyclization of 8 with ethylenediamine resulted in dehydrobromin-
ation and the formation of 10 (Scheme 2). Apparently, ethylenediamine behaved as
a base rather than a nucleophile in this reaction.

Med Chem Res (2010) 19:629–642
633
Scheme 1 Reagents: a morpholine, AcOH, C6H6, heat; b NH2NH2.H2O, AcOH; c Br2 (2 equiv.), AcOH;
d o-phenylenediamine, Et3 N, EtOH, heat; e Br2 (2 equiv.), AcOH
In vivo anti-inflammatory screening
The in vivo anti-inflammatory effect of quinoxaline derivatives 5a–c and 9a–c was
evaluated using Paw Volume method in carrageenan-induced rat paw edema model
(see experimental section for method details) (DiRosa and Willoughby, 1971). The
results of edema reduction are presented in Table 1 and Fig. 3.
Results and discussion
The quinoxaline scaffold represented by the compound 5a showed activity in both
docking model and in anti-inflammatory activity test compared with diclofenac
(Fig. 3). The two aryl groups are pointing to the same area of space; nevertheless,
the question was about opportunities and mode of binding of pyridazinone ring. As
shown in Fig. 2, Docking of 5a on the active site of COX-2 crystal structure
produced a complex with total energy comparable to the original crystal structure.
The compound 5a is situated in the active site without unfavorable interactions by
visual inspections as well as by docking energies (Fig. 2b).

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2
2
2
2
Scheme 2
Table 1 Inhibition of rat paw edema compared with the control at different time intervals after treatment
with a single dose (5 mg/kg) of diclofenac or test compounds
Compound Time intervals (hr)
After injection
1
2
3
4
Control
5a
1.95 0.03a
2.00 0.00a
1.97 0.02a
1.97 0.02a
2.00 0.00a
1.95 0.02a
2.00 0.00a
1.95 0.03c
1.97 0.02c
1.48 0.03b
1.55 0.09b
2.00 0.00c
1.93 0.03c
1.58 0.09b
0.92 0.03a
2.00 0.03d
1.37 0.06c
0.95 0.04b
1.50 0.10c
2.02 0.02d
1.93 0.03d
1.42 0.04c
0.57 0.03a
2.03 0.02e
1.00 0.07c
0.55 0.04b
1.38 0.06d
2.00 0.00e
2.00 0.04e
1.38 0.05d
0.3 0.03a
2.20 0.04d
0.38 0.07b
0.03 0.02a
1.27 0.08c
2.07 0.03d
2.12 0.05d
1.32 0.06c
0.03 0.02a
5b
5c
9a
9b
9c
Diclofenac 1.97 0.02a
F value
P value
0.88
50.20
102.87
0.000*
234.20
0.000*
321.97
0.000*
0.534
0.000*
All values are represented as mean standard error. a, b, and c = results with same letter has no
significant difference by using Turkey HSD multiple comparison test at P \ 0.06
The top of the active site gorge is occupied by the fused benzene ring, and the
pendent phenyl fits in the main hydrophobic channel of the active site. The
pyridazinone moiety is placed inside the hydrophobic pocket, which determines the
most selectivity of COX-2 inhibitors over COX-1 (Gierse et al., 1996). As seen
Fig. 4, there are effective hydrogen bonding network between the pyridazinone and

Med Chem Res (2010) 19:629–642
635
2.5
2.0
1.5
1.0
0.5
0.0
9c
Control
5a
5b
5c
9a
9b
Diclofenac
Fig. 3 Effect of test compounds on carrageenan-induced inflammation in rats after 4 h
Fig. 4 Image of docking of 5a on the active site to illustrate interactions of the pyridazine moiety with
residues in the hydrophilic side pocket. Numbers indicate H-Bond distances
His-90, Gln-192, and Phe-518 (main chain NH) (Kurumbail et al., 1996; Slater
et al., 2001). Due to dissimilarity of the two sides of the pyridazine ring, docking
experiments investigated the possibility of flipping of the heterocyclic ring inside
the hydrophilic channel. It was found that this situation results in poor docking
model because the polar side is interacting with the lipophilic phenyl group of Phe-
518 and some other nonpolar residues while the carbocyclic side loses hydrogen-
bonding contacts. As expected, the pendent phenyl group is properly filling the main
hydrophobic channel of the active site. This pocket is surrounded by a group of
aromatic and aliphatic nonpolar amino acid side chains, such as Val-528, Tyr-385,
Trp-387, Phe-518, and Leu-352. Note that the last two residues are interacting with
the nonpolar (carbocyclic) side of the pyridazinone ring (Fig. 5a).

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Fig. 5 Docking of the chloro compound 5b on the active site: a Interactions of p-chlorophenol moiety
with residues of some active site’s hydrophobic gorge; b interaction of the bicyclic quinoxaline moiety
with other hydrophobic residues
Regarding substituents on the pendent phenyl group, the model predicted that
bulky or polar groups are not desired on the pendent phenyl ring due to limitation in
pocket size and the lipophilic nature of the surrounding residues (Figs. 2 and 5b).
This might explains the poor activity of the methoxy analogue 5c in pharmaco-
logical screening. In contrast, the model predicted high potency of the chloro
analogue 5b as the chloro group interacts properly with the aromatic ring of Tyr-
385.
Our study confirmed the importance of the planar aromatic nature of the central
scaffold, a SAR feature that is not frequently discussed in COX-2 inhibitors
(Kalgutkar and Zhao, 2001; Garg et al., 2003). Loss of the planarity in the
nonaromatic analogue 9b resulted in complete loss of activity. Docking of trans
diastereomers of 9a produced very poor binding as compound did not fit with active
site and collided with residues surrounding it (data not shown).
Conclusions
Bicyclic quinoxaline nucleus attached to pyridazinone and phenyl substituents
formed potent anti-inflammatory novel structures especially the chloro analogue 5b.
The in vivo high potency of 5b is comparable to that of diclofenac. This
combination constitutes an important development of the nonclassic bicyclic
COX-2 inhibitors because it is a novel bicyclic nonsulfonated compound with high
in vivo anti-inflammatory activity. The structure-based molecular design accurately

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637
predicted the inherent activity of the scaffold and the rank of potency of the
compounds 5a–c. The study also illustrated the importance of the aromatic planar
structure of the central scaffold due to the poor activity of the nonaromatic
analogues 9a–c.
Experimental
All melting points were determined on a Stuart apparatus and are uncorrected. IR
spectra were determined as KBr discs on Shimadzu IR 435 spectrophotometer and
1
values are represented in cm-1. H-NMR spectra were performed using a Varian
Gemini 200 MHz spectrometer with TMS as internal standard. Chemical shift
values were recorded in ppm on d scale. Mass spectra were recorded on a Hewlett-
Packard 5988 spectrometer. Elemental analyses were performed at the Microan-
alytical Center, Cairo University, Egypt. Progress of the reactions was monitored by
TLC using TLC sheets precoated with UV florescent silica gel Merck 60 F 254 and
visualized using UV light.
(E)-6-Aryl-4-oxo-5-hexenoic acids (6a–c) and 4,5-dihydro-3(2H)-pyridazinones
(7a–c) were prepared according to reported procedures (Zaheer et al., 1956; Sircar
et al., 1989).
2-Aryl-3-(6-oxo-1,6-dihydropyridazin-3-yl)quinoxaline (5a–c)
To a vigorously stirred solution of 0.01 mol of the appropriate 9a–c (0.01 mol) in
glacial acetic acid (20 ml) kept at 70–80°C, bromine (0.02 mol) was added
portionwise for 15 minutes. The mixture was stirred further for 3 hours at the same
temperature and poured into ice-cold water (50 ml). The separated solid was
filtered, washed with water, and crystallized from ethanol to give 5a–c.
2-Phenyl-3-(6-oxo-1,6-dihydropyridazin-3-yl)quinoxaline (5a) was obtained
as white solid, yield: 70%; mp: [300°C. IR (m, cm-1): 3500–3300 (NH), 1680
(C=O). 1H-NMR (DMSO-d6) d: 7.06 (d, J = 9.8 Hz, 1H, pyridazinone H-4), 7.49–
7.71 (m, 9H, Ar–H), 7.96 (d, J = 9.8 Hz, 1H, pyridazinone H-5), 13.22 (s, 1H, NH
pyridazinone, D2O exchangeable). EIMS m/z: 300 (M?, 10.00%). Anal. Calcd. for
C18H12N4O: C, 71.52; H, 4.63; N, 18.54. Found: C, 71.84; H, 4.57; N, 18.92.
2-(4-Chlorophenyl)-3-(6-oxo-1,6-dihydropyridazin-3-yl)quinoxaline (5b) was
obtained as white solid, yield: 65%; mp: [300°C. IR (m, cm-1): 3500–3300 (NH),
1
1680 (C=O). H-NMR (DMSO-d6) d: 7.16 (d, 1H, pyridazinone H-4), 7.60–7.79
(dd, 4H, Ar–H), 8.01–8.24 (m, 5H, 4Ar–H and pyridazinone H-5), 13.27(s, 1H,
pyridazinone NH, D2O exchangeable). Anal. Calcd. for C18H11ClN4O: C, 64.19; H,
3.86; N, 16.64. Found: C, 64.32; H, 3.65; N, 16.40.
2-(4-Methoxyphenyl)-3-(6-oxo-1,6-dihydropyridazin-3-yl)quinoxaline
(5c)
was obtained as white solid, yield: 68%; mp: 200–201°C. IR (m cm-1): 3500–
3300 (NH), 1680 (C=O). 1H-NMR (DMSO-d6) d: 3.79 (s, 3H, OCH3), 6.87-7.92 (m,
10H, 8 Ar–H and 2 pyridazinone H-4, H-5), 12.95 (s, 1H, pyridazinone NH, D2O
exchangeable). Anal. Calcd. for C19H14N4O2: C, 68.67; H, 4.81; N, 16.86. Found:
C, 68.29; H, 5.26; N, 16.70.

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6-(2-Arylethyl-1,2-dibromo)pyridazin-3(2H)-one (8a–c)
General procedure: A vigorously stirred solution of 7 (0.01 mol) in glacial acetic
acid (20 ml) was heated to 70°C and then treated portionwise with bromine
(0.02 mol) for 15 minutes. The mixture was stirred further for 3 hours at the same
temperature and poured into ice-cold water (50 ml). The separated solid was
filtered, washed with water, and crystallized from ethanol to give the dibromo
derivatives 8a–c.
6-(2-Phenylethyl-1,2-dibromo)pyridazin-3(2H)-one (8a) was obtained as a
pale-yellow solid, yield: 60%; mp: 204–205°C. IR (m, cm-1): 3500–3300 (NH),
1680 (C=O); 1H-NMR (DMSO-d6) d 5.96 (d, J = 6 Hz, 1H, CHBr–CHBr), 6.02 (d,
J = 6 Hz, 1H, CHBr–CHBr), 7.04 (d, J = 4 Hz, 1H pyridazinone H-4), 7.34–7.66
(m, 5H, Ar–H), 7.83 (d, J = 4 Hz, 1H, pyridazinone H-5), 13.21 (s, 1H,
pyridazinone NH, D2O exchangeable). EIMS (m/z): 356 (M?, 0.16%), 358
(M ? 2, 0.29%), 360 (M ? 4, 0.19%). Anal. Calcd. for C12H10Br2N2O: C, 40.22;
H, 2.79; N, 7.82. Found: C, 40.38; H, 2.28; N, 8.27.
6-[2-(4-Chlorophenyl)ethyl-1,2-dibromo]pyridazin-3(2H)-one
(8b)
was
obtained as a pale-yellow solid; yield: 65%; mp: 223–224°C. IR (m, cm-1):
1
3500–3300 (NH), 1680 (C=O); H-NMR (DMSO-d6) d 5.99 (d, J = 6 Hz, 1H,
CHBr–CHBr), 6.05 (d, J = 6 Hz, 1H, CHBr–CHBr), 7.02 (d, J = 4 Hz, 1H,
pyridazinone H-4), 7.40–7.71 (dd, 4H, Ar–H), 7.9 (d, J = 4 Hz, 1H, pyridazinone
H-5), 13.20 (s, 1H, D2O exchangeable, NH pyridazinone). Anal. Calcd. for
C12H9Br2ClN2O: C, 36.68; H, 2.29; N, 7.13. Found: C, 36.98; H, 2.59; N, 7.18.
6-[2-(4-Methoxyphenyl)ethyl-1,2-dibromo]pyridazin-3(2H)-one (8c) was
obtained as a yellow solid; yield: 70%; mp: 221–222°C. IR (m, cm-1): 3500–
3300 (NH), 1680 (C=O); 1H-NMR (DMSO-d6) d: 3.82 (s, 3H, OCH3), 5.92 (br, 1H,
CHBr–CHBr), 6.14 (br, 1H, CHBr–CHBr), 6.83–8.14 (m, 6H, 4Ar–H and 2H
pyridazinone H-4, H-5), 12.95 (s, 1H, D2O exchangeable, NH pyridazinone). EIMS
(m/z): 386 (M?, 9.03%), 388 (M ? 2, 18.11%), 390 (M ? 4, 8.87%). Anal. Calcd.
for C13H12Br2N2O2: C, 40.20; H, 3.09; N, 7.21. Found: C, 39.85; H, 3.1; N, 7.50.
2-Aryl-3-(6-oxo-1,6-dihydropyridazin-3-yl)-1,2,3,4-tetrahydroquinoxaline
(9a–c)
The appropriate dibromo derivative 8a–c and o-phenylenediamine (0.001-mol each)
were placed in ethanol (25 ml) along with two drops of triethylamine and the
mixture was heated under reflux for 5 hours. The reaction mixture was filtered while
hot, concentrated to half its volume, and poured into ice-cold water (50 ml). The
separated solid was filtered, washed with water, dried, and crystallized from ethanol
to give the quinoxaines 9a–c.
2-Phenyl-3-(6-oxo-1,6-dihydropyridazin-3-yl)-1,2,3,4-tetrahydroquinoxaline
(9a) was obtained as an off-white solid; yield: 75%; mp: 144–145°C. IR (m, cm-1):
1
3600–3300 (NH), 1660 (C=O). H-NMR (DMSO-d6) d: 4.13 (d, 1H, J = 6.4 Hz,
quinoxaline N–CH–Ar), 4.32 (d, 1H, J = 6.4 Hz, quinoxaline N–CH-pyridazine),
6.01 (s, 1H, quinoxaline-NH, D2O exchangeable), 6.11 (s, 1H, D2O exchangeable,

Med Chem Res (2010) 19:629–642
639
quinoxaline-NH), 6.47–8.20 (m, 11H, 9Ar–H and 2pyridazinone-H), 12.74 (s, 1H,
NH pyridazinone, D2O exchangeable). EIMS (m/z): 304 (M?, 35.14%). Anal.
Calcd. for C18H16N4O: C, 71.05; H, 5.26; N, 18.42. Found: C, 71.37; H, 5.52; N,
18.35.
2-(4-Chlorophenyl)-3-(6-oxo-1,6-dihydropyridazin-3-yl)-1,2,3,4-tetrahydro-
quinoxaline (9b) was obtained as an off-white solid, yield: 76%, mp: 170–171°C.
1
IR (m, cm-1): 3500–3300 (NH), 1660 (C=O). H-NMR (DMSO-d6) d: 4.09 (d,
J = 6.4 Hz, 1H, quinoxaline N–CH–Ar), 4.36 (d, J = 6.4 Hz, 1H, quinoxaline N–
CH-pyridazine), 6.01 (s, 1H, quinoxaline-NH, D2O exchangeable), 6.11 (s, 1H,
quinoxaline-NH, D2O exchangeable), 6.94–8.15 (m, 10H, 8Ar–H and 2pyridaz-
inone-H), 12.74 (s, 1H, pyridazinone-NH, D2O exchangeable). EIMS (m/z): 338
(M?, 11.84%), 340 (M ? 2, 6.54). Anal. Calcd. for C18H15ClN4O: C, 63.81; H,
4.43; N, 16.50. Found: C, 63.56; H, 4.63; N, 16.7.
2-(4-Methoxyphenyl)-3-(6-oxo-1,6-dihydropyridazin-3-yl)-1,2,3,4-tetrahydro-
quinoxaline (9c) was obtained as a pale-yellow solid, yield: 76%; mp: 145–146°C.
1
IR (m, cm-1): 3600–3300 (NH), 1660 (C=O). H-NMR (DMSO-d6) d: 3.81 (s, 3H,
OCH3), 4.34 (d, 1H, J = 6.4 Hz, 1H, quinoxaline N–CH–Ar), 4.85 (d, 1H,
J = 6.4 Hz, 1H, quinoxaline N–CH–Ar), 5.80 (s, 1H, quinoxaline-NH, D2O
exchangeable), 5.95 (s, 1H, quinoxaline-NH, D2O exchangeable), 6.37–8.23 (m,
10H, 8Ar–H and 2H-pyridazinone-H), 12.76 (s, 1H, pyridazinone-NH, D2O
exchangeable). Anal. Calcd. for C19H18N4O2: C, 57.37; H, 3.18; N, 11.15. Found:
C, 57.10; H, 3.22; N, 10.93.
6-(2-Aryl-1-bromo-2-phenylethenyl)pyridazin-3-(2H)-one (10a–b)
A mixture of the appropriate 8b–c (0.001 mol) and ethylenediamine (0.06 g,
0.002 mol) was heated under reflux for 5 hours. The reaction mixture was filtered
while hot and concentrated to half its volume. The solid separated after cooling was
filtered, dried, and crystallized from ethanol to give 10a–b.
6-(2-(4-Chlorophenyl)-1-bromoethyl)pyridazin-3-(2H)-one (10a) was obtained
as a white solid, yield: 65%, mp: 278–280°C. IR (cm-1): 3600–3300 (NH), 1680
1
(C=O), 1620 (C=N); H-NMR (DMSO-d6) d: 3.79 (s, 1H, BrC=CH), 6.89–7.94
(m, 6H, 4Ar–H and 2-pyridazinone-H), 13.00 (s, 1H, NH-pyridazinone,
D2O exchangeable). EIMS (m/z): 311 (M?, 6.53%); 313 (M ? 2, 6.14%). Anal.
Calcd. for C12H8BrClN2O: C, 46.22; H, 2.56; N, 8.98; Found: C, 45.71; H, 3.10;
N, 8.90.
6-(2-(4-Methoxyphenyl)-1-bromoethyl)pyridazin-3-(2H)-one (10b) was obtained
as an off-white solid, yield 70%; mp: 218–219°C; IR(cm-1): 3600–3300 (NH), 1680
(C=O), 1620 (C=N). H-NMR (CDCl3): 3.84(s, 3H, OCH3), 4.72 (s, 1H, = CH),
1
6.80–7.64 (m, 6H, 4Ar–H and 2H-pyridazinone H-4, H-5), 10.80 (s, 1H, NH-
pyridazinone, D2O exchangeable). EIMS (m/z): 307 (M?, 3.62%), 309 (M ? 2,
3.41%). Anal. Calcd. for C13H11BrN2O2: C, 50.81; H, 3.58; N, 9.12; Found: C,
50.66; H, 3.45; N 9.16.

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Med Chem Res (2010) 19:629–642
Effect of test compounds on paw volume in carrageenan-induced rat paw
edema model (in vivo measurements)
Materials and methods
Male albino rats weighing 120–150 g (Pharmacological Department, Faculty of
Pharmacy, Cairo University) were used throughout the work. They were kept in the
animal house under standard conditions of light and temperature with free access to
food and water. The animals were randomly divided into groups of six rats each.
The paw edema was induced by subplantar injection of 50 ll of 2% carrageenan
solution in saline (0.9%). Diclofenac and the test compounds were dissolved in
DMSO and injected subcutaneously in 5 mg/kg body weight, 1 hour before
carrageenan injection. DMSO was injected to the control group. The volume of paw
edema was determined by means of water plethysmometer immediately after
injection of carrageenan and 1, 2, 3, and 4 hours later.
Statistical analysis of data
The percentage protection against inflammation was calculated as follows:
Vc - Vd/Vc 9 100, where Vc is the increase 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 expressed as the mean standard error ( SEM). Data
with P \ 0.05 value were considered to be significant (Table 1).
The % inhibition of the rat paw edema 4 hours after treatment with a single dose
(5 mg/kg) of the tested compounds and their activity relative to diclofenac is
presented in Table 1 and Fig. 5.
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