Synthesis, biological evaluation, and docking studies of novel heterocyclic diaryl compounds as selective COX-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.07.009

Three novel series of diaryl heterocyclic derivatives bearing the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole, and 1H-pyrazole moieties as the central heterocyclic ring were synthesized and their in vitro inhibitory activities on COX-1 and COX-2 isoforms were evaluated using a purified enzyme assay. The 2-oxo-5H-furan derivative 6b was identified as potent COX inhibitor with selectivity toward COX-1 (COX-1 IC₅₀ = 0.061 μM and COX-2 IC ₅₀ = 0.325 μM; selectivity index (SI) = 0.19). Among the 1H-pyrazole derivatives, 11b was found to be a potent COX-2 inhibitor, about 38 times more potent than Rofecoxib (COX-2 IC₅₀ = 0.011 μM and 0.398 μM, respectively), but showed no selectivity for COX-2 isoform. Compound 11c demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC ₅₀ = 1 μM, COX-2 IC₅₀ = 0.011 μM; SI = ～92). Molecular docking studies of compounds 6b and 11b-d into the binding sites of COX-1 and COX-2 allowed to shed light on the binding mode of these novel COX inhibitors.

Full text of DOI:10.1016/j.bmc.2010.07.009

Bioorganic & Medicinal Chemistry 18 (2010) 6367–6376
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and docking studies of novel heterocyclic diaryl
compounds as selective COX-2 inhibitors
a,
Gökçen Eren a, , Serdar Ünlü , Maria-Teresa Nuñez b, Luis Labeaga b, Francisco Ledo b, Antonio Entrena c,
*
Erden Banog˘lu a, Gabriele Costantino d, M. Fethi Sßahin a
a Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey
b Faes Farma, S.A., R&D and Innovation Department, 48940 Leioa, Spain
c Facultad de Farmacia, c/Campus de Cartuja s/n, 18071 Granada, Spain
d Dipartimento Farmaceutico, Via G.P. Usberti 27/A, Università degli Studi di Parma, 43100 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Three novel series of diaryl heterocyclic derivatives bearing the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole,
and 1H-pyrazole moieties as the central heterocyclic ring were synthesized and their in vitro inhibitory
activities on COX-1 and COX-2 isoforms were evaluated using a purified enzyme assay. The 2-oxo-5H-
furan derivative 6b was identified as potent COX inhibitor with selectivity toward COX-1 (COX-1
Received 17 May 2010
Revised 30 June 2010
Accepted 6 July 2010
Available online 5 August 2010
IC50 = 0.061
atives, 11b was found to be a potent COX-2 inhibitor, about 38 times more potent than Rofecoxib (COX-2
IC50 = 0.011 M and 0.398 M, respectively), but showed no selectivity for COX-2 isoform. Compound 11c
demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC50 = 1 M, COX-2 IC50 = 0.011 M;
lM and COX-2 IC50 = 0.325 lM; selectivity index (SI) = 0.19). Among the 1H-pyrazole deriv-
Keywords:
l
l
Diaryl heterocyclic
COX inhibition
Benzoxazole
Furanone
l
l
SI = ꢀ92). Molecular docking studies of compounds 6b and 11b–d into the binding sites of COX-1 and
COX-2 allowed to shed light on the binding mode of these novel COX inhibitors.
Ó 2010 Elsevier Ltd. All rights reserved.
Oxazolone
Pyrazole
1. Introduction
result of its long-term use in clinic. Despite this apparent failure,
there is still growing interest for the design of novel COX-2 inhib-
Cyclooxygenases (COXs), which catalyze the first step in arachi-
donic acid metabolism,1 are the molecular targets of the nonsteroi-
dal antiinflammatory drugs (NSAIDs).2–4 COX enzymes exist at
least in three isoforms, namely cyclooxygenase-1 (COX-1), a con-
stitutive enzyme, cyclooxygenase-2 (COX-2), an isoform induced
in response to a variety of pro-inflammatory stimuli,5,6 and cyclo-
oxygenase-3 (COX-3), present mainly in the cerebral cortex and
human heart.7 It was thought that while the antiinflammatory ef-
fect of NSAIDs occurs as a result of COX-2 inhibition, many of the
undesirable side effects including; gastric irritation were due to
the inhibition of COX-1 isoform.8 Therefore, several new inhibitors
directed towards COX-2 without interfering with COX-1 enzymatic
activity were developed during the last two decades. These mole-
cules, termed coxibs (i.e., Celecoxib,9 Rofecoxib,10 Valdecoxib,11
and Etoricoxib12) showed reduced gastrointestinal side effects as
compared to traditional NSAIDs.13 However, coxibs were recently
withdrawn from the market because of an increased risk of cardio-
vascular side effects particularly observed with Rofecoxib as a
itors owing to the reduced gastric and renal side effects as com-
pared to conventional NSAIDs and also due to the additional
therapeutic benefits of COX-2 inhibitors in several diseases includ-
ing certain types of cancer.14–18
Selective COX-2 inhibitors so far can be divided into three
classes, namely (i) vicinal diaryl heterocycle inhibitors (coxibs),
(ii) sulfonanilide inhibitors, and (iii) modified classical nonselective
NSAIDs. The pharmacophore of diaryl heterocycle inhibitors is
characterized by a vicinal diaryl substitution about a central carbo-
cyclic or heterocyclic ring system in which one of the aryl groups is
substituted by a methylsulfonyl or sulfonamido group at para
position.19
Although modifications on established nonselective NSAIDs20
such as lengthening of the carboxyl side chain21 of Indomethacin,
could let to effective COX-2 selective inhibitors, most of the suc-
cessful efforts have so far been directed to the diaryl heterocycle
class. In particular, structural variation of the central ring in the tri-
cyclic series is still a popular area of research.22
Therefore, we focused our attention to the design and synthesis
on three different diaryl heterocyclic ring systems in which the
2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole, and 1H-pyrazole moieties
served as the central ring template bearing 3-methyl-2-oxo-3H-
benzoxazole and 4-substituted phenyl group as vicinal aryl
* Corresponding author. Tel.: +90 312 2023235; fax: +90 312 2235018.
E-mail address: gokcene@gazi.edu.tr (G. Eren).
Present address: FARGEM (Pharmaceutical Research and Development Center)
Inc., Sancaklar Mevkii, 81100 Düzce, Turkey.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.009

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G. Eren et al. / Bioorg. Med. Chem. 18 (2010) 6367–6376
moieties. Several 2-oxo-3H-benzoxazole derivatives have been
previously reported as analgesic and antiinflammatory agents,
and some of them were reported to reduce PGE2 formation in the
exudates of paw edema.23–26 Therefore, we aimed to take advan-
tage of 3-methyl-2-oxo-3H-benzoxazole moiety as replacement
of one of the aryl groups within the diaryl heterocyclic system to
insure some additional structural benefits for COX-2 inhibition
(Fig. 1).
oxo-3H-benzoxazole (7) by acid catalyzed hydrolysis. Compound 7
was reacted with appropriate phenyl isocyanates to yield
2-(3-methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl phenyl car-
bamates(8a–e)whichwerethencyclizedtoprovidethecorrespond-
ing 2-oxo-3H-1,3-oxazoles (9a–e) in refluxing acetic acid. The
methylsulfonyl derivative (9f) was prepared by oxidation of methyl-
thio derivative with m-chloroperbenzoic acid (70%) in dichloro-
methane (Scheme 3).
4,4,4-Trifluoro-1-(3-methyl-2-oxo-3H-benzoxazole-6-yl)-1,3-di-
oxobutane (10) was obtained from the reaction between compound 4
and ethyl trifluoroacetate in the presence of potassium t-butoxide.
Subsequent, treatment of compound 10 with appropriate phen-
ylhydrazine hydrochlorides led to 1-phenyl-3-trifluoromethyl-5-(3-
methyl-2-oxo-3H-benzoxazole-6-yl)-1H-pyrazoles (11a–e) by the
ring closure reaction under microwave conditions (Scheme 4).
Structures of all target compounds were elucidated by spectro-
scopic data and confirmed by elemental analyzes.
2. Results and discussion
2.1. Synthesis
We focused our synthetic efforts on three different diaryl het-
erocyclic ring systems as illustrated in Schemes 1–4. The starting
compound, 3-methyl-2-oxo-3H-benzoxazole (2), was prepared by
methylation with dimethyl sulfate of 2-oxo-3H-benzoxazole (1)
which was readily synthesized via the reaction of o-aminophenol
and urea. Compound 2 was then converted to 6-bromoacetyl-
3-methyl-2-oxo-3H-benzoxazole (3) and to 6-acetyl-3-methyl-
2-oxo-3H-benzoxazole (4) via Friedel–Crafts acylation under
microwave conditions (Scheme 1).
To obtain the 2-oxo-5H-furans (6a–e), compound 3 was reacted
with appropriate phenylacetic acid derivatives to obtain 2-(3-
methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl phenyl acetates
(5a–e). For the ring closure reaction, compounds 5a–e were heated
in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to
furnish the 3-phenyl-4-(3-methyl-2-oxo-3H-benzoxazole-6-yl)-2-
oxo-5H-furans (6a–e) (Scheme 2).
2.2. Biological evaluation
The 3,4-diaryl-2-oxo-5H-furans (6a–e), the 3,4-diaryl-2-oxo-
3H-1,3-oxazoles (9a–d and 9f), and the 1,5-diaryl-1H-pyrazoles
(11a–e) were initially screened at 10 lM to determine their COX-
2 inhibitory activities using purified enzyme assay.27,28 Indometh-
acin and Rofecoxib were used as nonselective and COX-2 selective
inhibitor standards, respectively, under the same assay conditions.
The in vitro activity results are reported as a percentage of inhibi-
tion of the purified enzymes at 10
compounds exhibited inhibition more than 50% for COX-2, the
inhibition of COX-1 at 10 M and the IC50 values for both isoforms
lM (Table 1). For the
l
Reaction of compound 3 with sodium formate afforded 2-(3-
methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl formate (7i) which
was converted without purification to 6-hydroxyacetyl-3-methyl-2-
were also calculated from the concentration–response curves by
means of the PRISM 4.0, GraphPad Software.29
Figure 1. Structures of marketed COX-2 inhibitors and general structures of the synthesized compounds.

G. Eren et al. / Bioorg. Med. Chem. 18 (2010) 6367–6376
6369
Scheme 1. Reagents and conditions: (a) MW irradiation, 140 °C, 10 min; (b) dimethyl sulfate, NaOH, 30 min; (c) bromoacetic acid, polyphosphoric acid (PPA), MW irradiation,
120 °C, 70 min; (d) acetic acid, PPA, MW irradiation, 90 °C, 20 min.
Scheme 2. Reagents and conditions: (a) TEA, CH3CN, 4 h, reflux; (b) DBU, CH3CN, 1–2 h, rt.
Scheme 3. Reagents and conditions: (a) sodium formate, CH3CN, 4 h, reflux; (b) HCl, reflux; (c) appropriate phenyl isocyanate derivative, toluene, 7–8 h reflux; (d) acetic acid,
3–4 h reflux; (e) CH2Cl2, 5 h, rt.
Scheme 4. Reagents and conditions: (a) potassium t-butoxide, ethyl trifluoroacetate, benzene, 5 h reflux; (b) appropriate phenylhydrazine HCl derivative, HCl, MW
irradiation, 90 °C, 1 h.
Only compound 6b from the 3,4-diaryl-2-oxo-5H-furan class
(6a–e), significantly inhibited the activity of both COX-2 and
COX-1 at 10
lM screening concentration. The calculated IC50
values of compound 6b for COX-2 and COX-1 were found to be

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G. Eren et al. / Bioorg. Med. Chem. 18 (2010) 6367–6376
Table 1
In vitro purified COX-2 and COX-1 enzyme inhibition assay data for the synthesized compounds
A
Compound
R
% Inhibitiona
IC50
(
lM)
SI
COX-2
COX-1
COX-2
COX-1
6a
6b
6c
6d
6e
H
OCH3
Cl
F
0.13 6.16
ntb
80.94 3.77
nt
nt
nt
—
0.325
—
—
—
—
0.061
—
—
—
—
0.19
—
—
—
59.77 0.78
48.07 0.58
11.60 7.35
0.40 2.46
SO2CH3
9a
9b
H
OCH3
À22.34 7.90
nt
nt
—
—
—
—
—
—
47.10 1.05
9c
9d
9f
Cl
F
SO2CH3
36.96 3.13
0.00 2.60
15.55 5.25
nt
nt
nt
—
—
—
—
—
—
—
—
—
11a
11b
H
OCH3
21.27 1.34
94.90 0.32
nt
—
0.011
—
0.004
—
0.36
95.61 0.65
11c
11d
11e
Cl
F
SO2CH3
93.19 0.40
66.82 1.20
29.23 1.19
91.21 1.14
55.47 8.57
nt
0.011
1
—
1
6
—
91.90
6
—
—
—
Indomethacin
Rofecoxibc
—
—
89.94 0.29
59.34 3.39
71.89 6.31
13.66 6.66
0.537
0.398
0.069
>100
0.13
253
a
b
c
Data are indicated as percentage of inhibition at 10
l
M
SEM (n = 4).
Rofecoxib was assayed at 100
nt: not tested.
lM and 1 lM for COX-1 and COX-2, respectively.
0.325
l
M and 0.061
l
M, respectively. The selectivity index (SI) de-
dues) that is appropriate to accommodate such fragments. On
the other hand, the ligand is slightly destabilized by the insertion
of methoxyphenyl group into the hydrophobic pocket. Binding
energy and docking energy of À10.51 kcal/mol and À11.49 kcal/
mol are obtained, respectively. Figure 2b reports the binding mode
of compound 6b in COX-1 active site (binding energy: À11.03 kcal/
mol; docking energy: À10.95 kcal/mol). It is interesting to note
that the binding orientation of compound 6b in the active site of
COX-1 is different to the orientation of the same compound in
the binding site of COX-2. At this regard, it should be observed that
very recently Rimon et al.33 have reported the X-ray structure of
Celecoxib bound to COX-1. Although the disposition of compound
6b in our best pose is different from that Celecoxib in the X-ray
structure of COX-1, our result confirms that the synthesized diaryl
heterocyclic derivatives can productively be docked into the active
site of COX-1. Whether this binding results or not into a functional
inhibition is more complicated to predict, since it may be related to
kinetic of dissociation rather than to the stability of the complex.
Given our biological results, it can be speculated that the orienta-
tion we found for compound 6b into COX-1 is able to interfere with
the metabolism of arachidonic acid, at difference of what observed
for Celecoxib in Rimon et al.33
fined as [IC50(COX-1)]/[IC50(COX-2)] for compound 6b is 0.19 as
compared to Indomethacin (SI = 0.13) and Rofecoxib (SI = 250).
Generally, none of the compounds having the 2-oxo-3H-
1,3-oxazole as a central ring showed the desired activity profile
of COX-2. Only for compound 9b the COX-2 inhibitor activity
was found to be moderate (47%).
From the 1H-pyrazole series, compounds 11b, 11c, and 11d sig-
nificantly inhibited the activity of both COX-1 and COX-2 enzymes.
Although compound 11b is about 38 times more potent than
Rofecoxib when tested in the in vitro COX-2 purified enzyme assay,
it did not show any considerable selectivity towards COX-2. Addi-
tionally, compound 11c was about 36 times more potent than
Rofecoxib in the in vitro COX-2 purified enzyme assay and it
showed some selectivity for COX-2. Compound 11d is about 2.5
times less potent than Rofecoxib and was proved to be COX-2
selective.
2.3. Molecular modeling
Molecular docking studies of compounds 6b and 11b–d in the
active sites of COX-1 (PDB code: 1PGF)30 and COX-2 (PDB code:
6COX)31 were performed in order to get further insight into the
nature of interactions between the compounds and the active site
amino acids to rationalize the obtained biological results.
Docking of compound 6b, 2-oxo-5H-furan derivative, into COX-
2 active site, showed an orientation similar to that of Rofecoxib as
reported previously.32 The carbonyl oxygen of the furanone moiety
of compound 6b is placed close to the side chain of Ser530 to con-
sider the possible formation of hydrogen bond whereas the phenyl
ring has been accommodated in the hydrophobic pocket
(Fig. 2a, blue residues), and that the polar and bulky benzoxazole
moiety is inserted into the selectivity pocket (Fig. 2a, green resi-
The docking study showed that compound 11b is bound to the
primary binding site of COX-2 with the trifluoromethyl moiety
which interacts with the polar zone (so-called ‘trifluoromethyl
zone’) residue Arg120 (Fig. 3). The benzoxazole moiety lies in the
selectivity pocket and the methoxyphenyl ring is located in a
hydrophobic region (binding energy: À11.26 kcal/mol; docking
energy: À12.49 kcal/mol).
A similar placement of compounds 11c and 11d has also been
observed during their dockings in the active site of COX-2. Confor-
mational superposition of SC-558 (from the X-ray crystal structure
of SC-558:COX-2 complex) and compounds 11b–d (from the

G. Eren et al. / Bioorg. Med. Chem. 18 (2010) 6367–6376
6371
Figure 2. (a) Structure of compound 6b docked into the binding site of COX-2 (blue: hydrophobic pocket, red: polar zone, yellow: responsible for the COX-2 selectivity, green:
selectivity pocket). (b) Structure of compound 6b docked into the binding site of COX-1.
Figure 3. (a) Structure of compound 11b docked into the binding site of COX-2. (b) Compound 11b forms a H-bond with Arg120 (presented in magenta).
docking simulation) are shown in Figure 4. The superposition
showed their hydrophilic and hydrophobic groups overlapped with
each other. From the above mentioned data, the molecular model-
ing studies of the examined compounds 11b–d showed that they
bound to the COX-2 active site with position and orientation very
similar to that of the crystal structure of SC-558 complex with
COX-2. Consequently, these observations provided a good explana-
tion for the observed potent inhibitory activity of compounds
11b–d.
elli et al.34 who put forward not only the importance of the
stability of the binding mode, but also the importance of the dy-
namic path of ligand(s) to reach the binding pose. In this context,
Limongelli et al. proposed the existence of alternative binding
modes of diaryl heterocyclic derivatives to COX-2. It can be specu-
lated that the reason for the inactivity of compounds 6e and 11e
may reside in their inability to achieve one or more alternative
binding modes possibly relevant to functional inhibition.
Docking studies were also carried out on compounds 6e and
11e with the aim of explaining their inactivity in inhibiting
COX-2. As reported in Figure 5, the best obtained poses for both
compounds are very similar to the crystallographic disposition of
SC-558 in the binding pocket of COX-2. There are no apparent rea-
sons for the inactivity of compounds 6e and 11e. The failure of sta-
tic docking experiments in predicting the inactivity of these
analogs, closely related to COX-2 active diaryl heterocyclic deriva-
tives can be commented on the light of a recent paper by Limong-
3. Conclusion
The present study describes the synthesis of three new series of
COX inhibitors in which the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxa-
zole, or 1H-pyrazole scaffolds are present in the central part, to
which 3-methyl-2-oxo-3H-benzoxazole and 4-substituted phenyl
moieties have been attached at vicinal positions. The results of bio-
logical evaluation revealed that especially compounds 11c and 11d
belonging to 1H-pyrazole series exhibited the highest activity

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G. Eren et al. / Bioorg. Med. Chem. 18 (2010) 6367–6376
Figure 4. (a) The binding conformations found for 11b (gray); (b) for 11c (gray, binding energy: À11.35 kcal/mol, docking energy: À12.44 kcal/mol); (c) for 11d (gray, binding
energy: À10.88 kcal/mol, docking energy: À11.91 kcal/mol), in comparison with SC-558 (cyan) from the X-ray crystal structure of SC-558:COX-2 complex (PDB code: 6COX).
Figure 5. (a) The binding conformations found for 6e (gray); (b) for 11e (gray), in comparison with SC-558 (cyan) from the X-ray crystal structure of SC-558:COX-2 complex
(PDB code: 6COX).
compared to the analog series. Compound 11c was found to be
more potent on COX-2, but has lower selectivity compared to
Rofecoxib. In addition, the substitution of 1H-pyrazole as a suitable
central ring template with the benzoxazole moiety can be sug-
gested as an effective scaffold for further design of selective and
potent COX-2 inhibitors.
temperature was set to 140 °C. After the reaction was completed,
the flask was cooled to room temperature and the solid was solved
in 5% solution of sodium hydroxide. After acidification with con-
centrated HCl the desired product was obtained. 11.61 g, 86% yield;
mp 136 °C (Ref. 36; 137–138 °C).
4.1.2. 3-Methyl-2-oxo-3H-benzoxazole (2)
This was carried out by the described method,37 and the reac-
tion product was obtained with a yield of about 90%; mp 83 °C
(Ref. 38; 83–84 °C).
4. Experimental
4.1. Chemistry
The chemicals were purchased from the commercial vendors
and were used without purification. Thin-layer chromatography
(TLC) was performed on Merck 60F254 plates. Reactions were mon-
itored by TLC on silica gel, with detection by UV light (254 nm) or
charring Dragendorff reagent.35 All the melting points were taken
on an Electrothermal 9300 capillary apparatus and are uncor-
rected. IR spectra were recorded on a Bruker Vector 22 spectrom-
eter as KBr disks. 1H NMR spectra were obtained with a Varian
400 MHz spectrometer in d-chloroform (CDCl3) or d6-dimethylsulf-
oxide (DMSO-d6) and tetramethylsilane (TMS) was used as an
internal standard. LC/MS spectra were performed using Waters
Micromass ZQ by using ESI (+) method. Elemental analyzes were
performed with LECO-932 (C, H, N, S-Elemental Analyzer). Micro-
wave-assisted reactions were carried out with a Milestone MicroS-
YNTH Microwave Synthesis System.
4.1.3. 6-Bromoacetyl-3-methyl-2-oxo-3H-benzoxazole (3)
The dry flask charged with 3-methyl-2-oxo-3H-benzoxazole
(14.90 g, 0.1 mol), bromoacetic acid (16.68 g, 0.12 mol), and PPA
(200 g) was placed in MicroSYNTH Microwave Synthesis System
and irradiated at 300 W for 70 min while the temperature was
set to 120 °C. The reaction mixture was poured into ice-cold water.
The precipitated mixture was filtered off, dried, and purified by
washing with toluene. 21.6 g, 80% yield; mp 178 °C (Ref. 39;
178–180 °C).
4.1.4. 6-Acetyl-3-methyl-2-oxo-3H-benzoxazole (4)
The dry flask charged with 3-methyl-2-oxo-3H-benzoxazole
(7.45 g, 0.05 mol), acetic acid (3.15 mL, 0.055 mol), and PPA
(100 g) was placed in MicroSYNTH Microwave Synthesis System
and irradiated at 300 W for 22 min while the temperature was
set to 90 °C. The reaction mixture was poured into ice-cold water.
The precipitated mixture was filtered off, dried, and purified by
recrystallization from ethanol–water mixture. 8.5 g, 89% yield;
mp 167–168 °C (Ref. 40; 166–168 °C).
4.1.1. 2-Oxo-3H-benzoxazole (1)
The dry flask charged with o-aminophenol (10.91 g, 0.1 mol)
and urea (12.01 g, 0.2 mol) was placed in MicroSYNTH Microwave
Synthesis System and irradiated at 400 W for 15 min while the

G. Eren et al. / Bioorg. Med. Chem. 18 (2010) 6367–6376
6373
4.2. General procedure for the preparation of 2-(3-methyl-2-
oxo-3H-benzoxazole-6-yl)-2-oxoethyl phenyl acetates (5a–e)
0.011 mol) in acetonitrile was stirred for 1–2 h at room tempera-
ture. The precipitated mixture was filtered off, dried, and recrystal-
lized from an appropriate solvent.
A solution of 6-bromoacetyl-3-methyl-2-oxo-3H-benzoxazole
(2.70 g, 0.01 mol), the respective phenylacetic acids (0.01 mol),
and triethylamine (1.39 mL, 0.01 mol) in acetonitrile was stirred
for 4 h at reflux, diluted with water. The precipitated mixture
was filtered off, dried, and recrystallized from the appropriate
solvent.
4.3.1. 3-Phenyl-4-(3-methyl-2-oxo-3H-benzoxazole-6-yl)-2-
oxo-5H-furane (6a)
Recrystallized from ethanol. 1.41 g, 46% yield; mp 244 °C; IR
(KBr) 3064, 2938, 2875, 1776, 1743 cmÀ1 1H NMR (DMSO-d6,
;
400 MHz) 7.46–7.40 (m, 3H), 7.35–7.31 (m, 3H), 7.29 (d, 1H,
3
4
3JH–H = 8.0 Hz), 7.24 (dd, 1H, JH–H = 8.0 Hz, JH–H = 1.6 Hz), 5.40
(s, 2H), 3.32 (s, 3H). Anal. Calcd for C18H13NO4: C, 70.35; H, 4.26;
N, 4.56. Found: C, 69.74; H, 4.02; N, 4.61.
4.2.1. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
phenyl acetate (5a)
Recrystallized from ethanol. 1.79 g, 55% yield; mp 128–129 °C;
4.3.2. 3-(4-Methoxyphenyl)-4-(3-methyl-2-oxo-3H-benzoxa-
zole-6-yl)-2-oxo-5H-furane (6b)
IR (KBr) 3066, 3030, 2959, 2926, 1775, 1753, 1697, 1625,
3
1236 cmÀ1
;
1H NMR (DMSO-d6, 400 MHz) 7.90 (dd, 2H, JH–
4
4
Recrystallized from n-butanol. 1.72 g, 51% yield; mp 204 °C; IR
H = 8.4 Hz, JH–H = 1.6 Hz), 7.88 (d, 1H, JH–H = 1.6 Hz), 7.40 (d, 1H,
3JH–H = 8.2 Hz), 7.36–7.24 (m, 5H), 5.49 (s, 2H), 3.83 (s, 2H), 3.38
(s, 3H). Anal. Calcd for C18H15NO5: C, 66.46; H, 4.65; N, 4.31. Found:
C, 66.02; H, 4.58; N, 4.40.
(KBr) 3063, 2980, 2880, 1776, 1744, 1243, 1064 cmÀ1 1H NMR
;
(DMSO-d6, 400 MHz) 7.35 (s, 1H), 7.30–7.27 (m, 4H), 6.99 (d, 2H,
3JH–H = 6.8 Hz), 5.35 (s, 2H), 3.79 (s, 3H), 3.33 (s, 3H). Anal. Calcd
for C19H15NO5: C, 67.65; H, 4.48; N, 4.15. Found: C, 67.20; H,
4.32; N, 4.23.
4.2.2. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
(4-methoxyphenyl) acetate (5b)
4.3.3. 3-(4-Chlorophenyl)-4-(3-methyl-2-oxo-3H-benzoxazole-
6-yl)-2-oxo-5H-furane (6c)
Recrystallized from ethanol. 2.61 g, 74% yield; mp 136.9 °C; IR
(KBr) 3074, 3035, 2956, 2838, 1772, 1734, 1691, 1610, 1233,
Recrystallized from ethanol–water mixture. 2.35 g, 69% yield;
3
1029 cmÀ1
;
1H NMR (DMSO-d6, 400 MHz) 7.92 (d, 1H, JH–H
=
mp 207 °C dec; IR (KBr) 3063, 2935, 1794, 1743, 1612 cmÀ1
;
1H
3
8.4 Hz), 7.89 (s, 1H), 7.41 (d, 1H, JH–H = 8.2 Hz), 7.24 (d, 2H,
3
NMR (DMSO-d6, 400 MHz) 7.52 (d, 2H, JH–H = 8.2 Hz), 7.39–7.35
3JH–H = 8.4 Hz), 6.90 (d, 2H, JH–H = 8.4 Hz), 5.49 (s, 2H), 3.75 (s,
3
3
3
(m, 3H), 7.31 (d, 1H, JH–H = 8.0 Hz), 7.24 (dd, 1H, JH–H = 8.0 Hz,
2H), 3.74 (s, 3H), 3.39 (s, 3H). Anal. Calcd for C19H17NO6: C,
64.22; H, 4.82; N, 3.94. Found: C, 63.93; H, 4.86; N, 4.06.
4JH–H = 1.6 Hz), 5.39 (s, 2H), 3.33 (s, 3H). Anal. Calcd for
C18H12ClNO4: C, 63.26; H, 3.54; N, 4.10. Found: C, 62.66; H, 3.60;
N, 4.45.
4.2.3. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
(4-chlorophenyl) acetate (5c)
4.3.4. 3-(4-Fluorophenyl)-4-(3-methyl-2-oxo-3H-benzoxazole-
6-yl)-2-oxo-5H-furane (6d)
Recrystallized from ethanol. 2.54 g, 71% yield; mp 154–155 °C;
IR (KBr) 3084, 2946, 1783, 1749, 1686, 1611, 1212 cmÀ1 1H NMR
;
3
4
Recrystallized from acetic acid. 2.35 g, 69% yield; mp 207 °C
(DMSO-d6, 400 MHz) 7.90 (dd, 1H, JH–H = 8.4 Hz, JH–H = 1.2 Hz),
dec; IR (KBr) 3070, 2927, 1793, 1745, 1214 cmÀ1 1H NMR (CDCl3,
;
4
7.89 (s, 1H, JH–H = 1.2 Hz), 7.42–7.33 (m, 5H), 5.52 (s, 2H), 3.86
3
4
400 MHz) 7.41 (dd, 2H, JH–H = 8.4 Hz, JH–F = 5.6 Hz), 7.27 (s, 1H),
(s, 2H), 3.38 (s, 3H). Anal. Calcd for C18H14ClNO5: C, 60.09; H,
3.92; N, 3.89. Found: C, 59.41; H, 3.77; N, 4.06.
3
4
7.20 (dd, 1H, JH–H = 8.0 Hz, JH–H = 1.6 Hz), 7.16–7.06 (m, 2H),
6.95 (d, 1H, JH–H = 7.6 Hz), 5.18 (s, 2H), 3.42 (s, 3H). Anal. Calcd
3
for C18H12FNO4: C, 66.46; H, 3.72; N, 4.31. Found: C, 65.75; H,
3.61; N, 4.39; LC/MS (ES+) m/z: 326.14 [M+H], 348.14 [M+Na].
4.2.4. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
(4-fluorophenyl) acetate (5d)
Recrystallized from ethanol. 3.14 g, 92% yield; mp 160.4 °C; IR
(KBr) 3084, 3064, 2936, 1780, 1750, 1687, 1610, 1217 cmÀ1 1H
;
4.3.5. 3-(4-Methylsulfonylphenyl)-4-(3-methyl-2-oxo-3H-
benzoxazole-6-yl)-2-oxo-5H-furane (6e)
3
4
NMR (DMSO-d6, 400 MHz) 7.91 (dd, 1H, JH–H = 8.2 Hz, JH–H
=
3
Recrystallized from ethanol. 2.19 g, 57% yield; mp 276.5 °C dec;
1.6 Hz), 7.89 (s, 1H), 7.41 (d, 1H, JH–H = 8.0 Hz), 7.37 (dd, 2H,
IR (KBr) 3087, 3016, 2932, 1798, 1741, 1338, 1152, 1294 cmÀ1
;
1H
3JH–H = 7.2 Hz, JH–F = 5.4 Hz), 7.17 (m, 2H), 5.52 (s, 2H), 3.86 (s,
4
3
NMR (DMSO-d6, 400 MHz) 7.98 (d, 2H, JH–H = 6.4 Hz), 7.61 (d, 2H,
2H), 3.39 (s, 3H). Anal. Calcd for C18H14FNO5: C, 62.97; H, 4.11;
N, 4.08. Found: C, 62.52; H, 4.09; N, 4.18.
4
3
3JH–H = 6.6 Hz), 7.38 (d, 1H, JH–H = 1.2 Hz), 7.30 (d, 1H, JH–H
=
3
4
8.0 Hz), 7.21 (dd, 1H, JH–H = 8.2 Hz, JH–H = 1.4 Hz), 5.43 (s, 2H),
3.32 (s, 3H), 3.26 (s, 3H). Anal. Calcd for C19H15NO6S: C, 59.21; H,
3.92; N, 3.63; S, 8.32. Found: C, 59.17; H, 3.90; N, 3.72; S, 8.40.
4.2.5. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
(4-methylsulfonylphenyl) acetate (5e)
Recrystallized from ethanol. 3.58 g, 89% yield; mp 152–153 °C;
IR (KBr) 3064, 3015, 2925, 1776, 1755, 1694, 1622, 1303, 1152,
4.3.6. 6-Hydroxyacetyl-3-methyl-2-oxo-3H-benzoxazole (7)
A solution of 6-bromoacetyl-3-methyl-2-oxo-3H-benzoxazole
(8.10 g, 0.03 mol) and sodium formate (4.08 g, 0.06 mol) in aceto-
nitrile was stirred for 4 h at reflux, diluted with water. The precip-
itated ester derivative was filtered off, washed with cold ethanol,
and hydrolyzed by concentrated HCl. The crude was purified by
recrystallization from water. 3.5 g, 56% yield; mp 176–177 °C; IR
1236 cmÀ1 1H NMR (DMSO-d6, 400 MHz) 7.92–7.88 (m, 4H),
;
3
3
7.61 (d, 2H, JH–H = 8.4 Hz), 7.40 (d, 1H, JH–H = 8.0 Hz), 5.53 (s,
2H), 4.01 (s, 2H), 3.37 (s, 3H), 3.22 (s, 3H). Anal. Calcd for
C19H17NO7S: C, 56.57; H, 4.25; N, 3.47; S, 7.95. Found: C, 55.80;
H, 4.10; N, 3.81; S, 7.72.
4.3. General procedure for the preparation of 3-phenyl-4-(3-
methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxo-5H-furanes (6a–e)
(KBr) 3447, 3399, 3090, 2929, 1788, 1764 cmÀ1
;
1H NMR (DMSO-
3
4
d6, 400 MHz) 7.88 (dd, 1H, JH–H = 8.2 Hz, JH–H = 1.2 Hz), 7.85 (d,
4
3
1H, JH–H = 1.2 Hz), 7.38 (d, 1H, JH–H = 8.4 Hz), 5.08 (s, 1H), 4.78
(s, 2H), 3.38 (s, 3H). Anal. Calcd for C10H9NO4: C, 57.97; H, 4.38;
N, 6.76. Found: C, 57.75; H, 4.45; N, 6.75.
The solution of the respective 2-(3-methyl-2-oxo-3H-benzoxaz-
ole-6-yl)-2-oxoethyl phenyl acetate (0.01 mol) and DBU (1.6 mL,

6374
G. Eren et al. / Bioorg. Med. Chem. 18 (2010) 6367–6376
4.4. General procedure for the preparation of 2-(3-methyl-2-
oxo-3H-benzoxazole-6-yl)-2-oxoethyl N-phenyl carbamates
(8a–e)
4.5. General procedure for the preparation of 3-phenyl-4-(3-
methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxo-3H-1,3-oxazoles
(9a–e)
A solution of 6-hydroxyacetyl-3-methyl-2-oxo-3H-benzoxazole
(2.07 g, 0.01 mol), the respective phenyl isocyanates (0.03 mol) in
toluene was stirred for 7–8 h at reflux. After that petroleum ether
was added and the precipitated mixture was filtered off, dried, and
recrystallized from an appropriate solvent.
The solution of the respective 2-(3-methyl-2-oxo-3H-benzoxaz-
ole-6-yl)-2-oxoethyl N-phenyl carbamate (0.01 mol) in acetic acid
was stirred for 3–4 h at reflux, diluted with water. The precipitated
mixture was filtered off, dried, and recrystallized from an appropri-
ate solvent.
4.5.1. 3-Phenyl-4-(3-methyl-2-oxo-3H-benzoxazole-6-yl)-2-
oxo-3H-1,3-oxazole (9a)
4.4.1. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
N-phenyl carbamate (8a)
Recrystallized from ethanol. 2.98 g, 97% yield; mp 197–198 °C;
Recrystallized from ethanol. 1.98 g, 61% yield; mp 178–179 °C;
;
IR (KBr) 3153, 3054, 1791, 1745 cmÀ1 1H NMR (DMSO-d6,
IR (KBr) 3301, 3133, 3035, 2946, 1750, 1725, 1699, 1231 cmÀ1
;
=
400 MHz) 7.65 (s, 1H), 7.46–7.36 (m, 3H), 7.28 (dd, 2H,
3
1H NMR (DMSO-d6, 400 MHz) 9.99 (s, 1H), 7.96 (dd, 1H, JH–H
4
3
3JH–H = 6.7 Hz, JH–H = 1.6 Hz), 7.20 (d, 1H, JH–H = 8.0 Hz), 7.13 (d,
4
4
8.4 Hz, JH–H = 1.6 Hz), 7.94 (d, 1H, JH–H = 1.6 Hz), 7.48 (d, 2H,
4
3
4
1H, JH–H = 1.6 Hz), 6.95 (dd, 1H, JH–H = 8.0 Hz, JH–H = 1.6 Hz),
3.29 (s, 3H). Anal. Calcd for C17H12N2O4: C, 66.23; H, 3.92; N,
9.09. Found: C, 65.67; H, 3.94; N, 9.05.
3JH–H = 7.6 Hz), 7.43 (d, 1H, JH–H = 8.0 Hz), 7.29 (t, 2H, JH–H
7.5 Hz), 7.01 (t, 1H, JH–H = 7.2 Hz), 5.52 (s, 2H), 3.40 (s, 3H). Anal.
Calcd for C17H14N2O5: C, 62.57; H, 4.32; N, 8.59. Found: C, 62.36;
H, 4.36; N, 8.53.
=
3
3
3
4.5.2. 3-(4-Methoxyphenyl)-4-(3-methyl-2-oxo-3H-benzoxazole-
6-yl)-2-oxo-3H-1,3-oxazole (9b)
4.4.2. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
N-(4-methoxyphenyl) carbamate (8b)
Recrystallized from ethanol. 3.10 g, 92% yield; mp 212–213 °C;
IR (KBr) 3130, 2946, 1775, 1751 cmÀ1
400 MHz) 7.62 (s, 1H), 7.23–7.19 (m, 3H), 7.14 (d, 1H, JH–H
;
1H NMR (DMSO-d6,
4
Recrystallized from ethanol. 3.28 g, 92% yield; mp 183 °C; IR (KBr)
=
3446, 3396, 3074, 2926, 1764, 1699, 1611, 1093 cmÀ1
;
1H NMR
1.6 Hz), 6.99–6.94 (m, 3H), 3.76 (s, 3H), 3.29 (s, 3H). Anal. Calcd
for C18H14N2O5: C, 63.90; H, 4.17; N, 8.28. Found: C, 63.59; H,
4.10; N, 8.30.
3
(DMSO-d6, 400 MHz) 9.76 (s, 1H), 7.94 (dd, 1H, JH–H = 8.0 Hz,
4JH–H = 1.2 Hz), 7.90 (d, 1H, 4JH–H = 1.2 Hz), 7.40 (d, 1H,
3
3
3JH–H = 8.0 Hz), 7.36 (d, 2H, JH–H = 9.2 Hz), 6.86 (d, 2H, JH–H
=
8.8 Hz), 5.47 (s, 2H), 3.70 (s, 3H), 3.38 (s, 3H). Anal. Calcd for
18H16N2O6: C, 60.67; H, 4.53; N, 7.86. Found: C, 60.98; H, 4.44; N,
7.87.
4.5.3. 3-(4-Chlorophenyl)-4-(3-methyl-2-oxo-3H-benzoxazole-
6-yl)-2-oxo-3H-1,3-oxazole (9c)
C
Recrystallized from ethanol. 2.63 g, 77% yield; mp 218–220 °C;
IR (KBr) 3143, 3094, 2946, 1767 cmÀ1
;
1H NMR (DMSO-d6,
3
4
400 MHz) 7.66 (s, 1H), 7.51 (dd, 2H, JH–H = 6.8 Hz, JH–H = 2.0 Hz),
4.4.3. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
N-(4-chlorophenyl) carbamate (8c)
3
4
7.23 (d, 1H, JH–H = 8.0 Hz), 7.19 (d, 1H, JH–H = 1.6 Hz), 6.94 (dd,
3
4
1H, JH–H = 8.0 Hz, JH–H = 1.6 Hz), 3.30 (s, 3H). Anal. Calcd for
17H11ClN2O4: C, 59.57; H, 3.23; N, 8.17. Found: C, 59.02; H,
Recrystallized from ethanol. 2.98 g, 83% yield; mp 214–215 °C;
IR (KBr) 3338, 3074, 2936, 1790, 1775, 1696, 1222 cmÀ1
;
1H NMR
C
3
3.26; N, 8.12.
(DMSO-d6, 400 MHz) 10.17 (s, 1H), 7.96 (dd, 1H, JH–H = 8.4 Hz,
4
3
4JH–H = 1.6 Hz), 7.94 (d, 1H, JH–H = 1.6 Hz), 7.50 (d, 2H, JH–H
=
3
3
4.5.4. 3-(4-Fluorophenyl)-4-(3-methyl-2-oxo-3H-benzoxazole-
6-yl)-2-oxo-3H-1,3-oxazole (9d)
9.2 Hz), 7.44 (d, 1H, JH–H = 8.2 Hz), 7.36 (d, 2H, JH–H = 8.8 Hz),
5.53 (s, 2H), 3.40 (s, 3H). Anal. Calcd for C17H13ClN2O5: C, 56.60;
H, 3.63; N, 7.77. Found: C, 56.10; H, 3.56; N, 7.77.
Recrystallized from ethanol. 2.45 g, 75% yield; mp 216 °C; IR
(KBr) 3163, 3074, 1793, 1762 cmÀ1 1H NMR (DMSO-d6, 400 MHz)
;
7.65 (s, 1H), 7.37–7.26 (m, 4H), 7.22 (d, 1H, 3JH–H = 8.4 Hz), 7.16 (d,
1H, 4JH–H = 1.6 Hz), 6.94 (dd, 1H, 3JH–H = 7.8 Hz, 4JH–H = 1.6 Hz), 3.30
(s, 3H). Anal. Calcd for C17H11FN2O4: C, 62.58; H, 3.40; N, 8.59. Found:
C, 62.15; H, 3.38; N, 8.53.
4.4.4. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
N-(4-fluorophenyl) carbamate (8d)
Recrystallized from ethanol. 2.75 g, 80% yield; mp 204–205 °C;
IR (KBr) 3336, 3084, 2946, 1787, 1772, 1733, 1222 cmÀ1
;
1H NMR
3
(DMSO-d6, 400 MHz) 10.04 (s, 1H), 7.96 (dd, 1H, JH–H = 8.4 Hz),
4.5.5. 3-(4-Methylthiophenyl)-4-(3-methyl-2-oxo-3H-benzoxaz-
ole-6-yl)-2-oxo-3H-1,3-oxazole (9e)
3
4
7.93 (s, 1H), 7.48 (dd, 2H, JH–H = 8.4 Hz, JH–F = 5.2 Hz), 7.43 (d,
3
1H, JH–H = 8.0 Hz), 7.14 (m, 2H), 5.51 (s, 2H), 3.40 (s, 3H). Anal.
Recrystallizedfromethanol. 2.15 g, 61%yield;mp170 °C;IR(KBr)
Calcd for C17H13FN2O5: C, 59.30; H, 3.81; N, 8.14. Found: C,
59.01; H, 3.82; N, 8.16.
3065, 2924, 2854, 1768 cmÀ1; 1H NMR (DMSO-d6, 400 MHz) 7.62 (s,
4
1H), 7.29–7.18 (m, 5H), 7.16 (d, 1H, JH–H = 1.6 Hz), 6.95 (dd, 1H,
4
3JH–H = 8.0 Hz, JH–H = 1.6 Hz), 3.28 (s, 3H), 2.46 (s, 3H). Anal. Calcd
for C18H14N2O4S: C, 61.01; H, 3.98; N, 7.90; S, 9.05. Found: C,
61.33; H, 4.04; N, 7.89; S, 9.03.
4.4.5. 2-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)-2-oxoethyl
N-(4-methylthiophenyl) carbamate (8e)
Recrystallized from ethanol. 2.38 g, 64% yield; mp 185 °C; IR
(KBr) 3310, 3064, 2926, 1783, 1715, 1692, 1223 cmÀ1
;
1H NMR
4.5.6. 3-(4-Methylsulfonylphenyl)-4-(3-methyl-2-oxo-3H-benz-
oxazole-6-yl)-2-oxo-3H-1,3-oxazole (9f)
3
(DMSO-d6, 400 MHz) 9.99 (s, 1H), 7.93 (dd, 1H, JH–H = 8.0 Hz,
4
3
4JH–H = 1.2 Hz), 7.91 (d, 1H, JH–H = 1.2 Hz), 7.41 (d, 2H, JH–H
=
The solution of 3-(4-methylthiophenyl)-4-(3-methyl-2-oxo-3
H-benzoxazole-6-yl)-2-oxo-3H-1,3-oxazole (3.54 g, 0.01 mol) and
m-chloroperbenzoic acid (70%) (9.92 g, 0.04 mol) in dichlorometh-
ane was stirred for 5 h at room temperature, diluted with water
and extracted with dichloromethane. The combined organic layer
3
3
8.4 Hz), 7.20 (d, 2H, JH–H = 8.0 Hz), 7.08 (d, 1H, JH–H = 8.8 Hz),
5.48 (s, 2H), 3.37 (s, 3H), 2.41 (s, 3H). Anal. Calcd for C18H16N2O5S:
C, 58.05; H, 4.33; N, 7.52; S, 8.61. Found: C, 57.71; H, 4.45; N, 7.41;
S, 8.54.

G. Eren et al. / Bioorg. Med. Chem. 18 (2010) 6367–6376
6375
was washed with saturated solution of sodium bicarbonate and
4.6.3. 1-(4-Chlorophenyl)-3-trifluoromethyl-5-(3-methyl-2-
dried with magnesium sulfate and the residue obtained after sol-
vent evaporation was recrystallized from acetone–water mixture.
1.44 g, 41% yield; mp 264 °C dec; IR (KBr) 3152, 3070, 2956,
oxo-3H-benzoxazole-6-yl)-1H-pyrazole (11c)
Recrystallized from ethanol. 2.89 g, 75% yield; mp 178 °C; IR
(KBr) 3133, 2956, 1768, 1611 cmÀ1
;
1H NMR (DMSO-d6,
3
3
1797, 1750, 1327, 1151 cmÀ1
;
1H NMR (CDCl3, 400 MHz) 7.94 (d,
400 MHz) 7.54 (d, 2H, JH–H = 8.8 Hz), 7.40 (d, 2H, JH–H = 8.4 Hz),
3
3
3
2H, JH–H = 8.0 Hz), 7.39 (d, 2H, JH–H = 8.0 Hz), 7.05 (s, 1H), 7.03
7.38 (s, 1H), 7.28 (d, 1H, JH–H = 8.0 Hz), 7.22 (s, 1H), 7.10 (d, 1H,
3
3
(s, 1H), 6.91 (d, 1H, JH–H = 8.0 Hz), 6.86 (d, 1H, JH–H = 8.0 Hz),
3.42 (s, 3H), 3.07 (s, 3H). Anal. Calcd for C18H14N2O6S: C, 55.95;
H, 3.65; N, 7.25; S, 8.30. Found: C, 56.37; H, 3.58; N, 7.47; S, 8.75.
3JH–H = 8.0 Hz), 3.34 (s, 3H). Anal. Calcd for C18H11F3N3O2: C,
54.91; H, 2.82; N, 10.67. Found: C, 54.73; H, 2.80; N, 10.61.
4.6.4. 1-(4-Fluorophenyl)-3-trifluoromethyl-5-(3-methyl-2-
oxo-3H-benzoxazole-6-yl)-1H-pyrazole (11d)
4.5.7. 4,4,4-Trifluoro-1-(3-methyl-2-oxo-3H-benzoxazole-6-yl)-
1,3-dioxobutane (10)
Recrystallized from ethanol. 1.92 g, 51% yield; mp 171 °C; IR
6-Acetyl-3-methyl-2-oxo-3H-benzoxazole (3.00 g, 0.015 mol)
and potassium t-butoxide (4.2 g, 0.037 mol) were suspended in
benzene and to the agitated reaction mixture ethyltrifluoro acetate
(3.57 mL, 0.03 mol) was added in portions under ice-cold condi-
tions. The mixture was let to stir for 30 min at 0 °C and then was
stirred for 5 h at reflux. The solvent was removed and the residue
was stirred with ice-cold water. After acidification with concen-
trated HCl, the solution was extracted with diethyl ether. The dried
organic layer after solvent evaporation left behind an oily residue
which was stirred with saturated solution of sodium bicarbonate
for overnight at room temperature to afford the desired product.
The solid was filtered off and recrystallized from carbon tetrachlo-
ride. 2.25 g, 53% yield; mp 313 °C dec; IR (KBr) 3514, 3090, 2890,
(KBr) 3133, 3074, 2926, 1772, 1611 cmÀ1 1H NMR (DMSO-d6,
;
400 MHz) 7.44 (dd, 2H, 3JH–H = 8.8 Hz, 4JH–F = 5.2 Hz), 7.35–7.29 (m,
3
3H), 7.26 (d, 1H, JH–H = 8.4 Hz), 7.21 (s, 1H), 7.09 (dd, 1H,
4
3JH–H = 8.4 Hz, JH–H = 1.6 Hz), 3.24 (s, 3H). Anal. Calcd for
C18H11F4N3O2: C, 57.30; H, 2.94; N, 11.14. Found: C, 57.10; H, 3.07;
N, 11.12.
4.6.5. 1-(4-Methylsulfonylphenyl)-3-trifluoromethyl-5-(3-methyl-
2-oxo-3H-benzoxazole-6-yl)-1H-pyrazole (11e)
Recrystallized from n-hexane. 2.56 g, 68% yield; mp 213 °C; IR
(KBr) 3104, 3064, 2926, 1786, 1315, 1155, 1616 cmÀ1
;
1H NMR
3
(DMSO-d6, 400 MHz) 8.00 (d, 2H, JH–H = 8.8 Hz), 7.63 (d, 2H,
3
3JH–H = 8.4 Hz), 7.44 (s, 1H), 7.30 (d, 1H, JH–H = 8.4 Hz), 7.27 (s,
3
1775, 1635 cmÀ1
;
1H NMR (DMSO-d6, 400 MHz) 7.72 (dd, 1H,
1H), 7.13 (d, 1H, JH–H = 8.4 Hz), 3.29 (s, 3H), 2.51 (s, 3H). Anal.
4
4
3JH–H = 7.8 Hz, JH–H = 1.6 Hz), 7.66 (d, 1H, JH–H = 1.6 Hz), 7.23 (d,
Calcd for C19H14F3N3O4SÁ0.1C6H14: C, 52.78; H, 3.48; N, 9.42; S,
3
1H, JH–H = 8.0 Hz), 5.89 (s, 1H), 3.47 (s, 3H). Anal. Calcd for
7.19. Found: C, 52.83; H, 3.56; N, 9.53; S, 7.31.
C18H14N2O6S: C, 46.95; H, 2.59; N, 4.50. Found: C, 46.95; H, 2.39;
N, 4.60; LC/MS (ES+) m/z: 288.10 [M+H].
4.7. Biological activity
4.6. General procedure for the preparation of 1-phenyl-3-tri-
fluoromethyl-5-(3-methyl-2-oxo-3H-benzoxazole-6-yl)-1H-
pyrazoles (11a–e)
4.7.1. COX inhibition-purified enzyme assay
All compounds were tested for their ability to inhibit COX-1
and/or COX-2 using purified enzyme (PE) assay.27,28 For both of
these in vitro assays, compounds were tested against COX-1 and/
The dry flask charged with solution of 4,4,4-trifluoro-1-(3-
or COX-2 at 10 lM with n = 4 determinations. Compounds that
methyl-2-oxo-3H-benzoxazole-6-yl)-1,3-dioxobutane
(2.87 g,
showed a % inhibition >50% were also evaluated to calculate their
COX-1 and COX-2 IC50 values. IC50 values were obtained by nonlin-
ear regression from only one experiment with eight different con-
centrations with n = 4 determinations, using PRISM 4.0, GraphPad
Software. For each assay selectivity indexes (SI) were calculated
as the ratio [COX-1 IC50/COX-2 IC50].
0.01 mol), the respective phenylhydrazine HCl (0.011 mol) and
concentrated HCl (2 mL, 0.055 mol) was placed in MicroSYNTH
Microwave Synthesis System and irradiated at 300 W for 1 h while
the temperature was set to 90 °C. The solvent was removed by evap-
oration. The residue was solved in chloroform and washed with
water and the saturated solution of sodium bicarbonate, respec-
tively. The combined organic layer was dried with magnesium sul-
fate and the residue obtained after solvent evaporation was
recrystallized from an appropriate solvent.
4.7.2. Enzyme reactions
A commercial kit with ovine COX-1 and COX-2 enzymes (Man-
ufacturer, Cayman Chemical Co., Ann Arbor, MI) was used to assess
inhibition of each COX isoform. One unit of COX-1 or COX-2 en-
4.6.1. 1-Phenyl-3-trifluoromethyl-5-(3-methyl-2-oxo-3H-benz-
oxazole-6-yl)-1H-pyrazole (11a)
zyme in 50
(100 mM) containing hematin (1
(2 mM) as co-factors. The reaction medium was preincubated at
37 °C for 15 min with 50 L of the test compound, standard drugs
or vehicle. Immediately, the enzymatic reaction was started adding
50 L of 100 M final concentration of arachidonic acid substrate
and was incubated for 5 min at 37 °C. To stop the reaction, 1 N
HCl (50 L) was added. After that, 50 L of Tris-base (1 M) was also
l
L was suspended in 0.35 mL of pH 8 Tris–HCl buffer
lM), EDTA (5 mM), and phenol
Recrystallized from ethanol–water mixture. 2.94 g, 82% yield;
mp 160 °C; IR (KBr) 3132, 1771, 1613 cmÀ1
;
1H NMR (DMSO-d6,
l
4
400 MHz) 7.46–7.32 (m, 5H), 7.29 (d, 1H, JH–H = 1.6 Hz), 7.19 (d,
3
3
1H, JH–H = 8.0 Hz), 7.18 (s, 1H), 7.07 (dd, 1H, JH–H = 8.0 Hz,
4JH–H = 1.6 Hz), 3.29 (s, 3H). Anal. Calcd for C18H12F3N3O2: C,
60.17; H, 3.37; N, 11.69. Found: C, 59.97; H, 3.34; N, 11.55.
l
l
l
l
added. Immediately, prostaglandin (PGE2) production was quanti-
fied in samples diluted 1:10, by an specific enzyme-linked immu-
noassay (EIA) (Amersham Biosciences, RPN222) following
manufacturer’s instructions.
4.6.2. 1-(4-Methoxyphenyl)-3-trifluoromethyl-5-(3-methyl-2-
oxo-3H-benzoxazole-6-yl)-1H-pyrazole (11b)
Recrystallizedfromethanol–watermixture. 2.94 g, 82%yield;mp
177 °C; IR (KBr) 3133, 2966, 1773, 1611 cmÀ1
;
1H NMR (DMSO-d6,
3
400 MHz) 7.30–7.27 (m, 3H), 7.24 (d, 1H, JH–H = 8.4 Hz), 7.15 (s,
4.7.3. Drugs
3
4
1H), 7.08 (dd, 1H, JH–H = 8.2 Hz, JH–H = 1.6 Hz), 6.99 (d, 2H,
For all drugs, including Indomethacin and Rofecoxib using as
standard drugs, a 10 lM concentration in DMSO was prepared.
3JH–H = 8.4 Hz), 3.78 (s, 3H), 3.31 (s, 3H). Anal. Calcd for
C19H14F3N3O3: C, 58.61; H, 3.62; N, 10.79. Found: C, 58.46; H, 3.71;
Successive dilutions also in DMSO, were prepared to get the appro-
N, 10.69.
priate concentrations.

6376
G. Eren et al. / Bioorg. Med. Chem. 18 (2010) 6367–6376
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Potential maps were generated using the AUTOGRID program,46
available in the AUTODOCK 3.05 package, using a grid centered in
the COX-2 or COX-1 binding pockets, respectively (coordinates:
x = À24.000, y = À1.060, z = 8.600), with a size of 60 Â 60 Â 60
points and a grid spacing of 0.375 Å. Hundred runs of GA were per-
formed for each ligand using the standard conditions defined in the
AUTODOCK program. Final geometries of each run were compared
with the initial geometry and clustered as a function of the root
mean square deviation in relation to the initial geometry. Free en-
ergy of binding and docking energies was also calculated by the
AUTODOCK program for the most stable conformation of the most
populated cluster for each docked compound. The standard devia-
tion of each computed energy, reported in the text as the average
value of the chosen is always below 0.01, since the differences be-
tween the geometries included in each cluster are very small.
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