Synthesis and biological evaluation of 4,5-diphenyloxazolone derivatives on route towards selective COX-2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2008.10.039

A series of 3-unsubstituted/substituted-4,5-diphenyl-2-oxo-3H-1,3-oxazole derivatives were prepared as selective cyclooxygenase-2 (COX-2) inhibitors. Among the synthesized compounds, 4-(4-phenyl-3-methyl-2-oxo-3H-1,3-oxazol-5-yl)benzensulfonamide (compoun

Full text of DOI:10.1016/j.ejmech.2008.10.039

European Journal of Medicinal Chemistry 44 (2009) 1830–1837
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of 4,5-diphenyloxazolone derivatives on route
towards selective COX-2 inhibitors
,
a
a
b
c
Yasemin Du¨ndar a , Serdar Unlu¨ , Erden Banog˘lu , Antonio Entrena , Gabriele Costantino ,
*
¨
Maria-Teresa Nunez d, Francisco Ledo d, M. Fethi S¸ahin a, Ningur Noyanalpan a
a Department of Medicinal Chemistry, Faculty of Pharmacy, Gazi University, Taç Sk, 06330 Etiler, Ankara, Turkey
b Facultad de Farmacia, c/Campus de Cartuja s/n, 18071 Granada, Spain
c
`
Dipartimento Farmaceutico, ,Via G.P. Usberti 27/A Universita degli Studi di Parma, 43100 Parma, Italy
d Faes Farma, S.A., Departamanto de Investigacion, Apartado 555, 48080 Bilbao, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-unsubstituted/substituted-4,5-diphenyl-2-oxo-3H-1,3-oxazole derivatives were prepared as
selective cyclooxygenase-2 (COX-2) inhibitors. Among the synthesized compounds, 4-(4-phenyl-3-
methyl-2-oxo-3H-1,3-oxazol-5-yl)benzensulfonamide (compound 6) showed selective COX-2 inhibition
Received 20 June 2008
Received in revised form
23 October 2008
Accepted 30 October 2008
Available online 12 November 2008
with a selectivity index of >50 (IC50COX-1 ¼ >100
m
m, IC50COX-2 ¼ 2
mm) in purified enzyme (PE) assay.
Compound 6 also exhibited selective COX-2 inhibition in human whole blood assay. Molecular docking
studies showed that 6 can be docked into the COX-2 binding site thus providing the molecular basis for
its activity.
Keywords:
Ó 2008 Elsevier Masson SAS. All rights reserved.
4,5-Diphenyloxazolone
Cyclooxygenase inhibition
COX-1
COX-2
Docking
1. Introduction
phenomena in specific patient populations such as cardiovascular
disease patients challenging the benefits of selective COX-2 inhi-
Nonsteroidal anti-inflammatory drugs (NSAID) are among the
most frequently prescribed medications being the drugs of the first
choice for treatment of the inflammatory and rheumatic diseases.
The common mechanism of NSAIDs involves the nonselective
inhibition of cyclooxygenases (COXs) thereby preventing the
biosynthesis of prostaglandins (PG) which are the important lipid
mediators of inflammation as well as numerous homeostatic
physiological functions [1]. As it is now well appreciated, COXs exist
in two isoforms, namely COX-1 and COX-2 [2], while the existence
of a third isoforom (COX-3) is still into debate. In general terms,
COX-1 is the constitutive isoform providing normal production of
PGs having roles in homeostasis and gastroprotection, whereas
COX-2 is induced by proinflammatory stimuli at inflammatory sites
[3]. The discovery of inducible COX-2 at sites of inflammation led to
the development of selective COX-2 inhibitors with the hope of
dimished gastrointestinal side effects associated with traditional
NSAIDs [4,5]. However, recent studies have shown that COX-2
inhibitors are associated with increased thromboembolic
bition [6–8]. Moreover, there is currently no clear evidence that
COX-2 inhibitors represent an independent risk factor in patients at
low demographic risk of cardiovascular diseases and therefore,
clinical rationale for developing compounds with selective COX-2
inhibition still remains to be established [8,9]. Meantime, consid-
erable interest in the further potential clinical utilities of COX-2
inhibitors has emerged [10–12]. Recent studies indicating the place
of COX-2 inhibitors in cancer chemotherapy and neurological
diseases such as Alzheimer’s [13,14] and Parkinson [13,14] diseases
still continues to attract investigations on development of COX-2
inhibitors.
The structural information on the tricyclic COX-2 selective
inhibitors, for example vicinal diaryl substitution about a central
heterocyclic ring, is described in detail in literature and having the
characteristic sulfonyl group on one of the aryl rings are believed to
play a crucial role on selectivity [15,16]. For this purpose, vicinal
diaryl substituents on a central five- or six-membered ring
template such as pyrazole, 2-(5H)-furanone, isoxazole, pyridine
have been extensively investigated as selective COX-2 inhibitors [4]
(Fig. 1). In addition, some studies for developing COX-2 inhibitors
have concentrated on the preparation of the amide derivatives
of currently used NSAIDs such as indomethacin [17,18] and
* Corresponding author. Tel.: þ90 312 2023242; fax: þ90 312 2235018.
E-mail address: akkocysmn@gmail.com (Y. Du¨ndar).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.039

Y. Du¨ndar et al. / European Journal of Medicinal Chemistry 44 (2009) 1830–1837
1831
SO2NH2
SO2CH3
ammonium hydroxide to yield the sulfonamide derivatives (5, 6)
(Scheme 1). The methylsulfonyl derivative (8) was obtained by the
methylation of the sodium salt of 4 with dimethyl sulfate as
demonstrated in Scheme 1.
The preparation of the amide derivatives 15–21 are outlined in
Scheme 2. Alkylation of 1 with ethyl bromoacetate generated ethyl
2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-3-yl)ethanoate (9) [23].
Subsequent hydrolysis of the ester linkage under basic conditions
afforded 2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-3-yl)ethanoic acid
(10) [24,25]. Amidation of 10 with appropriate secondary and
tertiary amines in the presence of ethyl chloroformate in
dichloromethane at room temperature, resulted in the synthesis of
amide derivatives 15–21 with quantitative yields (51–66%).
The sulfonamide derivative having an acetic acid substitution on
the nitrogen of oxazolone was prepared as shown in Scheme 3.
Firstly, ester derivative 9 was reacted with chlorosulfonic acid to
yield sulfonyl chloride derivative (11) under mild reaction condi-
tions. After protection of sulfonyl chloride with dibenzylamine and
subsequent treatment with concentrated sulfuric acid resulted in
the target sulfonamide derivative (13), which was readily hydro-
lyzed under acid conditions to obtain the desired acid derivative 14.
N
N
CH3
O
F3C
O
Celecoxib
Rofecoxib
SO2NH2
SO2CH3
Cl
H3C
N
O
N
N
CH3
Valdecoxib
Etoricoxib
O
3. Results and discussion
CH3
Cl
NHR'
CH3
H3CO
The compounds reported herein were tested for their ability to
inhibit COX-2 and/or COX-1 using the purified enzyme assay
described by Futaki et al. [26] and Janusz et al. [27]. The in vitro
activity results are reported as a percentage of inhibition of the
N
N
H
Cl
O
NHR'
O
Cl
purified enzymes at 10
exhibited inhibition of more than 50% for COX-2, the inhibition of
COX-1 at 10 M and the IC50 values were also calculated from the
mM (Table 1). For compounds which
Indomethacin amides
Meclofenamic acid amides
m
concentration curves by means of the PRISM program. Further-
more, compound 6 was selected on the basis of its activity in the
cell-free assay for evaluation of inhibition of human COX-2 and
COX-1 using in vitro human whole blood assay described by Pat-
rignani et al. [28].
R1
N
O
O
In this preliminary study towards new potential COX-2 selective
compounds as novel drug candidates for inflammatory and related
diseases, we have introduced systematic modifications to the 4,5-
diphenyloxazolone core structure. It is well established that 3,4-
diaryloxazolones having a sulfone or sulfonamide on the 4-phenyl
is a good template for selective COX-2 inhibition [29,30]. Thus,
taking into account this structural feature, we planned a structure–
activity relationship study using the 4,5-diphenyloxazolone core as
a template. In particular, we envisaged a series of substitution on
the N3-nitrogen atom of the heterocylce in order to introduce more
flexibility to the template, while keeping the diphenyl portion
unsubstituted or substituted with sulfone/sulfonamide which was
required to maintain COX-2 selectivity. Results are shown in Table 1.
In general, none of the newly synthesized derivatives proved to
be endowed with the desired activity profile at COX-2, as none but
one of the compounds (compound 6) inhibited at least 50% of the
COX-2 isoform during preliminary screening.
Compound 6 is endowed with a N-methyl substitution and with
a p-sulfonamide on the 5-phenyl ring. We found that N-methyl
derivative with the unsubstituted 5-phenyl ring (2) had a dimin-
ished COX-2 activity (w5% inhibition) with respect to the sulfon-
amide (6, 70%) or methylsulfonyl (8, 49%) analogs. This preliminary
results indicated that the presence of p-sulfone/sulfamoyl is
important for COX-2 activity and sulfone on the 5-phenyl ring was
less effective with regard to sulfamoyl at the same position which
was in close agreement with literature results for related
compounds [29,30]. Within the sulfonamide analogs, introduction
of larger substituents on the N3 position, i.e., ester (13) or acid (14),
R2
General structure of the sythesized compounds
Fig. 1. Representative examples of selective COX-2 inhibitor compounds and the
general structure of the synthesized 4,5-diphenyl-2-oxo-3H-1,3-oxazole derivatives.
meclofenamic acid [19] (Fig. 1) and found that neutralization of
these NSAIDs by preparing the corresponding amide derivatives
resulted into compounds that selectively inhibited COX-2 but not
COX-1. As a part of our ongoing program to acquire structure–
function relationship data for COX-2 inhibitors, we hereby describe
the synthesis and the preliminary biological evaluation of a group
of diphenyl substituted oxazolone derivatives possessing a sulfonyl
group at the para-position of C-5 phenyl moiety in conjunction
with a variety of substituents at the nitrogen atom of the central
oxazolone ring (Fig. 1).
2. Chemistry
The synthetic routes for the synthesized compounds are out-
lined in Schemes 1–3. The starting compound, 4,5-diphenyl-2-oxo-
3H-1,3-oxazole (1), was readily prepared by the reaction of benzoin
and urethane under distillation conditions as shown in Scheme 1
[20]. Compound 1 was then reacted with dimethyl sulfate to obtain
4,5-diphenyl-3-methyl-2-oxo-3H-1,3-oxazole (2) [20–22]. Treat-
ment of 1 and 2 with chlorosulfonic acid yielded the sulfonyl
chloride derivatives (3, 4) which were subsequently reacted with
caused a decrease in the observed COX-2 activity at 10
mM screning
dose as compared to N-methyl analog (6).

1832
Y. Du¨ndar et al. / European Journal of Medicinal Chemistry 44 (2009) 1830–1837
CH3
N
H
O
N
OH
O
a
b
+
O
O
H2N
OC2H5
O
O
1
2
R
R
R
N
O
N
O
N
O
c
d
O
O
O
ClO2S
H2NO2S
R
R
R
1 = H
2 = CH3
3 = H
4 = CH3
5 = H
6 = CH3
CH3
CH3
CH3
N
N
O
N
O
e
f
O
O
O
O
ClO2S
NaO2S
H3CO2S
4
7
8
Scheme 1. Reagents: (a) dist.; (b) NaOH, dimethyl sulfate; (c) chlorosulfonic acid; (d) NH4OH, reflux then concentrated HCl; (e) Na2SO3, NaHCO3; (f) dimethyl sulfate, NaHCO3,
reflux.
Recent studies evaluating ester and amide derivatives of indo-
methacin and meclofenamic acid resulted in highly potent and
selective compounds and it was suggested that derivatization of the
carboxylate moiety in moderately selective COX-1 inhibitors can be
used as a novel strategy for generation of potent and selective COX-
2 inhibitors [17,19,31]. On the basis of this background, we decided
to introduce a carboxamide moiety in the 4,5-diphenyloxazolone
scaffold by amidation of the acid derivative (10) in which the COX-2
(85% for COX-2 and 7% for COX-1). Therefore, IC50 values of 6 for
both isoforms was further calculated using in vitro purified enzyme
assay from dose–response curve as >100 mM and 2 mM for COX-1
and COX-2, respectively, indicating that 6 behaves as a selective
COX-2 inhibitor with a selectivity index (SI) of >50, which was
about 5 times less potent than Rofecoxib (IC50COx-1 >100
IC50COx-2 ¼ 0.4 M, SI ¼ 250).
mM and
m
With the aim of getting insights into the structural basis for its
activity, compound 6 was docked into the active site of COX-2. The
X-ray structure of COX-2 complexed with the selective inhibitor
SC558 in the I222 space group (pdb code: 6cox) was selected for
this purpose. The docking studies were carried out using Glide, as
descibed in Section 5. In the top ranked pose, compound 6 adopts
a disposition exactly matching that of SC558 (Fig. 2a). In particular,
the unsubstituted phenyl moiety is inserted inside the COX-2
hydrophobic pocket (blue residues) while the sulfonamide group is
positioned into the selectivity pocket (magenta residues) as the
corresponding moiety of SC558. It can be appreciated from Fig. 2b
how compound 6 forms two hydrogen bonds, one of them between
the sulfonamide moiety and His90, situated at the bottom of the
selectivity pocket, and the second one between the CO bond and
Arg120 thus behaving in a similar manner like SC558 positioning
the oxazolone with its carbonyl group coordinated to Arg120 in the
inhibitor activity was shown to be moderate at 10 mM (w42%) with
the hope of obtaining better derivatives as selective COX-2 inhibi-
tors. However, this was not the case and all of the amide derivatives
(15–21) generally resulted in less potent COX-2 inhibition with
respect to parent acid analog (10) in a range from 22 to 28% at this
high screening concentration. This indicates that the impact of the
substitution pattern of the nitrogen of oxazolone ring is low since
the all tested derivatives show lower activity at 10
Since compound 6 was the only derivative that resulted in more
than 50% COX-2 inhibition at 10 M (w70%), it was further evalu-
mM.
m
ated for its inhibitory activity on COX-1 isoform and found that it
insignificantly inhibited COX-1 isoform, 4% and 19% at 10 and
100
m
M concentrations, respectively. Consequently, compound 6
M and showed
was also tested in human whole blood assay at 10
m
selective COX-2 inhibition under these assay conditions as well
O
O
O
H
N
OC2H5
OH
R
N
O
N
O
N
O
a
b
c
O
O
O
O
O
1
9
10
15-21
Scheme 2. Reagents: (a) K2CO3, ethyl bromoacetate, acetone, reflux; (b) KOH, water, reflux, then 1 N HCl; (c) ethyl chloroformate, Et3N, amine, CH2Cl2.

Y. Du¨ndar et al. / European Journal of Medicinal Chemistry 44 (2009) 1830–1837
1833
O
O
O
OC2H5
OC2H5
OC2H5
N
O
N
O
N
O
a
b
O
O
O
ClO2S
O
Bn2NO2S
9
11
12
O
OC2H5
OH
N
O
N
O
c
d
O
O
H2NO2S
H2NO2S
13
14
Scheme 3. Reagents: (a) chlorosulfonic acid, CHCl3; (b) Bn2NH, Et3N, CH2Cl2; (c) H2SO4; (d) HCl.
lower entrance constriction as SC558 does with is CF3 group. It can
also be appreciated from Fig. 2 how the N-methyl group is posi-
tioned into a small, mainly hydrophobic cavity where larger groups
won’t fit, thus providing the basis for explaining the poor activity of
compounds 9–21 (Table 1).
spectrometer using tetramethylsilane as the internal standard at
the NMR facility of Faculty of Pharmacy, Ankara University. All
chemical shifts were recorded as
d (ppm). Microanalyses for C, H,
and N were performed on a Leco-932 at Faculty of Pharmacy,
Ankara University, Ankara, Turkey, and they were within the range
of ꢁ0.4% of the theoretical value. The synthesis of 4,5-diphenyl-2-
oxo-3H-1,3-oxazole (1) [20], 4,5-diphenyl-3-methyl-2-oxo-3H-1,3-
oxazole (2) [20–22], 4-(4-phenyl-2-oxo-3H-1,3-oxazol-5-yl)benze
nsulfonyl chloride (3) [32], 4-(4-phenyl-2-oxo-3H-1,3-oxazol-5-yl)
benzensulfonamide (5) [33], ethyl 2-(4,5-diphenyl-2-oxo-3H-1,
Fig. 3 reports the binding mode of 6 as compared to 5-iodoin-
domethacin complexed with COX-1 (PDB entry: 1PGG). In the most
favourable orientation found by the docking program the sulfon-
amide moiety is projected towards the hydrophobic pocket (blue
residues) as Ile523 (yellow residue) closes the entry to selectivity
pocket in this enzyme. Because of the polarity of the sulfonamide
moiety, it is quite unlikely that this disposition is actually
a productive one, as it is also confirmed by the low value of the
Gscore (ꢀ11.20 kcal/mol as compared to ꢀ13.50 kcal/mol of the
best pose of 6 into COX-2). It can be concluded that compound 6
neatly fits the COX-2 binding site, giving a stable complex and
acting as a good inhibitor of this isoform. On the other hand, the
best obtained putative COX-1/compound 6 complex is much less
stable thus providing a clear explanation for the lack of activity of 6
at COX-1.
Table 1
In vitro inhibition of purified COX-2 by 4,5-diaryloxazolone derivatives.
R1
N
O
O
R2
4. Conclusion
Compound R1
R2
H
COX-2 inhibition COX-1 inhibition
(%)a
(%)a
The synthesis of a series of 4,5-diphenyloxazolone derivatives
substituted at N3 is described along with their preliminary evalu-
ation as potential COX-2 inhibitors. Unfortunatelly, most of the
compounds show no significant COX-2 inhibitory activity. Only
compound 6 displayed potent and selective COX-2 inhibition. In
conclusion, we feel that the preliminary in vitro activity results of
this class of compounds may possess potential for design of future
molecules with modifications on the aryl substituents as well as at
N3 side chain to specifically inhibit one or more of these enzymes.
Further studies are in progress.
2
5
6
CH3
H
CH3
5.31 ꢁ 8.25
ntc
nt
SO2NH2 47.84 ꢁ 3.05
SO2NH2 70.14 ꢁ 1.71
3.56 ꢁ 8.01
(10 mM)
19.28 ꢁ 7.02
(100
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
mM)
8
9
CH3
CH2COOC2H5
CH2COOH
CH2COOC2H5
SO2CH3 48.60 ꢁ 4.33
H
H
27.53 ꢁ 1.30
10
13
14
15
16
17
18
19
20
21
41.78 ꢁ 15.95
SO2NH2 32.84 ꢁ 5.29
SO2NH2 33.17 ꢁ 3.75
CH2COOH
CH2CONH(CH2)7CH3
CH2CONHCH2CH2C6H5
CH2CONH(4-ClC6H4)
CH2CONH(4-OCH3C6H4)
CH2CO(piperidin-1-yl)
CH2CONH(thiazol-2-yl)
CH2CO(morpholin-1-yl)
H
H
H
H
H
H
H
23.22 ꢁ 3.65
22.22 ꢁ 2.95
26.53 ꢁ 2.67
24.38 ꢁ 1.60
20.56 ꢁ 1.30
28.19 ꢁ 5.31
23.05 ꢁ 4.40
91.44 ꢁ 0.24
66.78 ꢁ 2.83
5. Experimental
5.1. Materials and methods
All chemicals and solvents were purchased locally from Merck
AG and Aldrich Chemicals. Melting points were determined with an
Electrothermal-9200 Digital Melting Point Apparatus and are
uncorrected. 1H NMR spectra were recorded in DMSO-d6 on a Var-
ian Mercury 400, 400 MHz High Performance Digital FT-NMR
Indomethacin
Rofecoxibb
71.89 ꢁ 6.31
13.66 ꢁ 6.66
a
Data are indicated as percentage of inhibition at 10
Rofecoxib was assayed at 100
nt: not tested.
m
M ꢁ SEM (n ¼ 4).
b
c
mM and 1 mM for COX-1 and COX-2, respectively.

1834
Y. Du¨ndar et al. / European Journal of Medicinal Chemistry 44 (2009) 1830–1837
Fig. 2. (a) The best pose obtained for compound 6 (gray) inside the COX-2 binding pocket compared with SC558 (green). Note the very similar orientation of the two ligands. (b)
Compound 6 makes two hydrogen bonds with Arg120 and His90. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
3-oxazol-3-yl)ethanoate (9) [23], 2-(4,5-diphenyl-2-oxo-3H-1,3-
oxazol-3-yl)ethanoic acid (10) [24,25] was previously reported.
4-(4-phenyl-3-methyl-2-oxo-3H-1,3-oxazol-5-yl)benzensulfonam-
ide separated. This was removed by filtration and dissolved in water
and converted to the free sulfonamide by acidification with 1 N HCl
to give a solid precipitate. The product was collected by suction
filtration, washed with water, dried and recrystallized from iso-
5.1.1. 4-(4-Phenyl-2-oxo-3H-1,3-oxazol-5-yl)benzensulfonamide
(5)
4-(4-Phenyl-2-oxo-3H-1,3-oxazol-5-yl)benzensulfonyl chloride
(3) (0.003 mol) was added to 30 mL of cold concentrated ammo-
nium hydroxide, and the mixture was heated to reflux and stirred
for 1 h. After cooling to room temperature, the mixture was acidi-
fied with concentrated HCl to give a solid precipitate. The product
was collected by suction filtration, washed with water, dried, and
crystallized from ethanol (yield 58%, m.p. 267 ꢂC).
propanol to yield 45%; m.p. 210–211 ꢂC. 1H NMR (DMSO-d6)
d: 7.67–
7.65 (2H, d, 5-phenyl H3, H5), 7.56–7.54 (5H, m, 4-phenyl), 7.31 (2H,
s, SO2-NH2), 7.28–7.26 (2H, d, 5-phenyl H2, H6), 2.95 (3H, s, N-CH3).
Anal. Calc. for C16H14N2O4S: C, 58.17; H, 4.27; N, 8.48; S, 9.71. Found:
C, 57.97; H, 4.29; N, 8.46; S, 9.52%.
5.1.5. 4-Phenyl-3-methyl-5-[4-(methylsulfonyl)phenyl]-2-oxo-3H-
1.3-oxazole (8)
5.1.2. Ethyl 2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-3-yl)ethanoate
(9)
The mixture of sodium sulfite (10 mmol) and sodium bicar-
bonate (10.5 mmol) in water (15 mL) was heated at 80 ꢂC and
sulfonyl chloride (4) (5.3 mmol) was added in portions of 20 mg
The mixture of 4,5-diphenyl-2-oxo-3H-1,3-oxazole (1)
(5 mmol), potassium carbonate (5.5 mmol) and ethyl bromoacetate
(5.5 mmol) in 30 mL acetone was heated to reflux and stirred for
6 h. 100 g ice water was added to the cooled (0–10 ꢂC) reaction
mixture. After stirring for 1 h, the precipitated solid product was
collected by suction filtration, washed with water, dried, and
recrystallized from ethanol (yield 85%, m.p. 64 ꢂC).
5.1.3. 2-(4,5-Diphenyl-2-oxo-3H-1,3-oxazol-3-yl)ethanoic acid (10)
Ethyl 2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-3-yl)ethanoate (9)
(0.05 mol) and potassium hydroxide (0.05 mol) in 200 mL water
was refluxed for 4 h. After cooling to room temperature, the
mixture was acidified with 1 N HCl to give a solid precipitate. The
product was collected by suction filtration, washed with water,
dried, and crystallized from ethanol/water (yield 85%, m.p. 184 ꢂC).
5.1.4. 4-(4-Phenyl-3-methyl-2-oxo-3H-1,3-oxazol-5-
yl)benzensulfonamide (6)
4,5-Diphenyl-3-methyl-2-oxo-3H-1,3-oxazole (2) (0.005 mol)
was added slowly to 0.025 mol of chlorosulfonic acid at 10–15 ꢂC.
The reaction mixture was stirred for 15 min after all the 4,5-
diphenyl-3-methyl-2-oxo-3H-1,3-oxazole (2) was added. The
temperature was then raised to 60 ꢂC and maintained at this
temperature for 2 h. The reaction mixture was poured onto ice. The
product was removed by filtration and dissolved in ether. The
ethereal solution was washed with water and dried with anhydrous
sodium sulfate and evaporated to dryness (yield 66%). 0.003 mol of
the solid residue (4-(4-phenyl-3-methyl-2-oxo-3H-1,3-oxazol-5-
yl)benzensulfonyl chloride (4)) was added to 30 mL of cold
concentrated ammonium hydroxide, and the mixture was heated
to reflux and stirred for 1 h. On cooling, the ammonium salt of the
Fig. 3. The best pose found for compound 6 (gray) inside the COX-1 binding site,
compared with the orientation of 5-iodoindomethacin (green) in the crystal structure
1PGG. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)

Y. Du¨ndar et al. / European Journal of Medicinal Chemistry 44 (2009) 1830–1837
1835
with stirring, during 3 h. After the addition was completed, the
mixture was heated and stirred at 80 ꢂC for 1 h. The crude sodium
sulfinate solution was allowed to cool for overnight. The solid
sodium 4-(4-phenyl-3-methyl-2-oxo-3H-1,3-oxazol-5-yl)benzen-
sulfinate (7) which separated was collected by filtration and mixed
with 10 mmol of NaHCO3 and 8.15 mmol of dimethyl sulfate in 2 mL
water. After heating under reflux for 20 h, the mixture was cooled
and water was added (20 mL). The precipitated solid product was
collected by suction filtration, washed with water, dried, and
crystallized from acetone/water to yield 20%; m.p. 196–198 ꢂC. 1H
m, piperidine H3(5)), 1.20–1.19 (2H, m, piperidine H4). Anal. Calc. for
C22H22N2O3: C, 72.91; H, 6.12; N, 7.73. Found: C, 72.90; H, 6.19; N,
7.70%.
5.1.6.6. N-(1,3-Thiazol-2-yl)-2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-
3-yl)acetamide (20). Recrystallized from ethanol to yield 33%; m.p.
185–186 ꢂC. 1H NMR (DMSO-d6)
d: 12.34 (1H, s, NH), 7.54–7.44 (6H,
m, 4-phenyl, thiazole H4), 7.33–7.18 (6H, m, 5-phenyl, thiazole H5),
4.35 (2H, s, –CH2–CO–). Anal. Calc. for C20H15N3O3S: C, 63.65; H,
4.01; N, 11.13; S, 8.50. Found: C, 63.53; H, 4.15; N, 11.10; S, 8.34%.
NMR (DMSO-d6) d
: 7.81–7.79 (2H, d, 5-phenyl H3, H5), 7.62–7.56
(5H, m, 4-phenyl), 7.37–7.34 (2H, d, 5-phenyl H2, H6), 3.16 (3H, s,
–SO2CH3), 2.97 (3H, s, N–CH3). Anal. Calc. for C17H15NO4S: C, 61.99;
H, 4.59; N, 4.25; S, 9.74. Found: C, 61.92; H, 4.70; N, 4.31; S, 9.71%.
5.1.6.7. 3-[2-Oxo-2-(morpholin-1-yl)ethyl]-4,5-diphenyl-2-oxo-3H-
1,3-oxazole (21). Recrystallized from ethanol to yield 51%; m.p.174–
176 ꢂC. 1H NMR (DMSO-d6) : 7.56–7.54 (3H, m, 4-phenyl H2, H4,
d
H6), 7.41–7.39 (2H, m, 4-phenyl H3, H5), 7.28–7.16 (5H, m, 5-phenyl),
4.30 (2H, s, –CH2–CO–), 3.43–3.41 (2H, m, morpholine H2(6)), 3.35–
3.33 (4H, m, morpholine H3, H5, H, H), 3.28–3.27 (2H, m, morpho-
line H2(6)). Anal. Calc. for C21H20N2O4: C, 69.22; H, 5.53; N, 7.69.
Found: C, 69.38; H, 5.58; N, 7.68%.
5.1.6. General procedure for the synthesis of 2-(4,5-diphenyl-2-oxo-
3H-1,3-oxazol-3-yl)acetamide derivatives (15–21)
2-(4,5-Diphenyl-2-oxo-3H-1,3-oxazol-3-yl)ethanoic acid (10)
(0.01 mol) in 40 mL dichloromethane at 0 ꢂC (ice-bath) was treated
with triethylamine (0.015 mol) and 0.01 mol of ethyl chlor-
oformate. After stirring the reaction mixture at 0 ꢂC for further
20 min, 0.011 mol of appropriate amine derivative (0.013 mol) was
added, and the final mixture was stirred at room temperature for
overnight. After evaporation to dryness, the product was solidified
with ice-cold water and crystallized from the appropriate solvent.
5.1.7. Ethyl 2-{4-phenyl-5-[4-(N,N-dibenzylaminosulfonyl)phenyl]-
2-oxo-3H-1,3-oxazol-3-yl}ethanoate (12)
Ethyl 2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-3-yl)ethanoate (9)
(0.01 mol) was dissolved in chloroform (4 mL). Chlorosulfonic acid
(0.05 mol) was added and the reaction mixture stirred at rt for
30 min. The mixture then was poured into a slush of 200 g of ice
and then added 50 mL ether. Organic phase was separated, washed
with water, dried with Na2SO4 and evaporated to dryness (yield
75%). 0.0075 mol of the solid residue (ethyl 2-{4-phenyl-5-[4-
(chlorosulfonyl)phenyl]-2-oxo-3H-1,3-oxazol-3-yl}ethanoate) (11)
was dissolved in 25 mL of CH2Cl2 and the resulting solution was
added dropwise to a mixture of 0.01 mol of dibenzylamine and
0.01 mol of triethylamine in 15 mL CH2Cl2 and being stirred at room
temperature. After the reaction was complete, CH2Cl2 was evapo-
rated to dryness, acetone was added to the residue and the
precipitate formed was filtered off. Acetone was evaporated and the
residue was crystallized from ethanol to yield 90%; m.p. 110 ꢂC. 1H
5.1.6.1. N-octyl-2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-3-yl)acetamide
(15). Recrystallized from ethanol to yield 61%; m.p. 112–114 ꢂC. 1H
NMR (DMSO-d6) d: 8.01 (1H, t, –NH–), 7.56–7.50 (3H, m, 4-phenyl
H2, H4, H6), 7.46–7.43 (2H, m, 4-phenyl H3, H5), 7.31–7.16 (5H, m, 5-
phenyl), 3.95 (2H, s, –CH2–CO–), 2.97 (2H, q, –NH–CH2–), 1.30–1.13
(12H, m, –CH2–), 0.86 (3H, t, –CH3). Anal. Calc. for C25H30N2O3: C,
73.86; H, 7.44; N, 6.89. Found: C, 73.97; H, 7.56; N, 6.88%.
5.1.6.2. N-Phenylethyl-2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-3-yl)a-
cetamide (16). Recrystallized from ethanol to yield 56%; m.p. 168–
170 ꢂC. 1H NMR (DMSO-d6)
d: 8.17 (1H, t, –NH–), 7.58–7.53 (3H, m,
4-phenyl H2, H4, H6), 7.45–7.43 (2H, m, 4-phenyl H3, H5), 7.31–7.10
(10H, m, phenyl, 5-phenyl), 3.95 (2H, s, –CH2–CO–), 3.21 (2H, q,
–NH–CH2–), 2.61 (2H, t, –CH2–C6H5). Anal. Calc. for C25H22N2O3: C,
75.36; H, 5.57; N, 7.03. Found: C, 74.94; H, 5.59; N, 6.99%.
NMR (DMSO-d6) d
: 7.76–7.74 (2H, d, 5-phenyl H3, H5), 7.62–7.60
(3H, m, 4-phenyl H2, H4, H6), 7.49–7.47 (2H, m, 4-phenyl H3, H5),
7.34–7.32 (2H, d, 5-phenyl H2, H6), 7.18–7.17 (6H, dd, –N–benzyl H2,
H4, H6), 7.05–7.02 (4H, dd, –N–benzyl H3, H5), 4.27 (2H, s, –CH2–CO–
), 4.25 (4H, s, C6H5–CH2–N–), 4.05 (2H, q, –O–CH2–CH3), 1.08 (3H, t,
–CH2–CH3). Anal. Calc. for C31H26N2O6S: C, 68.02; H, 5.19; N, 4.81; S,
5.50. Found: C, 67.56; H, 5.38; N, 4.81; S, 5.26%.
5.1.6.3. N-(4-Chlorophenyl)-2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-3-
yl)acetamide (17). Recrystallized from ethanol to yield 63%; m.p.
207–208 ꢂC. 1H NMR (DMSO-d6)
d: 10.25 (1H, s, –NH–), 7.55–7.53
(3H m, 4-phenyl H2, H4, H6), 7.50–7.48 (4H, m, 4-phenyl H3, H5, 4-
Cl-phenyl H2, H6), 7.36–7.34 (2H, d, 4-Cl-phenyl H3, H5), 7.30–7.19
(5H, m, 5-phenyl), 4.23 (2H, s, –CH2–CO–). Anal. Calc. for
C23H17ClN2O3: C, 68.23; H, 4.23; N, 6.92. Found: C, 68.15; H, 4.27; N,
6.90%.
5.1.8. Ethyl 2-{4-phenyl-5-[4-(aminosulfonyl)phenyl]-2-oxo-3H-
1,3-oxazol-3-yl}ethanoate (13)
A suspension of 1 mmol of ethyl 2-{4-phenyl-5-[4-(N,N-diben-
zylaminosulfonyl)phenyl]-2-oxo-3H-1,3-oxazol-3-yl}ethanoate (12)
in 1.3 mL concentrated H2SO4 was stirred at room temperature for
20 min. The mixture was poured into ice. The resulting solid was
filtered, washed with water, dried and crystallized from methanol/
5.1.6.4. N-(4-Methoxyphenyl)-2-(4,5-diphenyl-2-oxo-3H-1,3-oxazol-
3-il)acetamide (18). Recrystallized from ethanol to yield 55%. m.p.
water to yield 28%; m.p. 163–164 ꢂC. 1H NMR (DMSO-d6)
d: 7.73–
150–151 ꢂC. 1H NMR (DMSO-d6)
d
: 9.96 (1H, s, –NH–), 7.55–7.53
7.71 (2H, d, 5-phenyl H3, H5), 7.60–7.58 (3H, m, 4-phenyl H2, H4, H6),
7.48–7.45 (2H, m, 4-phenyl H3, H5), 7.36 (2H, s, disappeared after
D2O exchange, NH2), 7.34–7.32 (2H, d, 5-phenyl H2, H6), 4.28 (2H, s,
–CH2–CO–), 4.04 (2H, q, –O–CH2–CH3), 1.08 (3H, t, –CH2–CH3). Anal.
Calc. for C19H18N2O6S: C, 56.71; H, 4.51; N, 6.96; S, 7.97. Found: C,
56.72; H, 4.61; N, 6.94; S, 7.99%.
(3H, m, 4-phenyl H2, H4, H6), 7.49–7.48 (2H, m, 4-phenyl H3, H5),
7.37–7.35 (2H, d, J: 8.8 Hz, 4-CH3O-phenyl H2, H6), 7.30–7.20 (5H, m,
5-phenyl), 6.87–6.85 (2H, d, J: 8.8 Hz, 4-CH3O-phenyl H3, H5), 4.18
(2H, s, –CH2–CO–), 3.70 (3H, s, –OCH3). Anal. Calc. for C24H20N2O4:
C, 71.99; H, 5.03; N, 7.00. Found: C, 72.01; H, 5.11; N, 6.97%.
5.1.6.5. 3-[2-Oxo-2-(piperidin-1-yl)ethyl]-4,5-diphenyl-2-oxo-3H-1,
3-oxazole (19). Recrystallized from ethanol to yield 56%; m.p. 160–
5.1.9. 2-{4-Phenyl-5-[4-(aminosulfonyl)phenyl]-2-oxo-3H-1,3-
oxazol-3-yl} ethanoic acid (14)
Ethyl 2-{4-phenyl-5-[4-(aminosulfonyl)phenyll]-2-oxo-3H-1,3-
oxazol-3-yl}ethanoat (13) (0.01 mol) was heated up to reflux
temperature in concentrated HCl for 12 h. After cooling and diluted
with water, the precipitate formed was filtered off, washed with
161 ꢂC. 1H NMR (DMSO-d6)
d
: 7.56–7.54 (3H, m, 4-phenyl H2, H4,
H6), 7.42–7.39 (2H, m, 4-phenyl H3, H5), 7.29–7.17 (5H, m, 5-phenyl),
4.25 (2H, s, –CH2–CO–), 3.34 (1H, m, piperidine H2(6)), 3.21 (3H, t,
piperidine H2(6)), 1.49–1.47 (2H, m, piperidine H3(5)), 1.31–1.30 (2H,

1836
Y. Du¨ndar et al. / European Journal of Medicinal Chemistry 44 (2009) 1830–1837
water, dried and recrystallized from ethanol/water to obtain a yield
were incubated either with 1 L of test
m
L of vehicle (DMSO) or 1
m
of 20%; m.p. 224–226 ꢂC. 1H NMR (DMSO-d6)
d
: 71–7.69 (2H, d, 5-
compound solution (10 M) in the presence of LPS (10 mg/mL) for
m
phenyl H3,H5), 7.59–7.58 (3H, m, 4-phenyl H2, H4, H6), 7.47–7.44 (2H,
m, 4-phenyl H3, H5), 7.36 (2H, s, NH2), 7.32–7.30 (2H, d, 5-phenyl H2,
H6), 4.13 (2H, s, N–CH2–CO). Anal. Calc. for C17H14N2O6S: C, 54.54; H,
3.77; N, 7.48; S, 8.57. Found: C, 54.24; H, 4.12; N, 7.18; S, 7.83%.
24 h at 37 ꢂC to induce COX-2 expression. Plasma was separated by
centrifugation (5 min at 13000 rpm, 4 ꢂC) and PGE2 levels were
measured using the Correlate-EIAÔ PGE2 Enzyme Immunoassay
Kit from Assay Design Inc. (Ann Arbor, MI).
5.2. Biological assays
5.3. Computational details
5.2.1. Cyclooxygenase inhibition
Docking studies have been performed using Glide program
(Glide is available from Schro¨dinger Inc., http://www.schrodinger.
com). With this purpose, crystal structure of both COX-1/iodoin-
domethacin (a nonselective inhibitor) and COX-2/SC558 (a selective
inhibitor) complexes (PDB codes: 1PGG and 6COX, respectively)
were obtained from the Protein Data Bank in order to prepare both
proteins for docking studies. Compound 6 structure was built using
Sybyl7.3 program (Sybyl is available from Tripos Inc., www.tripos.
com), and after the proper minimization an optimization was
exported to Glide for docking. Docking procedure was followed
using the standard protocol implemented in Glide and the geom-
etry of resulting complexes was studied using the Glide’s Pose
Viewer utility.
5.2.1.1. In vitro assay of purified COX-1 and COX-2 with exogenous
substrate (arachidonic acid). The method described by Futaki et al.
[26] and by Janusz et al. [27] was followed with some modifications.
Briefly, the assay was carried out in a final volume of 0.5 ml with
Tris–HCl buffer (100 mM, pH 8) as the reaction medium containing
hematin (1
After adding 50
compound (1 or 100
m
M) phenol (2 mM) as cofactors, and EDTA (5 mM).
L of the test compound (10 M), the reference
M) or the vehicle (DMSO, 1%), a unit of ovine
m
m
m
purified COX-1 or COX-2 (Cayman Chemical) was suspended in the
reaction medium and preincubated at 37 ꢂC with continuous stir-
ring for 10 min. The reaction was initiated with 50
acid (100 M). After 5 min incubation in the same condition, the
reaction was stopped by addition of 50 L 1 N HCl followed by
neutralization with 50 L of Tris Base (1 M). 50 L of the final
solution were diluted to a final volume of 500 L with the immu-
noenzymatic assay buffer, and 50 L of the diluted sample were
mL of arachidonic
m
m
m
m
m
Acknowledgement
m
taken to test prostaglandin E2 (PGE2) by enzyme immunoassay
(EIA) (Amersham). Detection was carried out in a microplate reader
(Lansystems multiscan MS) at 450 nm, and data were processed by
means of GENESIS-LITE program (Windows-based Microplate
This study was supported by Gazi University BAP (Project
Number 02/2005-22).
References
Software). Indometacin (COX-1, IC50 ¼ 0.069
0.537
M) and rofexocib (COX-1, IC50 ¼ >100
IC50 ¼ 0.398 M) were used as nonselective COX inhibitor and
selective COX-2 inhibitor references in the assays. Rofecoxib tested
at 1 M final concentration for COX-2 and 100 M for COX-1. Results
m
M, COX-2, IC50
¼
m
mM, COX-2,
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