Amide Derivatives of [6-(5-Methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-2-yl]acetic Acids as Potential Analgesic and Anti-Inflammatory Compounds

Article abstract of DOI:10.1002/ardp.200200738

In this study, amide derivatives of [6-(5-methyl-3-phenyl-pyrazole-1-yl)-3(2H)-pyridazinone-2-yl]acetic acid were synthesized and tested for their in vivo analgesic and anti-inflammatory activity by using the p-benzoquinone-induced writhing test and carrageenan-induced hind paw edema model, respectively. The analgesic and anti-inflammatory activity of the compounds 6a, 6d, 6e, 6g, 6h and 6m were more potent than that of aspirin as an analgesic and indomethacin as an anti-inflammatory drug, respectively. The other derivatives generally resulted in comparable activity to reference compounds. Inhibitor activity of the active compounds on cyclooxygenase isoforms was also investigated by using in vitro human whole blood assay and found that these derivatives did not exert their analgesic and anti-inflammatory activities through COX inhibition and other mechanisms might be involved.

Full text of DOI:10.1002/ardp.200200738

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 7–14
Potential Analgesic and Anti-Inflammatory Compounds
7
Erden Banoglua,
Amide Derivatives of
[6-(5-Methyl-3-phenylpyrazole-1-yl)-3(2H)-
pyridazinone-2-yl]acetic Acids as Potential
Analgesic and Anti-Inflammatory Compounds
Çag¹ la Akog¹ lua,
Serdar Ünlüa,
Esra Küpelib,
Erdem Yes¸iladab,
M. Fethi S¸ahina
In this study, amide derivatives of [6-(5-methyl-3-phenyl-pyrazole-1-yl)-3(2H)-pyrid-
azinone-2-yl]acetic acid were synthesized and tested for their in vivo analgesic and
anti-inflammatory activity by using the p-benzoquinone-induced writhing test and
carrageenan-induced hind paw edema model, respectively.The analgesic and anti-
inflammatory activity of the compounds 6a, 6d, 6 e, 6 g, 6 h and 6 m were more po-
tent than that of aspirin as an analgesic and indomethacin as an anti-inflammatory
drug, respectively.The other derivatives generally resulted in comparable activity to
reference compounds.Inhibitor activity of the active compounds on cyclooxygenase
isoforms was also investigated by using in vitro human whole blood assay and found
that these derivatives did not exert their analgesic and anti-inflammatory activities
through COX inhibition and other mechanisms might be involved.
a
Department of
Pharmaceutical Chemistry,
Faculty of Pharmacy,
Gazi University, Ankara,
Tur key
b
Department of
Pharmacognosy,
Faculty of Pharmacy,
Gazi University, Ankara,
Tur key
Keywords: Pyridazinone; Analgesic; Anti-Inflammatory; COX-1; COX-2
Received: October 30, 2002; Accepted: November 15, 2002 [FP738]
DOI 10.1002/ardp.200200738
about the central 5 or 6-membered carbocyclic or het-
erocyclic ring for selective COX-2 inhibition [7, 8]. Howev-
er, a few reports also indicated that 1,3-diarylsubstitution
about an annulated pyrazole ring was also an effective
pattern for selective COX-2 inhibition (Figure 1) [9, 10].
Introduction
Most currently used nonsteroidal anti-inflammatory
drugs (NSAIDs) have limitations for their therapeutic use
since they cause gastrointestinal and renal side effects
which are inseparable from their pharmacological activi-
ties [1–3]. However, the discovery of the two distinct cy-
clooxygenase (COX) isoforms, namely cyclooxygenase-
1 (COX-1) and cyclooxygenase-2 (COX-2), made it pos-
sible to separate the pharmacological effects from the
side effects of NSAIDs since COX-1 is expressed consti-
tutively in most cells and tissues and responsible for the
synthesis of PGs that are important for gastric protection
and vascular homeostasis while COX-2 is mostly ex-
pressed as a result of mitogenic and inflammatory stimu-
li [2]. Many studies indicated that inhibition of COX-2 but
not COX-1 resulted compounds that lacked the gastroin-
testinal and renal side effects of currently used NSAIDs
[1–4]. Celecoxib [5] and rofecoxib [6] have been market-
ed as the first drugs that selectively inhibit COX-2 (Figure
1).
In addition, many studies also focused on 3(2H)-pyrid-
azinone derivatives for developing potent and safer
NSAIDs without gastric side effects [11–13]. Various
compounds incorporating 3(2H)-pyridazinone nucleus
have been synthesized and their analgesic and anti-in-
flammatory activity have been reported. Among these
compounds,
4-ethoxy-2-methyl-5-morpholino-3(2H)-
pyridazinone (emorfazone) is currently being marketed
in Japan as an analgesic and anti-inflammatory drug
[14]. Moreover, 3-O-substituted benzyl pyridazinone de-
rivatives were synthesized and shown that these com-
pounds demonstrated in vitro selective COX-2 inhibitory
activity and also potent anti-inflammatory activity using
carrageenan-induced rat paw edema assay [11]. Addi-
tionally, 2-substituted 4,5-functionalized 6-phenyl-
3(2H)-pyridazinone derivatives [12] have also been re-
ported to bear potent analgesic activity lacking the gen-
eral side effects of currently used NSAIDs. Meantime,
many authors reported that pyridazinone derivatives
bearing an arylpiperazine moiety at the side chain on the
lactam nitrogen of the ring had significant analgesic ac-
tivity and stressed the significance of this structural fea-
ture [15, 16]. Moreover, they claimed that these deriva-
tives exhibited better analgesic activity if they bear a car-
The structure-activity studies indicated that the spatial
orientation of the two aromatic rings of this tricyclic inhib-
itor drug class was critical, that is, two aryl rings must re-
side on adjacent positions (1,2-disubstitution pattern)
Correspondence:
Erden
Banoglu,
Department
of
Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi Uni-
versity, 06330 Etiler, Ankara, Turkey. Phone: +90 312 2126645,
Fax: +90 312 2235018; e-mail: banoglu@gazi.edu.tr
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

8
Banoglu et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 7–14
Figure 1. Structures of COX-2 inhibitors.
analgesic and anti-inflammatory activity. Therefore, we
synthesized amide derivatives of [6-(5-methyl-3-phen-
ylpyrazole-1-yl)-3(2H)-pyridazinone-2-yl]acetic acid
(Figure 2) to test their in vivo analgesic and anti-inflam-
matory activities and also their in vitro inhibitory activity
on COX-1 and COX-2 enzymes.
Results and discussion
Chemistry
Figure 2. General structure of the synthesized com-
pounds.
The title amide derivatives were synthesized by the reac-
tion of appropriate amine derivatives with [6-(5-methyl-
3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-2-yl]acetic
acid in the presence of N,N-bis(2-oxo-3-oxazolidinyl)-
phosphorodiamidic chloride (BOP-Cl) [19] as the car-
boxylate activator. Analytical data for structure elucida-
tion were given in Experimental Part.The preparation of
the resulting amide derivatives 6 a–m were outlined in
Scheme 1.
bon chain between the nitrogen atom of the lactam and
the amine component of the side chain. Dogruer et al.
subsequently synthesized [6-(4-methoxyphenyl)-3(2H)-
pyridazinone-2-yl]acetamide and propanamide deriva-
tives and reported that these compounds showed poten-
tial analgesic activity [13]. Meantime, some studies for
developing COX-2 inhibitors have concentrated on the
preparation of the amide derivatives of currently used
NSAIDs such as indomethacin [17] and meclofenamic
acid [18] and found that neutralization of these NSAIDs
by preparing amide derivatives resulted compounds that
selectively inhibited COX-2 but not COX-1.
In the synthesis of the amide derivatives (6 a–m), com-
mercially available 3,6-dichloropyridazine was used as
the starting material, and 6-(5-methyl-3-phenylpyrazole-
1-yl)-3(2H)-pyridazinone (3) were prepared by adapted
procedures according to the previously published meth-
ods [20, 21]. 3 was then treated with ethyl bromoacetate
to prepare the ethyl [6-(5-methyl-3-phenylpyrazole-1-yl)-
3(2H)-pyridazinone-2-yl]acetate (4) which was subse-
quently acid-hydrolysed to obtain [6-(5-methyl-3-phen-
ylpyrazole-1-yl)-3(2H)-pyridazinone-2-yl]acetic acid (5).
By treatment of 5 with appropriate amines in the pres-
ence of BOP-Cl in dichloromethane at room tempera-
ture, resulting amide derivatives (6 a–m) were prepared
in moderate to good yield (52–70 %).
These findings stimulated us to search for new com-
pounds with 1,3-diaryl substitution pattern about a cen-
tral pyrazole ring. Based on the information that 3(2H)-
pyridazinone derivatives bear potent analgesic and
anti-inflammatory activities [11–16], we have chosen the
pyridazinone ring as one of the aryl substituents about
the central pyrazole ring. We also aimed to explore
the presence of the acetamide side chain that is linked
to the lactam nitrogen of pyridazinone ring to deter-
mine the contribution of this structural feature to the
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 7–14
Potential Analgesic and Anti-Inflammatory Compounds
9
Scheme 1.Synthetic pathways of [6-(5-methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-2-yl]acetamide derivatives.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

10 Banoglu et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 7–14
azinone-2-yl]acetamide and propanamide derivatives,
the highest analgesic activity was observed with the 4-
fluorophenylpiperazine derivative at the amide portion of
the compounds [13]. In their work 2-pyridylpiperazine
amide derivative also showed potential analgesic activi-
ty. Kalgutkar et al. also reported that N-octyl and N-4-
methoxyphenylamide derivatives of indomethacin were
potent selective inhibitors of COX-2 [17]. However, other
secondary amide derivatives including 2-aminopyridine
(6i), 4-bromophenyl (6j) and 4-chlorophenyl (6k) substit-
uents caused a decrease in the analgesic activity. In ad-
dition, 6-(5-methyl-3-phenylpyrazole-1yl)-3-chloropyri-
dazine (2), 6-(5-methyl-3-phenylpyrazole-1-yl)-3(2H)-
pyridazinone (3), ethyl [6-(5-methyl-3-phenyl-pyrazole-
1-yl)-3(2H)-pyridazinone-2-yl]acetate (4) and [6-(5-
methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-2-
yl]acetic acid (5) derivatives were also tested for their an-
algesic activity and found that they exhibited moderate
analgesic activities which is comparable to aspirin but
lower than some of the corresponding amide derivatives.
These results indicated that certain amide derivatives
contributed to the higher analgesic activity of the com-
pounds.
Pharmacology
Analgesic and anti-inflammatory activities of the synthe-
sized amide derivatives (6 a–m) and intermediate 6-(5-
methyl-3-phenylpyrazole-1-yl)-3-chloropyridazine (2),
6-(5-methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone
(3), ester (4) and acid (5) derivatives were assessed by
p-benzoquinone-induced writhing test [22] and carra-
geenan-induced hind paw edema model [23], respec-
tively.As seen inTable 1, analgesic and anti-inflammato-
ry activity of the compounds 6 a–m were comparable to
the reference compounds aspirin and indomethacin, re-
spectively. Analgesic activity results indicated that com-
pounds having 4-fluorophenylpiperazine (6 a), phenyl-
piperazine (6 d), 2-pyridylpiperazine (6 e), octyl (6h) and
4-methoxyphenyl (6 m) substituents at the amide portion
of the synthesized compounds showed superior analge-
sic activity to that of aspirin in the p-benzoquinone-in-
duced writhing test. These results correlated well with
the previous results indicating that the presence of aryl-
piperazine moiety at the side chain of the pyridazinone
ring increases the analgesic activity [15, 16]. Dogruer
et al.reported that in [6-(4-methoxyphenyl)-3(2H)-pyrid-
Table 1. Analgesic and anti-inflammatory activity of the synthesized compounds.
Compound
Analgesic
activity#
Antiinflammatory activity#
Inhibition of edema (%)
Inhibition of
writhing (%)
90 min
180 min
270 min
360 min
6 a
6 b
6 c
6 d
6 e
6 f
6 g
6 h
6 i
6 j
6 k
6 m
2
3
4
5
62.5
41.1
17.3
66.9
65.6
44.9
54.1
71.1
38.5
44.9
44.9
64.6
50.9
44.4
55.6
50.4
56.8
–
23.7
10.3
8.4
28.1
29.2
6.6
18.7
35.6
16.5
16.0
14.7
22.4
19.1
10.5
24.2
10.3
–
17.8
8.3
6.7
21.9
27.4
9.1
17.2
34.1
11.8
12.4
10.3
19.3
13.4
7.7
26.6
17.3
11.7
24.3
31.3
12.3
27.3
39.6
14.9
17.3
10.7
20.4
15.1
12.3
22.2
14.9
–
31.4
16.6
10.5
28.5
37.1
15.6
26.9
37.6
15
16.1
13.4
22.9
15.3
10.9
22.1
16.6
–
21.5
6.3
–
Aspirin
Indomethacin
38.0
33.7
34.9
36.3
#
Analgesic and anti-inflammatory activity of the compounds were tested at 100-mg/kg doses. Analgesic acitivity of
aspirin was tested at 100-mg/kg and anti-inflammatory activity of indomethacin was tested at 10-mg/kg dose as
described in Experimental Part. P < 0.05 was found for all testing as in comparison with control group.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 7–14
Potential Analgesic and Anti-Inflammatory Compounds 11
Table 2. COX inhibitory activity of the selected com-
pounds.
bear potent analgesic and anti-inflammatory activity at
100 mg/kg doses in mice, and selected for further stud-
ies to investigate their COX inhibitory activities by using
in vitro human whole blood assay [25] to elucidate if the
COX inhibition is present relating to their analgesic and
anti-inflammatory activities.
Compound COX-1 inhibition, COX-2 inhibition,
(%) 10 µM
(%) 10 µM
6 a
6 d
6 e
6 g
6 h
6 m
0
0
1.5
0
1.4
1.6
14
3
14
16
8
Although we hypothesized that the selected compounds
would have inhibitory activity on COX-enzymes to shut
down prostaglandin synthesis to exert their analgesic
and anti-inflammatory activities, this was not the case
and none of the selected in vivo active compounds have
resulted considerable inhibition at 10 µM neither in
COX-2 nor in COX-1 (Table 2).Therefore, we concluded
that these [6-(5-methyl-3-phenylpyrazole-1-yl)-3(2H)-
pyridazinone-2-yl]acetamide derivatives do not exert
their analgesic and anti-inflammatory activities through
COX inhibition and other mechanisms might be involved.
The mechanism that underlie the analgesic and anti-in-
flammatory activities of the resulting amide derivatives
are currently under investigation in our laboratory.
5
Inhibitor activity of the selected compounds at 10-µM
concentrations on COX-1 and COX-2 were tested by us-
ing in vitro human whole blood assay as described in Ex-
perimental Part. Indomethacin (COX-1, IC50 = 0.19 µM
and COX-2, IC50 = 0.22 µM) and celexocib (COX-1, IC50
=
12.2 µM and COX-2, IC50 = 1.1 µM) were used as nonse-
lective COX inhibitor and selective COX-2 inhibitor refer-
ences in the assays.
Experimental
Analgesic activity results of the compounds also showed
good correlation with their anti-inflammatory activities
tested by using the carrageenan-induced hind paw ede-
ma model. As seen in Table 1, the same amide deriva-
tives (6 a, 6 d, 6 e, 6 h and 6 m) exhibited at 100 mg/kg as
potent anti-inflammatory activity as indomethacin. The
N-octylamide derivative (6 h) especially showed high
anti-inflammatory activity that was comparable to in-
domethacin. It is known that an edema produced by car-
rageenan is a biphasic event and it is reported that the
inhibitory effects of agents which act on the first stage of
the carrageenan-induced hind paw inflammation are at-
tributable to the inhibition of the chemical mediators
such as histamine, serotonin and bradykinin [24].On the
other hand, the second stage of the edema might be re-
lated to the arachidonic acid metabolites since it is inhib-
ited by aspirin, indomethacin and other cyclooxygenase
inhibitors [24].As seen inTable 1, the tested compounds
exhibited considerable anti-inflammatory activity both in
the first and second phases of edema and the activity did
not show a significant increase in the second phase of
the edema indicating that these compounds might exert
their anti-inflammatory activities through the mecha-
nisms that involve the inhibition of chemical mediators
such as histamine and serotonin and also presumably
the COX isoforms. In addition, none of the compounds
except 6 f (in one of the 6 animals caused gastric le-
sions) and 6 j (in 2 of the 6 animals caused gastric le-
sions) showed gastric lesions in the stomach of mice uti-
lized in the in vivo assays. Based on these results, com-
pounds 6 a, 6 d, 6 e, 6 g, 6 h, and 6 m were considered to
3,6-dichloropyridazine, hydrazine hydrate, benzoylacetone,
ethyl bromoacetate, triethylamine, N,N-bis(2-oxo-3-oxazolidi-
nyl)phosphorodiamidic chloride and amine derivatives were
obtained from Aldrich (Germany) and Merck (Germany).
3-Chloro-6-hydrazinopyridazine was synthesized according to
a previously published procedure (mp 136–137 °C; compared
to literature mp 137–138 °C [20]). All other chemicals were ob-
tained from commercial sources. IR spectra were recorded on
a Bruker Vector 22 IR (Opus Spectroscopic Software Version
2.0) spectrometer (KBr, υ, cm–1).1H-NMR spectra were record-
ed on a Bruker 400 FT-NMR spectrometer using TMS as an in-
ternal standard in DMSO-d6. LC-MS analysis of the com-
pounds was performed on ThermoQuest Finnigan AQA Mass
Spectrometry equipped with ThermoQuest Liquid Chromato-
graph.
Synthesis of 6-(5-methyl-3-phenylpyrazole-1-yl)-3-chloro-
pyridazine (2)
A mixture of 3-chloro-6-hydrazinopyridazine (0.01 mol) and
benzoylacetone (0.01 mol) in 30 mL ethanol was heated to re-
flux for 5 h. After cooling, the separated crystals are filtered off,
washed with ice-cold ethanol, dried and recrystallized from eth-
anol to yield 71.2 %.mp 150 °C.LC-MS (+ESI) m/z 271.5 (M+᭹
+
1) – 1H-NMR (CDCl3): δ 7.83 (d, 1 H); 7.45 (d, 1 H); 7.38–7.08
(m, 5 H); 6.28 (s, 1 H); 2.3 (s, 3 H). – IR νmax cm–1 (KBr): 3064,
2627, 1570, 763, 697.
Synthesis of 6-(5-methyl-3-phenylpyrazol-1yl)-3(2H)-pyridazi-
none (3)
A solution of 2 (0.01 mol) in 20 mL of glacial acetic acid was re-
fluxed for 18 h. After cooling, it was poured into ice-water
(50 mL) and the precipitate formed was filtered off, washed
with water and dried and recrystallized from ethanol (yield
96 %). mp 210 °C. LC-MS (+ESI) m/z 253 (M+᭹ + 1). – 1H-NMR
(CDCl3): δ 12.7 (s, 1 H); 7.75 (d, 1 H); 7.45–7.27 (m, 5 H); 7.06
(d, 1 H); 6.5 (s, 1 H); 2.27 (s, 3 H). – IR νmax cm–1 (KBr): 3063,
2948, 1679, 1602, 762.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

12 Banoglu et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 7–14
1-[2-[6-(5-Methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-
2-yl]acetyl]-4-phenylpiperazine (6 d)
Synthesis of ethyl [6-(5-methyl-3-phenylpyrazole-1-yl)-3(2H)-
pyridazinone-2-yl]acetate (4)
Recrystallized from 2-propanol (yield 70 %, mp 161 °C).LC-MS
To the solution of 3 (0.01 mol) and potassium carbonate
(0.04 mol) in 40 mL dimethylformamide was added ethyl-
bromoacetate (0.015 mol) and stirred at room temperature for
90 min. The reaction mixture was then poured into ice-water
and the precipitate formed filtered off, washed with water, dried
and recrystallized from ethanol to yield 85 %, mp 88 °C. LC-MS
1
(-ESI) m/z 453 (M+᭹ – 1) – H-NMR (CDCl3): δ 7.47 (d, 1 H);
7.35–7.12 (m, 7 H); 6.98–6.88 (m, 4 H); 6.23 (s, 1 H); 4.68 (s,
2 H); 3.68 (t, 2 H); 3.45 (t, 2 H); 3.1 (m, 4 H); 2.29 (s, 3 H). – IR
νmax cm–1 (KBr): 2995, 2905, 2816, 1671, 1665, 1595, 1230,
757, 693.
1
(-ESI) m/z 337 (M+᭹ – 1). – H-NMR (CDCl3): δ 7.53 (d, 1 H);
1-[2-[6-(5-Methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-
2-yl]acetyl]-4-(2-pyridyl)piperazine (6 e)
7.34–7.14 (m, 5 H); 6.92 (d, 1 H); 6.23 (s, 1 H); 4.5 (s, 2 H); 4.1
(q, 2 H); 2.29 (s, 3 H); 1.17 (t, 3 H). – IR νmax cm–1 (KBr): 3058,
2999, 2958, 1758, 1671, 1597, 1204, 853, 762, 695.
Recrystallized from methanol (yield 64 %, mp 171 °C). LC-MS
(+ESI) m/z 456 (M+᭹ + 1) – 1H-NMR (DMSO-d6):δ 8.28 (m, 1 H),
7.86 (d, 1 H); 7.8–7.66 (m, 1 H); 7.61–7.42 (m, 5 H); 7.26 (d,
1 H);7.03 (d, 1 H);6.92 (m, 1 H);6.69 (s, 1 H);4.93 (s, 2 H);3.69
(m, 8 H); 2.45 (s, 3 H). – IR νmax cm–1 (KBr): 3032, 2982, 2912,
2843,1662, 1593, 1233, 758.
Synthesis of 2-[6-(5-methyl-3-phenylpyrazole-1-yl)-3(2H)-pyr-
idazinone-2-yl]acetic acid (5)
Compound 4 (0.01 mol) was heated up to reflux temperature in
NaOH (10 % w/v) for 90 min. After cooling, the reaction mixture
was acidified with concentrated HCl and the precipitate formed
was filtered off, washed with water, dried and recrystallized
from ethanol-water to yield 96 %, mp 170–171 °C. LC-MS
1-[2-[6-(5-Methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-
2-yl]acetyl]-4-(4-nitrophenyl)piperazine (6 f)
Recrystallized from methanol (yield 57 % mp 216 °C). LC-MS
(-ESI) m/z 498 (M+᭹ – 1) – 1H-NMR (CDCl3): δ 8.1 (d, 2 H), 7.45
(d, 1 H); 7.35–7.13 (m, 5 H); 6.88 (d, 1 H); 6.75 (d, 2 H); 6.24 (s,
1 H);4.68 (s, 2 H);3.7 (t, 2 H);3.52 (t, 2 H);3.39 (m, 4 H);2.29 (s,
3 H). – IR νmax cm–1 (KBr): 3054, 2986, 2909,1668, 1595, 1236,
767.
1
(+ESI) m/z 311 (M+᭹ + 1). H-NMR (CDCl3): δ 7.36 (d, 1 H);
7.29–7.19 (m, 5 H); 6.92 (d, 1 H); 6.24 (s, 1 H); 5.8–4.7 (w, 1 H);
4.6 (s, 2 H); 2.3 (s, 3 H). IR νmax cm–1 (KBr): 3005, 2873, 2568,
2477, 1732, 1648, 1572, 1248, 848, 763, 695.
General Procedure for the amidation of [6-(5-methyl-3-phen-
ylpyrazole-1-yl)-3(2H)-pyridazinone-2-yl]acetic acid (6 a–m)
N-Phenylethyl-2-[6-(5-methyl-3-phenylpyrazole-1-yl)-3(2H)-
pyridazinone-2-yl]acetamide (6 g)
A reaction mixture containing [6-(5-methyl-3-phenylpyrazole-
1-yl)-3(2H)-pyridazinone-2-yl]acetic acid (0.01 mol) and bis-(2-
oxazolidinyl)phosphinic chloride (0.01 mol) in 10 mL anhydrous
CH2Cl2 was treated with Et3N (0.01 mol) and allowed to stir at
room temperature for 30 min.The mixture was then treated with
appropriate amine derivatives and continued to stir at room
temperature overnight. After evaporation to dr8yness, the resi-
due was solidified by stirring in ice-cold water, and the precipi-
tate formed was recrystallized from the appropriate solvent.
Recrystallized from ethanol (yield 63 %, mp 130 °C). LC-MS
(-ESI) m/z 412 (M+᭹ – 1) – 1H-NMR (CDCl3):δ 7.63 (d, 1 H);7.27
(m, 3 H); 7.17 (m, 5 H); 7.02 (m, 2 H); 6.9 (d, 1 H); 6.23 (s, 1 H);
5.76 (t, 1 H);4.33 (s, 2 H);3.37 (q, 2 H);2.67 (t, 2 H);2.3 (s, 3 H).
– IR νmax cm–1 (KBr): 3286, 3084, 3027, 2926, 1683, 1655,
1599, 757, 695.
N-Octyl-2-[6-(5-methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazi-
none-2-yl]acetamide (6 h)
Recrystallized from ethanol-water (yield 57.8 %, mp 105 °C).
LC-MS (-ESI) m/z 420 (M+᭹ – 1) – 1H-NMR (CDCl3): δ 7.61 (d,
1 H); 7.4–7.1 (m, 5 H); 6.95 (d, 1 H); 6.23 (s, 1 H); 5.9 (t, 1 H);
4.33 (s, 2 H); 3.1 (q, 2 H); 2.29 (s, 3 H); 1.36 (m, 2 H); 1.27–1.03
(m, 10 H); 0.8 (t, 3 H). – IR νmax cm–1 (KBr): 3320, 2932, 2851,
1677, 1653, 1603, 760.
1-[2-[6-(5-Methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-
2-yl]acetyl]-4-(4-fluorophecyl)piperazine (6 a)
Recrystallized from 2-propanol (yield 52,6 %, mp 152 °C). LC-
MS (-ESI) m/z 471 (M+᭹ – 1) – 1H-NMR (CDCl3): δ 7.45 (d, 1 H);
7.32–7.21 (m, 5 H); 6.98–6.86 (m, 3 H); 6.85–6.77 (m, 2 H);
6.23 (s, 1 H); 4.68 (s, 2 H); 3.68 (t, 2 H); 3.45 (t, 2 H); 3.01 (m,
4 H);2.29 (s, 3 H).– IR νmax cm–1 (KBr):3055, 2923, 1668, 1591,
1236, 760, 695.
N-(3-Pyridyl)-2-[6-(5-methyl-3-phenylpyrazole-1-yl)-3(2H)-
pyridazinone-2-yl]acetamide (6 i)
Recrystallized from toluene (yield 61 %, mp 173 °C). LC-MS
(+ESI) m/z 387 (M+᭹ + 1) – 1H-NMR (CDCl3): δ 9.2 (s, 1 H); 8.61
(s, 1 H);8.25 (m, 1 H);8.16 (m, 1 H);7.65 (d, 1 H);7.34–7.21 (m,
6 H); 6.97 (d, 1 H); 6.24 (s, 1 H); 4.65 (s, 2 H); 2.29 (s, 3 H). – IR
νmax cm–1 (KBr): 3587, 3071, 1701, 1674, 1587, 760.
1-[2-[6-(5-Methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-
2-yl]acetyl]-4-(2-fluorophenyl)piperazine (6 b)
Recrystallized from 2-propanol (yield 51 %, mp 121 °C).LC-MS
1
(-ESI) m/z 471 (M+᭹ – 1) – H-NMR (CDCl3): δ 7.46 (d, 1 H);
7.36–7.1 (m, 5 H);7.1–6.78 (m, 5 H);6.23 (s, 1 H);4.68 (s, 2 H);
3.7 (t, 2 H); 3.46 (t, 2 H); 3.01 (m, 4 H); 2.29 (s, 3 H). – IR νmax
cm–1 (KBr): 3052, 2929, 1669, 1595, 1234, 757, 696.
N-(4-Bromophenyl)-2-[6-(5-methyl-3-phenylpyrazole-1-yl)-
3(2H)-pyridazinone-2-yl]acetamide (6 j)
Recrystallized from ethanol (yield 70 %, mp 199 °C). LC-MS
1
(+ESI) m/z 465 (M+᭹ + 1) – H-NMR (CDCl3): δ 8.45 (s, 1 H);
1-[2-[6-(5-Methyl-3-phenylpyrazole-1-yl)-3(2H)-pyridazinone-
2-yl]acetyl]-4-(4-piperonyl)piperazine (6 c)
7.65 (d, 1 H); 7.39–7.13 (m, 9 H); 6.96 (d, 1 H); 6.23 (s, 1 H);
4.66 (s, 2 H); 2.29 (s, 3 H). – IR νmax cm–1 (KBr): 3278, 3054,
1684, 1666, 1588, 759.
Recrystallized from methanol (yield 58 %, mp 99 °C). LC-MS
1
(+ESI) m/z 513 (M+᭹ + 1) – H-NMR (CDCl3): δ 7.41 (d, 1 H);
N-(4-Chlorophenyl)-2-[6-(5-methyl-3-phenylpyrazole-1-yl)-
3(2H)-pyridazinone-2-yl]acetamide (6 k)
7.34–7.13 (m, 5 H); 6.88 (d, 1 H); 6.77 (s, 1 H); 6.66 (d, 2 H);
6.22 (s, 1 H); 5.87 (s, 2 H); 4.62 (s, 2 H); 3.51 (t, 2 H); 3.36 (s,
2 H); 3.27 (t, 2 H); 3.01 (m, 4 H); 2.27 (s, 3 H). – IR νmax cm–1
(KBr): 3024, 2960, 2882, 1671, 1645, 1586, 1033, 764.
Recrystallized from methanol (yield 64 %, mp 200 °C). LC-MS
(-ESI) m/z 418.5 (M+᭹ – 1) – 1H-NMR (CDCl3): δ 8.38 (s, 1 H);
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 7–14
Potential Analgesic and Anti-Inflammatory Compounds 13
Gastric-side effects
7.66 (d, 1 H); 7.4–7.1 (m, 9 H); 6.98 (d, 1 H); 6.24 (s, 1 H); 4.64
(s, 2 H); 2.29 (s, 3 H). – IR νmax cm–1 (KBr): 3284, 3100, 3047,
1713, 1665, 1589, 759.
After 8 h the analgesic activity experiments, mice were killed
under deep ether anesthesia and stomachs were removed.
Then the abdomen of each mouse was opened through the
great curvature and examined under dissecting microscope for
lesions or bleedings.
N-(4-Methoxyphenyl)-2-[(5-methyl-3-phenylpyrazole-1-yl)-
3(2H)-pyridazinone-2-yl]acetamide (6 m)
Recrystallized from ethanol (yield 57 %, mp 172 °C). LC-MS
(-ESI) m/z 414 (M+᭹ – 1) – 1H-NMR (CDCl3):δ 8.02 (s, 1 H);7.65
(d, 1 H); 7.38–7.13 (m, 7 H); 6.96 (d, 1 H); 6.73 (d, 2 H); 6.24 (s,
1 H);4.63 (s, 2 H);3.7 (s, 3 H);2.29 (s, 3 H).– IR νmax cm–1 (KBr):
3282, 3136, 3084, 1703, 1663, 1591, 766.
Statistical analysis of data
Data obtained from animal experiments were expressed as the
mean standard error ( SEM). Statistical differences between
the treatments and the control were tested by ANOVA test.Data
with p < 0.05 value was considered to be significant.
Pharmacology
Effect of compounds on human whole blood COX-1 and COX-2
activities
Male Swiss albino mice (The Animal Breeding Laboratories of
Refik Saydam Hifzisihha Institute Ankara, Turkey) weighing
20–25 g were used for all experiments. The animals were
housed in colony cages (6 mice each), maintained on standard
pellet diet and water ad libitum and left for two days for acclima-
tization before the experimental sessions. The food was with-
drawn on the day before the experiment, but free access to wa-
ter was allowed. All experiments were carried out according to
the suggested ethical guidelines for the care of laboratory ani-
mals.
The human whole blood assay, originally developed by Patrig-
nani et al [25], is considered to be the more biologically relevant
way to assess the inhibition of the cyclooxygenase isoen-
zymes, COX-1 and COX-2, by a test compound [2]. In this as-
say, platelets stimulated by calcium ionophore are believed to
be the main source of COX-1, whereas monocytes stimulated
with LPS are thought to be the source of COX-2. COX-1 activity
is determined by the production of thromboxane B2 (TXB2),
while COX-2 activity is determined by the production of pros-
taglandin E2 (PGE2).
Preparation of test samples for bioassay
Test samples were given orally to test animals after suspending
in a mixture of distilled H2O and 0.5% sodium carboxymethyl
cellulose (CMC).The control group animals received the same
experimental handling as those of the test groups except that
the drug treatment was replaced with appropriate volumes of
the dosing vehicle. Either indomethacin (10 mg/kg) or aspirin
(100 mg/kg) in 0.5 % CMC was used as reference drug.
Acknowledgement
The authors greatly acknowledge Dr. Joan Caturla for
providing the COX-1 and COX-2 inhibition assays for the
selected compounds. This investigation was supported
by a Scientific Research Grant 02/2002-13 awarded by
Gazi University BAP.
p-Benzoquinone-induced writhing test [22]
60 min after the oral administration of test samples, the mice
were intraperitoneally injected with 0.1 mL/10 g body weight of
2.5 % (v/v) p-benzoquinone (PBQ; Merck) solution in distilled
H2O.Control animals received an appropriate volume of dosing
vehicle. The mice were then kept individually for observation
and the total number of abdominal contractions (writhing move-
ments) was counted for the next 15 min, starting on the 5th min
after the PBQ injection. The data represent the average of the
total number of writhing observed. The analgesic activity was
expressed as the percentage change from writhing controls.
References
[1] C. J. Hawkey, The Lancet 1999, 353, 307–314.
[2] P.Brooks, P.Emery, J. F.Evans,H.Fenner, C.J. Hawkey, C.
Patrono, J.Smolen, F.Breedveld, R.Day, M.Dougados, E.
W. Ehrich, J. Gijon-Banos, T. K. Kvien, M. H..Van Rijswijk,
T. Warner, H. Zeidler, Rheumatology 1999, 38, 779–788.
[3] E.A.Meade, W.L.Smith, D.L.DeWitt, J.Biol.Chem.1993,
268, 6610–6614.
Carrageenan-induced hind paw edema test
[4] J. Meyer-Kirchrath, K. Schrör, Curr. Med. Chem. 2000, 7,
1121–1129.
The method of Kasahara et al. [23] was used.The difference in
footpad thickness between the right and left foot was measured
with a pair of dial thickness gauge callipers (Ozaki Co., Tokyo,
Japan). Mean values of treated groups were compared with
mean values of a control group and analyzed using statistical
methods. 60 min after the oral administration of test sample or
dosing vehicle, each mice was injected with freshly prepared
(0.5 mg/25 µL) suspension of carrageenan (Sigma, St. Louis,
Missouri, USA) in physiological saline (154 mM NaCl) into sub-
plantar tissue of the right hind paw and 25 µL of saline solution
into that of the left as secondary control. Measurements were
done and evaluated as described above in every 90 min during
360 min.
[5] T.D.Penning, J.J.Talley, S.R.Bertenshaw, J.S.Carter, P.W.
Collins, S. Docter, M. J. Graneto, L. F. Lee, J.W. Malecha, J.
M.Miyashiro, R.S.Rogers, D.J.Rogier, S.S.Yu, G.D.Ander-
son, E. G. Burton, J. N. Cogburn, S. A. Gregory, C. M.
Koboldt, W. E. Perkins, K. Seibert, A. W. Veenhuizen, Y. Y.
Zhang, P. C. Isakson, J. Med. Chem. 1997, 40, 1347–1365.
[6] P. Prasit, Z.Wang, C. Brideau, C. C. Chan, S. Charleson, W.
Cromlish, D. Ethier, J. F. Evans, A.W. Ford-Hutchinson, J.Y.
Gauthier, R. Gordon, J. Guay, M. Gresser, S. Kargman, B.
Kennedy, Y. Leblanc, S. Leger, J. Mancini, G. P. O’Neill, M.
Ouellet, M.D.Percival, H.Perrier, D.Riendeau, I.Rodger, R.
Zamboni, Bioorg. Med. Chem. Lett. 1999, 9, 1773–1778.
Acute toxicity
[7] G. Dannhardt, S. Laufer, Curr. Med. Chem. 2000, 7,
Animals employed in the carrageenan-induced paw edema ex-
periment were observed during 24 h and mortality if present
was recorded for each group at the end of the observation
period.
1101–1112.
[8] J. Van Ryn, G. Trummlitz, M. Pairet, Curr. Med. Chem.
2000, 7, 1145–1161.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

14 Banoglu et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 7–14
[18] A.S.Kalgutkar, S.W.Rowlinson, B.C.Crews, L.J.Marnett,
[9] Z. Sui, J. Guan, M. P. Ferro, K. McCoy, M. P.Wachter, W.V.
Murray, M.Singer, M.Steber, D.M.Ritchie, D.C.Argentieri,
Bioorg. Med. Chem. Lett. 2000, 10, 601–604.
Bioorg. Med. Chem. Lett. 2002, 12, 521–524.
[19] J. Cabre, A. L. Palomo, Synthesis 1984, 413–417.
[10] H. h. Kim, J. G. Park, T. C. Moon, H. W. Chang, Y. Jahng,
[20] J. Druey, Kd. Meier, K. Eichenberger, Helv. Chim. Acta
1954, 37, 121–133.
Arch. Pharm. Res. 1999, 22, 372–379.
[11] V.K.Chintakunta, V.Akella, M.S.Vedula, P.K.Mamnoor, P.
Mishra, S. R. Casturi, A.Vangoori, R. Rajagopalan, Eur. J.
Med. Chem. 2002, 37, 339–347.
[21] G. Szilagyi, E. Kasztreiner, L. Tardos, E. Kosa, L. Jaszlits,
G.Cseh, I.Kovacs, N.Szabo, P.Tolnay, S.Elek, I.Elekes, I.
Polgari (Richter Gedeon Vegyeszeti Gyar Rt.). US
4,224,325. 1980.
[12] S. Pieretti, V. Dal Piaz, R. Matucci, M. P. Giovannoni, A.
Galli, Life Sci. 1999, 65, 1381–1394.
[22] R. Okun, S. C. Liddon, L. Lasagnal, J. Pharmacol. Exp.
Ther. 1963, 139, 107–114.
[13] D. S. Dogruer, M. F. ahin, S. Ünlü, S. Ito, Arch. Pharm.
Pharm. Med. Chem. 2000, 333, 73–104.
[23] Y. Kasahara, H. Hikino, S. Tsurufuji, M. Watanabe, K.
Ohuchi, Planta Med. 1985, 51, 325–331.
[14] M. Takaya, M. Sato, K. Terashima, h. Tanizawa, J. Med.
Chem. 1979, 22, 53–58.
[24] R. Viniegra, W. Schreiber, R. Hugo, J. Pharmacol. Exp.
Ther. 1969, 166, 96–103.
[15] M. C. Viaud, P. Jamoneau, C. Flouzat, J. G. B. Espiard, B.
Pfeiffer, P. Renard, D. H. Caignard, G. Adam, G. J. Guil-
laumet, J. Med. Chem. 1995, 38, 1278–1286.
[25] P. Patrignani, M. R. Panara, A. Greco, O. Fusco, C. Natoli,
S. Iacobelli, F. Chipollone, A. Ganci, C. Crèminon, J.
Maclouf, C. Patrono, J. Pharmacol. Exp. Ther. 1994, 271,
1705–1712.
[16] F. Rohet, C. Rubat, P. Coudert, E. Albuisson, J. Couquelet,
Chem. Pharm. Bull. 1996, 44, 980–986.
[17] A. S. Kalgutkar, A. B. Marnett, B. C. Crews, R. P. Remmel,
L. J. Marnett, J. Med. Chem. 2000, 43, 2860–2870.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim