Synthesis, molecular modeling and biological evaluation of 2-(benzylthio)-5-aryloxadiazole derivatives as anti-tumor agents

Article abstract of DOI:10.1016/j.ejmech.2011.11.015

A series of 2-(benzylthio)-5-aryloxadiazole derivatives have been designed and synthesized, and their biological activities are also evaluated for EGFR inhibitory activity. Fourteen compounds among the twenty compounds are reported for the first time. Their chemical structures are characterized by ¹H NMR, MS, and elemental analysis. Anti-proliferative and EGFR inhibition assay results have demonstrated that compound 3e shows the most potent biological activity (IC₅₀ = 1.09 μM for MCF-7 and IC₅₀ = 1.51 μM for EGFR). Docking simulation has been performed to position compound 3e into the EGFR active site to determine the probable binding model, with an estimated binding free energy value of -10.7 kcal/mol. Compound 3e with potent inhibitory activity in tumor growth inhibition may be a promising anti-tumor leading compound for the further research.

Full text of DOI:10.1016/j.ejmech.2011.11.015

European Journal of Medicinal Chemistry 47 (2012) 473e478
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, molecular modeling and biological evaluation
of 2-(benzylthio)-5-aryloxadiazole derivatives as anti-tumor agents
*
Kai Liu, Xiang Lu, Hong-Jia Zhang, Juan Sun, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2011
Received in revised form
9 November 2011
Accepted 9 November 2011
Available online 18 November 2011
A series of 2-(benzylthio)-5-aryloxadiazole derivatives have been designed and synthesized, and their
biological activities are also evaluated for EGFR inhibitory activity. Fourteen compounds among the
twenty compounds are reported for the first time. Their chemical structures are characterized by 1H
NMR, MS, and elemental analysis. Anti-proliferative and EGFR inhibition assay results have demonstrated
that compound 3e shows the most potent biological activity (IC50 ¼ 1.09
mM for MCF-7 and
IC50 ¼ 1.51
m
M for EGFR). Docking simulation has been performed to position compound 3e into the EGFR
active site to determine the probable binding model, with an estimated binding free energy value of
ꢀ10.7 kcal/mol. Compound 3e with potent inhibitory activity in tumor growth inhibition may be
a promising anti-tumor leading compound for the further research.
Keywords:
Oxadiazole
Anti-tumor
EGFR
Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
Kinase insert Domain containing Receptor, KDR) kinase inhibitors.
The structure-activity relationships as well as the enzyme binding
The epidermal growth factor receptor (EGFR), a transmembrane
protein tyrosine kinase (PTK) that is activated by ligand-induced
dimerization, plays a critical role in regulating cell proliferation,
differentiation, and migration [1]. The up-regulated activity of EGFR
correlates with many human tumors, including head and neck,
lung, breast, bladder, prostate, and kidney cancers [2e7]. It has
been also observed the overexpression of EGFR in breast cancer,
ovarian cancer and so on [8]. Therefore, EGFR tyrosine kinase
represents an attractive target for the development of novel anti-
cancer agents. In the past few years, large numbers of small organic
molecules have been employed to inhibit kinase function by tar-
geting the ATP binding site of EGFR tyrosine kinase. Gefitinib (Ire-
ssa) and erlotinib (Tarceva) are the representative drugs for this
kind of inhibitors and have been approved by US FDA for treatment
of patients with non small-cell-lung cancer (NSCLC) [9,10].
model are also illustrated. In this model, 2-anilino-5-aryloxazoles
bonds to the ATP binding site of KDR kinase. The central oxazole
ring comprises the core structure of the compound and interacts
with Cys919 of the peptide backbone via hydrogen acceptor
bonding with the oxazole nitrogen [15].
To our knowledge, 2-thiol-5-aryloxadiazoles as a potential anti-
fungal agent attracts much attention. However, there are few
reports involving in the anti-tumor activity, especially for EGFR
inhibitory activity of 2-(benzylthio)-5-aryloxadiazole derivatives.
We have performed a virtual screening of these substituted oxa-
diazole derivatives by AutoDock Tools, including ortho-substitution,
meta-substitution and para-substitution. The results reveal that all
compounds would have potent EGFR inhibitory activity, with an
estimated binding free energy ranging from about ꢀ8 kcal/mol
to ꢀ13 kcal/mol. The corresponding inhibition constant can come
to nM level. Virtual screening results also predict that ortho-
substitution at the 5-phenyl ring would access active site and might
have better EGFR inhibitory activity. As a result, we hope to discover
Compounds containing an oxazole or oxadiazole ring, which is
as a building element can be seen in many natural products, have
various activities such as hypoglycemic [11], anti-fungal [12], anti-
inflammatory [13], and anti-bacterial activities [14]. In the area of
anti-tumor agents, for example, Harris et.al have reported that 2-
a
potential effective compound from these 2-(benzylthio)-5-
aryloxadiazole derivatives that can be as the leading compound
for the further optimal research.
anilino-5-aryloxazoles can be as
a novel class of VEGFR2
(Vascular Endothelial Growth Factor Receptor 2, also called the
Herein, in order to extend our research on anticancer
compounds with EGFR inhibitory activity, a series of 2-(ben-
zylthio)-5-aryloxadiazole derivatives are synthesized and evalu-
ated with EGFR inhibitory activity. Thirteen compounds(3de3j,
3le3n and 3re3t) are reported for the first time, 3a-3c, 3k and
* Corresponding author. Tel./fax: þ86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.015

474
K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 473e478
3oe3q have been published [16,17]. The structure-activity rela-
tionships and possible enzyme binding mode which is predicted by
molecular docking research are also illustrated.
2. Results and discussion
2.1. Chemistry
The general synthesis of various substituted 2-(benzylthio)-5-
phenyl-1,3,4-oxadiazole derivatives were depicted in Scheme 1,
2. Most substituted benzoylhydrazine were obtained from
commercial resource, but a few of them still needed to be prepared
using the published methods from corresponding acids or esters in
about medium yields [16e19]. It was a reaction of two steps to
obtain the key intermediate 1. Firstly, benzoylhydrazine, carbon
disulfide and potassium hydroxide were reacted in 95% ethanol for
24 h, cyclizing to produce potassium salt intermediate. Then the
potassium salt intermediate converted to 1 by adjusting the pH
value. Finally, the direct condensation between 1 and 2 led to the
formation of end product 3ae3t (Table 1) under basic condition in
room temperature with a high yield. However, the total yields
were moderate due to the low yields of intermediate. All of the
synthetic compounds gave satisfactory analytical and spectro-
scopic data, which were in full accordance with their depicted
structures.
Scheme 2. General synthesis of 2-(benzylthio)-5-aryloxadiazole derivatives 3ae3t.
Reagents and conditions: (a) i KOH/CS2, 95% ethanol, reflux, 24h; ii HCl/pH5-6; (b)
Benzyl bromide, NaOH, 80% ethanol, rt, 3h.
cell lines. The IC50 values of most 5-aryloxadiazole derivatives
ranged from 1.09
3e displayed the most potent anti-proliferative activity with IC50 of
1.09 M, comparable to the positive control gefitinib
(IC50 ¼ 12.05 M). Nevertheless, IC50 values would have a 2e18
mM to 15.1 mM in MCF-7. Among them, compound
m
m
folds decrease in A549 and 1e10 folds in B16-F10 on average. More
potent anti-proliferative activity for MCF-7 together with virtual
screening results both indicated that the over-expressed EGFR
might be a potential target which these 5-aryloxadiazole deriva-
tives interacted with.
2.2. The anti-proliferative activity and structure-activity
relationship (SAR) study
Structure-activity relationships at cellular level for these 5-
aryloxadiazole derivatives demonstrated that compounds with
substitution of benzene ring (3ae3p) showed more potent activi-
ties than those with substitution of pyridine ring (3qe3t). For
benzene substituent, substituents at ortho position (3be3g) might
have an increase of activity than those at the para position. In
particular, compound 3e exhibited the most potent anti-
All the compounds 3ae3t were evaluated for cytotoxic proper-
ties on two human tumor cell lines, breast adeno carcinoma cell
line MCF-7 and lung adeno carcinoma cell line A549, one mouse
skin melanoma cell line B16-F10 with gefitinib as a standard. The
inhibitory effects on cell proliferation were determined by MTT
assay [20]. The inhibitory potency (IC50) of compounds 3ae3t were
given in Table 2. The IC50 values were the average of at least three
independent experiments.
As shown in Table 2, it was observed that 5-aryloxadiazole
derivatives had been found to show the inhibition of growth of
three tumor cell lines with moderate IC50 values. For MCF-7 cell line
which over-expressed EGFR that previously references had re-
ported [21], 5-aryloxadiazole derivatives in particular displayed
more potent anti-proliferative activity than the other two tumor
proliferative activity with IC50 value of 1.09 mM. A comparison of
ortho position substitution on benzene ring indicated that a ortho
electron donating group had slightly improved anti-proliferative
activity. Compared to 3a, substitution at the para position led to
Table 1
Structure of 2-(benzylthio)-5-aryloxadiazole derivatives 3ae3t.
Compd.
R
A
B
Compd.
R
A
B
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
2-F
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3k
3l
4-Cl
CH
CH
CH
CH
CH
CH
N
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
2eOH, 4-Me
2eOH, 4-OMe
3,5-di-OMe
4-F
4-NMe2
H
2eNH2
2-Cl
2eNH2
2eCH3
2eNO2
2eNH2
2-OEt
2-OMe
4-t-Bu
4-i-Pr
4eNH2
3m
3n
3o
3p
3q
3r
Scheme 1. General synthesis of substituted benzoylhydrazine from corresponding
substituted acids and esters. Reagents and conditions: (a) 98% H2SO4, methanol, reflux,
6h; (b) 85% NH2NH2$H2O, ethanol, reflux, 3h.
3s
3t
3j

K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 473e478
475
Table 2
Anti-proliferative activity in 3 tumor cell lines and EGFR inhibitory activity of top 10 compounds.
Compd.
IC50
,
mM
Compd.
IC50, mM
MCF-7
A549
B16-F10
EGFR
MCF-7
A549
B16-F10
EGFR
Gefitinib
12.05
3.89
2.11
2.69
5.92
1.09
5.31
8.11
3.29
15.6
38.4
10.08
30.3
22.9
31.2
47.0
18.2
34.0
42.7
18.6
29.7
76.5
e
0.02
16.63
5.11
5.72
38.32
1.51
38.43
12.32
7.44
nt
3a
3b
3c
3d
3e
3f
3g
3h
3i
24.7
23.0
19.9
20.3
10.3
17.9
19.3
9.2
3k
3l
31.7
9.12
15.1
7.81
13.3
39.0
38.5
30.0
29.2
41.8
52.1
29.2
27.9
20.0
30.7
75.5
78.5
68.7
65.6
60.0
21.0
9.0
13.7
6.8
22.0
41.1
48.2
45.1
35.1
44.6
nt
34.25
nt
26.06
nt
nt
nt
nt
nt
nt
3m
3n
3o
3p
3q
3r
8.8
41.3
3s
3t
3j
nt
nt, not test.
a significant loss of activity, whether with substitution of electron
donating groups (3h, 3i) or electron withdrawing (3k, 3o) groups.
In addition, considering the effect of the number of substituents of
benzene ring, we designed and evaluated the compounds with two
substituents at 2,4-position (3l, 3m) or 3,5-position (3n). Never-
theless, there was also no notable improvement compared to the
other mono-substituted derivatives.
In order to gain more understanding of the interaction with
EGFR, molecular docking of the most potent inhibitor 3e into ATP
binding site of EGFR kinase was performed on a binding model
based on the EGFR complex structure (1M17.pdb). The binding
model of compound 3e and EGFR was depicted in Fig. 2.
Previously research had revaled that the binding model of
gefitinib was strikingly similar to erlotinib. Both quinazoline ring of
gefitinib and erlotinib were oriented with the 1-N in the back of the
ATP binding pocket. Interestingly, unlike most kinase inhibitors,
both they formed only a single hydrogen bond. Gefitinib’s hydrogen
bond formed with the mainchain amide of Met 793 while the N1 of
the quinazoline of erlotinib accepted an H-bond from the Met769
amide nitrogen [22,23].
Compared to the binding model of gefitinib and erlotinib,
compound 3e was also projected into the ATP binding pocket with
a quite smooth conformation, and was nicely bound to the EGFR
kinase with its N atom forming a H-bond interaction with Pro770.
This H-bond might be favor to the EGFR inhibitory activity. The 2-
benzyl group orientated toward the front of the pocket. As the
same as the quinazoline ring of erlotinib, the 5-phenyl ring was also
in the back of the pocket and was much close to the active residue
Met769. Met769 was a critical amino residue in the active site
which was previously mentioned for erlotinib. Though direct H-
bond interaction with Met769 was not observed in this model, NH2
group of 5-phenyl ring was such close to this residue that we could
believe that the further modification of this group might bring
improved EGFR inhibitory activity.
2.3. EGFR inhibitory activity and molecular docking study
Based on the potent anti-proliferative activity in MCF-7 cell line,
furthermore, we had selected the top 10 compounds (3ae3h, 3l,
3n), which had the best anti-proliferative activity results among the
20 compounds for MCF-7, and tested their EGFR inhibitory activity
to get an insight whether these compounds acted through EGFR-
TKI pathway. As shown in Table 2, all compounds had EGFR
inhibitory activity. Compound 3e, in particular, exhibited the most
potent inhibitory activity consistent with anti-proliferative assay
results (IC50 ¼ 1.51
mM). Then, an analysis between EGFR enzyme
inhibition and inhibition of MCF-7 cellular proliferation of the top
10 compounds indicated that there was a moderate correlation
between EGFR enzyme inhibition and inhibition of MCF-7 cellular
proliferation, as evidenced in Fig. 1, with a correlation coefficient of
0.72, R square value 0.805. However, a discrepancy was also
observed between enzyme inhibition and cell anti-proliferation
that the anti-proliferative activity of these dioxazole derivatives
could be comparable to gefinitib but EGFR inhibitory activity is
much less than it. This might suggested that they were acting as
multi-kinase inhibitor, EGFR was one of their targets. In general,
these compounds could inhibit EGFR enzyme, subsequently
inhibited the proliferation of MCF-7.
Fig. 2. Molecular docking modeling of compound 3e with EGFR kinase shows intra-
molecular hydrogen bonds with Pro770. Note: For clarity, only interacting residues are
displayed. The H-bond is displayed as dot line.
Fig. 1. Correlation between -pIC50’s for the inhibition of EGFR enzyme and MCF-7
cellular proliferation, r ¼ 0.72, R2 ¼ 0.805.

476
K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 473e478
3. Conclusions
water, filtered and recrystallized from ethanol, washed with cool
ethanol for three times.
A series of 2-(benzylthio)-5-aryloxadiazole derivatives have
been designed and synthesized, and their biological activities have
also been evaluated as potent EGFR inhibitor. Most compounds
showed moderate inhibition against MCF-7, A549 and B16-F10 cell
lines. Compound 3e demonstrated the most potent anti-
proliferative activity and EGFR inhibitory activity. Docking simu-
lation result shows compound 3e was stabilized by hydrogen
bonding interactions. In general, our current findings regarding
EGFR tyrosine kinase inhibition of compounds containing (ben-
zylthio)oxadiazole framework are completely new. This study
further presents (benzylthio)oxadiazole framework as new class of
anti-tumor agents and it may serve as model compounds for design
and development of therapeutic based anticancer agents.
4.3.1. 2-(Benzylthio)-5-phenyl-1,3,4-oxadiazole (3a)
White powder. Yield, 95%. Mp 94e95 ꢁC. 1H NMR (300 MHz,
CDCl3):
d 4.53 (s, 2H, CH2), 7.30e7.38 (m, 3H), 7.45e7.53 (m, 5H),
7.97e8.00 (m, 2H). MS (ESI): 268.1 (C15H12N2OS [M þ H]þ). Anal.
Calcd for C15H11N2OS: C, 67.14; H, 4.51; N, 10.44. Found: C, 67.36; H,
4.63; N, 10.73%.
4.3.2. 2-(Benzylthio)-5-(2-fluorophenyl)-1,3,4-oxadiazole (3b)
White crystal. Yield, 55%. Mp 61e62 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
4.54 (s, 2H, CH2), 7.22 (d, J ¼ 8.5 Hz,1H), 7.28e7.36 (m, 4H),
7.46 (d, J ¼ 7.2 Hz, 2H), 7.50e7.54 (m, 1H), 7.98e8.01 (m, 1H). MS
(ESI): 286.1 (C15H11FN2OS [M þ H]þ). Anal. Calcd for C15H10FN2OS:
C, 62.92; H, 3.87; N, 9.78. Found: C, 63.06; H, 4.03; N, 9.53%.
4. Experimental section
4.3.3. 2-(Benzylthio)-5-o-tolyl-1,3,4-oxadiazole (3c)
White powder. Yield, 70%. Mp 105e106 ꢁC. 1H NMR (300 MHz,
4.1. General
CDCl3):
d 2.68 (s, 3H), 4.53 (s, 2H, CH2), 7.30e7.40 (m, 6H), 7.47 (d,
J ¼ 6.2 Hz, 2H), 7.84 (d, J ¼ 6.75 Hz,1H). MS (ESI): 282.1 (C16H14N2OS
[M þ H]þ). Anal. Calcd for C16H13N2OS: C, 68.06; H, 5.00; N, 9.92.
Found: C, 67.86; H, 5.13; N, 9.73%.
All commercially available reagents were obtained from various
producers and used without further purification. The reactions
were monitored and the purity of the compounds was checked by
thin layer chromatography (TLC) using aluminum sheets with Silica
Gel 60 F254 (Merck). Melting points (uncorrected) were determined
on a XT4MP apparatus (Taike Corp., Beijing, China). ESI mass
spectra were obtained on a Mariner System 5304 mass spectrom-
eter, and 1H NMR spectra were recorded on a Bruker DPX300
spectrometer at 25 ꢁC with TMS and solvent signals allotted as
4.3.4. 2-(Benzylthio)-5-(2-nitrophenyl)-1,3,4-oxadiazole (3d)
Yellow crystal. Yield, 90%. Mp 120e121 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
4.52 (s, 2H, CH2), 7.30e7.38 (m, 3H), 7.45 (d, J ¼ 7.68 Hz,
2H), 7.70e7.78 (m, 2H), 7.92e7.95 (m, 1H), 8.01e8.04 (m, 1H). MS
(ESI): 313.1 (C15H11N3O3S [M þ H]þ). Anal. Calcd for C15H10N3O3S: C,
57.50; H, 3.54; N, 13.41. Found: C, 57.66; H, 3.68; N, 13.63%.
internal standards. Chemical shifts were reported in ppm (d).
Elemental analyses were performed on a CHN-O-Rapid instrument
and were within ꢂ0.4% of the theoretical values.
4.3.5. 2-(5-(benzylthio)-1,3,4-oxadiazol-2-yl)aniline (3e)
White powder. Yield, 95%. Mp 86e87 ꢁC. 1H NMR (300 MHz,
CDCl3):
d 4.52 (s, 2H, CH2), 5.77(brs, 2H), 6.71e6.79(m, 2H),
4.2. General procedure for the synthesis of 5-phenyl-1,3,4-
oxadiazole-2-thiol derivatives
7.29e7.39 (m, 4H), 7.46(d, J ¼ 7.5 Hz, 2H), 7.65(d, J ¼ 7.86 Hz,1H). MS
(ESI): 283.1 (C15H13N3OS [M þ H]þ). Anal. Calcd for C15H12N3OS: C,
63.58; H, 4.62; N, 14.83. Found: C, 63.71; H, 4.68; N, 14.63%.
For the synthesis of substituted benzoylhydrazines, a mixture of
corresponding esters (20 mmol), 85% hydrazine hydrate (20 mmol)
in ethanol (35 ml) was heated to reflux for 5 h. After that, the
solution was poured into ice-water. The precipitate was filtered and
crystallized from ethanol.
Then equimolar portions of the appropriately substituted ben-
zoylhydrazine (5 mmol) and potassium hydroxide (5 mmol) were
dissolved in 20 mL of 95% ethanol. The mixture was allowed to stir
for several minutes at room temperature and then carbon disulfide
(7.5 mmol) was slowly added dropwise to the reaction system via
a self-equalizing addition funnel while the mixture was heated to
reflux. After 24 h, the solvent was completely removed under
reduced pressure. The residue obtained was dissolved in water
(50 mL) and diluted hydrochloric acid was added to adjust the pH
values of the solution to 5e6. Then the precipitate was collected by
filtering under reduced pressure, washed with water for several
times and dried without further purification.
4.3.6. 2-(Benzylthio)-5-(2-ethoxyphenyl)-1,3,4-oxadiazole (3f)
Light yellow powder. Yield, 93%. Mp 69e70 ꢁC. 1H NMR
(300 MHz, CDCl3):
J ¼ 6.93 Hz, 2H), 4.52 (s, 2H, CH2), 7.03 (t, J ¼ 8.05 Hz, 2H), 7.29e7.37
(m, 3H), 7.43e7.49 (m, 3H), 7.86e7.89 (m, 1H). MS (ESI): 312.1
(C17H16N2O2S [M þ H]þ). Anal. Calcd for C17H15N2O2S: C, 65.36; H,
5.16; N, 8.97. Found: C, 65.21; H, 5.28; N, 8.78%.
d
1.47 (t, J ¼ 6.94 Hz, 3H), 4.13e4.20 (q,
4.3.7. 2-(Benzylthio)-5-(2-methoxyphenyl)-1,3,4-oxadiazole (3g)
White powder. Yield, 80%. Mp 48e50 ꢁC. 1H NMR (300 MHz,
CDCl3):
7.29e7.37 (m, 3H), 7.46e7.52 (m, 3H), 7.84e7.87 (m, 1H). MS (ESI):
298.1 (C16H14N2O2S [M þ H]þ). Anal. Calcd for C16H13N2O2S: C,
64.41; H, 4.73; N, 9.39. Found: C, 64.23; H, 4.84; N, 9.58%.
d
3.95 (s, 3H), 4.52(s, 2H, CH2), 7.06 (t, J ¼ 7.41 Hz, 2H),
4.3.8. 2-(Benzylthio)-5-(4-tert-butylphenyl)-1,3,4-oxadiazole (3h)
White powder. Yield, 52%. Mp 66e67 ꢁC. 1H NMR (300 MHz,
4.3. General procedure for the synthesis of 2-(benzylthio)-5-
CDCl3): d 1.35 (s, 9H), 4.52 (s, 2H, CH2), 7.29e7.37 (m, 3H), 7.44e7.47
phenyl-1,3,4-oxadiazole derivatives (3ae3t)
(m, 2H), 7.50 (d, J ¼ 8.4 Hz, 2H), 7.91 (d, J ¼ 8.43 Hz, 2H). MS (ESI):
324.1 (C19H20N2OS, [M þ H]þ). Anal. Calcd for C19H19N2OS: C, 70.34;
H, 6.21; N, 8.63. Found: C, 70.48; H, 6.35; N, 8.51%.
The synthesis of all compounds adopted the following proce-
dure: to a solution of substituted 5-phenyl-1,3,4-oxadiazole-2-thiol
(1 mmol) in 80% ethanol benzyl bromide (1.1 mmol) was added
slowly and stirred at room temperature for 10 min, then 5 mL (8%,
w/w) sodium hydroxide solution was added dropwise to the
reaction mixture, stirred for another 3 h [16]. Most commonly,
a precipitate was formed when the solution was poured into ice-
4.3.9. 2-(Benzylthio)-5-(4-isopropylphenyl)-1,3,4-oxadiazole (3i)
Yellow powder. Yield, 53%. Mp 49e50 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
1.28 (d, J ¼ 6.93 Hz, 6H), 2.94e2.99 (m, 1H), 4.52 (s, 2H,
CH2), 7.29e7.35 (m, 5H), 7.45 (d, J ¼ 7.68 Hz, 2H), 7.91 (d, J ¼ 8.25 Hz,
2H). MS (ESI): 310.1 (C18H18N2OS [M þ H]þ). Anal. Calcd for

K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 473e478
477
C18H17N2OS: C, 69.65; H, 5.84; N, 9.02. Found: C, 69.78; H, 5.95; N,
8.89%.
4.3.18. 4-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)pyridin-2-amine
(3r)
Light yellow powder. Yield, 72%. Mp 187e188 ꢁC. 1H NMR
(300 MHz, CDCl3): 4.54 (s, 2H, CH2), 4.66 (s, 2H), 7.05 (s, 1H),
7.17e7.19 (m, 1H), 7.30e7.38 (m, 3H), 7.44e7.47 (m, 2H), 8.21 (d,
J ¼ 5.31 Hz, 1H). MS (ESI): 284.1 (C14H12N4OS [M þ H]þ). Anal. Calcd
for C14H11N4OS: C, 59.14; H, 4.25; N, 19.70. Found: C, 59.35; H, 4.35;
N, 19.49%.
4.3.10. 4-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)aniline (3j)
d
Yellow powder. Yield, 85%. Mp 123e124 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
4.49 (s, 2H, CH2), 6.71 (d, J ¼ 8.79 Hz, 2H), 7.28e7.37 (m,
3H), 7.45 (d, J ¼ 6.39 Hz, 2H), 7.78 (d, J ¼ 6.75 Hz, 2H). MS (ESI):
283.1 (C15H13N3OS [M þ H]þ). Anal. Calcd for C15H12N3OS: C, 63.58;
H, 4.62; N, 14.83. Found: C, 63.78; H, 4.75; N, 14.99%.
4.3.19. 2-(Benzylthio)-5-(2-chloropyridin-4-yl)-1,3,4-oxadiazole
(3s)
4.3.11. 2-(Benzylthio)-5-(4-chlorophenyl)-1,3,4-oxadiazole (3k)
White powder. Yield, 78%. Mp 119e120 ꢁC. 1H NMR (300 MHz,
White powder. Yield, 98%. Mp 80e81 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
4.53 (s, 2H, CH2), 7.30e7.38 (m, 3H), 7.45e7.49 (m, 4H),
CDCl3): d 4.56 (s, 2H, CH2), 7.32e7.39 (m, 3H), 7.45e7.48 (m, 2H),
7.91e7.94 (m, 2H). MS (ESI): 302.1 (C15H11ClN2OS [M þ H]þ). Anal.
Calcd for C15H10ClN2OS: C, 59.50; H, 3.66; N, 9.25. Found: C, 59.65;
H, 3.75; N, 9.09%.
7.77e7.79 (m, 1H), 7.87 (s, 1H), 8.56 (d, J ¼ 5.1 Hz, 1H). MS (ESI):
303.1 (C14H10ClN3OS [M þ H]þ). Anal. Calcd for C14H9ClN3OS: C,
55.35; H, 3.32; N, 13.83. Found: C, 59.48; H, 3.45; N, 13.59%.
4.3.12. 2-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)-5-methylphenol
(3l)
4.3.20. 5-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)pyridin-2-amine
(3t)
White powder. Yield, 97%. Mp 93e94 ꢁC. 1H NMR (300 MHz,
Yellow powder. Yield, 76%. Mp 156e158 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
2.37 (s, 3H), 4.52 (s, 2H, CH2), 6.80 (d, J ¼ 8.04 Hz, 1H), 6.92
CDCl3):
d
4.50 (s, 2H, CH2), 4.83 (s, 2H), 6.55 (d, J ¼ 8.79 Hz, 1H),
(s, 1H), 7.30e7.39 (m, 3H), 7.46 (d, J ¼ 6.39 Hz, 2H), 7.55 (d,
J ¼ 8.04 Hz, 1H), 9.80 (s, 1H). MS (ESI): 298.1 (C16H14N2O2S,
[M þ H]þ). Anal. Calcd for C16H13N2O2S: C, 64.41; H, 4.73; N, 9.39.
Found: C, 64.58; H, 4.85; N, 9.19%.
7.27e7.39 (m, 3H), 7.44 (d, J ¼ 7.68 Hz, 2H), 7.98e8.02 (m, 1H), 8.65
(s, 1H). MS (ESI): 284.1 (C14H12N4OS [M þ H]þ). Anal. Calcd for
C14H11N4OS: C, 59.14; H, 4.25; N, 19.70. Found: C, 59.27; H, 4.38; N,
19.55%.
4.3.13. 2-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)-5-methoxyphenol
(3m)
4.4. Anti-proliferative assay
Colorless crystal. Yield, 91%. Mp 139e140 ꢁC. 1H NMR (300 MHz,
The anti-proliferative activities of compounds 3ae3t were
determined using a standard (MTT)-based colorimetric assay
(Sigma). Briefly, cell lines were seeded at a density of 7 ꢃ 103 cells/
well 96-well microtiter plates (Costar). After 24 h, exponentially
growing cells were exposed to the indicated compounds at final
concentrations ranging from 0.1 to 100
survival was determined by the addition of an MTT solution (10
of 5 mg/mL MTT in PBS). After 4 h, 100 L of 10% SDS in 0.01 N HCl
CDCl3):
d 3.84 (s, 3H), 4.51 (s, 2H, CH2), 6.53e6.57 (m, 1H), 6.60 (s,
1H), 7.31e7.36 (m, 3H), 7.44e7.47 (m, 2H), 7.56 (d, J ¼ 8.79 Hz, 1H),
10.00 (s, 1H). MS (ESI): 314.1 (C16H14N2O3S [M þ H]þ). Anal. Calcd
for C16H13N2O3S: C, 61.13; H, 4.49; N, 8.91. Found: C, 61.38; H, 4.35;
N, 9.09%.
mg/mL. After 48 h, cell
mL
4.3.14. 2-(Benzylthio)-5-(3,5-dimethoxyphenyl)-1,3,4-oxadiazole
(3n)
m
was added, and the plates were incubated at 37 ꢁC for a further
18 h; optical absorbance was measured at 570 nm on an LX300
Epson Diagnostic microplate reader. Survival ratios are expressed in
percentages with respect to untreated cells. IC values were deter-
mined from replicates of six wells from at least two independent
experiments.
White powder. Yield, 93%. Mp 109e110 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
3.85 (s, 6H), 4.52 (s, 2H, CH2), 6.60 (t, J ¼ 2.38 Hz, 1H), 7.13
(d, J ¼ 2.19 Hz, 2H), 7.30e7.38 (m, 3H), 7.45e7.47 (m, 2H). MS (ESI):
328.1 (C17H16N2O3S [M þ H]þ). Anal. Calcd for C17H15N2O3S: C,
62.18; H, 4.91; N, 8.53. Found: C, 62.35; H, 4.75; N, 8.69%.
4.3.15. 2-(Benzylthio)-5-(4-fluorophenyl)-1,3,4-oxadiazole (3o)
Colorless crystal. Yield, 60%. Mp 96e97 ꢁC. 1H NMR (300 MHz,
4.5. EGFR inhibitory assay
CDCl3):
d
4.53 (s, 2H, CH2), 7.18 (t, J ¼ 8.6 Hz, 2H), 7.30e7.38 (m, 3H),
A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain (EGFR-
CD, amino acids 645e1186) were cloned into baculoviral expression
vector pFASTBacHTc. A sequence that encodes (His)6 was located at
the 50 upstream to the EGFR sequence. Sf-9cells were infected for 3
days for protein expression. Sf-9 cell pellets were solubilized at 0 ꢁC
in a buffer at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1%
7.44e7.47 (m, 2H), 7.97e8.01 (m, 2H). MS (ESI): 286.1 (C15H11FN2OS
[M þ H]þ). Anal. Calcd for C15H10FN2OS: C, 62.92; H, 3.87; N, 9.78.
Found: C, 62.72; H, 4.01; N, 9.59%.
4.3.16. 4-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)-N,N-
dimethylaniline (3p)
Triton, 10
m
M ammonium molybdate 100
g/mL aprotinin, 10 g/mL leupeptin, 10
mg/mL benzamidine HCl for 20 min followed by 20 min centri-
m
M sodium vanadate,
Yellow crystal. Yield, 88%. Mp 158e159 ꢁC. 1H NMR (300 MHz,
10
16
m
m
mg/mL pepstatin, and
CDCl3):
d
3.06 (s, 6H), 4.53 (s, 2H, CH2), 6.56 (d, J ¼ 8.79 Hz, 1H), 6.79
(s, 2H), 7.29e7.36 (m, 3H), 7.45 (d, J ¼ 6.78 Hz, 2H), 7.83e7.86 (m,
1H). MS (ESI): 311.1 (C17H17N3OS [M þ H]þ). Anal. Calcd for
C17H16N3OS: C, 65.57; H, 5.50; N, 13.49. Found: C, 65.72; H, 4.91; N,
13.69%.
fugation. Crude extract supernatant was passed through an equil-
ibrated Ni-NTA superflow packed column and washed with 10 mM
and then100 mM imidazole to remove nonspecifically bound
material. Histidine tagged proteins were eluted with 250 and
500 mM imidazole and dialyzed against 50 mM NaCl, 20 mM
4.3.17. 2-(Benzylthio)-5-(pyridin-4-yl)-1,3,4-oxadiazole (3q)
Dark green crystal. Yield, 65%. Mp 103e104 ꢁC. 1H NMR
HEPES, 10% glycerol, and 1 mg/mL each of aprotinin, leupeptin, and
pepstatin for 2 h. The entire purification procedure was performed
(300 MHz, CDCl3):
d 4.56 (s, 2H, CH2), 7.31e7.39 (m, 3H), 7.45e7.48
at 4 ꢁC or on ice.
(m, 2H), 7.83e7.85 (m, 2H). MS (ESI): 269.1 (C14H11N3OS [M þ H]þ).
Anal. Calcd for C14H10N3OS: C, 62.43; H, 4.12; N, 15.60. Found: C,
62.25; H, 4.25; N, 15.79%.
The EGFR kinase assay was set up to assess the level of auto-
phosphorylation based on DELFIA/Time-Resolved Fluorometry.
Compounds 3ae3g, 3l, 3n were dissolved in 100% DMSO and

478
K. Liu et al. / European Journal of Medicinal Chemistry 47 (2012) 473e478
diluted to the appropriate concentrations with 25 mM HEPES at pH
7.4. In each well, 10 L of compound was incubated with 10 L (5 ng
for EGFR) of recombinant enzyme (1:80 dilution in 100 mM HEPES)
for 10 min at room temperature. Then, 10 L of 5 mM buffer
(containing 20 mM HEPES, 2 mM MnCl2, 100 M Na3VO4, and 1 mM
DTT) and 20 L of 0.1 mM ATP-50 mM MgCl2 was added for 1 h.
Positive and negative controls were included in each plate by
incubation of enzyme with or without ATP-MgCl2. At the end of
incubation, liquid was aspirated, and plates were washed three
Acknowledgments
m
m
This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239).
m
m
m
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