Amide derivatives of [6-acyl-2-benzothiazolinon-3-yl] acetic acids as potential analgesic and anti-inflammatory compounds

Article abstract of DOI:10.1007/s00044-009-9168-x

In this study, we investigated the analgesic and anti-inflammatory activities of [6-acyl-2-benzothiazolinon-3-yl]acetic acids by the derivatization of the carboxylate moiety into amides. We have tested the analgesic and anti-inflammatory activities of the synthesized compounds in vivo by using p-benzoquinone-induced writhing test and carrageenan-induced hind paw edema model, respectively. Compounds 4h, 4i, 4n, and 4o showed comparable analgesic and anti-inflammatory activities to the references without gastric lesions in the tested animals. In addition, all compounds also tested for their inhibitory activity against cyclooxygenase (COX)-1, COX-2 and 5-lipoxygenase (LOX), but no significant inhibition was observed under assayed conditions.

Full text of DOI:10.1007/s00044-009-9168-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:11–24
DOI 10.1007/s00044-009-9168-x
ORIGINAL RESEARCH
Amide derivatives of [6-acyl-2-benzothiazolinon-3-yl]
acetic acids as potential analgesic and anti-
inflammatory compounds
¨
Tijen Onkol Æ Erden Banoglu Æ Yasemin Du¨ndar Æ
Esra Ku¨peli Æ M. Fethi S¸ahin
Received: 9 May 2007 / Accepted: 6 February 2009 / Published online: 14 March 2009
¨
Ó Birkhauser Boston 2009
Abstract In this study, we investigated the analgesic and anti-inflammatory
activities of [6-acyl-2-benzothiazolinon-3-yl]acetic acids by the derivatization of the
carboxylate moiety into amides. We have tested the analgesic and anti-inflammatory
activities of the synthesized compounds in vivo by using p-benzoquinone-induced
writhing test and carrageenan-induced hind paw edema model, respectively.
Compounds 4h, 4i, 4n, and 4o showed comparable analgesic and anti-inflammatory
activities to the references without gastric lesions in the tested animals. In addition,
all compounds also tested for their inhibitory activity against cyclooxygenase
(COX)-1, COX-2 and 5-lipoxygenase (LOX), but no significant inhibition was
observed under assayed conditions.
Keywords Benzothiazolinone Á Analgesic Á Anti-inflammatory Á
5-LOX Á COX-1 Á COX-2
Introduction
Arachidonic acid (AA) stored in cell membranes is metabolized by two enzymatic
families namely cyclooxygenases (COX-1, -2, and -3) and lipoxygenases (5-, 8-, 12-,
and 15-LOX). These enzymes convert AA into prostaglandins, prostacyclines, and
leukotrienes, which are involved in physiological processes as well as pathological
responses such as inflammation and cancer formation (Funk 2001). Currently used
¨
T. Onkol (&) Á E. Banoglu Á Y. Du¨ndar Á M. F. S¸ahin
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University,
06330 Etiler, Ankara, Turkey
e-mail: tijen@gazi.edu.tr
E. Ku¨peli
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Etiler, Ankara,
Turkey

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R
O
S
N
O
CH2CONHR1
R: H, F, Cl
R1: phenyl, 4-chlorophenyl, 3-chlorophenyl, 2-pyridinyl,
pyridinyl
4-methyl-2- pyridinyl, 6-methyl-2-
Fig. 1 General structure of the synthesized compounds
nonsteroidal anti-inflammatory drugs (NSAIDs) act through the nonselective
inhibition of cyclooxygenase isoforms (COX-1 and COX-2) and show some side-
effects including gastrointestinal (GI) toxicity, which appears to occur as a result of
the inhibition of COX-1 isoenzyme which is involved in many physiological
¨
processes including gastric cytoprotection (Meyer-Kirchrath and Schror 2000).
Moreover, COX inhibition alone may lead to upregulation of the 5-LOX pathway,
causing various side-effects especially in the GI tract and kidney (Charlier et al.,
2003). Therefore, developing potential drugs with high analgesic and anti-
inflammatory activities lacking the general side-effects of currently used NSAIDs
is still a debate.
We have long been interested in developing compounds with potent analgesic
and anti- inflammatory activity without GI liabilities of currently utilized NSAIDs
(Banoglu et al., 2003; Gu¨lcan et al., 2003; Cakır et al., 1999). Our recent studies
indicated that (2-benzothiazolinon-3-yl)acetamides (Onkol et al., 2000) and (2-
benzothiazolinon-3-yl)propionamides (Onkol et al., 2001, 2004) alleviated the
induced pain and suppressed the induced inflammation with no observed acute
gastric toxicity in the tested animals. Furthermore, 5-chloro-6-acyl-2-benzoxazoli-
none nucleus as an aryl/heteroaryl ketone class was determined as important
structure for potent analgesic and anti-inflammatory activity. Considering that
amide derivatives of certain NSAIDs such as indomethacin and ketoprofen showed
some preference for COX-2 over COX-1 (Kalgutkar et al., 2000a), we designed and
synthesized a series of compounds (Fig. 1) which might lead to further studies to
develop better candidates for selective inhibition of COX-2 and 5-LOX to obtain
compounds with better analgesic and anti-inflammatory activities and less gastric
side-effects.
Results and discussion
In the synthesis of resulting amide derivatives, 2-benzothiazolinone, 6-acyl-2-
benzothiazolinones (1a–s) and (6-acyl-2-benzothiazolinon-3-yl)acetic acids (3a–s)
were prepared according to the previously published procedures (Fife et al., 1975;

Med Chem Res (2010) 19:11–24
13
R
O
R
O
COOH
S
S
N
S
b
R
a
+
O
O
O
N
H
N
H
1
2
CH2COOC2H5
c
R
O
R
O
S
N
S
N
d,e
O
O
CH2CONHR1
4
CH2COOH
3
Scheme 1 Synthetic route of the title compounds. a benzoic acid, PPA, b ethyl bromoacetate, potassium
carbonate, acetone, c HCl, d oxalyl chloride, benzene, e primer amine, potassium carbonate, THF
Unlu¨ et al., 2003a). Compounds 3a–s were then treated with oxalyl chloride to
prepare the corresponding acid chlorides, which were then reacted without
subsequent purification with appropriate amine derivatives to obtain resulting novel
amide derivatives (4a–s). The synthetic route for the resulting amide derivatives
(4a–s) is outlined in Scheme 1.
The ketone function as a link between an aryl and a heterocyclic ring is long
known in the structural classes of NSAIDs such as indomethacin and ketoprofen. In
addition, extensive structure–activity relationship (SAR) studies have been done by
preparing amide derivatives of these type of compounds and found that, by
preparing certain amide derivatives, it was possible to achieve more potent
antiinflammatory compounds which inhibited COX enzymes (Kalgutkar et al.,
2000a, b, 2002, 2005). Moreover, antiinflammatory and analgesic activity of the
benzoxazolinone ring and its 6-acyl derivatives have been described previously
¨
(Gokhan et al., 1999; Palaska et al., 1995; Dogruer et al., 1998). Therefore, we have
used the bioisosteric 6-acyl-benzothiazolinone ring as a building block which might
have advantage with regard to metabolic stability as compared with benzoxazoli-
none ring, and synthesized amide derivatives to obtain compounds with potential
activity.
Analgesic activity of the synthesized amide derivatives was determined by
p-benzoquinone-induced writhing test (Okun et al., 1963). As seen in Table 1, four
of the amide derivatives of the (6-acyl-2-benzothiazolinon-3-yl)acetic acids (4h, 4i,
4n, and 4o) resulted in compounds with comparable analgesic activities to aspirin.
Especially, [6-(2-fluorobenzoyl)-2-benzothiazolinon-3-yl]acetamide derivatives
having 4-chlorophenyl and 3-chlorophenyl substituents at the amide portion (4h
and 4i) exhibited analgesic activity equal to that of aspirin in p-benzoquinone-
induced writhing test. The same was also true for compounds having the
2-chlorobenzoyl substituents in place of 2-flurobenzoyl at 6 position of benzo-
thiazolinone ring (4n and 4o). Interestingly, replacement of fluoro or chlorobenzoyl
with unsubstituted benzoyl at 6-position led to a decrease in the analgesic activity.
Apart from the benzamide derivatives, the amide derivatives prepared with
2-aminopyridine demonstrated lower activity, indicating that benzamides were more

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Med Chem Res (2010) 19:11–24

Med Chem Res (2010) 19:11–24
15

16
Med Chem Res (2010) 19:11–24
important in terms of the analgesic activity and no significant differences between
the [6-(2-fluorobenzoyl)- or [6-(2-chlorobenzoyl)-2-benzothiazolinon-3-yl]acetam-
ide derivatives were observed.
Anti-inflammatory activities of the title amide derivatives were assessed by
utilizing carrageenan-induced hind paw edema model (Kasahara et al., 1985). Since
carrageenan edema has been used in the development of indomethacin, many
researchers adapted this procedure for in vivo screening of the potential anti-
inflammatory activity of compounds. It is known that an edema produced by
carrageenan 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
attributable to the inhibition of the chemical mediators such as histamine, serotonin,
and bradykinin, while the second stage of the edema might be related to the
arachidonic acid metabolites since it is inhibited by aspirin, indomethacin, and other
cyclooxygenase inhibitors (Vinegar et al., 1969, 1987).
Anti-inflammatory activities of the synthesized compounds also demonstrated
parallel results with their corresponding analgesic activities in which the compounds
4h, 4i, 4n, and 4o demonstrated comparable activity to indomethacin (Table 1).
There were no considerable differences between the 6-(2-chlorobenzoyl)- or 6-(2-
fluorobenzoyl)- derivatives of the title amide derivatives in terms of their anti-
inflammatory activities, as seen with their analgesic activities. Table 1 shows that
these compounds exhibited increasing anti-inflammatory activities in the second
phase of carrageenan-induced edema, indicating that these compounds may exert
their activities through the inhibition of enzymes which are important in the
arachidonic acid cascade, thereby preventing the formation of inflammatory
prostaglandins from arachidonic acid. Additionally, microscopic examination of
the stomachs of tested animals showed no obvious gastric lesions or bleeding after
treatment with most of the compounds in acute phase, also supporting this
hypothesis.
Besides cyclooxygenases (COXs), 5-lipoxygenase (5-LOX) is also an important
metabolic enzyme for formation of leukotrienes from AA, leading to inflammation
and other pathological responses. Products of the 5-LOX pathway, LTB4 and LTE4,
are indicated in inflammation and inflammation related-diseases such as athero-
sclerosis and cerebrovascular diseases (Werz and Steinhilber 2006; Radmark et al.,
2007). Combined with the earlier studies that COX inhibition alone may lead to an
upregulation of AA metabolism by the 5-LOX pathway, it is now appreciated that
dual inhibitors against both COXs and 5-LOX might present an enhanced anti-
inflammatory potency without risks of serious side-effects including gastric damage
(Coruzzi et al., 2007; Leone et al., 2007). Therefore, a single agent inhibiting both
enzymes has been of interest to medicinal chemists. Based on these results, we
decided to screen the title amide derivatives for possible inhibitory activity on 5-
LOX and COXs at 10 lM screening concentrations. Unfortunately, none of the
compounds resulted in considerable inhibition of both enzymes at the tested high
screening concentrations (Table 1).
We conclude that benzamide derivatives with 2-chloro or 2-fluorobenzoyl
substituents in the acyl portion exhibited analgesic and anti-inflammatory
activities in vivo at the second stage of inflammation in carrageenan-induced

Med Chem Res (2010) 19:11–24
17
edema model. The higher biological activity of 2-substituted benzoyl derivatives
might be attributed to the effect of halogen substituent, which may force the
phenyl ring to take out-of-plane conformation to the benzothiazolinone ring,
causing a structure resembling indomethacin derivatives as hypothesized previ-
ously (Unlu¨ et al., 2003b). Further studies are underway in our laboratory in
order to investigate the effect of different alkanoic acid derivatives of the
synthesized compounds on chronic inflammatory models and in vitro inhibition
on COXs and 5-LOX.
Experimental
Chemistry
Benzoic acid, 2-chlorobenzoic acid, 2-fluorobenzoic acid, ethyl bromoacetate,
polyphosphoric acid (PPA), and oxalyl chloride were obtained from Aldrich Co.
(Germany). 2-Oxo-benzothiazoline, 6-acyl-2-benzothiazolinone (1), and [6-(2-
chloro/fluorobenzoyl)-2-benzothiazolinon-3-yl]acetic acids (3) were prepared
according to the previously published procedures (Unlu¨ et al., 2003a; Cakır et al.,
1997; Dalaeva et al., 1994; Petrov et al., 1994). Infrared (IR) spectra were recorded
on a Bruker Vector 22 IR (Opus Spectroscopic Software Version 2.0) spectrometer
(KBr, r, cm-1). 1H-nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker 400 FT-NMR spectrometer using trimethylsilane (TMS) as an internal
standard in CDCl3 or dimethyl sulfoxide (DMSO)-d6. All chemical shifts were
reported as d (ppm) values. Elemental analyses were performed with Leco-932 (C,
H, N, S, O Elemental Analyzer) at the Instrumental Analysis Center of the Scientific
and Technical Research Council of Turkey (Ankara, Turkey) and were within the
range of 0.4% of the theoretical value.
Synthesis of 3-(6-acyl-2- benzothiazolinone-3-yl)acetamide (4a–s)
(6-acyl-2-benzothiazolinon-3-yl)acetic acid (5 mmole) was dispersed in benzene
(50 mL), and oxalyl chloride (1.5 mmole) was added. The final mixture was
refluxed for 3 h and stirred for another 1 h at room temperature. After evaporating
to dryness, tetrahydrofuran (50 mL) was added to the crude (6-acyl-2-benzothiaz-
olinon-3-yl)acetyl chloride derivative. After adding potassium carbonate
(15 mmole) and the appropriate primer amine derivative (15 mmole), the reaction
mixture was refluxed for 3–8 h and then poured into ice-water. The crude product
precipitated was filtered and crystallized from the appropriate solvents.
N-(Phenyl)-2-[6-(benzoyl)-2-benzothiazolinone-3-yl]acetamide
(4a) Recrystal-
1
lized from ethanol (yield 69%). mp 241–243°C. H-NMR (CDCl3) d; 10.49 (1H,
s, NH), 8.17 (1H, d, H7), 7.75–7.73 (3H, m, H5, benzoyl-H2,6), 7.66 (1H, t, benzoyl-
H4), 7.58–7.54 (4H, m, phenyl-H2,6, benzoyl-H3,5), 7.47 (1H, d, H4), 7.31 (2H, d,

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Med Chem Res (2010) 19:11–24
phenyl-H3,5), 7.06 (1H, t, phenyl-H4), 4.91 (2H, s, CH2) – IR mmax cm-1 (KBr):
3315,1686, 1645 – Anal.: C22H16N2O3S.
N-(4-Chlorophenyl)-2-[6-(benzoyl)-2-benzothiazolinone-3-yl]acetamide (4b) Re-
crystallized from ethanol (yield 68%). mp 237–239°C. 1H-NMR (CDCl3) d;
10.65(1H, s, NH), 8.17 (1H, dd, H7), 7.75–7.72 (3H, m, H5, benzoyl-H2,6), 7.67 (1H,
t, benzoyl-H4), 7.60 (2H, d, phenyl-H2,6), 7.56 (2H, t, benzoyl-H3,5), 7.47 (1H, d,
H4), 7.38 (2H, d, phenyl-H3,5), 4.91 (2H, s, CH2) – IR mmax cm-1 (KBr): 3321, 1693,
1636– Anal.: C22H15ClN2O3S.
N-(3-Chlorophenyl)-2-[6-(benzoyl)-2-benzothiazolinone-3-yl]acetamide (4c) Re-
1
crystallized from ethanol–toluene (yield 50%). mp 205–207°C. H-NMR (DMSO-
d6) d; 10.71(1H, s, NH), 8.17 (1H, d, H7), 7.77–7.73 (4H, m, H5, benzoyl-H2,6
,
phenyl-H2), 7.67 (1H, t, benzoyl-H4) 7.56 (2H, t, benzoyl-H3,5), 7.47 (1H, d, H4),
7.43 (1H, d, phenyl-H6), 7.35 (1H, t, phenyl-H5), 7.14 (1H, d, phenyl-H4), 4.92 (2H,
s, CH2) – IR mmax cm-1 (KBr): 3312, 1693, 1644 – Anal.: C22H15ClN2O3S.
N-(2-Pyridyl)-2-[6-(benzoyl)-2-benzothiazolinone-3-yl]acetamide (4d) Recrystal-
1
lized from Methanol (yield 48%). mp 232–234°C. H-NMR (DMSO-d6) d; 11.00
(1H, s, NH), 8.35 (1H, d, pyr-H6), 8.17 (1H, d, H7), 7.96 (1H, d, pyr-H3), 7.79–7.72
(4H, m, H5, benzoyl-H2,6, pyr-H4), 7.66 (1H,t, benzoyl-H4), 7.55 (1H, t, benzoyl-
H3,5), 7.48 (1H, d, H4), 7.12 (1H, t, pyr-H5), 4.99 (2H, s, CH2) – IR mmax cm-1
(KBr): 3253, 1677, 1655 – Anal.: C21H15N3O3S.
N-(4-Methyl-2-Pyridyl)-2-[6-(benzoyl)-2-benzothiazolinone-3-yl]acetamide
(4e) Recrystallized from Ethanol (yield 55%). mp 248°C. 1H-NMR (DMSO-d6) d;
10.92 (1H, s, NH), 8.20 (1H, d, pyr-H6), 8.17 (1H, d, H7), 7.83 (1H, d, pyr-H3),
7.74–7.72 (3H, m, H5, benzoyl-H2,6), 7.67 (1H, t, benzoyl-H4), 7.56 (2H, t, benzoyl-
H3,5), 7.46 (1H, d, H4), 6.97 (1H, t, pyr-H5), 4.98 (2H, s, CH2), 1.73 (3H, s, CH3) –
IR mmax cm-1 (KBr): 3208, 1682, 1644 – Anal.: C22H17N3O3S.
N-(6-Methyl-2-Pyridyl)-2-[6-(benzoyl)-2-benzothiazolinone-3-yl]acetamide
1
(4f) Recrystallized from Ethanol (yield 48%). mp 200–203°C. H-NMR (DMSO-
d6) d; 10.96 (1H, s, NH), 8.16 (1H, d, H7), 7.74–7.72 (4H, m, H5, pyr-H3, benzoyl-
H2,6), 7.67–7.65 (2H, m, benzoyl-H4, pyr-H4), 7.55 (2H, t, benzoyl-H3,5), 7.45 (2H,
t, H4), 6.99 (1H, d, pyr-H5), 4.97 (2H, s, CH2), 2.42 (3H, s, CH3) – IR mmax cm-1
(KBr): 3263, 1671, 1655 – Anal.: C22H17N3O3S.
N-(Phenyl)-2-[6-(2-Fluorobenzoyl)-2-benzothiazolinone-3-yl]acetamide (4g) Re-
1
crystallized from Ethanol (yield 54%). mp 245–246°C. H-NMR (DMSO-d6) d;
10.49 (1H, s, NH), 8.20 (1H, s, H7), 7.75 (1H, d, H5), 7.67–7.65 (1H, m, benzoyl-
H4), 7.59 (1H, d, benzoyl-H6), 7.55 (2H, d, phenyl-H2,6), 7.46 (1H, d, H4), 7.40–7.36
(2H, m, benzoyl-H3,5), 7.31 (2H, t, phenyl-H3,5), 7.06 (1H, t, phenyl-H4), 4.90 (2H,
s, CH2) – IR mmax cm-1 (KBr): 3339, 1666 – Anal.: C22H15FN2O3S.

Med Chem Res (2010) 19:11–24
19
N-(4-Chlorophenyl)-2-[6-(2-Fluorobenzoyl)-2- benzothiazolinone -3-yl]acetamide
(4h) Recrystallized from acetone (yield 46%). mp 247–249°C. 1H-NMR (DMSO-
d6) d; 10.64 (1H, s, NH), 8.20 (1H, s, H7), 7.75 (1H, d, H5), 7.67–7.65 (1H, m,
benzoyl-H4), 7.59–7.56 (3H, m, benzoyl-H6, phenyl-H2,6), 7.45 (1H, d, H4), 7.40–
7.36 (4H, m,benzoyl-H3,5, phenyl-H3,5), 4.90 (2H, s, CH2) – IR mmax cm-1 (KBr):
3336, 1685, 1661 – Anal.: C22H14ClFN2O3S.
N-(3-Chlorophenyl)-2-[6-(2-Fluorobenzoyl)-2-benzothiazolinone -3-yl]acetamide
(4i) Recrystallized from acetone (yield 82%). mp 241-243°C. 1H-NMR
(DMSO-d6) d; 10.71 (1H, s, NH), 8.20 (1H, s, H7), 7.76–7.74 (2H, m, H5,
phenyl-H2), 7.67–7.65 (1H, m, benzoyl-H4), 7.57 (1H, m, benzoyl-H6), 7.47 (1H, d,
H4), 7.43 (1H, d, phenyl-H6), 7.40–7.33 (3H, m, benzoyl-H3,5, phenyl-H5), 7.13
(1H, dd, phenyl-H4), 4.91 (2H, s, CH2) – IR mmax cm-1 (KBr): 3334, 1667 – Anal.:
C22H14ClFN2O3S.
N-(2-Pyridyl)-2-[6-(2-Fluorobenzoyl)-2-benzothiazolinone-3-yl]acetamide
1
(4j) Recrystallized from ethanol (yield 27%). mp 249–251°C. H-NMR (DMSO-
d6) d; 11.00 (1H, s, NH), 8.35 (1H, d, pyr-H6), 8.19 (1H, s, H7), 7.95 (1H, d, pyr-
H3), 7.78–7.73 (2H, m, H5, pyr-H4), 7.68–7.63 (1H, m, benzoyl-H4), 7.57 (1H, t,
benzoyl-H6), 7.47 (1H, t, H4), 7.39–7.36 (2H, m, benzoyl-H3,5), 7.13–7.11 (1H, m,
pyr-H5), 4.98 (2H, s, CH2) – IR mmax cm-1 (KBr): 3251, 1671 – Anal.:
C21H14FN3O3S.
N-(4-Methyl-2-Pyridyl)-2-[6-(2-Fluorobenzoyl)-2-benzothiazolinone-3-yl]acetam-
ide (4k) Recrystallized from acetone (yield 36%). mp 231–233°C. 1H-NMR
(DMSO-d6) d; 10.92 (1H, s, NH), 8.19 (1H, s, H7), 8.18 (1H, s, pyr-H6), 7.82 (1H, d,
pyr-H3), 7.74 (1H, d, H5), 7.68–7.64 (1H, m, benzoyl-H4), 7.57 (1H, t, benzoyl-H6),
7.45 (1H, d, H4), 7.40–7.36 (2H, m, benzoyl-H3,5), 6.96 (1H, d, pyr-H5), 4.97 (2H, s,
CH2), 2.26 (3H, s, CH3) – IR mmax cm-1 (KBr): 3208, 1680, 1655 – Anal.:
C22H16FN3O3S.
N-(6-Methyl-2-Pyridyl)-2-[6-(2-Fluorobenzoyl)-2-benzothiazolinone-3-yl]acetam-
ide (4l) Recrystallized from acetone (yield 45%). mp 263–265°C. 1H-NMR
(DMSO-d6) d; 10.96 (1H, s, NH), 8.19 (1H, d, H7), 7.74 (1H, s, pyr-H3), 7.73 (1H, s,
H5), 7.66–7.63 (2H, m, pyr-H4, benzoyl-H4), 7.57 (1H, t, benzoyl-H6), 7.45 (2H, d,
H4), 7.39–7.35 (2H,m, benzoyl-H3,5), 6.98 (1H, d, pyr-H5), 4.96 (2H, s, CH2), 2.41
(3H, s, CH3) – IR mmax cm-1 (KBr): 3262, 1671, 1654 – Anal.: C22H16FN3O3S.
N-(Phenyl)-2-[6-(2-chlorobenzoyl)-2-benzothiazolinone-3-yl]acetamide (4m) Re-
1
crystallized from ethanol (yield 66%). mp 214-215°C. H-NMR (DMSO-d6) d;
10.48 (1H, s, NH), 8.15 (1H, s, H7), 7.69 (1H, dd, H5), 7.61–7.54 (4H, m, benzoyl-
H4,5, phenyl-H2,6), 7.50 (2H, d, benzoyl-H3,6), 7.46 (1H, d, H4), 7.30 (2H, t, phenyl-
H3,5), 7.05 (1H, t, phenyl-H4), 4.89 (2H, s, CH2) – IR mmax cm-1 (KBr): 3311, 1684,
1644 – Anal.: C22H15ClN2O3S.

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Med Chem Res (2010) 19:11–24
N-(4-Chlorophenyl)-2-[6-(2-chlorobenzoyl)-2-benzothiazolinone-3-yl]acetamide
(4n) Recrystallized from ethanol (yield 57%). mp 218–219°C. 1H-NMR (DMSO-
d6) d; 10.64 (1H, s, NH), 8.15 (1H, d, H7), 7.69 (1H, dd, H5), 7.61–7.56 (4H, m,
benzoyl-H4,5, phenyl-H2,6), 7.50 (2H, d, benzoyl-H3,6), 7.46 (1H, d, H4), 7.37 (2H,
d, phenyl-H3,5), 4.90 (2H, s, CH2) – IR mmax cm-1 (KBr): 3260, 1690, 1667 – Anal.:
C22H14Cl2N2O3S.
N-(3-Chlorophenyl)-2-[6-(2-chlorobenzoyl)-2-benzothiazolinone-3-yl]acetamide
1
(4o) Recrystallized from ethanol (yield 61%). mp 235–236°C. H-NMR (DMSO-
d6) d; 10.71 (1H, s, NH), 8.16 (1H, d, H7), 7.75 (1H, s, phenyl-H2), 7.69 (1H, dd,
H5), 7.61–7.57 (2H, m, benzoyl-H4,5), 7.50 (2H, d, benzoyl-H3,6), 7.46 (1H, d, H4),
7.42 (1H, d, phenyl-H6), 7.34 (1H, t, phenyl-H5), 7.13 (2H, d, phenyl-H4), 4.90 (2H,
s, CH2) – IR mmax cm-1 (KBr): 3291, 1699, 1650 – Anal.: C22H14Cl2N2O3S.
N-(2-Pyridyl)-2-[6-(2-chlorobenzoyl)-2-benzothiazolinone-3-yl]acetamide
1
(4p) Recrystallized from ethanol (yield 38%). mp 184–185°C. H-NMR (DMSO-
d6) d; 11.00 (1H, s, NH), 8.34 (1H, d, pyr-H6), 8.15 (1H, d, H7), 7.93 (1H, s, pyr-
H3), 7.76 (1H, t, pyr-H4), 7.68 (1H, dd, H5), 7.61–7.56 (2H, m, benzoyl-H4,5), 7.50
(2H, d, benzoyl-H3,6), 7.46 (1H, d, H4), 7.12 (1H, t, pyr-H5), 4.98 (2H, s, CH2) – IR
mmax cm-1 (KBr): 3253, 1676, 1649 – Anal.: C21H14ClN3O3S.
N-(4-Methyl-2-pyridyl)-2-[6-(2-chlorobenzoyl)-2-benzothiazolinone-3-yl]acetamide
1
(4r) Recrystallized from ethanol (yield 34%). mp 203–205°C. H-NMR (DMSO-
d6) d; 10.32 (1H, s, NH), 8.19 (1H, d, pyr-H6), 8.15 (1H, d, H7), 7.82 (1H, d, pyr-
H3), 7.67 (1H, dd, H5), 7.59–7.57 (2H, m, benzoyl-H4,5), 7.50 (2H, d, benzoyl-H3,6),
7.45 (1H, d, H4), 6.96 (1H, d, pyr-H5), 4.96 (2H, s, CH2), 2.26 (3H, s, CH3) – IR
mmax cm-1 (KBr): 3205, 1685, 1654 – Anal.: C22H16ClN3O3S.
N-(6-Methyl-2-pyridyl)-2-[6-(2-chlorobenzoyl)-2-benzothiazolinone-3-yl]acetamide
(4s) Recrystallized from acetone (yield 55%). mp 250°C. 1H-NMR (DMSO-d6) d;
10.96(1H, s, NH), 8.15 (1H, d, H7), 7.73 (1H, s, pyr-H3), 7.68 (1H, dd, H5), 7.64
(1H, t, pyr-H4), 7.61–7.56 (2H, m, benzoyl-H4,5), 7.50 (2H, d, benzoyl-H3,6), 7.44
(1H, d, H4), 6.98 (1H, d, pyr-H5), 4.95 (2H, s, CH2), 2.41 (3H, s, CH3) – IR mmax
cm-1 (KBr): 3264, 1692, 1669 – Anal.: C22H16ClN3O3S.
Pharmacology
Animals
Male Swiss albino mice (20–25 g) were used for all experiments. The animals were
kept in colony cages (six mice each), maintained on standard pellet diet, water ad
libitum, and left for 2 days for acclimatization before the experimental session. The
food was withdrawn on the day before the experiment, but free access of water was
allowed. All experiments were carried out according to the suggested ethical
guidelines for the care of laboratory animals.

Med Chem Res (2010) 19:11–24
21
Preparation of test samples for bioassay
Test samples were suspended in a mixture of distilled H2O and 0.5% sodium
carboxymethyl cellulose (CMC) and were given orally to the test animals. The
animals of the control group received the same experimental handling except that
the drug treatment was replaced with appropriate volumes of the dosing vehicle.
Either indomethacin (10 mg/kg) or acetyl salicylic acid (ASA) in 0.5% CMC
(100 mg/kg) was used as reference drug.
p-Benzoquinone-induced writhing test (Okun et al., 1963)
Sixty minutes after oral administration of test samples, the mice were injected
intraperitoneally with 0.1 mL/10 g body weight 2.5% (v/v) p-benzoquinone (PBQ)
solution in distilled H2O (PBQ, Merck, Darmstadt, Germany). 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
movements) was counted for the next 15 min, starting on the fifth min after the PBQ
injection. The data represent average values of the total number of writhes observed.
The analgesic activity was expressed as percentage change from writhing controls.
Carrageenan-induced hind paw edema test
The test was performed according to the method of Kasahara et al. (1985). The
difference in footpad thickness between the right and left foot was measured with a
pair of dial thickness gauge calipers (Ozaki Co., Tokyo, Japan). Mean values of
treated groups were compared with mean values of a control group and analyzed
using statistical methods. Sixty minutes after the oral administration of the test
sample or dosing vehicle each mouse was injected with freshly prepared (0.5 mg/
25 mL) suspension of carrageenan (Sigma, St. Louis, Mo, USA) in physiological
saline (154 mM NaCl) into subplantar tissue of the right hind paw and 25 lL saline
solution was injected into that of the left hind paw as secondary control.
Measurements were done and evaluated every 90 min during 360 min after
induction of inflammation, as described above.
Gastric side ulceration effects
After 2 h the analgesic activity experiment was done; mice were killed under deep
ether anesthesia and stomachs were removed. Then the abdomen of each mouse was
opened through great curvature and examined under dissecting microscope for
lesion or bleedings.
Acute toxicity
Animals employed in the carrageenan-induced paw edema experiment were
observed during 48 h and mortality was recorded where present for each group at
the end of observation period.

22
Med Chem Res (2010) 19:11–24
Statistical analysis of data
Data obtained from the animal experiments were expressed as the mean standard
error standard error on the mean (SEM). Statistical differences between the
treatments and the control were tested by analysis of variance (ANOVA) test. Data
with p-value \ 0.05 was considered to be significant.
COX-1 and COX-2 inhibitory activity
All compounds were tested for their ability to inhibit human COX-1 and COX-2
using in vitro human whole blood assay as described by Patrignani et al. (1994).
Each drug was evaluated at 10 lM concentration in triplicate determinations. DuP-
697 and SC-560 were used as selective COX-2 inhibitor and selective COX-1
inhibitor references, respectively, and assayed at 1 lM for both COX-2 and COX-1
assay.
COX-1 activity in human whole blood
Fresh blood from healthy volunteers who had not taken any NSAIDs for at least
7 days prior to blood extraction was collected in tubes containing no anticoagulants.
Aliquots of 500 lL blood were incubated either with 1 lL vehicle (DMSO) or 1 lL
test compound solution (10 lM) for 1 h at 37°C. Plasma was separated by
centrifugation (5 min at 13,000 rpm, 4°C) and TXB2 levels were measured using
the Correlate-EIATM TXB2 Enzyme Immunoassay Kit from Assay Design Inc. (Ann
Arbor, MI, USA).
COX-2 activity in human whole blood
Fresh blood from healthy volunteers who had not taken any NSAIDs for at least
7 days prior to blood extraction was collected in ethylene diamine tetraacetic acid
(EDTA)-containing tubes. Aliquots of 500 lL blood were incubated either with
1 lL vehicle (DMSO) or 1 lL test compound solution (10 lM) in presence of LPS
(10 lg/mL) for 24 h at 37°C to induce COX-2 expression. Plasma was separated by
centrifugation (5 min at 13000 rpm, 4°C) and PGE2 levels were measured using the
Correlate-EIATM PGE2 Enzyme Immunoassay Kit from Assay Design Inc. (Ann
Arbor, MI).
5-LOX inhibitory activity
The inhibitor activity of the title compounds was screened at 10 lM in Merck
Research Laboratories, Canada as a courtesy of Dr. Zhaoyin Wang.
Acknowledgements This study was supported by a grant from Research Foundation of Gazi University
(EF-02/2004-16). We greatly appreciate Dr. Zhaoyin Wang for testing the compounds for 5-LOX
inhibitory activity.

Med Chem Res (2010) 19:11–24
23
References
¨
Banoglu E, Okc¸elik B, Kupeli E, Unlu¨ S, Yes¸ilada E, Amat M, Caturla JF, Sahin MF (2003) Amide
derivatives of [5-chloro-6-(2-chloro/fluorobenzoyl)-2-benzoxazolinone-3-yl]acetic acids as potential
analgesic and anti-inflammatory compounds. Arch Pharm Pharm Med Chem 336:251–257. doi:
10.1002/ardp.200300723
Cakır B, Dogruer DS, Unlu¨ S, Yesilada E, Sahin MF (1997) Biochemical and pharmacological
characterization of the cyclooxygenase activity in human blood prostaglandin endoperoxide
synthases. J Fac Pharm Gazi 14(2):103–109
Cakır B, Ulucay A, Dogruer DS, Isımer A, Sahin MF (1999) Synthesis and antinociceptive activity of
some 3-substituted benzothiazolone derivatives. Farmaco 54:846–851. doi:10.1016/S0014-
827X(99)00111-1
Charlier C, Michaux C, Charlier C (2003) Dual inhibition of cyclooxygenase-2 (COX-2) and 5-
lipoxygenase (5-LOX) as a new strategy to provide safer non-steroidal anti-inflammatory drugs. Eur
J Med Chem 38:645–659. doi:10.1016/S0223-5234(03)00115-6
Coruzzi G, Venturi N, Spaggiari S (2007) Gastrointestinal safety of novel nonsteroidal antiinflammatory
drugs: selective COX-2 inhibitors and beyond. Acta Biomed 78(2):96–110
Dalaeva L, Petrov O, Antonova A, Kalcheva V (1994) Neuropharmacological screening of 2(3H)-
benzothiazolone derivatives. Dokl Bolg Akad Nauk 47(4):121–124
Dogruer DS, Unlu¨ S, Sahin MF, Yesilada E (1998) Anti-nociceptive and anti-inflammatory activity of
some (2-benzoxazolone-3-yl and 2-benzothiazolone-3-yl)acetic acid derivatives. Farmaco 53:80–84.
doi:10.1016/S0014-827X(97)00017-7
Fife TH, Hutchins JEC, Wang MS (1975) Highly efficient intramolecular nucleophilic reactions.
Cyclization of p-nitrophenyl N-(2-mercaptophenyl)-N-methylcarbamate and phenyl N-(2-amino-
phenyl)-N-methylcarbamate. J Am Chem Soc 97:5878–5882. doi:10.1021/ja00853a038
Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science
294(5548):1871–1875. doi:10.1126/science.294.5548.1871
¨
Gokhan N, Erdogan H, Durlu NT, Demirdamar R (1999) Analgesic activity of acylated 2-benzoxaz-
olinone derivatives. Farmaco 54:112–115. doi:10.1016/S0014-827X(98)00111-6
¨
Gu¨lcan HO, Unlu¨ S, Banoglu E, Sahin MF, Ku¨peli E, Yes¸ilada E (2003) Synthesis of new 4-(5-chloro-2-
oxo-3H-benzoxazol-3-yl)butanamide derivatives and their analgesic and anti-inflammatory proper-
ties. Turk J Chem 27:467–476
Kalgutkar AS, Marnett AB, Crews BC, Remel RP, Marnett LJ (2000a) Ester and amide derivatives of the
nonsteroidal antiinflammatory drug, indomethacin, as selective cyclooxygenase-2 inhibitors. J Med
Chem 43:2860–2870. doi:10.1021/jm000004e
Kalgutkar AS, Crews BC, Rowlinson SW, Marnett AB, Kozak KR, Remmel RP, Marnett LJ (2000b)
Biochemically based design of cyclooxygenase-2 (COX-2) inhibitors: facile conversion of
nonsteroidal antiinflammatory drugs to potent and highly selective COX-2 inhibitors. Proc Natl
Acad Sci USA 97(2):925–930. doi:10.1073/pnas.97.2.925
Kalgutkar AS, Rowlinson SW, Crews BC, Marnett LJ (2002) Amide derivatives of meclofenamic acid as
selective cyclooxygenase-2 inhibitors. Bioorg Med Chem Lett 12:521–524. doi:10.1016/
S0960-894X(01)00792-2
Kalgutkar AS, Crews BC, Saleh S, Prudhomme D, Marnett LJ (2005) Indolyl esters and amides related to
indomethacin are selective COX-2 inhibitors. Bioorg Med Chem 13(24):6810–6822. doi:
10.1016/j.bmc.2005.07.073
Kasahara Y, Hikino H, Tsurufiji S, Watanabe M, Ohuchi K (1985) Studies on the constitutents of ephedra.
18. Antiinflammatory actions of ephedrines in acute inflammations. Planta Med 51:325–331. doi:
10.1055/s-2007-969503
Leone S, Ottani A, Bertolini A (2007) Dual acting anti-inflammatory drugs. Curr Top Med Chem
7(3):265–275. doi:10.2174/156802607779941341
¨
Meyer-Kirchrath J, Schror K (2000) Cyclooxygenase-2 inhibition and side-effects of non-steroidal anti-
inflammatory drugs in the gastrointestinal tract. Curr Med Chem 7:1121–1129
Okun R, Liddon SC, Lasagnal L (1963) The effects of aggregation, electric shock, and adrenergic
blocking drugs on inhibition of the writhing syndrome. J Pharmacol Exp Ther 139:107–114
Onkol T, Dogruer DS, Ito S, Sahin MF (2000) Synthesis and antinociceptive activity of (5-chloro-2-
benzothiazolinon-3-yl)acetamide derivatives. Arch Pharm Pharm Med Chem 333:337–340. doi:
10.1002/1521-4184(200010)333:10\337::AID-ARDP337[3.0.CO;2-A

24
Med Chem Res (2010) 19:11–24
Onkol T, Ito S, Yıldırım E, Erol K, Sahin MF (2001) Synthesis and antinociceptive activity of (2-
benzazolon-3-yl)propionamide derivatives. Arch Pharm Pharm Med Chem 334:17–20. doi:
10.1002/1521-4184(200101)334:1\17::AID-ARDP17[3.0.CO;2-Z
Onkol T, Yildirim E, Erol K, Ito S, Sahin MF (2004) Synthesis and antinociceptive activity of (5-chloro-
2(3H)-benzothiazolon-3-yl)propanamide derivatives. Arch Pharm Pharm Med Chem 337:475–481.
doi:10.1002/ardp.200300863
¨
¨
Palaska E, Unlu¨ S, Ozkanli F, Pilli G, Erdogan H, Safak C, Demirdamar R, Gu¨mu¨sel B (1995) 3-
Substituted piperazinomethyl benzoxazolinones – analgesic and antiinflammatory compounds
inhibiting prostaglandin E(2). Arzneim-Forsch Drug Res 45:693–696
Patrignani P, Panara MR, Greco A, Fusco O, Natoli C, Iacobelli S, Cipollone F, Ganci A, Creminon C,
Maclouf J, Patrono C (1994) Biochemical and pharmacological characterization of the cycloox-
ygenase activity in human blood prostaglandin endoperoxide synthases. J Pharmacol Exp Ther
271(3):1705–1712
Petrov OI, Antonova AT, Kalcheva VB, Veleva Ch G (1994) Synthesis of 6-benzoyl-2(3H)-
benzothiazolones and compounds of their reduction and alkylation. Dokl Bolg Akad Nauk
47(3):31–34
Radmark O, Werz O, Steinhilber D, Samuelsson B (2007) Lipoxygenase: regulation of expression and
enzyme activity. Trends Biochem Sci 32(7):332–341. doi:10.1016/j.tibs.2007.06.002
Unlu¨ S, Onkol T, Du¨ndar Y, Okc¸elik B, Ku¨peli E, Yesilada E, Noyanalpan N, Sahin MF (2003a)
Synthesis and analgesic and anti-inflammatory activity of some new (6-acyl-2-benzoxazolinone and
6-acyl-2-benzothiazolinone derivatives with acetic acid and propanoic acid residues. Arch Pharm
Pharm Med Chem 336:353–361. doi:10.1002/ardp.200300746
Unlu¨ S, Nacak-Baytas S, Kupeli E, Yesilada E (2003b) Studies on novel 7-acyl-5-chloro-2-oxo-3H-
benzoxazole derivatives as potential analgesic and anti-inflammatory agents. Arch Pharm Pharm
Med Chem 336:310–321
Vinegar R, Schreiber W, Hugo R (1969) Biphasic development of carrageenin edema in rats. J Pharmacol
Exp Ther 166:96–103
Vinegar R, Truax JF, Selph JL, Johnston PR, Venable AL, McKenzie KK (1987) Pathway to carrageenan-
induced inflammation in the hindlimb of the rat. Fed Proc 46:118–126
Werz O, Steinhilber D (2006) Therapeutic options for 5-lipoxygenase inhibitors. Pharmacol Ther
112(3):701–718. doi:10.1016/j.pharmthera.2006.05.009