Investigations of new pyridazinone derivatives for the synthesis of potent analgesic and anti-inflammatory compounds with cyclooxygenase inhibitory activity

Article abstract of DOI:10.1002/ardp.200300778

In this study we describe the synthesis of two novel 4-phenyl- and 4-(2-chlorophen-yl)-6-(5-chloro-2-oxo-3H-benzoxazol-7-yl)-3(2H)-pyridazinone derivatives (compounds 8 a and b) and their testing as inhibitors of cyclooxygenases (COX-1 and COX-2). Both compounds inhibited COX-1 (by 59% and 61% for compounds 8 a and 8 b respectively) and COX-2 (by 37% and 28% for compounds 8 a and 8 b respectively) at a concentration of 10 μM. Furthermore, we tested the analgesic and anti-inflammatory activities of the synthesized compounds in vivo by using the p-benzoquinone-induced writhing test and the carrageenan-induced hind paw edema model, respectively. Compounds 8 a and b showed potent analgesic and anti-inflammatory activities without causing gastric lesions in the tested animals.

Full text of DOI:10.1002/ardp.200300778

406 Ökçelik et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 406–412
Berna Ökçelika,
Serdar Ünlüa,
Investigations of New Pyridazinone Derivatives for
the Synthesis of Potent Analgesic and
Anti-Inflammatory Compounds with
Erden Banoglua,
Esra Küpelib,
Erdem Yes¸iladab,
M. Fethi S¸ ahina
Cyclooxygenase Inhibitory Activity
In this study we describe the synthesis of two novel 4-phenyl- and 4-(2-chlorophen-
yl)-6-(5-chloro-2-oxo-3H-benzoxazol-7-yl)-3(2H)-pyridazinone derivatives (com-
pounds 8 a and b) and their testing as inhibitors of cyclooxygenases (COX-1 and
COX-2). Both compounds inhibited COX-1 (by 59 % and 61 % for compounds 8 a
and 8 b respectively) and COX-2 (by 37 % and 28 % for compounds 8 a and 8 b re-
spectively) at a concentration of 10 µM. Furthermore, we tested the analgesic and
anti-inflammatory activities of the synthesized compounds in vivo by using the p-
benzoquinone-induced writhing test and the carrageenan-induced hind paw edema
model, respectively. Compounds 8 a and b showed potent analgesic and anti-in-
flammatory activities without causing gastric lesions in the tested animals.
a
Division of Pharmaceutical
Sciences, Department of
Pharmaceutical Chemistry,
Faculty of Pharmacy,
Gazi University,
6330 Etiler, Ankara, Turkey
Division of Pharmaceutical
Sciences, Department of
Pharmacognosy, Faculty of
Pharmacy, Gazi University,
06330 Etiler, Ankara, Turkey
b
Keywords: 2-Oxo-3H-benzoxazole; Pyridazinone; Analgesic; Anti-inflammatory;
COX-1; COX-2
Received: January 23, 2003; Accepted: April 1, 2003 [FP778]
DOI 10.1002/ardp.200300778
COX-2 inhibitors have the common structural properties
of two aromatic moieties attached to adjacent atoms
Introduction
Cyclooxygenases (COX-1 and COX-2) catalyze the
committed step in prostaglandin (PG) synthesis and are
of particular interest because they are the major targets
of nonsteroidal anti-inflammatory drugs (NSAIDs) [1–3].
Inhibition of PGs by NSAIDs acutely reduces inflamma-
tion, pain and fever.The COX-2 isoform is responsible for
the production of PGs which mediate the inflammatory
response. Therefore, the development of new com-
pounds which selectively inhibit COX-2, without modify-
ing the physiological levels of constitutive COX-1, has
emerged as a growing research area for generation of
new anti-inflammatory drugs lacking the side-effects of
traditional NSAIDs [4–7]. However, COX-2 is also in-
volved in delayed ulcer healing, renal physiology and fe-
male reproduction processes indicating that the func-
tions of COX-1 and COX-2 might be more complex then
originally thought [8].
(1,2-diarylsubstituted) in a bridging carbocyclic or
heterocyclic five-membered ring [2, 4–6] but do not show
the side effects inherent to traditional inhibitors. Some
1,3-diarylpyrazoles fused to a cycloalkane have also
been reported to function as selective COX-2 inhibitors
(1 in Figure 1) [9, 10].The most effective inhibitor, deriva-
tive (2), had an IC50 of 0.64 µM against COX-2 and
>10 µM against COX-1.
Our research group has been interested for some time in
studying the effect of substituting selected aromatic
rings in current NSAIDs with alternative heteroaromatic
moieties such as 2-oxo-3H-benzoxazole, 2-oxo-3H-ben-
zothiazole and 3(2H)-pyridazinone [11–15].We are par-
ticularly interested in whether the biological activity of
the derivatives can be preserved by isosteric replace-
ment of a key aromatic ring.With this purpose and based
on the fact that heteroaryl acetic acid derivatives bear
potential analgesic and anti-inflammatory activities, we
have previously studied the 2-oxo-3H-benzoxazole alka-
noic acid derivatives and found that 6- or 7-acyl-2-oxo-
3H-benzoxazole alkanoic acids had potent analgesic
and anti-inflammatory activities [16, 17]. We then pro-
ceeded to investigate the 3(2H)-pyridazinones and de-
rivatives carrying acetamide and propanamide moieties
at position 2 of the pyridazinone ring and found that
these compounds also showed good analgesic and anti-
inflammatory properties [14, 15]. Furthermore, many
pyridazinone derivatives have been reported to function
as novel potent analgesic and anti-inflammatory agents
The majority of NSAIDs currently used in the therapy of
inflammatory conditions belong to the chemical class of
arylacetic and arylpropionic acids. Their side effects re-
sult from inhibition of the constitutive COX-1 isoform
which is responsible for the production of prostaglandins
(PGs) important for gastroprotection and vascular
homeostasis [3, 8]. The newly developed selective
Correspondence: Serdar Ünlü, Division of Pharmaceutical
Sciences, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Gazi University, 06330 Etiler, Ankara, Turkey.
Phone: +90-312-2126645, Fax: +90-312-2235018, email:
sunlu@gazi.edu.tr.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 406–412 New Pyridazinone Derivatives with Cyclooxygenase Inhibitory Activity 407
Figure 1. 1,3-Diarylpyrazoles as selective COX-2 inhibitors.
For the synthesis of compounds 8a and 8 b, commercial-
ly available 2-amino-4-chlorophenol was first converted
to 2-acetylamino-4-chlorophenol to maintain the further
acylation of this compound at position 6. Acylation of 2-
acetylamino-4-chlorophenol, using Friedel-Crafts acyla-
tion conditions, allowed us to obtain the acetyl group in
the appropriate position to synthesize 7-acetyl-5-chloro-
2-oxo-3H-benzoxazole (3). Treatment of 3 with the ap-
propriate benzaldehyde derivative, under base-cata-
lysed reaction conditions, resulted in compounds 4 a–b.
Synthesis of the corresponding 4-oxobutyronitrile
(5 a–b) and 4-oxobutyric acid (6 a–b) derivatives was
Figure 2. The general structure of the compounds syn-
achieved by treatment of 4 a–b with potassium cyanide
thesized.
followed by acid-catalyzed hydrolysis. The 4,5-dihydro-
pyridazinone derivatives (7 a–b) were obtained by treat-
ment of 6 a–b with hydrazine hydrate, followed by subse-
quent oxidization to produce 4-phenyl- and 4-(2-chlo-
rophenyl)-6-(5-chloro-2-oxo-3H-benzoxazol-7-yl)-3(2H)-
and some have been shown to selectively inhibit COX-2
function [18–20].
These studies prompted us to search for novel lead com-
pounds for use as selective COX-2 inhibitors. In the
present study, we describe the biological consequences
of incorporation of a 2-oxo-3H-benzoxazole ring as one
of the aryl substituents and the effect of a 1,3-diarylsub-
stitution pattern around the pyridazinone ring (Figure 2)
on the in vitro and in vivo activity of the resulting deriva-
tives.Since 2-oxo-3H-benzoxazoles and 3(2H)-pyridazi-
nones have good analgesic and anti-inflammatory prop-
erties and the 1,3-diaryl/heteroaryl structures might also
be important for these activities, we have combined
2-oxo-3H-benzoxazole and 3(2H)-pyridazinone rings in
the same structure and investigated the ability of the re-
sulting derivates to inhibit cyclooxygenase. Here we de-
scribe the methodology we employed to the synthesis of
the derivatives and their resulting in vivo and in vitro
biological activity.
pyridazinone derivatives (8 a–b). Synthesis of the inter-
medite compounds 2–7 have not been previously report-
ed in the literature.
Pharmacology
The inhibitory activity of compounds 8a and 8b on COX-
1 and COX-2 was assayed using the COX Inhibitor
Screening Assay Kit (Cayman No: 560131) according to
the protocol recommended by the supplier. Preliminary
screening of both title compounds (8a and b) and refer-
ences (indomethacin and DFU) was performed at a final
concentration of 10 µM to determine the percent inhibi-
tion of the COX-1 and COX-2 isoforms.The inhibitor ac-
tivity of indomethacin, at a 10 µM final concentration in
the test system, deviated from previously published re-
ports [21, 22].However, the results in our assay were ba-
sically reproducible and the average of typical sets of da-
ta are represented here. Previously published literature
reports on the inhibitory activity of indomethacin indicate
that depending on the in vitro assay used, the COX-2/
COX-1 ratio can vary from 1.31 to 107.1 [3, 4].Differenc-
es in the published results obtained for indomethacin in
Results and discussion
Chemistry
The synthetic methodology used in the synthesis of
compounds 8 a and b is shown in Scheme 1.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

408 Ökçelik et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 406–412
Scheme 1. Synthetic route for the synthesis of compounds 8 a–b.
various test systems illustrate how the determined inhib-
itory values can vary for a single compound. Variability
between assays was reported to be the consequence of
many factors including incubation time, the use of exoge-
nous or endogenous substrate, use of whole cells, mi-
crosomes or recombinant enzymes, and the presence or
absence of plasma proteins in the medium [3]. There-
fore, our results from the in vitro screening assay utilized
serve only as a guide to the relative selectivity of different
compounds in the same assay system.
The percent inhibition of COX-1 and COX-2 by in-
domethacin was determined to be 69 % and 78 %, re-
spectively suggesting that indomethacin was functioning
as a non-selective inhibitor as previously reported [16].
DFU inhibited COX-2 (86 %) but did not have any inhibi-
tory effect on COX-1 in the assay system also in accord-
ance with a previously published report [23]. Com-
pounds 8 a and b showed inhibitor activity against the
COX-1 and COX-2 enzymes but the selectivity was ei-
ther not very pronounced or only slightly selective for the
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 406–412 New Pyridazinone Derivatives with Cyclooxygenase Inhibitory Activity 409
Table 1. Anti-inflammatory and analgesic activities of compounds 8 a and 8 b.
Compound
Anti-inflammatory activity
Thickness of Edema SD
(Inhibition %)
Analgesic activity
Number of writhings SD
(Inhibition %)
Ratio of
gastric
lesions
90 min
180 min
270 min
360 min
8 a
8 b
31 2.29
(17.9)
33.5 1.61
35 2.11
(39.4)**
33.8 1.47
(49.7)***
11.3 1.52
(70.4)***
0/6
0/6
0/6
2/6
0/8
(28.4)*
30.3 2.01 33.8 1.35
(19.8) (27.7)*
35 2.24
(39.4)**
31.2 2.81
(53.6)***
17 2.96
(55.5)**
Indomethacin 26.5 2.06 27.3 1.91 27.2 2.76
29 1.07
(56.8)***
–
(29.9)*
(41.7)**
(52.9)***
Aspirin
Control
–
–
–
–
16.3 2.26
(57.3)**
37.8 3.57 46.8 5.39 57.8 5.15 67.2 3.69
38.2 4.29
The analgesic and anti-inflammatory activity of compounds 8 a and b were tested at 100 mg/kg doses.The analgesic
activity of aspirin was tested at a 100 mg/kg dose and the anti-inflammatory activity of indomethacin was tested at a
10 mg/kg dose as described in the Experimental section. *: p < 0.05, **: p < 0.01, ***: p < 0.001.
COX-1 isoform.The inhibitory activity of compounds 8 a
and 8 b against COX-1 was 59 % and 61 %, respectively,
while the inhibitory activity against COX-2 was 37 % and
28 %, respectively.
360 min).This supports our in vitro COX inhibitory activi-
ty results indicating that these compounds may also ex-
ert their activities in vivo through the inhibition of COX
enzymes, thereby preventing the formation of inflamma-
tory prostaglandins from arachidonic acid.
Compounds 8 a and b were also tested, at a single dose
of 100 mg/kg in mice for their analgesic and anti-inflam-
matory activities using the p-benzoquinone-induced
writhing test [24] and carrageenan-induced hind paw
edema model [25], respectively. As shown in Table 1,
compounds 8 a and 8 b showed equal or higher analge-
sic activity than that of aspirin at a 100 mg/kg dose.Com-
pound 8 a had the highest analgesic activity (70.4 %).
Additionally, compounds 8 a and 8 b at 100 mg/kg dose
showed a reasonable anti-inflammatory activity, but the
overall activity was lower than that observed with in-
domethacin at a 10 mg/kg dose. It is known that edema
produced by carrageenan is a biphasic event and that
the inhibitory effects of agents which act during the first
stage of the carrageenan-induced hind paw inflamma-
tion are attributable to inhibition of chemical mediators
such as histamine, serotonin and bradykinin. The sec-
ond stage of the edema might be related to arachidonic
acid metabolites since it is inhibited by aspirin, in-
domethacin and other cyclooxygenase inhibitors [26,
27].As shown inTable 1, compounds 8 a and b exhibited
considerable anti-inflammatory activity in the second
phase of carrageenan-induced edema (270 and
In conclusion, the 4-phenyl-6-(5-chloro-2-oxo-3H-ben-
zoxazol-7-yl)-3(2H)-pyridazinone derivatives represent
a promising new diarylheterocyclic structure for cyclo-
oxygenase inhibition and may provide the structural ba-
sis for the development of compounds with better anal-
gesic and anti-inflammatory activity capable of selective
COX-2 inhibition. Further analyses with derivatives of
these compounds, including those containing a 1,2-dis-
ubstitution pattern around the pyridazinone ring, are cur-
rently ongoing research in our laboratory.This may allow
the development of compounds with greater selectivity
in inhibition of COX-1 and COX-2 activity.
Experimental
Chemical methods
2-Amino-4-chlorophenol, sodium acetate, acetic anhydride,
aluminium chloride, acetyl chloride, benzaldehyde, 2-chlo-
robenzaldehyde, and ethyl chloroformate were purchased from
Merck Co. (Germany).The starting compounds 2-acetylamino-
4-chlorophenol and 2-amino-6-acetyl-4-chlorophenol were
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

410 Ökçelik et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 406–412
synthesized according to previously reported procedures [28].
2-Acetylamino-6-acetyl-4-chlorophenol was synthesized ac-
cording to the previously reported procedure with modifications
[29].All other chemicals were obtained from commercial sourc-
es.COX Inhibitor Screening Assay Kits (No:560131), including
recombinant ovine COX-1 and recombinant human COX-2,
were purchased from Cayman Chemical (France). The selec-
tive COX-2 inhibitor reference compound DFU (5,5-dimethyl-
3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-
furanone) was obtained from Merck Research Laboratories
(USA). IR spectra were recorded on a Bruker Vector 22 IR
(Opus Spectroscopic SoftwareVersion 2.0) spectrometer (KBr,
υ, cm–1). 1H-NMR spectra were recorded on a Bruker 400 FT-
NMR spectrometer using TMS as an internal standard in
DMSO-d6 or CDCl3. All chemical shifts were reported as δ
(ppm) values.Elemental analyses were performed with a Leco-
932 (C,H,N,S-O-Elemental analyzer) at the Instrumental
Analysis Center of the Scientific andTechnical Research Coun-
cil of Turkey (Ankara, Turkey), and were within the range of
0.4 %.
cm–1:νmax 3406–3140 (NH, lactam), 3062 (CH, aromatic), 1779
(C=O, lactam), 1669 (C=O, ketone), 1594 (C=C, aromatic).
Anal. (C16H10ClNO3) C, H, N.
Compound 4 b 1H-NMR (CDCl3-DMSO-d6): δ = 12.2 (s, 1 H,
NH); 8.25 (dd, 3J3,4 = 7.06 Hz, 4J3,5 = 2.31 Hz, 1 H, phH-3); 8.23
3
3
(d, J = 15.71 Hz, 1 H, Ph-CH=); 7.99 (d, J = 15.63 Hz, 1 H,
4
=CH-CO); 7.86 (d, J6,4 = 2.14 Hz, 1 H, 2-oxo-3H-benzoxa-
zoleH-6); 7.79 (dd, 3J6,5 = 7.57 Hz, 4J6,4 = 1.92 Hz, 1 H, phH-6);
4
7.70 (m, 2 H, phH-4,H-5); 7.61 (d, J4,6 = 2.12 Hz, 1 H, 2-oxo-
3H-benzoxazoleH-4); IR (KBr) cm–1: νmax 3442–3096 (NH,
lactam), 3076 (CH, aromatic), 1828–1791 (C=O, lactam), 1653
(C=O, ketone). Anal. (C16H9Cl2NO3) C, H, N.
2-Phenyl- and 2-(2-chlorophenyl)-4-(5-chloro-2-oxo-3H-benz-
oxazol-7-yl)-4-oxobutyronitrile (5 a–b)
A reaction mixture of 3-phenyl- or 3-(2-chlorophenyl)-1-(5-
chloro-2-oxo-3H-benzoxazol-7-yl)-2-propene-1-one (0.03 mol)
and potassium cyanide (0.075 mol) in 250 mL methanol con-
taining 25 g glycerin was refluxed for 2.5 h.The reaction mixture
was then poured into ice-water containing 5 mL concentrated
HCl and the precipitate formed filtered off, washed with water
and recrystallized from methanol-water or ethanol to yield
92.5 % of 5 a or 98.33 % of 5 b, respectively.
2-Acetylamino-6-acetyl-4-chlorophenol (1)
A reaction mixture of N,N-dimethylformamide (0.22 mol) and
aluminium chloride (0.8 mol), obtained in an ice-bath, was
treated with acetyl chloride (0.2 mol) and stirred for 10 min.The
mixture was then treated with 2-acetylamino-4-chlorophenol,
heated to 120 °C and left to incubate for 2 h.The reaction mix-
ture was poured into 1 L ice-water and 10 mL concentrated HCl
was added. The formed precipitate was filtered off and re-
crystallized from 2-propanol to yield 96 % of 1. 1H-NMR
(DMSO-d6): δ = 12.67 (s, 1 H, OH); 9.56 (s, 1 H, NH-CO); 8.29
Compound 5 a 1H-NMR (DMSO-d6):δ = 11.91 (s, 1 H, NH);7.33
4
(d, J6,4 = 2.14 Hz, 1 H, 2-oxo-3H-benzoxazoleH-6); 7.28 (m,
4
2 H, phH-2,H-6); 7.21 (m, 2 H, phH-3,H-5); 7.19 (d, J4,6
=
2.05 Hz, 1 H, 2-oxo-3H-benzoxazoleH-4); 7.13 (m, 1 H, phH-
4); 4.42 (dd, 3Jcb = 8.99 Hz, 3Jca = 5.11 Hz, 1 H, Hc); 3.67 (dd,
3Jba = 18.6 Hz, 3Jbc = 9.04 Hz, 1 H, Hb); 3.47 (dd, 3Jab = 18.6 Hz,
3Jac = 5.14 Hz, 1 H, Ha); IR (KBr) cm–1: νmax 3279 (NH, lactam),
3060 (CH, aromatic), 2947 (CH, aliphatic), 2250 (CN), 1818–
1782 (C=O, lactam), 1677 (C=O, ketone), 1618 (C=C,
aromatic). Anal. (C17H11ClN2O3) C, H, N.
4
4
(d, J5,3 = 2.2 Hz, 1 H, H-5); 7.71 (d, J3,5 = 2,5 Hz, 1 H, H-3);
2.68 (s, 3 H, CH3-CO);2.14 (s, 3 H, CH3-CO-NH);IR (KBr) cm–1:
ν
max 3265 (NH), 1670 (C=O, amide), 1641 (C=O, ketone). Anal.
(C10H10ClNO3) C, H, N.
Compound 5 b 1H-NMR (DMSO-d6): δ = 12.2 (s, 1 H, NH); 7.78
(m, 1 H, phH-3); 7.64 (m, 1 H, phH-4); 7.63 (d, 4J6,4 = 2.25 Hz,
1 H, 2-oxo-3H-benzoxazoleH-6); 7.51 (m, 3 H, 2-oxo-3H-benz-
7-Acetyl-5-chloro-2-oxo-3H-benzoxazole (3)
2-Amino-6-acetyl-4-chlorophenol (0.1 mol) was dissolved in
100 mL pyridine and cooled to 0 °C. Ethylchloroformate
(0.2 mol) was added dropwise and the mixture was stirred at
this temperature for 10 min.The reaction mixture was then incu-
bated at 100 °C for 4 h, cooled, poured into iced-water and acid-
ified with 115 mL concentrated HCl.The precipitate formed was
filtered off and recrystallized from toluene to yield 62.8 % of 3.
1H-NMR (CDCl3-DMSO-d6):δ = 11.74 (s, 1 H, NH);7.42 (d, 4J6,4
= 2.17 Hz, 1 H, 2-oxo-3H-benzoxazoleH-6);7.13 (d, 4J4,6 = 2.15
Hz, 1 H, 2-oxo-3H-benzoxazoleH-4); 2.63 (s, 3 H, CO-CH3); IR
(KBr) cm–1:νmax 3312–2890 (NH, lactam), 3094 (CH, aromatic),
1831 (C=O, lactam), 1658 (C=O, ketone), 1623 (C=C, aromat-
ic). Anal. (C9H6ClNO3) C, H, N.
3
3
oxazoleH-4, phH-5,H-6); 4.93 (dd, Jcb = 8.63 Hz, Jca
=
5.31 Hz, 1 H, Hc); 4.10 (dd, 3Jba = 18.73 Hz, 3Jbc = 8.7 Hz, 1 H,
Hb); 3.85 (dd, 3Jab = 18.75 Hz, 3Jac = 5.32 Hz, 1 H, Ha); IR (KBr)
cm–1: νmax 3423–3098 (NH, lactam), 3097,3062 (C-H, aromat-
ic), 2974–2929 (CH, aliphatic), 2192 (CN), 1819–1782 (C=O,
lactam), 1688 (C=O, ketone), 1627 (C=C, aromatic). Anal.
(C17H10Cl2N2O3) C, H, N.
3-Phenyl- and 3-(2-chlorophenyl)-1-(5-chloro-2-oxo-3H-benz-
oxazol-7-yl)-2-propen-1-one (4 a–b)
A reaction mixture of 7-acetyl-5-chloro-2-oxo-3H-benzoxazole
(0.01 mol) in 100 mL distilled water containing NaOH
(0.025 mol) was treated with a solution of benzaldehyde
(0.01 mol) or 2-chlorobenzaldehyde (0.01 mol) in 100 mL etha-
nol and allowed to stir at room temperature for 8 h. It was then
poured into 1 L ice-water and neutralized with HCl (10 %, w/v).
The formed precipitates were filtered off and recrystallized
from ethanol to yield 97.4 % of 4 a or 96.4 % of 4 b.
2-Phenyl- and 2-(2-chlorophenyl)-4-(5-chloro-2-oxo-3H-benz-
oxazol-7-yl)-4-oxobutanoic acid (6 a–b)
A reaction mixture of 2-phenyl- or 2-(2-chlorophenyl)-4-(5-chlo-
ro-2-oxo-3H-benzoxazol-7-yl)-4-oxobutyronitrile (0.02 mol) in
150 mL water-sulphuric acid-DMF (45:45:60) was refluxed for
4.5 h, cooled, and poured into 250 mL ice-water.The precipitate
was filtered off, washed with water and recrystallized from ace-
tonitrile to yield 58.17 % of 6 a or 51.3 % of 6 b.
Compound 4 a 1H-NMR (DMSO-d6):δ = 11.83 (s, 1 H, NH);7.60
(m, 2 H, phH-2,H-6); 7.57 (d, 3J = 15.49 Hz, 1 H, Ph-CH=); 7.49
(d, 1 H, 3J = 15.74 Hz, =CH-CO); 7.39 (d, 4J6,4 = 1.93 Hz, 1 H, 2-
oxo-3H-benzoxazoleH-6); 7.26 (m, 3 H, phH-3,H-4,H-5); 7.17
Compound 6 a 1H-NMR (DMSO-d6): δ = 12.6–11.5 (wide, 2 H,
4
4
(d, J4,6 = 1.89 Hz, 1 H, 2-oxo-3H-benzoxazoleH-4); IR (KBr)
NH, COOH); 7.28 (d, J6,4 = 2.17 Hz, 1 H, 2-oxo-3H-benzoxa-
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 406–412 New Pyridazinone Derivatives with Cyclooxygenase Inhibitory Activity 411
zoleH-6);7.16 (d, 4J4,6 = 2.15 Hz, 1 H, 2-oxo-3H-benzoxazoleH-
4);7.12 (m, 4H, phH-2,H-3,H-5,H-6);7.06 (m, 1 H, phH-4);3.90
4-Phenyl- and 4-(2-chlorophenyl)-6-(5-chloro-2-oxo-3H-benz-
oxazol-7-yl)-3(2H)-pyridazinone (8 a–b)
3
3
3
(dd, Jcb = 10.25 Hz, Jca = 4.06 Hz, 1 H, Hc); 3.60 (dd, Jba
=
A reaction mixture of 4-phenyl- or 4-(2-chlorophenyl)-6-(5-
chloro-2-oxo-3H-benzoxazol-7-yl)-4,5-dihydro-3(2H)-pyridazi-
none (0.005 mol) in 15 mL acetic acid was treated with bromine
(0.0055 mol in 5 mL acetic acid) by drop wise addition over a
period of 1.5 h. The mixture was stirred for an additional half
hour and then poured into ice-water. The precipitate formed
was filtered off and recrystallized from dimethylsulfoxide to
yield 44.19 % of 8 a or 51.8 % of 8 b.
18.60 Hz, 3Jbc = 10.31 Hz, 1 H, Hb); 3.12 (dd, 3Jab = 18.64 Hz,
3Jac = 4.12 Hz, 1 H, Ha); IR (KBr) cm–1: νmax 3191 (NH, lactam),
1776–1763 (C=O, lactam), 1698 (C=O, COOH), 1680 (C=O,
ketone), 1626 (C=C, aromatic). Anal. (C17H12ClNO5) C, H, N.
Compound 6 b 1H-NMR (CDCl3-DMSO-d6): δ = 11.80 (wide,
2 H, NH, COOH); 7.44 (d, 4J6,4 = 2.19 Hz, 1 H, 2-oxo-3H-benz-
oxazoleH-6); 7.36 (dd, 1 H, phH-3); 7.31 (dd, 1 H, phH-6), 7.21
4
(m, 2 H, phH-4,H-5); 7.17 (d, J4,6 = 2.16 Hz, 1 H, 2-oxo-3H-
Compound 8 a 1H-NMR (DMSO-d6): δ = 13.61 (s, 1 H, pyridazi-
noneNH); 11.99 (s, 1 H, 2-oxo-3H-benzoxazoleNH); 8.12 (s,
benzoxazoleH-4); 4.63 (dd, 3Jcb = 9.95 Hz, 3Jca = 3.93 Hz, 1 H,
Hc); 3.80 (dd, 3Jba = 18.73 Hz, 3Jbc = 9.96 Hz, 1 H, Hb); 3.19 (dd,
3Jab = 18.72, 3Jac = 3.97, 1 H, Ha). IR (KBr) cm–1: νmax 3163 (NH,
lactam), 3062 (CH, aromatic), 1775 (C=O, lactam), 1702 (C=O,
COOH), 1680 (C=O, ketone),1622 (C=C, aromatic). Anal.
(C17H11Cl2NO5) C, H, N.
1 H, pyridazinoneH-5);7.94 (m, 2 H, phH-2,H-6);7.61 (d, 4J6,4
=
2.06 Hz, 1 H, 2-oxo-3H-benzoxazoleH-6); 7.51 (m, 3 H, phH-
4
3,H-4,H-5); 7.24 (d, J4,6 = 2.08 Hz, 1 H, 2-oxo-3H-benzoxa-
zoleH-4). IR (KBr) cm–1: νmax 3192-3151 (NH, 2-oxo-3H-benz-
oxazole, pyridazinone), 3059 (CH, aromatic), 1767 (C=O,
2-oxo-3H-benzoxazole), 1649 (C=O, pyridazinone). Anal.
(C17H10ClN3O3) C, H, N.
Compound 8 b 1H-NMR (DMSO-d6): δ = 13.69 (s, 1 H, pyridazi-
noneNH); 12.04 (s, 1 H, 2-oxo-3H-benzoxazoleNH); 8.02 (s,
4
1 H, pyridazinoneH-5); 7.61 (m, 1H, phH-3); 7.58 (d, J6,4
=
2.06 Hz, 1 H, 2-oxo-3H-benzoxazoleH-6); 7.49 (m, 3 H, phH-
4
4,H-5,H-6); 7.24 (d, J4,6 = 2.03 Hz, 1 H, 2-oxo-3H-benzoxa-
zoleH-4). IR (KBr) cm–1: νmax 3203–3140 (NH, 2-oxo-3H-benz-
oxazole, pyridazinone), 1770 (C=O, 2-oxo-3H-benzoxazole),
1646 (C=O, pyridazinone). Anal. (C17H9Cl2N3O3) C, H, N.
4-Phenyl- and 4-(2-chlorophenyl)-6-(5-chloro-2-oxo-3H-benz-
oxazol-7-yl)-4,5-dihydro-3(2H)-pyridazinone (7 a–b)
Pharmacology
Male Swiss albino mice (weighing 20–25 g), from the animal
breeding Laboratories of the Refik Saydam Hifzisihha Insti-
tute of Ankara Turkey, were used for all experiments. The ani-
mals were housed in colony cages (6 mice per cage), main-
tained on a standard pellet diet with water given ad-lib and left
for two days for acclimatization before the experimental ses-
sions.The food was withheld the day before the experiment but
animals were allowed free access to water. All experiments
were carried out according to the suggested ethical guidelines
for the care of laboratory animals.
2-Phenyl- or 2-(2-chlorophenyl)-4-(5-chloro-2-oxo-3H-benz-
oxazol-7-yl)-4-oxobutanoic acid (0.005 mol) was dissolved in
hot ethanol and then hydrazine hydrate (0.0055 mol) was add-
ed. The reaction mixture was refluxed for 4.5 h. After cooling,
the mixture was poured into ice-water. The precipitate formed
was filtered off and recrystallized from ethanol to yield 47.6 %
of 7 a or 57.44 % of 7 b.
1
Compound 7 a H-NMR (DMSO-d6): δ = 11.76 (s, 1 H, 2-oxo-
3H-benzoxazoleNH); 11.30 (s, 1 H, pyridazinoneNH); 7.30 (d,
4J6,4 = 2.09 Hz, 1 H, 2-oxo-3H-benzoxazoleH-6); 7.21 (m, 5 H,
Preparation of test samples for bioassay
4
phH-2,H-3,H-4,H-5,H-6); 7.07 (d, J4,6 = 2.09 Hz, 1 H, 2-oxo-
3
3
3H-benzoxazoleH-4); 3.80 (dd, Jcb = 10.13 Hz, Jca = 7.2 Hz,
1 H, Hc); 3.29 (dd, 3Jab = 17.25 Hz, 3Jac = 7.22 Hz, 1 H, Ha); 3.18
(m, Hb, and DMSO-d6). IR (KBr) cm–1: νmax 3250–3114 (NH, 2-
oxo-3H-benzoxazole, pyridazinone), 3025 (CH, aromatic),
1782–1751 (C=O, 2-oxo-3H-benzoxazole), 1677 (C=O, pyri-
dazinone). Anal. (C17H12ClN3O3) C, H, N.
Test samples, suspended in a mixture of distilled H2O and
0.5 % sodium carboxymethyl cellulose (CMC), were given oral-
ly to the animals. Control animals received the same experi-
mental handling as the test groups with the exception that the
drug treatment was replaced with an appropriate volumes of
the dosing vehicle. Either indomethacin (10 mg/kg) or aspirin
(100 mg/kg) in 0.5 % CMC was used as the reference drug.
1
Compound 7 b H-NMR (DMSO-d6): δ = 12.19 (wide, 1 H, 2-
oxo-3H-benzoxazoleNH); 11.44 (s, 1 H, pyridazinoneNH); 7.50
p-Benzoquinone-induced writhing test [22]
(m, 1 H, phH-3); 7.41 (d, 4J6,4 = 2.16 Hz, 1 H, 2-oxo-3H-benzox-
60 Minutes after oral administration of test samples, the mice
were intraperitoneally injected with 2.5 % (v/v) p-benzoquinone
solution in distilled water (0.1 mL/10 g body weight).Control an-
imals received an appropriate volume of dosing vehicle. The
mice were housed individually for observation and starting the
5th min after p-benzoquinone injection, the total number of ab-
dominal contractions (writhing movements) was counted for a
15 min period. The data represent an average of the total
number of writhing movements observed.The analgesic activi-
ty was expressed as the percentage change compared to
writhing controls.
4
azoleH-6); 7.36 (m, 3 H, phH-4,H-5,H-6); 7.16 (d, J4,6 = 2.13
Hz, 1 H, 2-oxo-3H-benzoxazoleH-4); 4.27 (dd, 3Jcb = 12.87 Hz,
3Jca = 7.48 Hz, 1 H, Hc);3.39 (dd, 3Jab = 17.15 Hz, 3Jac = 7.48 Hz,
1 H, Ha); 3.28 (dd, 3Jba = 17.10 Hz, 3Jbc = 12.89 Hz, 1 H, Hb). IR
(KBr) cm–1: νmax 3207–3117 (NH, 2-oxo-3H-benzoxazole, pyri-
dazinone), 1822–1775 (C=O, 2-oxo-3H-benzoxazole), 1694
(C=O, pyridazinone). Anal. (C17H11Cl2N3O3) C, H, N.
Carrageenan-induced hind paw edema test
For the Carrageenan-induced hind paw edema test the method
of Kasahara et al.[23] was used.The difference in footpad thick-
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

412 Ökçelik et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 406–412
ness between the right and left foot was measured using a pair
of dial thickness gauge callipers (Ozaki Co., Tokyo, Japan).
Mean values of treated versus control groups were compared
and analyzed using statistical methods. 60 min after oral ad-
ministration of test sample or dosing vehicle, each mouse was
injected with a freshly prepared (0.5 mg/25 µl) suspension of
carrageenan (Sigma, St.Louis, Missouri, USA) in physiological
saline (154 mM NaCl) into the subplantar tissue of the right hind
paw. A saline solution (25 µl) was injected into the left paw as a
secondary control. Measurements were performed and evalu-
ated as described above every 90 min during a 360 min period.
[8] J. Meyer-Kirchrath, K. Schrör, Curr. Med. Chem. 2000, 7,
1121–1129.
[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.
[10] H. H. Kim, J. G. Park, T. C. Moon, H. W. Chang, Y. Jahng,
Arch. Pharm. Res. 1999, 22, 372–379.
[11] D. S. Dogruer, S. Ünlü, E. Yes¸ilada, M.F. S¸ ahin, Farmaco
1997, 52, 745–750.
[12] D. S. Dogruer, S. Ünlü, M. F. S¸ ahin, E.Yes¸ilada, Farmaco
1998, 53, 80–84.
Acute toxicity
Animals employed in the carrageenan-induced paw edema ex-
periment were observed for 24 h and the mortality rate was re-
corded for each group at the end of the observation period.
[13] T. Önkol, D. S. Dogruer, M. F. S¸ ahin, S. Ito, Arch. Pharm.
Pharm. Med. Chem. 2000, 333, 337–340.
[14] D. S. Dogruer, M. F. S¸ ahin, S. Ünlü, S. Ito, Arch. Pharm.
Pharm. Med. Chem. 2000, 333, 79–86.
Gastric-lesions inducing effect
Eights hours after the analgesic activity experiment, mice un-
der deep ether anesthesia were killed and their stomachs were
removed. The abdomen of each mouse was opened through
great curvature and examined for lesions or bleedings using a
dissecting microscope.
[15] M. Gökçe, D. S. Dogruer, M. F. S¸ ahin, Farmaco 2001, 56,
233–237.
[16] E. Banoglu, B. Okçelik, E. Küpeli, S. Ünlü, E.Yes¸ilada, M.
Amat, J. F.Caturla, M. F.Sahin, Arch.Pharm. Pharm. Med.
Chem. 2003, 336, 251–257.
Statistical analysis of data
[17] S. Ünlü, S. Nacak, E. Küpeli, E. Yes¸ilada, Arch. Pharm.
Pharm. Med. Chem. 2003, 336, 310–321.
Data obtained from animal experiments were expressed as the
mean standard error ( SEM). Statistical differences between
treatment and control groups was determined using the ANO-
VA test. Data with p < 0.05 value was considered to be signifi-
cant.
[18] F. Rohet, C. Rubat, P. Coudert, E. Albuisson, J. Couquelet,
Chem. Pharm. Bull. 1996, 44, 980–986.
[19] P.Coudert, C. Rubat, F. Rohet, F. Leal, J.Fialip, J. Couque-
let, Pharm. Pharmacol. Commun. 2000, 6, 387–396.
[20] 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.
References
[1] C. J. Hawkey, The Lancet 1999, 353, 307–314.
[21] A. S. Kalgutkar, A. B. Marnett, B. C. Crews, R. P. Remmel,
[2] J. Van Ryn, G. Trummlitz, M. Pairet, Curr. Med. Chem.
2000, 7, 1145–1161.
L. J. Marnett, J. Med. Chem. 2000, 43, 2860–2870.
[22] J. K. Gierse, C. M. Koboldt, M. C. Walker, K. Seibert, P. C.
[3] 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.
Isakson, Biochem. J. 1999, 339, 607–614.
[23] D.Riendeau, M.D.Percival, S.Boyce, C.Brideau, S.Char-
leson, W. Cromlish, D. Ethier, J. Evans, J.-P. Falgueyret, A.
W. Ford-Hutchinson, R. Gordon, G. Greig, M. Gresser, J.
Guay, S. Kargman, S. Leger, J. A. Mancini, G. O’Neill, M.
Quellet, I. W. Rodger, M. Therien, Z. Wang, J. K. Webb, E.
Wong, L. Xu, R. N. Young, R. Zamboni, P. Prasit, C.-C.
Chan, Brit. J. Pharmacol. 1997, 121, 105–117.
[4] G. Dannhardt, S. Laufer, Curr. Med. Chem. 2000, 7,
1101–1112.
[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. Ma-
lecha, J. M. Miyashiro, R. S. Rogers, D. J. Rogier, S. S.Yu,
G.D.Anderson, 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.
[24] R. Okun, S. C. Liddon, L. Lasagnal, J. Pharmacol. Exp.
Ther. 1963, 139, 107–114.
[25] Y. Kasahara, H. Hikino, S. Tsurufuji, M. Watanabe, K.
Ohuchi, Planta Med. 1985, 51, 325–331.
[26] R.Vinegar, J.F.Truax, J.L.Selph, P.R.Johnston, A.L.Ven-
[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. Rod-
ger, R. Zamboni, Bioorg. Med. Chem. Lett. 1999, 9, 1773–
1778.
able, K. K. McKenzie, Fed. Proc. 1987, 46, 118–126.
[27] R. Vinegar, W. Schreiber, R. Hugo, J. Pharmacol. Exp.
Ther. 1969, 166, 96–103.
[28] V. M. Guran, U. K. Jagwani, J. Indian Chem. Soc. 1979, 56,
325–328
[7] C. H. Park, X. Siomboing, S.Yous, B. Gressier, M. Luyckx,
[29] D. R. Patel, S. R. Patel, J. Indian Chem. Soc. 1968, 45,
P. C h a v a t t e , Eur. J. Med. Chem. 2002, 37, 461–468.
703–708.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim