3-O-Substituted benzyl pyridazinone derivatives as COX inhibitors

Article abstract of DOI:10.1016/S0223-5234(02)01336-3

New 3-O-substituted benzyl pyridazinone compounds have been synthesised and evaluated for their cyclooxygenase inhibitory activity and COX-2 selectivity. Among the compounds synthesised, three compounds (11b-11d) have shown in vitro COX-2 selectivity. These compounds have been evaluated for their in vivo potential using carrageenan-induced rat paw edema assay. One compound (11b) showed 32% anti-inflammatory activity at 30 mg kg^-1 dose.

Full text of DOI:10.1016/S0223-5234(02)01336-3

Eur. J. Med. Chem. 37 (2002) 339–347
www.elsevier.com/locate/ejmech
Short communication
3-O-Substituted benzyl pyridazinone derivatives as COX
inhibitorsꢀ
Vamsee Krishna Chintakunta a, Venkateswarlu Akella a, Manohar Sharma Vedula a,*,
Prem Kumar Mamnoor b, Parimal Mishra b, Seshagiri Rao Casturi b,
Akhila Vangoori b, Ramanujam Rajagopalan b
a Disco6ery Chemistry, Dr. Reddy’s Research Foundation, Bollaram Road, Miyapur, Hyderabad 500050, India
b Disco6ery Biology, Dr. Reddy’s Research Foundation, Bollaram Road, Miyapur, Hyderabad 500050, India
Received 18 June 2001; received in revised form 14 January 2002; accepted 17 January 2002
Abstract
New 3-O-substituted benzyl pyridazinone compounds have been synthesised and evaluated for their cyclooxygenase inhibitory
activity and COX-2 selectivity. Among the compounds synthesised, three compounds (11b–11d) have shown in vitro COX-2
selectivity. These compounds have been evaluated for their in vivo potential using carrageenan-induced rat paw edema assay. One
compound (11b) showed 32% anti-inflammatory activity at 30 mg kg−1 dose. © 2002 Editions scientifiques et me´dicales Elsevier
´
SAS. All rights reserved.
Keywords: 3-O-Substituted benzyl pyridazinone; COX inhibitors; Anti-inflammatory activity
COX-1 is expressed in many normal tissues and is the
major form present in platelets, kidneys, gastrointesti-
nal tract and play a key role in physiological processes
[7], whereas COX-2 is an inducible form by many
pro-inflammatory cytokines and mitogens. The second
isoform is generally not detectable in normal tissues,
but is elevated in inflammatory condition [8] and is also
implicated in colon cancers [9], and Alzheimer’s disease
[10]. COX-2 is also constitutively expressed in kidneys
[11], brain [12] spinal cord [13] and in mucosa of
stomach [14]. As most of the NSAIDs inhibit both the
isoforms of COX enzyme, currently the major focus of
inflammatory research is to discover agents, which se-
lectively inhibit the inducible COX-2 with little or no
effect on COX-1. Synthesis of selective COX-2 in-
hibitors has been made possible due to X-ray crystal
structure of COX enzyme [15]. Recently, Allen et al.
[16] [17] claimed 3-aroyl benzyl pyridazinones with the
following general formula as selective COX-2 in-
hibitors. The compounds, even though have shown
good COX-2 activity in in vitro, the in vivo activity was
not comparable to that of selective COX-2 inhibitors
such as celecoxib and rofecoxib. In the present
1. Introduction
Non-steroid anti-inflammatory drugs (NSAIDs) are
widely used for the treatment of pain, inflammation,
rheumatoid arthritis and osteoarthritis. The common
dose limiting toxicity of NSAIDs is the increased risk of
gastrointestinal ulceration, perforation and haemor-
rhage [1]. The enzyme cyclooxygenase (COX) cataly-
ses the biooxygenation of arachidonic acid to
prostaglandin G2, which serves as a precursor for the
synthesis of prostaglandins, prostacyclins and throm-
boxanes which are collectively termed as prostanoids
[2]. The cyclooxygenase activity of the enzyme is the
site of action of NSAIDs [3,4]. However, inhibition of
prostanoid biosynthesis is associated with side effects
such as ulceration and impairment of renal functions
[5]. It has been well established that the cells express
two isoforms of cyclooxygenases, namely cyclooxyge-
nase-1 (COX-1) and cyclooxygenase-2 (COX-2) [6].
ꢀ DRF Publication No. 154.
* Corresponding author.
E-mail address: sarmavm@drreddys.com (M.S. Vedula).
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(02)01336-3

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V.K. Chintakunta et al. / European Journal of Medicinal Chemistry 37 (2002) 339–347
communication we describe our exploratory study to
modify the structural motif of this compound by incor-
porating hetero atoms like N and O with an assump-
tion that these may provide additional sites of
interaction, thus giving the desired selectivity with in-
creased potency Fig. 1. The compounds synthesised
were initially tested for their in vitro COX inhibition at
100 mM followed by determination of IC50 for COX-1/
COX-2 activity for interesting compounds.
3. Results and discussion
The compounds synthesised were screened at 100 mM
using in vitro spectrophotometric peroxidase assay in
the initial screens for potency and COX-2 selectivity.
Subsequently selected compounds exhibiting COX-2 se-
lectivity were further evaluated and IC50 values were
determined for COX-1 and COX-2. The COX-1 activity
was assessed using Ram seminal vesicles as a source for
COX-1 enzyme, whereas for COX-2 the enzyme source
was the human recombinant cyclooxygenase-2 ex-
pressed in Sf9 cells infected with baculo virus. The test
compounds were dissolved in DMSO and the enzyme
activity was measured by the estimation of N,N,N,N-te-
tramethyl-p-phenylene diamine (TMPD) oxidation du-
ring the reduction of PGG2 to PGH2. Compounds,
which showed better in vitro activity, were tested for
their in vivo activity in carrageenan-induced rat paw
edema model.
Using I and II as templates, a limited study has been
initiated by changing substituents on phenyl ring of the
phenacyl moiety in template I where X is oxygen. All
these compounds were tested in vitro. In general, intro-
duction of a bromo or fluoro in the fourth position of
phenacyl ring resulted in desired COX-2 inhibition.
This inhibitory activity observed was more in the case
of bromo substituent 9a. When the substituent was
fluoro (9e) COX-2 specificity was observed to some
extent. Other substituents like methyl (9c), tert-butyl
(9d), have shown poor COX-2 selectivity at 100 mM
concentration. In contrast, disubstituted compounds
like 4-chloro-3-methyl (9g), 4-fluoro-3-methoxy (9h),
and 2,4-dichloro (9i) analogues failed to show apprecia-
ble COX activity.
2. Chemistry
Synthesis of these compounds is very concise and is
outlined in Fig. 2. In the first step commercially avai-
lable 3-methoxy benzaldehyde (1), was reduced with
sodium borohydride to yield 3-methoxy benzyl alcohol
(2), which was converted to the chloride 3 with thionyl
chloride in dichloromethane and further to the nitrile 4
using potassium cyanide in dimethyl sulphoxide at
room temperature. The obtained 3-methoxy benzo ni-
trile (4) was condensed with 3,6 dichloropyridazine to
yield
2-(6-chloro-3-pyridazinyl)-2-(3-methoxyphenyl)
acetonitrile (5) which on hydrolysis and decarboxyla-
tion gave 3-(3-methoxy benzyl)-1,6-dihydro-6-pyridazi-
none (6) which was demethylated using 48% aq. HBr.
After purification by chromatography on silica gel 3-(3-
hydroxybenzyl)-1,6-dihydro-6-pyridazinone (7) was iso-
lated. Now this intermediate was reacted with various
phenacyl bromides 8a–j in the presence of sodium
hydride in dimethyl formamide to obtain compounds
9a–j. On the other hand, compounds 11a–e were syn-
thesised by reacting 7 with various N1-phenyl-2-chloro
acetamides 10a–e (prepared from substituted anilines
and chloroacetyl chloride in presence of triethyl amine)
in the presence of sodium hydride in DMF.
At this point further modification was carried out to
synthesise A ring-substituted analogues of template II.
Fig. 1.

V.K. Chintakunta et al. / European Journal of Medicinal Chemistry 37 (2002) 339–347
341
Fig. 2. Synthetic routes to compounds 9a–j and 11a–e.
Even though no clear SAR has been evolved, four
compounds 11a–d have shown better COX-2 selectivity
at 100 mM concentration. These compounds were fur-
ther tested and IC50 values were determined for COX-1
and COX-2. These results are summarised in Table 1.
Three of these compounds (11b–11d), which showed
desired COX-2 inhibition were evaluated further in
carrageenan-induced rat paw edema model at 30

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V.K. Chintakunta et al. / European Journal of Medicinal Chemistry 37 (2002) 339–347
Table 1
COX-1 and COX-2 inhibitory data at 100 mM concentration

V.K. Chintakunta et al. / European Journal of Medicinal Chemistry 37 (2002) 339–347
343
Table 1 (Continued)
NT=Not tested.
Enzyme source: a COX-1-Ram seminal vesicles; b COX-2-Human (expressed in Sf9 cells using baculo virus).
*Average of three determinations at 100 mM.
**IC50 tested at seven concentrations namely 0.1, 0.3, 1.0, 3.0, 10, 30 and 100 mM.
***Rofecoxib was tested up to 500 mM for COX-1 activity.
mg kg−1 by oral route (Table 2). Only one compound
11b showed 32% inhibition at this dose whereas other
derivatives in spite of having good in vitro COX-2
activity were found inactive in in vivo screen.
none derivatives were synthesised and screened for their
in vitro COX-2 selectivity and in vivo anti-inflamma-
tory activity. Even though few compounds have shown
good in vitro COX-2 selectivity, this was not translated
in in vivo studies.
In conclusion novel 3-O-substituted benzyl pyridazi-

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4. Experimental protocols
4.1.4. Synthesis of 3-(3-methoxybenzyl)-1,6-dihydro-
6-pyridazinone (6)
2-(6-Chloro-3-pyridazinyl)-2-(3-methoxyphenyl) ace-
tonitrile (5) (2.5 g, 0.009 mol) was suspended in a 20
mL mixture of 2:1:1 HCl, acetic acid and water. The
contents were refluxed for 12 h, cooled to r.t. and
extracted with dichloromethane. The organic layer was
washed with water and concentrated. The residue was
filtered and washed with diethyl ether to get the pyri-
dazinone as a colourless solid (1.35 g, 67.5%).
4.1. Chemistry
The reported molecules were synthesised in the drug
discovery Department of Dr. Reddy’s research founda-
tion. All other chemicals and reagents used in the
synthesis were of reagent grade. Melting points were
determined in an Electro thermal melting point appara-
tus (Buchi 535) and were uncorrected. Pre-coated silica
gel plates (20×20 cm, silica gel 60 F254, Merck) were
used for TLC. 1H-NMR spectra were recorded in a
Varian-200 MHz, Gemini-200-software spectrometer
(Varian USA). TMS was used as an internal standard
and chemical shifts are given in ppm. IR spectra were
4.1.5. Synthesis of 3-(3-hydroxybenzyl)-1,6-dihydro-
6-pyridazinone (7)
3-(3-Methoxybenzyl)-1,6-dihydro-6-pyridazinone (3.0
g, 0.0138 mol) was taken in 48% aq. HBr (15 mL) and
the contents were refluxed for 2 h. The reaction mass
was poured into ice and extracted with ethyl acetate.
The ethyl acetate extract was washed with water and
concentrated. The residue was purified by chromatogra-
phy over 100–200 mesh silica gel. Elution of the
column with 80% ethyl acetate in petroleum ether re-
sulted in the pure compound as a pale brown solid (1.7
g, 60.9%)
recorded in
a Perkin–Elmer 1600 series FT-IR
(Perkin–Elmer, USA). Mass spectra were recorded in a
Hewlett Packard 5989-A mass spectrometer. Starting
materials were either commercially available or synthe-
sised according to known literature methods.
4.1.1. General procedure for the synthesis of the interme-
diates 9a–j
Bromine (1 mol) was added at room temperature to
the appropriate acetophenone (1 mol) in 10 mL of
acetic acid. Subsequently 0.5 mL of 48% aq. HBr was
added and the contents were stirred at room tempera-
ture (r.t.) for 4 h. The reaction mixture was poured into
ice and the precipitated solid was filtered, washed with
water and dried under vacuum to get the phenacyl
bromide.
4.1.6. Synthesis of 1-(4-bromophenyl)-2-[3-(6-oxo-1,6-
dihydro-3-pyridazinylmethyl)phenoxy]-1-ethanone (9a)
3-(3-Hydroxybenzyl)-1,6-dihydro-6-pyridazinone
(0.310 g, 0.00154 mol) was dissolved in dry DMF (5
mL), 60% NaH (0.160 g, 0.00308 mol) was added at
5 °C, then 4-bromophenacyl bromide (0.427 g, 0.00154
mol) was added and contents were stirred at r.t. for 15
min. The reaction mixture was poured on ice, extracted
with dichloromethane, washed with water, the organic
layer was dried over sodium sulphate and concentrated
to dryness. The residue was purified by chromatogra-
phy over 100–200 mesh silica gel. Elution with 75%
ethyl acetate in petroleum ether afforded the pure com-
pound. Yield (32.4%); m.p. 218–219 °C; IR (KBr,
cm−1): 3262, 2928, 1696, 1661, 1580, 1524, 1484, 1460,
1349, 1281, 1223, 1153, 1107, 1072; 1H-NMR
(CDCl3+DMSO-d6) lH: 8.8 (1H, s, ꢀNH), 7.9 (2H, d,
J=6.6 Hz, phenylethanone), 7.7 (2H, d, J=6.6 Hz,
phenylethanone), 7.2 (1H, d, J=9.4 Hz, pyridazine),
7.1 (1H, m, phenoxy), 6.8 (1H, d, J=9.4 Hz, pyrida-
zine), 6.6 (3H, m, phenoxy), 5.5 (2H, s, ꢀOCH2), 3.8
(2H, s, ꢀCH2). MS; m/z (DIP): 400 [M+], 291, 215, 185
(100%).
4.1.2. General procedure for the synthesis of intermedi-
ates 10a–e
Triethyl amine (1 mol) and chloroacetyl chloride (1
mol) were added at 0–5 °C to the appropriately substi-
tuted aniline (1 mol) in dichloromethane. The contents
were stirred at 5 °C for 0.5 h and the precipitated solid
was filtered and washed with diethyl ether to yield
N1-phenyl-2-chloro acetamides as solid.
4.1.3. Synthesis of 2-(6-chloro-3-pyridazinyl)-2-
(3-methoxyphenyl) acetonitrile (5)
To 3-methoxy benzonitrile (4) (5.0 g) in dry DMF
(25 mL), at 0–5 °C was added 60% sodium hydride
(1.14 g, 0.0341 mol). The contents were stirred there for
5 min and 3,6-dichloro pyridazine (5.025 g, 0.0341 mol)
was added at one lot. The reaction mixture was stirred
at 5–10 °C for 2 h, poured into ice and extracted with
dichloromethane. The organic layer was washed with
water, dried and concentrated. The residue was purified
by chromatography over 100–200 mesh silica gel. Elu-
tion with 30% ethyl acetate in petroleum ether
yielded the pure compound as a colourless solid (2.6 g,
30%).
4.1.7. Synthesis of 1-(4-chlorophenyl)-2-[3-(6-oxo-1,6-
dihydro-3-pyridazinylmethyl)phenoxy]-1-ethanone (9b)
Yield (22.7%); m.p. 222–223 °C; IR (KBr, cm−1):
3276, 2939, 1696, 1661, 1580, 1525, 1486, 1460, 1352,
1281, 1225, 1153, 1096, 1006; 1H-NMR (CDCl3+
DMSO-d6) lH: 8.6 (1H, s, ꢀNH), 7.9 (2H, d, J=8.4 Hz,
phenylethanone), 7.5 (2H, d, J=8 Hz, phenyl-
ethanone), 7.3 (1H, m, phenoxy), 7.2 (1H, d, J=9.2

V.K. Chintakunta et al. / European Journal of Medicinal Chemistry 37 (2002) 339–347
345
Hz, pyridazine), 6.8 (1H, d, J=9.2 Hz, pyridazine), 6.7
4.1.12. Synthesis of 1-(3-methyl-4-chlorophenyl)-2-[3-
(3H, m, phenoxy), 5.5 (2H, s, ꢀOCH2), 3.8 (2H, s,
ꢀCH2). MS; m/z (DIP): 354 [M+], 247, 217, 215, 186,
158, 139 (100%).
(6-oxo-1,6-dihydro-3-pyridazinylmethyl)phenoxy]-1-
ethanone (9g)
Yield (22.1%); m.p. 232–234 °C; IR (KBr, cm−1):
3428, 2926, 1662,1 1582, 1486, 1347, 1281, 1235, 1153;
1H-NMR (CD3OD) lH: 7.9 (1H, d, J=8.6 Hz,
phenylethanone), 7.26 (3H, m, pyridazine, 1H-phenoxy,
1H-phenylethanone), 7.0 (1H, m, phenylethanone), 6.8
(1H, d, J=9.4 Hz, pyridazine), 6.6 (3H, m, phenoxy),
5.5 (2H, s, ꢀOCH2), 3.8 (2H, s, ꢀCH2), 2.4 (3H, s,
ꢀCH3); MS; m/z (DIP): 368 [M+], 261, 237, 215, 153
(100%).
4.1.8. Synthesis of 1-(4-methylphenyl)-2-[3-(6-oxo-
1,6-dihydro-3-pyridazinylmethyl)phenoxy]-1-ethanone
(9c)
Yield (19.3%); m.p. 228–229 °C; IR (KBr, cm−1):
3264, 2942, 1692, 1663, 1581, 1525, 1486, 1452; 1H-
NMR (CDCl3+DMSO-d6) lH: 7.8 (2H, d, J=8.2 Hz,
phenylethanone), 7.3 (2H, d, J=8.2 Hz, phenyl-
ethanone), 7.2 (1H, d, J=9.4 Hz, pyridazine), 7.1 (2H,
m, phenoxy), 6.9 (1H, d, J=9.4 Hz, pyridazine), 6.8
(2H, m, phenoxy), 5.6 (2H, s, ꢀOCH2), 3.8 (2H, s,
ꢀCH2) 2.2 (3H, s, ꢀCH3); MS; m/z (DIP): 334 [M+],
227, 215, 119 (100%).
4.1.13. Synthesis of 1-(3-methoxy-4-fluorophenyl)-2-[3-
(6-oxo-1,6-dihydropyridazinylmethyl)phenoxy]-1-
ethanone (9h)
Yield (16.42%); m.p. 211–212 °C; IR (KBr, cm−1):
3247, 2941, 1661, 1685, 1608, 1580, 1522, 1488, 1357,
1
1291, 1240; H-NMR (CDCl3+CD3OD) lH: 7.8 (2H,
4.1.9. Synthesis of 1-(4-tert-butylphenyl)-2-[3-(6-oxo-
1,6-dihydro-3-pyridazinylmethyl)phenoxy]-1-ethanone
(9d)
m, phenylethanone), 7.2 (3H, m, phenylethanone, pyri-
dazine, phenoxy), 6.9 (1H, d, J=9.8 Hz, pyridazine),
6.7 (3H, m, phenoxy), 5.5 (2H, s, ꢀOCH2), 4.1 (3H, s,
OCH3), 3.8 (2H, s, ꢀCH2); MS; m/z (DIP): 368 [M+],
261, 215, 186, 153 (100%).
Yield (20.6%); m.p. 115 °C; IR (KBr, cm−1): 3300,
2961, 1698, 1661, 1581, 1525, 1487, 1405, 1345, 1277,
1
1236, 1153; H-NMR (CD3OD) dH: 8.0 (2H, d, J=8.2
Hz, ditert-butylphenylethanone), 7.56 (2H, d, J=8.2
Hz, ditert-butylphenylethanone), 7.3 (1H, d, J=9.4
Hz, pyridazine), 7.1(2H, m, phenoxy), 6.8 (1H, d, J=
9.4 Hz, pyridazine), 6.8 (2H, m, phenoxy), 5.6( 2H, s,
ꢀOCH2), 3.8 (2H, s, ꢀCH2), 1.3 (9H, s, C(CH3)3); MS;
m/z (DIP): 376 [M+], 215, 161 (100%), 146.
4.1.14. Synthesis of 1-(2,4,dichlorophenyl)-2-
[3-(6-oxo-1,6-dihydro-3-pyridazinylmethyl)phenoxy]-1-
ethanone (9i)
Yield (20.7%); m.p. 110–111 °C; IR (KBr, cm−1):
1
3272, 2927, 1709, 1661, 1581, 1487; H-NMR (DMSO-
d6) lH: 9.3 (1H, s, pyridazineꢀNH), 7.89 (2H,
m,phenylethanone), 7.2 (1H, m, phenoxy), 7.60 (1H, d,
J=8.2 Hz, phenylethanone), 7.35 (1H, d, J=9.6 Hz,
pyridazine),7.2 (1H, m), 6.9 (1H, d, J=9.6 Hz, pyrida-
zine), 6.6 (3H, m, phenoxy), 5.6 (2H, s, ꢀOCH2), 3.8
(2H, s, ꢀCH2); MS; m/z (DIP): 388 [M+],353, 325, 316,
281, 270, 230,197, 186, 173 (100%).
4.1.10. Synthesis of 1-(4-fluorophenyl)-2-[3-(6-oxo-1,6-
dihydro-3-pyridazinylmethyl)phenoxy]-1-ethanone (9e)
Yield (26.1%); m.p. 204–205 °C; IR (KBr, cm−1):
3283, 2941, 1696, 1662, 1582,1525, 1507, 1487, 1412,
1
1353, 1280, 1155; H-NMR (CD3OD) lH: 8.1 (2H, m,
phenylethanone), 7.4 (3H, m, phenylethanone, pyrida-
zine), 7.2 (2H, m, phenoxy), 7.0 (1H, d, J=9.2 Hz,
pyridazine), 6.8 (2H, m, phenoxy), 5.4 (2H, s, ꢀOCH2),
3.8 (2H, s, ꢀCH2); MS; m/z (DIP): 338 [M+], 215, 123
(100%).
4.1.15. Synthesis of N1-(4-methoxy-2-methylphenyl)-2-
[3-(6-oxo-1,6-dihydro-3-pyridazinylmethyl)phenoxy]
acetamide (11a)
Yield (41.27%); m.p. 172–173 °C; IR (KBr, cm−1):
3259, 2924, 1656, 1579, 1548, 1501, 1447, 1390, 1280,
1
1234, 1156, 1112; H-NMR (CD3OD) lH: 7.28 (1H, d,
4.1.11. Synthesis of 1-(3,4-dimethoxyphenyl)-2-[3-(6-
oxo-1,6-dihydro-3-pyridazinylmethyl)phenoxy]-1-
ethanone (9f)
J=9.6 Hz,pyridazine), 7.0 (3H, m, 2-methyl-3-methoxy
aniline), 6.8 (1H, d, J=9.6 Hz, pyridazine), 6.7 (4H, m,
phenoxy), 4.8 (2H, s, ꢀOCH2), 3.8 (3H, s, OCH3), 3.7
(2H, s, ꢀCH2), 2.2 (3H, s, ꢀCH3); MS; m/z (DIP): 379
[M+], 243, 229, 215, 205, 137 (100%).
Yield (33.8%); m.p. 220–221 °C; IR (KBr, cm−1):
3188, 2941, 1659, 1581, 1520, 1484, 1356, 1280, 1255,
1
1210, 1175, 1136, 1105; H-NMR (CDCl3+DMSO-d6)
lH: 9.0 (1H, s, ꢀNH), 7.6 (3H, m, dimethoxy
phenylethanone), 7.1 (3H, m, phenoxy, pyridazine), 6.8
(1H, d, J=9.4 Hz, pyridazine), 6.6 (1H, m, phenoxy),
5.5 (2H, s, ꢀOCH2), 3.9 (6H, s, 2*OMe), 3.8 (2H, s,
ꢀCH2); MS; m/z (DIP): 380 [M+], 165 (100%), 149,
137.
4.1.16. Synthesis of N1-(3,4,difluorophenyl)-2-
[3-(6-oxo-1,6-dihydro-3-pyridazinyl methyl)phenoxy]
acetamide (11b)
Yield (16.3%); m.p. 118–119 °C; IR (KBr, cm−1):
3282, 2925, 1660, 1588, 1510, 1437, 1336, 1240, 1206,
1155; 1H-NMR (CD3OD) lH: 7.70 (3H, m, difluoro

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V.K. Chintakunta et al. / European Journal of Medicinal Chemistry 37 (2002) 339–347
aniline), 7.2 (3H, m,phenoxy, pyridazine), 6.8 (3H, m,
phenoxy, pyridazine), 4.8 (2H, s, ꢀOCH2), 3.3 (2H, s,
ꢀCH2); MS; m/z (DIP): 371 [M+], 243 (100%), 229,
215, 201, 186, 170, 159.
4.2. Pharmacology
4.2.1. Spectrophotometric assay of COX-1 and COX-2
Microsomal fractions of Ram seminal vesicles were
used as a source of COX-1 enzyme [18] and microsomes
from Sf9 cells infected with baculo virus containing
human COX-2 cDNA was used as a source of COX-2
enzyme [19]. Enzyme activity was measured using a
chromogenic assay based on the oxidation of
4.1.17. Synthesis of N1-(4-trifluoromethylphenyl)-2-
[3-(6-oxo-1,6-dihydro-3-pyridazinylmethyl)phenoxy]
acetamide (11c)
Yield (28.7%); m.p. 136–138 °C; IR (KBr, cm−1):
3296, 1653, 1582, 1610, 1549, 1413, 1327, 1260, 1181,
N,N,N%,N%-tetramethyl-p-phenylenediamine
(TMPD)
1
1115, 1080; H-NMR (CD3OD) dH: 7.5 (2H, d, J=8.6
during the reduction of PGG2 to PGH2 as per the
procedure described by Copeland et al. [20] with the
following modifications. The assay mixture (1000 mL)
contained 100 mM Tris pH 8.0, 3 mM EDTA, 15 mM
hematin, 150 units enzyme and 8% DMSO. The mix-
ture was pre-incubated at 25 °C for 15 min before
initiation of enzyme reaction in presence of compound/
vehicle. The reaction was initiated by the addition of 10
mM arachidonic acid and 120 mM TMPD. The enzyme
activity was measured by estimation of the reaction
followed from increase in absorbance at 603 nm.
Hz, trifluoromethyl phenyl), 7.3 (2H, d, J=8.6 Hz,
4-trifluoromethyl phenyl), 7.2 (1H, d, J=9.4 Hz,
pyridazine), 7.0 (2H, m, phenoxy), 6.6 (2H, m,
phenoxy), 4.8 (2H, s, ꢀOCH2), 3.8 (2H, s, ꢀCH2); MS;
m/z (DIP): 403 [M+], 257, 243 (100%), 229, 215.
4.1.18. Synthesis of N1-(4-ethylphenyl)-2-
[3-(6-oxo-1,6-dihydro-3-pyridazinylmethyl)phenoxy]
acetamide (11d)
Yield (31%); m.p. 94–95 °C; IR (KBr, cm−1): 3291,
2964, 1652, 1581, 1544, 1486, 1454, 1415, 1339, 1309;
1H-NMR (CD3OD) lH: 7.4 (2H, d, J=8.4 Hz, ethyl
aniline), 7.2(1H, d, J=9.8 Hz, pyridazine), 7.1 (2H, d,
J=8.4 Hz, ethylbenzene), 7.0 (2H, m, phenoxy), 6.8
(1H, d, J=9.8 Hz, pyridazine), 6.6 (2H, m, phenoxy),
4.8 (2H, s, ꢀOCH2), 3.8 (2H, s, ꢀCH2), 2.5 (2H, q,
J=14, 9 Hz, ꢀCH2 CH3), 1.9 (3H, t, J=9 Hz, ꢀCH2
CH3); MS; m/z (DIP): 363 [M+], 257, 243, 215, 202 121
(100%).
4.2.2. Carrageenan-induced rat paw edema assay
Male/female Wistar rats (120–140 g) were fasted for
16 h before the experiment. Compounds were sus-
pended in 0.25% carboxy methylcellulose and adminis-
tered orally in a volume of 10 mL kg−1 2 h before
carrageenan injection. Edema was induced in rats by
intradermal injection of 50 mL of 1% l-carrageenan in
saline into the plantar surface of the right hind paw
[21]. The paw volume was monitored before and 3 h
after carrageenan injection using plethysmometer (Ugo-
Basile, Italy). The paw edema was compared with the
vehicle treated group and the percent inhibition was
calculated (Table 2).
4.1.19. Synthesis of N1-benzo[d][1,3]dioxo[5-yl-2-[3-
(6-oxo-1,6-dihydro-3-pyridazinylmethyl)phenoxy]
acetamide (11e)
Yield (17.7%); m.p. 219–220 °C; IR (KBr, cm−1):
3249, 2942, 1681, 1574, 1502, 1450, 1390; 1H-NMR
(DMSO-d6) lH: 10.2 (1H, s, ꢀNHꢀCO), 9.4 (1H, s,
ꢀNH, pyridazine), 7.33 (3H, m, methylene dioxy ani-
line), 7.1 (1H, m, phenoxy), 7.0 (2H, m, pyridazine,
phenoxy), 6.9 (3H, m, phenoxy, pyridazine) 5.9 (2H, s,
ꢀOꢀCH2ꢀO), 5.8 (2H, s, ꢀCH2), 4.8 (2H, s, ꢀOCH2);
MS; m/z (DIP): 379 [M+], 243, 215, 176 137 (100%).
Acknowledgements
We thank the analytical department for the spectral
data.
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