Synthesis, molecular docking study, and anticancer activity of triaryl-1,2,4-oxadiazole

Article abstract of DOI:10.1007/s00044-012-0436-9

This study describes synthesis of a new group of triaryl-1,2,4-oxadiazole derivatives and their anticancer activities. The target compounds were prepared from reaction of different imines and 4-substituted benzohydroxyiminoyl chlorides. All the synthesize

Full text of DOI:10.1007/s00044-012-0436-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4253–4262
DOI 10.1007/s00044-012-0436-9
ORIGINAL RESEARCH
Synthesis, molecular docking study, and anticancer activity
of triaryl-1,2,4-oxadiazole
•
Parisa Miralinaghi Mona Salimi Amirali Amirhamzeh
•
•
•
Mahnaz Norouzi Hirsa Mostafapour Kandelousi
•
•
Abbas Shafiee Mohsen Amini
Received: 15 August 2012 / Accepted: 15 December 2012 / Published online: 3 January 2013
ꢀ Springer Science+Business Media New York 2012
Abstract This study describes synthesis of a new group
of triaryl-1,2,4-oxadiazole derivatives and their anticancer
activities. The target compounds were prepared from
reaction of different imines and 4-substituted benzohydr-
oxyiminoyl chlorides. All the synthesized compounds were
screened for antiproliferative activities against MCF7 and
K562 cell lines using MTT assay at 50-lM concentration.
Four compounds that showed more than 50 % cytotoxicity
were selected for determination of IC50. Out of these, 6c-1y
showed remarkable inhibitory cytotoxicity activity against
MCF7 and K562 cell lines with IC50 6.50 and 21.66 lM,
respectively. A molecular modeling study where 6c-1y was
Keywords 1,2,4-Oxadiazole Á Anticancer Á Cytotoxicity Á
Molecular docking Á COX-2
Introduction
Over, the past few decades, the importance of chemother-
apy has increased in the treatment of cancer. In this
regards, drug resistance put forward as one the most rea-
sons in failure chemotherapy. Therefore, there is a great
interest to introduce new cytotoxic agents and also deter-
mine or predict their mechanisms of action.
˚
docked in the binding site of COX-2 showed a 2.3-A
The role of microtubules in cell proliferating has been
recognized as a validated target in cancer chemotherapy
(Aryapour et al., 2011). Three distinct binding sites were
recognized on b-subunit of tubulin that has been targeted
by many useful anticancer drugs (Alvarez et al., 2008). A
105972 and combretastatin are vicinal-diaryl stillben-like
structure that binds to b-tubulin and strongly inhibit tubulin
polymerization by binding to the colchicine site (Kumar
et al., 2011). Combretastatin A-4, a cis-conformation
stillben-like structure (Fig. 1a), was isolated from bark of
the South African tree Combretum caffrum. Isomerization
to the less active trans-form of combretastatin A-4
encouraged preparing di-aryl heterocyclic compounds
(Fig. 1c) owing cis-restricted analogous (Odlo et al., 2008).
In the other hand, di-aryl heterocyclic compounds have
been classically recognized as selective cyclooxygenase-2
(COX-2) inhibitors. Celecoxib (Fig. 1b), valdecoxib, and
several related structures have been used for the treatment
of inflammation-associated disorders in clinic. Previous
studies have indicated that celecoxib is chemopreventive
both in animal tumor models and in cancer patients
(Steinbach et al., 2000; Reddy et al., 2000). In a clinical
trial, celecoxib was found to reduce the number and size of
hydrogen bond forming via hydroxyl group of Ser516
residue and oxygen of central oxadiazole ring and triaryl
moiety of 6c-1y oriented toward the hydrophobic pockets
of COX-2. Our data indicate that these derivatives may
present promising chemotherapeutic agents, possibly tar-
geting COX-2 pathway.
P. Miralinaghi Á A. Amirhamzeh Á A. Shafiee Á M. Amini
Department of Medicinal Chemistry, Faculty of Pharmacy,
Tehran University of Medical Sciences, Tehran, Iran
M. Salimi Á H. M. Kandelousi
Physiology & Pharmacology Department, Pasteur Institute
of Iran, Tehran, Iran
M. Norouzi
Department of Biology, Faculty of Science, Kharazmi
University, Tehran, Iran
M. Amini (&)
Drug Design & Development Research Center, Tehran
University of Medical Sciences, P.O. Box 14155-6451,
Tehran, Iran
e-mail: moamini@sina.tums.ac.ir
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Med Chem Res (2013) 22:4253–4262
OH
model suggested that an additional aryl group adjoin to
central heterocycle ring enhanced the interaction of the
ligand in active site. Therefore, triaryl heterocycles have
been proposed might better fit in COX-2 active site. Con-
sequently, a series of triaryl imidazole derivatives (Fig. 1d)
was introduced as selective COX-2 inhibitors with IC50 %
ranging from 0.15 to 0.40 lM (Zarghi et al., 2012).
Keeping the above facts in mind, it was of our interest to
synthesize a series of triaryl-1,2,4-oxadiazole and evalu-
ated their anticancer activity against two different cancer
cell lines with the MTT assay. Some active compounds
were docked in the active site of murine COX-2 and their
probable mechanisms of action have been discussed.
H2NO2S
H
3CO
N
N
CF3
H
H
3CO
3CO
H
3C
OCH3
A
B
Ar
Ar
Ar
N
Het
Ar
N
H
Ar
C
D
Results and discussion
Chemistry
Fig. 1 Structure of combretastatin A-4, celecoxib and di- or triaryl
heterocyclic compounds
Synthesis of 1,2,4-oxadiazoles via the 1,3-dipolar cyclo-
addition of benzonitrile oxide and imines were reported
(Alcaide et al., 1987). Benzonitrile oxide tends to dimerize
within a few minutes to several days related to nature of
substituted group in aryl ring (Liu et al., 1980). In this
study, an easy method for preparation of new triaryl oxa-
diazole derivatives (6) from condensation of 4-substituted
benzohydroxyiminoyl chlorides (5) and imines (3) in die-
thyl ether was developed. In Scheme 1, the preparation of
compound 6 has been illustrated. 4-Substituted ben-
zohydroxyiminoyl chlorides that are stable and storable
were prepared from arylaldehyde oximes and N-chloro-
succinimide in DMF as reported procedure (Liu et al.,
1980). All final compounds were pure and stable. The
compounds were characterized by 1H and 13C- nuclear
polyps in-patients with familial adenomatous polyposis
(FAP) (Steinbach et al., 2000). Recent studies have shown
that celecoxib induces apoptosis in human chronic myeloid
leukemia, K562 and breast cancer cell lines (Subhashini
et al., 2005; Wang et al., 2012). The effects of celecoxib on
the hematopoietic cancer cell line like Jukrat, HL60, and
U937 revealed dose-dependent inhibition in growth of the
cell with arrest of cell cycle at G0/G1 phase. Previous
findings also suggested that COX-2 inhibition decreases
cell growth and promotes chemotherapy-induced apoptosis
in breast cancer cells (Wang et al., 2012). Collectively,
these reports reveal that celecoxib could be a potential
candidate for developing anticancer drug for the treatment
of chronic myeloid leukemia and other types of cancer.
The cytotoxicity effects of several di- or triaryl hetero-
cyclic rings such as pyrroles (Banwell et al., 2006), oxa-
diazole (Kiselyov et al., 2010), triazoles (Odlo et al., 2008)
and imidazole (Schobert et al., 2010), and triazine (Krauth
et al., 2010) have already been reported. From structure
points of view, selective COX-2 inhibitors are divided into
several classes. 1,2-di-aryl heterocyclic compounds,
Coxibs, are one of the extensive groups that have a five-
membered ring including furanone, pyrazole, oxazole,
isoxazole, pyrole,… or six-membered ring such as pyra-
none, pyridine, and pyridazinone (Joo et al., 2004) in the
center of the molecules that aryl groups is located as 1,2-
substituted. Guathier showed that human recombinant
COX-2 has been inhibited by 1,3-diaryl heterocyclic
compounds (Fig. 1c). Molecular modeling findings also
highlighted drug interaction with active site of both COX-1
and COX-2 suggested some modifications to enhance the
selectivity of the compounds (Gauthier et al., 2006). Their
1
magnetic resonance and CHN analysis. HNMR spectra of
the triaryl-1,2,4-oxadiazole derivatives (6) show a typical
proton signal for the H5-oxadiazole ring at 6.45–6.52-ppm
range. In 13CNMR spectra of compound 6, the signals at
*123 and 100 ppm are attributed to C3 and C5 of oxadi-
azole ring, respectively.
Biological study
Inhibitory effects of compound (6) on viability of breast
cancer and leukemia cells
To evaluate the effect of synthetic compound (6) on
human breast adenocarcinoma (MCF-7) and human eryth-
roleukemia K562 cells, cytotoxicity was measured using
the MTT assay. For this purpose, cultured cells were
incubated in the absence and in the presence of 50 lM of
different compounds for 24 h. A considerable difference
in percent of cytotoxicity of compounds was evident in
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Med Chem Res (2013) 22:4253–4262
4255
R1
R2
R1
+
R2
CHO
1
NH2
2
HC
N
3
_
NCS
TEA
OH
+
:N O
Cl
H
O N C
R3
R3
C N
H
R3
5
4
R1
R2
R2
R1
HC
N
..
N
_
R3
+
O
O
N
6
N
C
R3
R1= H; a / CH3; b / F; c / OCH3; d / Cl; e
R2 = H; 1 / F; 2 / Cl; 3
R3 = F; x / Cl; y
Scheme 1 Synthesis of 1,2,4-oxadiazole derivatives
MCF7- and K562-treated cells. Based on the desirable
cytotoxic effects of compounds 6e-3y, 6a-1y, 6c-1y, and
6e-1y (more than 50 % of cell cytotoxicity at 50 lM), we
examined the effect of these compounds on the prolifer-
ation of MCF-7 and K562 cells to obtain IC50 values.
The dose–response curves for four tested compounds are
shown in Figs. 2 and 3. The results revealed that all
compounds caused a dose-dependent inhibition of cell
proliferation. It is also noteworthy that for both tested cell
lines, compounds 6c-1y and 6e-1y showed a better cytotoxic
activity than two clinically established chemotherapeutics;
doxorubicin and paclitaxel which depend on the cell type
(Table 1). As it shown in Table 1, a more potent anti-
proliferative activity was documented for compounds 6c-
1y and 6e-1y in MCF-7 cells compared with doxorubicin.
It is interesting to suggest that MCF-7 cells reveal some
resistance against doxorubicin, whereas these two com-
pounds decreased the cell viability significantly (IC50
value *10 lM). Moreover, comparison of cytotoxic
effects of compounds 6c-1y and 6e-1y with reference drug
in K562 cells demonstrated that these compounds are more
potent than paclitaxel in this cell line. On the other hand, the
leukemia-resistant cell line (K562) against this chemo-
therapeutic (higher IC50) showed a remarkable chemosen-
sitivity to the two tested compounds. Considering all data,
compounds 6c-1y and 6e-1y were found to exhibit the
highest cytotoxic activity in comparison with other deriv-
atives against two human cancer cells studied here and
the order of inhibition against both cell lines are as follows:
6c-1y [ 6e-1y [ 6e-3y [ 6a-1y, which might be attributed
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Med Chem Res (2013) 22:4253–4262
to the presence of F or Cl as R1 and R3. With respect to H
substituent at R2 position, replacement of the hydrogen
substituent as R1 side chain (6a-1y) with fluoro and chloro
(6c-1y, 6e-1y) resulted in a remarkable increase in cytotoxic
activity. Moreover, 5-fluoro derivative is more potent than
chloro analog (6c-1y vs 6e-1y). It is worth noting that both
of the analogs (IC50: 6.5–10.1 lM) are more potent than
two reference drugs which depend on the cell type, MCF-7
or K562. Another interesting point is the presence of
chlorine atom in R3 for all the active compounds. The
substitution of F instead of Cl at R3 (compounds 6(a–e)-3x)
caused to decrease the activity.
A summary of HYDE reported free energy of binding of
compounds with COX-2 could be found in Table 2. Dock-
ing of these compounds was also accomplished on COX-1
but neither of them docked on it which may indicate the
predicted selectivity toward COX-2 for these structures.
The obtained results were matching with cytotoxicity
assay findings. It is evident that the compounds 6c-1y,
which was predicted in silico to be active compounds, has
been observed the most potent. As seen in Fig. 4, com-
pound 6c-1y was having strong H bonding via –OH moiety
of Ser516 residue and oxygen of central oxadiazole ring.
Although the MTT screening protocol did not conclude
of any possible mechanisms for the observed anticancer
activity of the test compounds, activity of 6c-1y, 6e-1y may
be attributed to apoptosis pathway due to the inhibition of
COX-2 route that require to the further studies.
Molecular modeling
In summary, our work provides new analogous of COX-2
inhibitors with a 1,2,4-oxadiazole in center of the molecules
as the heterocycle moiety, showing effective anticancer
activity and it present a promising lead for the generation of
new chemotherapeutic agents in selected cancer cells which
needs further research.
All four active compounds (6e-3y, 6a-1y, 6c-1y, and 6e-1y)
were docked in a similar manner in the active site of COX-
2. As exemplified by 6c-1y and could be seen in Fig. 4 a
˚
2.3-A hydrogen bond formed via –OH moiety of Ser516
residue and oxygen of central oxadiazole ring. The chlo-
rophenyl moiety of 6c-1y oriented toward a hydrophobic
pocket constructed by lipophilic side chains of Val335,
Leu345, Val102, Tyr341, and Leu517. In the middle, the
phenyl lies in the vicinity of Ser339, Tyr341, Val509,
Phe504, and Leu338 residues. The fluorophenyl group well
fitted in another hydrophobic side pocket of COX-2 con-
taining Leu338, Phe504, Trp375, Met508, Leu370, and Tyr
371 lipophilic side chains.
Materials and methods
General
Melting points were determined using a Thomas Hoover
melting point apparatus. A Bruker FT-500 MHz instrument
Fig. 2 Percentage of
cytotoxicity of MCF7 cells
after treatment with compounds
6e-3y (a), 6a-1y (b), 6c-1y (c),
and 6e-1y (d) for 24 h. Data
represent the means of
experiments performed in
triplicate
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Med Chem Res (2013) 22:4253–4262
4257
Fig. 3 Percentage of
cytotoxicity of K562 cells after
treatment with compounds
6e-3y (a), 6a-1y (b), 6c-1y (c),
and 6e-1y (d) for 24 h. Data
represent the means of
experiments performed in
triplicate
Table 1 IC50 values (lM) for antiproliferative activity of compounds 6e-3y, 6a-1y, 6c-1y and 6e-1y toward MCF7 and K562 cells
R2
R1
N
R3
O
N
Compounds
R1
R2
R3
IC50 (lM)
MCF-7
K562
6e-3y
Cl
H
Cl
H
H
H
Cl
Cl
Cl
Cl
22.60 1.25
23.70 1.27
6.50 1.09
10.10 1.20
[100
51.24 1.11
97.01 1.11
21.66 1.17
22.23 1.09
4.49 1.39
[100
6a-1y
6c-1y
F
6e-1y
Cl
Doxorubicin
Paclitaxel
1.86 1.40
Values are presented as mean SE of three independent experiments, performed in triplicate
1
Chemistry
was used to acquire HNMR spectra. The instrument was
set at 125 MHz for acquiring 13CNMR spectra. Coupling
constant (J) values are presented in hertz (Hz) and spin
multiples are given as s (singlet), d (double), t (triple), and
m (multiple). Elemental analyses were carried out with a
Perkin Elmer Model 240-C apparatus. The results of the
elemental analysis (C, H, N) were within 0.4 % of the
calculated amounts.
General procedure for synthesis of 3,4,5-triaryl-4,5-
dihydro-1,2,4-oxadiazole
To a solution of N-benzylideneaniline derivative (Alcaide
et al., 1987) (1 eq) in ether was added N-hydroxybenzimi-
doyl chloride (1 eq) and triethylamine (1.2 eq) successively.
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Med Chem Res (2013) 22:4253–4262
Fig. 4 Orientation of 6c-1y
(carbons in purple) in the active
site of COX-2. As could be seen
˚
a 2-A hydrogen bond formed
via hydroxyl group of Ser516
and oxygen of oxadiazole ring
(The pictures were prepared by
the Molsoft ICM-Browser.)
(Color figure online)
136.76 (C1-Ar5), 135.31 (C4-Ar3), 131.42 (C4-NAr), 129.63
[C2,6-Ar5 (C3,5-Ar5)], 129.45 [C3,5-Ar5 (C2,6-Ar5)], 129.22
[C3,5-Ar3 (C2,6-Ar3)], 129.13 [C2,6-Ar3 (C2,6-Ar3)], 127.16
(C3,5-NAr), 125.52 (C2,6-NAr), 123.65 (C3-oxadiazole),
100.53 (C5-oxadiazole), 21.37 (CH3). Anal. Calcd. For
C21H16Cl2N2O: C, 65.81; H, 4.21; N, 7.31. Found: C, 65.66;
H, 4.09; N, 7.25.
Table 2 Hyde scoring reported affinities of ligands toward COX-2
Compounds
HYDE reported DGintramolecular(kJ/mol)
6e-3y
6a-1y
6c-1y
6e-1y
-59
-52
-57
-55
3,4-Bis(4-chlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1,
2,4-oxadiazole (6c-3y) Yield: 73 %. Mp: 110–112 ꢁC.
1HNMR (CDCl3) d: 6.47 (s, 1H, H5-oxadiazole), 6.72 (d,
J = 8.35 Hz, 2H, H2,6-NAr), 7.13–7.20 (m, 4H, H3,5-NAr
and H3,5-Ar5), 7.34 (d, J = 8.15 Hz, 2H, H3,5-Ar3), 7.51 (d,
The reaction mixture was stirred at room temperature over-
night. The solvent was evaporated under reduced pressure.
The residue was crystallized in acetic acid–water to yield
3,4,5-triaryl-4,5-dihydro-1,2,4-oxadiazoles.
J = 8.15 Hz, 2H, H2,6-Ar3), 7.55 (t, J = 7.50 Hz, 2H, H2,6
-
Ar5). 13CNMR (CDCl3) d: 161.71 (d, JCF = 247.5 Hz, C4-
Ar5), 154.20 (C1-NAr), 139.28 (C1-Ar3), 136.93 [C4-Ar3
(C1-Ar5)], 134.17 [C1-Ar5 (C4-Ar3)], 131.85 (C4-NAr),
129.62 (C3,5-Ar3), 129.19 [C2,6-Ar5 and C3,5-NAr(d,
JCF = 7.30 Hz)], 125.74 (C2,6-NAr), 123.38 (C3-oxadiaz-
ole), 116.00 (d, JCF = 21.25 Hz, C3,5-Ar5), 99.95 (C5-
oxadiazole). Anal. Calcd. For C20H13Cl2FN2O: C, 62.03; H,
3.38; N, 7.23. Found: C, 62.28; H, 3.51; N, 7.41.
3,4-Bis(4-chlorophenyl)-5-phenyl-4,5-dihydro-1,2,4-oxadi-
1
azole (6a-3y) Yield: 88 %. Mp: 143–144 ꢁC. HNMR
(CDCl3) d: 6.49(s, 1H, H5-oxadiazole), 6.73(d, J = 7.45 Hz,
2H, H2,6-NAr), 7.16 (d, J = 7. 45 Hz, 2H, H3,5-NAr), 7.34
(d, J = 7.50 Hz, 2H, H3,5-Ar3), 7.46 (m, 3H, Ar5), 7.53 (d,
J = 7.50 Hz, 2H, H2,6-Ar3), 7.57 (m, 2H, Ar5). 13CNMR
(CDCl3) d: 154.10 (C1-NAr), 139.45 [C1-Ar3 (C1-Ar5)],
138.37 [C1-Ar5 (C1-Ar3)], 136.84 (C4-Ar3), 131.54 (C4-
NAr), 130.06 (C4-Ar5), 129.51 (C2,6-Ar5), 129.23 (C3,5-Ar5),
129.16 (C3,5-Ar3), 128.96 (C2,6-Ar3), 127.16 (C3,5-NAr),
125.53 (C2,6-NAr), 123.55 (C3-oxadiazole), 100.53 (C5-
oxadiazole). Anal. Calcd. For C20H14Cl2N2O: C, 65.06; H,
3.82; N, 7.59. Found: C, 65.26; H, 3.61; N, 7.44.
3,4-Bis(4-chlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-
1,2,4-oxadiazole (6d-3y) Yield: 66 %. Mp: 80–82 ꢁC.
1HNMR (CDCl3) d: 3.85 (s, 3H, OCH3), 6.45 (s, 1H, H5-
oxadiazole), 6.71 (d, J = 9.00 Hz, 2H, H2,6-NAr), 6.96 (d,
J = 9.00 Hz, 2H, H3,5-Ar5), 7.15 (d, J = 9.00 Hz, 2H,
H
3,5-NAr), 7.34 (d, J = 9.00 Hz, 2H, H2,6-Ar3), 7.48–7.51
3,4-Bis(4-chlorophenyl)-5-(p-tolyl)-4,5-dihydro-1,2,4-oxa-
1
(m, 4H, H2,6-Ar3 and H2,6-Ar5). 13CNMR (CDCl3) d: 160.97
(C4-Ar5), 154.12 (C1-NAr), 139.29 (C1-Ar3), 136.74 (C4-Ar3),
131.48 (C4-NAr), 130.21 (C1-Ar5), 129.47,129.20, 129.13,
128.75 [C2,6-Ar3 (C3,5-Ar3, C3,5-Ar5, C3,5-NAr), 123.67
(C3-oxadiazole), 55.38 (OCH3). Anal. Calcd. For
C21H16Cl2N2O2: C, 63.17; H, 4.04; N, 7.02. Found: C,
63.36; H, 3.91; N, 7.29.
diazole (6b-3y) Yield: 86 %. Mp: 120–122 ꢁC. HNMR
(CDCl3) d: 2.41 (s, 3H, CH3), 6.46 (s, 1H, H5-oxadiazole),
6.72 (d, J = 8.45 Hz, 2H, H2,6-NAr), 7.16 (d, J = 8.45 Hz,
2H, H3,5-NAr), 7.26 (d, J = 7.7 Hz, 2H, H3,5-Ar5), 7.34 (d,
J = 8.30 Hz, 2H, H3,5-Ar3), 7.45 (d, J = 7.7 Hz, 2H, H2,6
-
Ar5), 7.52 (d, J = 8.30 Hz, 2H, H2,6-Ar3). 13CNMR (CDCl3)
d: 154.06 (C1-NAr), 140.12 (C4-NAr), 139.41 (C1-Ar3),
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Med Chem Res (2013) 22:4253–4262
4259
3,4,5-Tris(4-chlorophenyl)-4,5-dihydro-1,2,4-oxadiazole (6e-
3y) Yield: 79 %. Mp: 97–99 ꢁC.1HNMR (CDCl3) d: 6.50
(s, 1H, H5-oxadiazole), 6.80 (d, J = 7.50 Hz, 2H, H2,6-NAr),
7.21 (d, J = 7.50 Hz, 2H, H3,5-NAr), 7.32 [d, J = 8.00 Hz,
2H, H3,5-Ar3 (H3,5-Ar5)], 7.43 [d, J = 8.00 Hz, 2H, H3,5-Ar5
(H3,5-Ar3)], 7.52 (m, 4H, H2,6-Ar3 and H2,6-Ar5). 13CNMR
(CDCl3) d: 154.20 (C1-NAr), 139.28 (C4-Ar5), 136.98 [C4-
Ar3(C1-Ar3)], 136.82 [C1-Ar3(C4-Ar3)], 136.04 (C1-Ar5),
131.90 (C4-NAr), 129.66 (C2,6-Ar3 and C2,6-Ar5), 129.21
(C3,5-Ar3 and C3,5-Ar5), 128.57 (C3,5-NAr), 125.71 (C2,6-
NAr), 123.30 (C3-oxadiazole), 99.86 (C5-oxadiazole). Anal.
Calcd. For C20H13Cl3N2O: C, 59.50; H, 3.25; N, 6.94. Found:
C, 59.81; H, 3.21; N, 7.12.
(C1-NAr), 140.80 (C1-Ar3), 136.68 (C4-Ar3), 134.67 (C1-
Ar5), 129.45, 129.24, 129.04 [C2,6-Ar3, (C3,5-NAr, C3,5
-
Ar3)], 129.15 (d, JCF = 8.75 Hz, C3,5-Ar5), 126.17
(C4-NAr), 124.52 (C2,6-NAr), 123.77 (C3-oxadiazole),
115.89 (d, JCF = 22.5 Hz, C2,6-Ar5), 100.00 (C5-oxadiaz-
ole). Anal. Calcd. For C20H14ClFN2O: C, 68.09; H, 4.00;
N, 7.94. Found: C, 68.21; H, 3.82; N, 7.69.
4-Phenyl-3-(4-chlorophenyl)-5-(4-methoxyphenyl)-4,5-dihy-
dro-1,2,4-oxadiazole
119–122 ꢁC. 1HNMR (CDCl3) d: 3.85 (s, 3H, OCH3), 6.49
(s, 1H, H5-oxadiazole), 6.79 (d, J = 7.50 Hz, 2H, H2,6
NAr), 6.96 (d, J = 8.00 Hz, 2H, H3,5-Ar5), 7.13 (t,
J = 7.50 Hz, 1H, H4-NAr), 7.19 (t, J = 7.50 Hz, 2H, H3,5
NAr), 7.31 (d, J = 8.00 Hz, 2H, H3,5-Ar3), 7.51 (d,
(6d-1y) Yield:
72 %.
Mp:
-
-
4,5-Diphenyl-3-(4-chlorophenyl)-4,5-dihydro-1,2,4-oxadi-
1
azole (6a-1y) Yield: 73 %. Mp: 133–135 ꢁC. HNMR
J = 8.00 Hz, 2H, H2,6-Ar5), 7.54 (d, J = 8.00 Hz, 2H, H2,6
-
Ar3). 13CNMR (CDCl3) d: 160.83 (C4-Ar5), 154.36 (C1-
NAr), 140.86 (C1-Ar3), 136.49 (C4-Ar3), 130.80 (C1-Ar5),
(CDCl3) d: 6.53 (s, 1H, H5-oxadiazole), 6.81 (d, J = 7.5 Hz,
2H, H2,6-NAr), 7.10–7.14 (m, 1H, H4-NAr), 7.19–7.22 (m,
2H, H3,5-NAr), 7.32 (d, J = 8.20 Hz, 2H, H3,5-Ar3), 7.45 (m,
3H, H3,4,5-Ar5), 7.55 (d, J = 8.20 Hz, 2H, H2,6-Ar3), 7.59
(m, 2H, H2,6-Ar5). 13CNMR (CDCl3) d: 154.36 (C1-NAr),
141.04 (C1-Ar3), 138.78 (C4-Ar3), 136.59 (C1-Ar5), 129.87
129.30, 129.23, 128.97, 128.70 [C3,5-Ar3, (C3,5-Ar5, C2,6
-
Ar3, C3,5-NAr)] 125.86 (C4-NAr), 124.44 (C2,6-NAr),
124.06 (C3-oxadiazole), 114.21 (C2,6-Ar5), 100.52 (C5-
oxadiazole), 55.35 (OCH3). Anal. Calcd. For C21H17ClN2
O2: C, 69.14; H, 4.70; N, 7.68. Found: C, 69.26; H, 4.55; N,
7.54.
(C4-Ar5), 129.36, 129.29, 129.03, 128.88 [C2,6-Ar5 (C3,5
-
Ar5, C3,5-Ar3, C3,5-NAr), 127.16 (C2,6-Ar3), 125.91 (C4-
NAr), 124.32 (C2,6-NAr), 123.95 (C3-oxadiazole), 100.59
(C5-oxadiazole). Anal. Calcd. For C20H15ClN2O: C, 71.75;
H, 4.52; N, 10.59. Found: C, 71.54; H, 4.63; N, 10.47.
4-Phenyl-3,5-bis(4-chlorophenyl)-4,5-dihydro-1,2,4-oxadi-
azole (6e-1y) Yield: 88 %. Mp: 124–126 ꢁC. 1HNMR
(CDCl3) d: 6.50 (s, 1H, H5-oxadiazole), 6.80 (d,
J = 7.50 Hz, 2H, H2,6-NAr), 7.16 (t, J = 7.00 Hz, 1H, H4-
NAr), 7.22 (t, J = 8.00 Hz, 2H, H3,5-NAr), 7.32 (d,
J = 8.50 Hz, 2H, H3,5-Ar5), 7.42 (d, J = 8.50 Hz, 2H,
4-Phenyl-3-(4-chlorophenyl)-5-(p-tolyl)-4,5-dihydro-1,2,4-
oxadiazole (6b-1y) Yield: 77 %. Mp: 116–118 ꢁC.
1HNMR (CDCl3) d: 2.41 (s, 3H, CH3), 6.50 (s, 1H, H5-
oxadiazole), 6.80 (d, J = 7.60 Hz, 2H, H2,6-NAr), 7.16 (t,
H
3,5-Ar3), 7.53 (d, J = 8.50 Hz, 4H, H2,6-Ar3 and H2,6-
Ar5). 13CNMR (CDCl3) d: 154.45 (C1-NAr), 140.80 (C1-
Ar3), 137.29, 136.72 [C4-Ar5 (C4-Ar3)], 135.01 (C1-Ar5),
129.98, 129.25, 129.12, 129.05, 128.57 [C2,6-Ar5 (C2,6-Ar3,
J = 7.20 Hz, 1H, H4-NAr), 7.18 (t, J = 7.20 Hz, 2H, H3,5
NAr), 7.25 (d, J = 8.00 Hz, 2H, H3,5-Ar5), 7.32 (d,
J = 8.00 Hz, 2H, H3,5-Ar3), 7.48 (d, J = 7.6 Hz, 2H, H2,6
-
-
Ar5), 7.55 (d, J = 8.00, 2H, H2,6-Ar3). 13CNMR (CDCl3)
d: 154.30 (C1-NAr), 140.99 (C4-Ar5), 139.96 (C1-Ar3),
136.51 [C1-Ar5 (C4-Ar3)], 135.95 [C4-Ar3 (C1-Ar5)],
129.54, 129.31, 129.25, 128.90, 128.44 [C3,5-Ar5 (C2,6-Ar3,
C
3,5-Ar5, C3,5-Ar3, C3,5-NAr)], 126.22 (C4-NAr), 123.67
(C3-oxadiazole), 99.96 (C5-oxadiazole). Anal. Calcd. For
C20H14Cl2N2O: C, 65.06; H, 3.82; N, 7.59. Found: C, 65.23;
H, 3.97; N, 7.50.
C3,5-Ar3, C2,6-Ar5, C3,5-NAr)], 126.44 (C4-NAr), 125.71
(C2,6-NAr), 124.05 (C3-oxadiazole), 100.50 (C5-oxadiaz-
ole), 21.36 (CH3). Anal. Calcd. For C21H17ClN2O: C,
72.31; H, 4.91; N, 8.03. Found: C, 72.45; H, 4.69; N, 8.23.
4-(4-Chlorophenyl)-3-(4-fluorophenyl)-5-phenyl-4,5-dihy-
dro-1,2,4-oxadiazole (6a-3x) Yield: 68 %. Mp: 112–
1
115 ꢁC. HNMR (CDCl3) d: 6.50 (s, 1H, H5-oxadiazole),
6.73 (d, J = 9.00 Hz, 2H, H2,6-NAr), 7.06 (t, J = 7.50 Hz,
2H, H3,5-Ar3), 7.16 (d, J = 9.00 Hz, 2H, H2,6-NAr),
7.46–7.48 (m, 3H, H3,4,5-Ar5), 7.56–7.61 (m, 4H, H2,6-Ar5
4-Phenyl-3-(4-chlorophenyl)-5-(4-fluorophenyl)-4,5-dihy-
dro-1,2,4-oxadiazole (6c-1y) Yield: 62 %. Mp: 106–
1
108 ꢁC. HNMR (CDCl3) d: 6.51 (s, 1H, H5-oxadiazole),
and H2,6-Ar3). 13CNMR (CDCl3) d: 164.01 (d, JCF
=
6.79 (d, J = 7.50 Hz, 2H, H2,6-NAr), 7.13–7.16 (m, 3H,
H3,4,5-NAr), 7.21 (t, J = 7.50 Hz, 2H, H3,5-Ar5), 7.31 (d,
J = 8.50 Hz, 2H, H3,5-Ar3), 7.53 (d, J = 8.50 Hz, 2H,
250.00 Hz, C4-Ar3), 154.08 (C1-NAr), 139.51 (C1-Ar3),
138.35 (C1-Ar5), 131.45 (C4-NAr), 130.12 (C4-Ar5),130.04
(d, JCF = 6.25 Hz, C2,6-Ar3), 129.46 (C2,6-Ar5), 128.95
(C3,5-Ar5), 127.15 (C3,5-NAr),125.52 (C2,6-NAr), 121.19
(C3-oxadiazole), 116.08 (d, JCF = 21.25 Hz, C3,5-Ar3),
H
2,6-Ar3), 7.57 (t, J = 7.50 Hz, H2,6-Ar5). 13CNMR
(CDCl3) d: 160.00 (d, JCF = 247.5 Hz, C4-Ar5), 154.45
123

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Med Chem Res (2013) 22:4253–4262
4,5-Bis(4-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1,
2,4-oxadiazole (6e-3x) Yield: 64 %. Mp: 80–82 ꢁC.
1HNMR (CDCl3) d: 6.46 (s, 1H, H5-oxadiazole), 6.73 (d,
100.38 (C5-oxadiazole). Anal. Calcd. For C20H14ClFN2O:
C, 68.09; H, 4.00; N, 7.94. Found: C, 68.32; H, 4.12; N, 7.78.
J = 8.50 Hz, 2H, H2,6-NAr), 7.06 (t, J = 8.50 Hz, 2H, H3,5
-
4-(4-Chlorophenyl)-3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihy-
Ar3), 7.18 (d, J = 8.50 Hz, 2H, H3,5-NAr), 7.43 (d, J =
8.00 Hz, 2H, H3,5-Ar5), 7.52 (d, J = 8.0 Hz, 2H, H2,6-Ar3),
7.55–7.59 (dd, J = 7.00 Hz and J = 3.5 Hz, 2H, H2,6-Ar5).
13CNMR (CDCl3) d: 164.03 (d, JCF = 250.0 Hz, C4-Ar3),
154.18 (C1-NAr), 139.33 (C1-Ar3), 136.90,135.99 [C4-NAr
dro-1,2,4-oxadiazole (6b-3x) Yield: 86 %. Mp: 80–
1
82 ꢁC. HNMR (CDCl3) d: 2.43 (s, 3H, CH3), 6.46 (s, 1H,
H5-oxadiazole), 6.72 (d, J = 8.50 Hz, 2H, H2,6-NAr), 7.05
(t, J = 8.00 Hz, 2H, H3,5-Ar3), 7.15 (d, J = 8.50 Hz, 2H,
H
3,5-NAr), 7.27 (d, J = 8.50 Hz, 2H, H3,5-Ar5), 7.46 (d,
(C4-Ar5)], 131.79 (C1-Ar5), 130.08 (d, JCF = 8.75 Hz, C2,6
-
J = 8.00 Hz, 2H, H2,6-Ar5), 7.56–7.60 (m, 2H, H2,6-Ar3).
13CNMR (CDCl3) d: 163.50 (d, JCF = 252.0 Hz, C4-Ar3),
154.04 (C1-NAr), 140.07 (C4-Ar5), 139.46 (C1-Ar3), 135.39
(C4-NAr), 131.31 (C1-Ar5), 130.8 (d, JCF = 8.75
Hz, C2,6-Ar3), 129.62, 129.41 [C3,5-NAr (C2,6-Ar5)],
127.16 (C3,5-Ar5), 125.50 (C2,6-NAr),121.27 (C3-oxadi-
azole), 116.08 (d, JCF = 21.25 Hz, C3,5-Ar3), 100.37 (C5-
oxadiazole). Anal. Calcd. For C21H16ClFN2O: C, 68.76; H,
4.40; N, 7.64. Found: C, 68.39; H, 3.99; N, 7.78.
Ar3), 129.61, 129.21, 128.56 [C2,6-Ar5 (C3,5-Ar5, C3,5
-
NAr)], 125.70 (C2,6-NAr), 120.92 (C3,-oxadiazole), 116.15
(d, JCF = 21.75 Hz, C3,5-Ar3), 99.71 (C5-oxadiazole). Anal.
Calcd. For C20H13Cl2FN2O: C, 62.03; H, 3.38; N, 7.23.
Found: C, 62.41; H, 3.50; N, 7.47.
3-(4-Chlorophenyl)-4-(4-fluorophenyl)-5-phenyl-4,5-dihydro-
1,2,4-oxadiazole (6a-2y) Yield: 78 %. Mp: 143–145 ꢁC.
1HNMR (CDCl3) d: 6.45 (s, 1H, H5-oxadiazole), 6.78–6.82
(m, 2H, H2,6-NAr), 6.90 (t, J = 9.00 Hz, 2H, H3,5-NAr),
7.32 (d, J = 8.50 Hz, 2H, H3,5-Ar3), 7.45–7.48 (m, 3H,
4-(4-Chlorophenyl)-3,5-bis(4-fluorophenyl)-4,5-dihydro-1,
2,4-oxadiazole (6c-3x) Yield: 55 %. Mp: 92–94 ꢁC.
1HNMR (CDCl3) d: 6.47 (s, 1H, H5-oxadiazole), 6.72 (d,
J = 9.00, Hz, 2H, H2,6-NAr), 7.06 (t, J = 9.00 Hz, 2H,
H
3,4,5-Ar5), 7.52 (d, J = 8.50 Hz, 2H, H2,6-Ar3), 7.55–7.60
(m, 2H, H2,6-Ar5). 13CNMR (CDCl3) d: 160.70 (d, JCF
=
251.0 Hz, C4-NAr), 154.60 (C1-NAr), 138.30 (C1-Ar5),
136.89, 136.69 [C1-Ar3 (C4-Ar3)], 130.00 (C4-Ar5), 129.24
(C2,6-Ar5), 129.05 (C3,5-Ar5), 128.89 (C3,5-Ar3), 127.29
(C2,6-Ar3), 126.80 (d, JCF = 7.55 Hz, C2,6-NAr),, 123.61
(C3-oxadiazole), 116.36 (d, JCF = 22.5Hz, C3,5-NAr),
100.99 (C5-oxadiazole). Anal. Calcd. For C20H14ClFN2O:
C, 68.09; H, 4.00; N, 7.94. Found: C, 68.31; H, 3.91; N,
7.67.
H
3,5-NAr), 7.12–7.20 (m, 4H, H3,5-Ar5 and H3,5-Ar3),
7.54–7.59 (m, 4H, H2,6-Ar3 and H2,6-Ar5). 13CNMR
(CDCl3) d: 164.04 [d, JCF = 251.25 Hz, C4-Ar5 (C4-Ar3)],
163.71 [d, JCF = 247.50 Hz, C4-Ar3 (C4-Ar5)], 154.18 (C1-
N–Ar), 139.33 (C1-Ar), 134.27 (C4-Ar4), 131.75 (C1-Ar5),
130.06 [d, JCF = 8.75 Hz, C2,6-Ar3 (C2,6-Ar5)], 129.57
(C4-NAr), 129.16 [d, JCF = 8.75 Hz, C2,6-Ar5 (C2,6-Ar3)],
125.73 (C1-NAr), 121.02 (C3-oxadiazole), 116.12 [d,
JCF = 21.25 Hz, C3,5-Ar3 (C3,5-Ar5)], 115.91 [d, JCF
=
3-(4-Chlorophenyl)-4-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihy-
dro-1,2,4-oxadiazole
(CDCl3) d: 2.46 (s, 3H, CH3), 6.42 (s, 1H, H5-oxadiazole),
6.79 (m, 2H, H2,6-NAr), 6.89 (t, J = 7.50 Hz, 2H, H3,5
(6b-2y) Yield:
65 %.1HNMR
21.25 Hz, C3,5-Ar5 (C3,5-Ar3)], 99.79 (C5-oxadiazole).
Anal. Calcd. For C20H13ClF2N2O: C, 64.79; H, 3.53; N,
7.56. Found: C, 64.41; H, 3.82; N, 7.68.
-
NAr), 7.27 (d, J = 8.00 Hz, 2H, H3,5-Ar5), 7.32 (d,
J = 8.50 Hz, 2H, H3,5-Ar3), 7.45 (d, J = 8.00 Hz, 2H,
4-(4-Chlorophenyl)-3-(4-fluorophenyl)-5-(4-methoxyphenyl)-
4,5-dihydro-1,2,4-oxadiazole (6d-3x) Yield: 71 %. Mp:
80–83 ꢁC. 1HNMR (CDCl3) d: 3.88 (s, 3H, OCH3), 6.45 (s,
1H, H5-oxadiazole), 6.71 (d, J = 8.50 Hz, 2H, H2,6-NAr),
6.97 (d, J = 8.50 Hz, 2H, H3,5-NAr), 7.05 (t, J = 9.00 Hz,
2H, H3,5-Ar3), 7.17 (d, J = 9.00 Hz, 2H, H2,6-Ar5), 7.49 (d,
H
2,6-Ar5), 7.51 (d, J = 8.50 Hz, 2H, H2,6-Ar3).13CNMR
(CDCl3) d; 160.67 (d, JCF = 246.0 Hz, C4-NAr), 154.57
(C1-NAr), 140.06 (C1-Ar3), 136.88, 136.62 [C4-Ar5 (C4-
Ar3)], 135.34 (C1-Ar5), 129.57, 129.23, 129.03 [C2,6-Ar5
(C3,5-Ar3, C3,5-Ar5)], 127.30 (C2,6-Ar3), 126.79 (d, JCF
=
J = 9.00 Hz, 2H, H3,5-Ar5), 7.55 (t, J = 7.5 Hz, 2H, H2,6
-
8.75 Hz, C2,6-NAr), 123.72 (C3-oxadiazole), 116.30 (d,
JCF = 22.50 Hz, C3,5-NAr), 100.99 (C5, oxadiazole), 21.37
(CH3). Anal. Calcd. For C21H16ClFN2O: C, 68.76; H, 4.40;
N, 7.64. Found: C, 68.46; H, 4.19; N, 7.39.
Ar3). 13CNMR (CDCl3) d: 163.01 (d, JCF = 251.0 Hz, H4-
Ar3), 161.2 (C4-Ar5), 154.04 (C1-NAr), 139.46 (C1-Ar3),
131.31, 130.04 [C1-Ar5 (C4-NAr)], 130.11 (d, JCF = 8.70 Hz,
C2,6-Ar3), 129.62, 128.75 [C2,6-Ar5 (C3,5-N–Ar)], 125.51
(C2,6-NAr), 121.37 (C3-oxadiazole), 115.55 (d, JCF
=
3-(4-Chlorophenyl)-4,5-bis(4-fluorophenyl)-4,5-dihydro-1,
2,4-oxadiazole (6c-2y) Yield: 70 %. Mp: 113–115 ꢁC.
1HNMR (CDCl3) d: 6.42 (s, 1H, H5-oxadiazole), 6.78–
21.25 Hz, C3,5-Ar3), 114.05 (C3,5-Ar5), 100.37 (C5-oxadi-
azole), 54.90 (OCH3). Anal. Calcd. For C21H16ClFN2
O2: C, 65.89; H, 4.21; N, 7.32. Found: C, 65.73; H, 3.98; N,
7.40.
6.80 (m, 2H, H2,6-NAr), 6.92 (t, J = 9.00 Hz, 2H, H3,5
-
NAr), 7.15 (t, J = 9.00 Hz, 2H, H3,5-Ar5), 7.32 (d,
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Med Chem Res (2013) 22:4253–4262
4261
J = 9.00 Hz, 2H, H3,5-Ar3), 7.49 (d, J = 9.00 Hz, 2H,
2,6-Ar3), 7.53–7.55 (m, 2H, H2,6-Ar5). 13CNMR (CDCl3) d:
163.72 [d, JCF = 248.5 Hz, C4-NAr (C4-Ar5)], 160.84 [d,
JCF = 245.0 Hz, C4-Ar5 (C4-NAr)], 154.69 (C1-NAr),
136.79, 136.55 [C4-Ar3 (C1-Ar3)],134.22 (C1-Ar5), 129.27 [d,
3LN1) was retrieved from the Brookhaven Protein Data
Bank (http://www.rcsb.org/pdb/home/home.do). The FlexX
program interfaced with LeadIT 2.1.2 (BioSolveIT GmbH,
Sankt Augustin, Germany) was used to dock all com-
pounds. FlexX is an automated docking program, which
considers ligand conformational flexibility by an incre-
mental fragment placing method (Rarey et al., 1996). The
active site for docking was determined as all atoms within
H
JCF = 8.75 Hz, C2,6-Ar5 (C2,6-NAr)], 129.21, 129.08 [C2,6
-
-
Ar3 (C3,5-Ar3)], 126.92 [d, JCF = 7.5 Hz, C2,6-NAr (C2,6
Ar5)], 123.44 (C3-oxadiazole), 116.48 [d, JCF = 22.50 Hz,
3,5-Ar5 (C3,5-NAr)], 115.94 [d, JCF = 21.25 Hz, C3,5-NAr
˚
7-A radius of the cocrystallized ligand. Validation of the
C
(C3,5-Ar5)], 100.39 (C5-oxadiazole). Anal. Calcd. For
C20H13ClF2N2O: C, 64.79; H, 3.53; N, 7.56. Found: C, 64.78;
H, 3.72; N, 7.66.
docking procedure was accomplished by energy minimi-
zation of celecoxib structure by the above-mentioned
method and the optimized geometry structure was
redocked in the active site of COX-2 which resulted in
predicted docking pose with RMSD 0.723 within the best
scored poses.
4-(4-Fluorophenyl)-3-(4-chlorophenyl)-5-(4-methoxyphenyl)-
4,5-dihydro-1,2,4-oxadiazole (6d-2y) Yield: 76 %. Mp:
1
98–102 ꢁC. HNMR (CDCl3) d: 3.88 (s, 3H, OCH3), 6.40
(s, 1H, H5-oxadiazole), 6.76–6.79 (m, 2H, H2,6-NAr), 6.89
For evaluation of the ligands affinity toward docked
receptor, the Hyde assessment facility of LeadIT software
was implemented to report the free energy of binding (DG)
and ligand efficiency of some of the best dock scored
poses. HYDE is an empirical scoring function, which
assesses protein–ligand complex by considering hydrogen
bond interactions and also hydrophobic and desolvation
effects and provides estimation for the binding affinity
(Reulecke et al., 2008; Schneider et al., 2011).
(t, J = 9.00 Hz, 2H, H3,5-NAr), 6.97 (d, J = 8.50 Hz, 2H,
H
3,5-Ar5), 7.32 (d, J = 8.50 Hz, 2H, H3,5-Ar3), 7.49 (d,
2,6-Ar3 and H3,5-Ar5). 13CNMR
J = 8.00 Hz, 4H,
H
(CDCl3) d: 160.95 (C4-Ar5), 160.69 (d, JCF = 245.0 Hz,
C4-NAr), 154.64 (C1-NAr), 136.77, 136.40 [C4-Ar3 (C1-Ar3)],
130.25 (C1-Ar5), 129.21, 129.02 [C2,6-Ar3 (C3,5-Ar3)], 128.87
(C2,6-Ar5), 126.92 (d, JCF = 8.75 Hz, C2,6-NAr), 123.75 (C3-
oxadiazole), 116.32 (d, JCF = 22.50 Hz, C3,5-NAr), 114.24
(C3,5-Ar5), 100.94 (C5-oxadiazole), 55.36 (OCH3). Anal.
Calcd. For C21H16ClFN2O2: C, 65.89; H, 4.21; N, 7.32.
Found: C, 65.53; H, 4.11; N, 7.46.
Biological study
Chemicals and reagent
4-(4-Fluorophenyl)-3,5-bis(4-chlorophenyl)-4,5-dihydro-1,
2,4-oxadiazole (6e-2y) Yield: 70 %. Mp: 107–109 ꢁC.
1HNMR (CDCl3) d: 6.50 (s, 1H, H5-oxadiazole), 6.79–6.81
(m, 2H, H2,6-NAr), 6.92 (t, J = 8.50 Hz, 2H, H3,5-NAr),
7.32 [d, J = 8.50 Hz, 2H, H3,5-Ar3 (H3,5-Ar5)], 7.43 [d,
J = 8.50 Hz, 2H, H3,5-Ar5 (H3,5-Ar3)], 7.33–7.52 (m, 4H,
Dulbecco’s Modified Eagle’s Medium (DMEM) was pur-
chased from Gibco-BRL (Rockville, IN, USA). Fetal
bovine serum (FBS) was supplied by Gibco-BRL. All other
chemicals were from Merck (Darmstadt, Germany) and
Sigma-Aldrich (St Louis, MO, USA).
H
2,6-Ar3 and H2,6-Ar5). 13CNMR (CDCl3) d: 160.87 (d,
Cell culture and in vitro cytotoxicity assay
JCF = 245.0 Hz, C4-NAr), 154.70 (C1-NAr), 136.77, 136.60
[C4-Ar3 (C1-Ar3)], 129.21, 129.02 [C2,6-Ar3 (C3,5-Ar3)],
128.69 (C2,6-Ar5), 126.95 (d, JCF = 8.75 Hz, C2,6-NAr),
Human breast adenocarcinoma (MCF-7) and human ery-
thromyeloblastoid leukemia (K562) cell lines were obtained
from the cell bank of Pasture Institute of Iran (NCBI), and
routinely cultured in DMEM medium supplemented with
10 % FBS, 100-U/mL penicillin and 100-lg/mL strepto-
mycin under the conditions of 5 % CO2 at 37 ꢁC. Cell
cytotoxicity was determined by 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The
MCF-7 (7 9 103) and K562 (2 9 104) cells/well were
cultured in 96-well plates. Then, cells were treated with
various concentrations of each compound (0.1–200 lM)
and incubated for 24 h. Afterward, 20 lL of MTT (5 mg/
mL in PBS) was added to each well and the cells were
incubated for another 4 h at 37 ꢁC. The supernatants were
then aspirated carefully and 200 lL of dimethyl sulfoxide
(DMSO) was added to each well. The plates were shaken
123.75 (C3-oxadiazole), 116.32 (d, JCF = 22.75 Hz, C3,5
-
NAr), 114.24 (C3,5-Ar5),100.94 (C5-oxadiazole), 55.36
(OCH3). Anal. Calcd. For C20H13Cl2FN2O: C, 62.03; H,
3.38; N, 7.23. Found: C, 62.19; H, 3.50; N, 7.46.
Docking study
The molecular geometry of compounds 6e-3y, 6a-1y, 6c-
1y, and 6e-1y which have most prominent biological
results was fully minimized by MMFF94 force field using
ChemBio3D Ultra 12.0 (Cambridgesoft), setting the ter-
minal condition as the RMS of potential energy smaller
-1
than 0.0001 kcal A mol-1. The crystal structure of
˚
cyclo-oxygenase-2 in complex with celecoxib (entry code
123

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Med Chem Res (2013) 22:4253–4262
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read by the Microplate Reader (Star Fax-2100, ST. Louis,
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cell cytotoxicity = [1 - Absdrug/Abscontrol] 9 100.
The IC50 value was measured by plotting the percentage
cytotoxicity versus concentration on a logarithmic graph.
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