Synthesis, molecular modeling and biological evaluation of 2-aminomethyl-5-(quinolin-2-yl)-1,3,4-oxadiazole-2(3H)-thione quinolone derivatives as novel anticancer agent

Article abstract of DOI:10.1016/j.ejmech.2012.11.039

A series of quinoline derivatives (4a-4o) have been synthesized and their biological activities were also evaluated as potential telomerase inhibitors. Bioassay tests demonstrated that most of the compounds exhibited substantial broad-spectrum antitumor a

Full text of DOI:10.1016/j.ejmech.2012.11.039

European Journal of Medicinal Chemistry 60 (2013) 23e28
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-aminomethyl-5-
(quinolin-2-yl)-1,3,4-oxadiazole-2(3H)-thione quinolone derivatives as novel
anticancer agent
*
Juan Sun, Hui Zhu, Zhong-Ming Yang, Hai-Liang Zhu
School of Life Sciences, Shandong University of Technology, Shandong 255049, 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:
A series of quinoline derivatives (4ae4o) have been synthesized and their biological activities were also
evaluated as potential telomerase inhibitors. Bioassay tests demonstrated that most of the compounds
exhibited substantial broad-spectrum antitumor activity against the three cancer cell lines (HepG2, SGC-
7901 and MCF-7). Moreover, all the title compounds were assayed for telomerase inhibition using the
TRAP-PCR-ELISA assay. Compounds 4d and 4i displayed the most potent anticancer activities, which
were comparable to the positive control. Docking simulation was performed to position compounds 4d
and 4i into the telomerase structure active site to determine the probable binding model. Compounds 4d
and 4i with potent inhibitory activity in tumor growth inhibition may be potential anticancer agents.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 15 May 2012
Received in revised form
16 October 2012
Accepted 26 November 2012
Available online 4 December 2012
Keywords:
Quinoline
1,3,4-Oxadiazole
Antiproliferative
Telomerase inhibitor
Molecular docking
1. Introduction
particularly, among these, a few differently substituted 1,3,4-
oxadiazoles have exhibited potent antitumor activities [19e21].
Cancer, second cause of mortality in the world, is continuing to
be a major health problem in developing as well as undeveloped
countries [1]. Although the overall survival rate of cancer patients
has substantially increased during the last decades, this is mainly
due to early tumor detection [2e4]. In normal human somatic cells,
critically short telomeres are suggested to cause irreversible cell
growth arrest and cellular senescence [5]. In contrast, most human
cancer cells have mechanisms that compensate for telomerase
shortening, mainly through the activation of telomerase [6]. Telo-
merase can allow them to stably maintain their telomeres and grow
permanently. The essential role of telomerase in cancer and aging
makes it an important target for the development of therapies to
treat cancer and other age-associated disorders.
Besides, quinoline derivatives have attracted significant interest in
pharmaceutical field as anticancer agents [22e25].
Therefore, in this paper, we described the synthesis and the
structural relationships (SAR) of a series of novel quinoline analogs
possessing 1,3,4-oxadiazole moiety as potential antitumor agents,
which were based on molecular modeling and the investigation of
SAR between new inhibitors and the X-ray crystallographic struc-
ture of the telomerase. To the best of our knowledge, the synthesis
and anticancer activities of these compounds have not been
reported so far.
2. Results and discussion
Oxadiazole derivatives play a significant role in various phar-
maceutical applications [7e11]. As an important class of hetero-
cyclic compound, 1,3,4-oxadiazoles show a broad spectrum of
bioactivities [12e17]. Further, 1,3,4-oxadiazole heterocycles are
very good bioisosteres of amides and esters, which can contribute
substantially in increasing pharmacological activity by partici-
pating in hydrogen bonding interactions with the receptors [18]. In
2.1. Chemistry
Nineteen quinoline analogs were synthesized to screen for the
antitumor activity. All of them were synthesized for the first time.
The synthesis of compounds 4ae4o followed the general pathway
outlined in Scheme 1. The key compound 5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione 3 was prepared as previously described
[26,27]. Firstly, the quinoline-2-carboxylic acid on treatment with
methanol containing concentrated H2SO4 was refluxed overnight.
This step can yield the corresponding ester. Secondly, the ester was
treated with hydrazine hydrate in ethanol overnight, refluxing.
* Corresponding author. Tel.: þ86 25 8359 2572; fax: þ86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.039

24
J. Sun et al. / European Journal of Medicinal Chemistry 60 (2013) 23e28
Scheme 1. General synthesis of compounds 4ae4o. Reagents and conditions: (a) 85% NH2NH2$H2O, EtOH, reflux, 8 h; (b) CS2/KOH, ethanol (95%), reflux, 24 h; (c) HCl, pH ¼ 5e6;
(d) HCHO 40%, different amines.
Thirdly, treatment of the hydrazide 2 with carbon disulfide in the
presence of 95% ethanol and potassium hydroxide provided the key
intermediate 3. Then, compounds 4ae4o were obtained by treated
with different primary and secondary amines in the presence of
formaldehyde in anhydrous ethanol.
All of the synthetic compounds gave satisfactory analytical and
spectroscopic data, which were full accordance with their depicted
structures.
the tested compounds displayed a potent telomerase inhibitory
effect. Among them, compound 4d and 4i showed the most potent
inhibitory with IC50 ranging from 0.8 to 0.9
mM. The results of
telomerase inhibitory activity of the tested compounds correlated
with the structural relationships (SAR) of their inhibitory effects on
the cell proliferation assay. This suggests that the potent inhibitory
effects of the synthetic compounds on the cell proliferation assay
were causally related to their telomerase inhibitory activities.
2.3. Binding model of compounds into telomerase structure
2.2. Biological activity
In an effort to elucidate the possible mechanism by which the
title compounds can induce anticancer activity and guide further
SAR studies, molecular docking of the potent inhibitors 4d and 4i
2.2.1. Cell proliferation assay (MTT method)
All the synthesized derivatives 4ae4o were evaluated for their
antiproliferative activity against HepG2 (human hepatoma cells),
SGC-7901 (human gastric cancer cells) and MCF-7 (human breast
cancer cells) cell lines. The results were shown in Table 1. As evi-
denced from Table 1, in general our compounds showed comparable
activities to 5-Fluorouracil (5-Fu), with the exception of compounds
4de4f, 4i and 4l that showed a greater anticancer activity against
one or more of the cell lines used. Most significantly, compounds 4d
and 4i displayed the most potent anticancer activities.
Structureeactivity relationships in these oxadiazole derivatives
demonstrated that compounds having halogen atom substituent
exhibited high activity, ranging from 0.8 to 26.9 mM, with regard to
broad-spectrum antitumor activity. Meanwhile, a comparison of the
substitution on the benzene ring was demonstrated as follows:
when the compounds were fluorine-substituted derivatives, the
potency order was ortho > meta > para, while when the compounds
were chlorine-substituted derivatives, the potency order was
para > meta > ortho. Interestingly, among the halogen-substituent
compounds, the potency order was fluorine > chlorine > bromine.
Table 1
In vitro anticancer activities (IC50, mM) of compounds 4ae4o against human tumor
cell lines.
Compounds
IC50 (mM)
HepG2
SGC-7901
MCF-7
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
35.6 ꢀ 2.3
29.8 ꢀ 1.8
13.7 ꢀ 1.1
1.2 ꢀ 0.2
41.8 ꢀ 3.0
32.7 ꢀ 1.7
31.1 ꢀ 2.0
8.3 ꢀ 1.6
35.8 ꢀ 2.5
20.2 ꢀ 1.3
19.3 ꢀ 0.8
6.8 ꢀ 0.5
11.3 ꢀ 1.5
17.3 ꢀ 2.0
25.7 ꢀ 1.9
18.1 ꢀ 2.1
0.8 ꢀ 0.2
12.9 ꢀ 1.3
18.5 ꢀ 0.8
26.9 ꢀ 2.3
17.7 ꢀ 1.6
7.6 ꢀ 1.0
15.0 ꢀ 0.9
20.7 ꢀ 1.4
24.8 ꢀ 2.8
16.8 ꢀ 2.3
7.1 ꢀ 0.8
30.7 ꢀ 2.5
28.1 ꢀ 2.3
20.6 ꢀ 0.5
27.6 ꢀ 1.9
24.9 ꢀ 1.8
15.2 ꢀ 1.3
21.9 ꢀ 1.4
45.5 ꢀ 2.9
40.8 ꢀ 2.5
22.7 ꢀ 1.7
40.3 ꢀ 2.8
39.6 ꢀ 2.7
34.8 ꢀ 2.5
28.5 ꢀ 2.0
34.6 ꢀ 1.7
22.1 ꢀ 1.9
15.3 ꢀ 1.2
24.8 ꢀ 2.2
20.5 ꢀ 1.8
18.9 ꢀ 1.7
17.2 ꢀ 1.5
2.2.2. Telomerase inhibitory assay
The telomerase inhibitory potency of the quinoline derivatives
was examined and the results were summarized in Table 2. Most of
5-Fluorouracila
a
Used as a positive control.

J. Sun et al. / European Journal of Medicinal Chemistry 60 (2013) 23e28
25
Table 2
compounds having fluoro substituent at ortho position (4d) and
having chloro substituent at the para position (4i) of anilines dis-
played the most potent anticancer activities. Docking simulation
was performed to position compounds 4d and 4i into the active site
of telomerase (3DU6) to determine the probable binding model.
Antiproliferative and enzyme assay results suggested that
compounds 4d and 4i were potential antitumor agents. Consid-
ering the results mentioned above, it could be concluded that some
quinoline derivatives are good candidates for antitumor agents
screening and research. The template quinoline with 1,3,4-
oxadiazole moiety was suitable to reconstruct and design for
development of more potential therapeutic drugs against cancer.
Further research would be ongoing for the synthesis and discovery
of more enhanced quinoline derivatives with outstanding anti-
cancer activity and low toxicity and side-effects.
Telomerase inhibitory activity of compounds 4ae4o.
Compounds
Telomerase inhibitation
IC50
M) ꢀ SD
(m
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
12.8 ꢀ 0.5
7.3 ꢀ 0.3
2.8 ꢀ 0.2
0.8 ꢀ 0.1
1.1 ꢀ 0.3
3.1 ꢀ 0.8
5.5 ꢀ 0.7
3.0 ꢀ 0.1
0.9 ꢀ 0.0
10.8 ꢀ 0.5
9.3 ꢀ 0.2
5.1 ꢀ 0.4
3.1 ꢀ 0.4
2.9 ꢀ 0.3
4.8 ꢀ 0.1
8.3 ꢀ 0.8
Staurosporinea
4. Experimental
a
Used as a positive control.
4.1. Chemistry
All chemicals and reagents used in the current study were of
analytical grade. The reactions were monitored by thin layer
chromatography (TLC) on Merck pre-coated silica GF254 plates.
Melting points (uncorrected) were determined on a XT4MP appa-
ratus (Taike Corp., Beijing, China). ESI mass spectra were obtained
on a Mariner System 5304 mass spectrometer, and 1H NMR spectra
into the binding site of telomerase were performed on the binding
model based on the telomerase complex structure (3DU6, pdb).
Binding models of compounds 4d and 4i and telomerase were
depicted in Figs. 1 and 2. In the binding model, compound 4d is
nicely bound to the telomerase with its nitrogen atom of the
quinoline ring project toward the amino hydrogen of ARG 194, with
the hydroxyl group forming a more optimal H-bond interaction.
Besides, both the fluorine atom of the benzene ring with amino
hydrogen of LYS 189 and the nitrogen atom of NH with an oxygen
atom of ASP 343 have optimal H-bond interactions. Compound 4i is
nicely bound to the telomerase with its nitrogen atom of oxadiazole
ring project toward the amino hydrogen of GLN 308, with the
hydroxyl group forming a more optimal H-bond interaction.
Besides, oxygen atom of oxadiazole ring project toward the amino
hydrogen of ARG 194, with the hydroxyl group forming a more
were collected on
temperature with TMS and solvent signals allotted as internal
standards. Chemical shifts are reported in ppm ( ). Elemental
a Bruker DPX300 spectrometer at room
d
analyses were performed on a CHN-O-Rapid instrument, and were
within ꢀ0.4% of the theoretical values.
4.1.1. General procedure for the preparation of target compounds
4ae4o
Formaldehyde (40%,1.5 mmol) was added to a stirred solution of
5-(quinolin-2-yl)-1,3,4-oxadiazole-2(3H)-thione 3 (1.5 mmol) in
anhydrous ethanol (20 mL). Anhydrous ethanol (5 mL) containing
the appropriate amine (1.5 mmol) was added dropwise to the
reaction mixture stirred for 10e15 h and then left overnight at
room temperature. The resulting solid was collected and washed
with cold ethanol, dried and crystallized from anhydrous ethanol to
get the desired compounds 4ae4o.
optimal H-bond interaction as well. Meanwhile, p-cation interac-
tion was shown as orange line (in the web version).
3. Conclusion
A
series of 5-(quinolin-2-yl)-1,3,4-oxadiazole-2(3H)-thione
quinoline derivatives have been synthesized and evaluated for their
antitumor activities against HepG2, SGC-7901 and MCF-7 cell lines.
Preliminary results showed that most of the compounds displayed
enhanced inhibitory activities. Of all the studied compounds,
4.1.1.1. 3-((Phenylamino)methyl)-5-(quinolin-2-yl)-1,3,4-oxadiazole-
2(3H)-thione (4a). White crystals, yield: 72%, mp: 153e155 ꢁC. 1H
NMR (500 MHz, CDCl3) d: 5.27 (brs, 1H), 5.66 (s, 2H), 6.85 (t,
Fig. 1. Molecular docking modeling of compound 4d with telomerase: for clarity, only interacting residues are displayed. Left: 3D model of the interaction between compound 4d
and the telomerase binding site. Right: 2D model of the interaction between compound 4d and the telomerase binding site.

26
J. Sun et al. / European Journal of Medicinal Chemistry 60 (2013) 23e28
Fig. 2. Molecular docking modeling of compound 4i with telomerase: for clarity, only interacting residues are displayed. Left: 3D model of the interaction between compound 4i
and the telomerase binding site. The -cation interactions are shown as orange lines. Right: 2D model of the interaction between compound 4i and the telomerase binding site. The
-cation interactions are shown as orange line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
p
p
J ¼ 7.5 Hz, 1H), 6.98 (d, J ¼ 7.5 Hz, 2H), 7.25 (t, J ¼ 7.5 Hz, 2H), 7.69 (t,
J ¼ 7.5 Hz, 1H), 7.84 (t, J ¼ 7.5 Hz, 1H), 7.91 (d, J ¼ 8.0 Hz, 1H), 8.03 (d,
J ¼ 8.5 Hz, 1H), 8.29 (d, J ¼ 8.5 Hz, 1H), 8.35 (d, J ¼ 8.5 Hz, 1H). 13C
4.1.1.5. 3-(((3-Fluorophenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4e). White powder, yield: 73%, mp: 191e
193 ꢁC. 1H NMR (300 MHz, CDCl3)
d
: 5.31 (t, J ¼ 8.0 Hz, 1H), 5.60 (d,
NMR (300 MHz, CDCl3)
d: 177.4, 150.3, 150.2, 149.6, 148.3, 137.4,
J ¼ 8.0 Hz, 2H), 6.52 (t, J ¼ 7.3 Hz, 1H), 6.68e6.72 (m, 2H), 7.12e7.20
(m, 1H), 7.66 (t, J ¼ 7.3 Hz, 1H), 7.82 (t, J ¼ 7.3 Hz, 1H), 7.89 (d,
J ¼ 8.5 Hz, 1H), 8.02 (d, J ¼ 8.6 Hz, 1H), 8.27 (d, J ¼ 8.4 Hz, 1H), 8.34
134.5,129.6,129.5,129.3,127.7,127.4,126.9,123.2,118.7,118.3,112.9,
60.3. MS (ESI): 334.11, 228.33, 170.15, 128.23, 106.07, 76.11.
(C18H15N4OS, [M þ H]þ). Anal. Calcd for C18H14N4OS: C, 64.65; H,
4.22; N, 16.75; Found: C, 64.58; H, 4.16; N, 16.84.
(d, J ¼ 8.6 Hz, 1H). 13C NMR (300 MHz, CDCl3)
d: 175.4, 163.7, 152.2,
150.6, 149.3, 136.6, 135.4, 129.8, 129.2, 128.0, 127.7, 127.2, 126.9,
116.2, 115.9, 111.7, 108.8, 60.3. MS (ESI): 352.11, 228.31, 170.22,
128.24, 124.26, 76.12. (C18H14FN4OS, [M þ H]þ). Anal. Calcd for
C18H13FN4OS: C, 61.35; H, 3.72; N, 15.90; Found: C, 61.46; H, 3.70; N,
15.83.
4.1.1.2. 5-(Quinolin-2-yl)-3-((p-tolylamino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (4b). Yellow crystals, yield: 75%, mp: 170e
172 ꢁC. 1H NMR (500 MHz, CDCl3)
d: 2.24 (s, 3H), 5.17 (brs, 1H), 5.64
(d, J ¼ 7.0 Hz, 2H), 6.89 (d, J ¼ 8.0 Hz, 2H), 7.05 (d, J ¼ 8.0 Hz, 2H),
7.68 (t, J ¼ 7.5 Hz, 1H), 7.84 (t, J ¼ 7.5 Hz, 2H), 7.90 (d, J ¼ 8.0 Hz, 1H),
8.02 (d, J ¼ 8.5 Hz, 1H), 8.29 (d, J ¼ 8.5 Hz, 1H), 8.34 (d, J ¼ 8.5 Hz,
4.1.1.6. 3-(((4-Fluorophenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4f). White powder, yield: 70%, mp: 177e
1H). 13C NMR (300 MHz, CDCl3)
d: 178.5, 149.6, 149.3, 149.1, 147.4,
179 ꢁC. 1H NMR (500 MHz, CDCl3)
d: 5.19 (brs, 1H), 5.62 (s, 2H),
137.6, 135.5,130.6, 129.2, 128.4, 127.7, 127.3, 125.9, 124.2, 119.2, 117.3,
113.4, 59.3, 22.3. MS (ESI): 348.22, 228.17, 170.22, 128.09, 120.17,
76.21. (C19H17N4OS, [M þ H]þ). Anal. Calcd for C19H16N4OS: C, 65.50;
H, 4.63; N, 16.08; Found: C, 65.61; H, 4.62; N, 16.01.
6.90e6.97 (m, 4H), 7.69 (t, J ¼ 7.0 Hz,1H), 7.83 (t, J ¼ 7.0 Hz,1H), 7.91
(d, J ¼ 8.0 Hz, 1H), 8.03 (d, J ¼ 8.5 Hz, 1H), 8.29 (d, J ¼ 8.5 Hz, 1H),
8.36 (d, J ¼ 8.5 Hz, 1H). 13C NMR (300 MHz, CDCl3)
d: 176.2, 155.8,
152.3, 149.6, 149.1, 137.4, 134.5, 129.6, 129.5, 129.3, 128.7, 127.4,
124.8,124.4,116.9,116.3,115.9, 61.4. MS (ESI): 352.28, 228.18,170.15,
128.09, 124.21, 76.08. (C18H14FN4OS, [M þ H]þ). Anal. Calcd for
C18H13FN4OS: C, 61.35; H, 3.72; N, 15.90; Found: C, 61.22; H, 3.78; N,
15.92.
4.1.1.3. 3-(((2-Ethoxyphenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4c). Light yellow crystals, yield: 80%, mp:
184e185 ꢁC. 1H NMR (300 MHz, CDCl3)
d
: 1.25 (t, J ¼ 7.1 Hz, 3H), 4.12
(q, J ¼ 7.1 Hz, 2H), 5.68 (s, 2H), 5.85 (s,1H), 6.75e6.79 (m, 2H), 6.87e
6.91 (m, 1H), 7.19 (d, J ¼ 8.4 Hz, 1H), 7.65 (t, J ¼ 7.0 Hz, 1H), 7.81 (t,
J ¼ 7.0 Hz, 1H), 7.88 (d, J ¼ 8.5 Hz, 1H), 8.01 (d, J ¼ 8.5 Hz, 1H), 8.26
(d, J ¼ 8.4 Hz,1H), 8.32 (d, J ¼ 8.5 Hz,1H). 13C NMR (300 MHz, CDCl3)
4.1.1.7. 3-(((2-Chlorophenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4g). White powder, yield: 77%, mp: 174e
175 ꢁC. 1H NMR (500 MHz, CDCl3)
d
: 5.72 (d, J ¼ 7.5 Hz, 2H), 5.83 (t,
d: 175.4, 154.2, 152.2, 150.6, 149.3, 139.5, 137.4, 130.8, 130.3, 129.5,
J ¼ 7.5 Hz, 1H), 6.78 (t, J ¼ 7.0 Hz, 1H), 7.21e7.32 (m, 3H), 7.69 (t,
J ¼ 7.5 Hz, 1H), 7.84 (t, J ¼ 7.5 Hz, 1H), 7.91 (d, J ¼ 8.0 Hz, 1H), 8.04 (d,
J ¼ 8.5 Hz, 1H), 8.29 (d, J ¼ 8.5 Hz, 1H), 8.36 (d, J ¼ 8.5 Hz, 1H). 13C
128.8, 127.6, 125.4, 122.8, 121.9, 118.3, 111.4, 61.2. MS (ESI): 378.39,
349.41, 228.31, 128.10, 121.20, 76.45. (C20H19N4O2S, [M þ H]þ). Anal.
Calcd for C20H18N4O2S: C, 63.47; H, 4.79; N, 14.80; Found: C, 63.40;
H, 4.80; N, 14.88.
NMR (300 MHz, CDCl3) d: 177.2, 153.3, 149.6, 148.4, 145.3, 136.4,
131.7, 131.4, 131.1, 129.5, 129.2, 127.9, 124.6, 123.2, 120.2, 119.3, 115.7,
58.5. MS (ESI): 368.68, 228.45, 170.32, 140.50, 128.12, 76.16.
(C18H14ClN4OS, [M þ H]þ). Anal. Calcd for C18H13ClN4OS: C, 58.61;
H, 3.55; N, 15.19; Found: C, 58.75; H, 3.55; N, 15.10.
4.1.1.4. 3-(((2-Fluorophenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4d). Light yellow powder, yield: 71%, mp:
182e183 ꢁC. 1H NMR (500 MHz, CDCl3)
d: 5.48 (brs, 1H), 5.69 (d,
J ¼ 7.5 Hz, 2H), 6.76e6.81 (m, 1H), 7.00e7.04 (m, 1H), 7.07 (t,
J ¼ 8.0 Hz, 1H), 7.29e7.31 (m, 1H), 7.69 (t, J ¼ 8.0 Hz, 1H), 7.84 (t,
J ¼ 8.0 Hz, 1H), 7.91 (d, J ¼ 8.5 Hz, 1H), 8.04 (d, J ¼ 8.5 Hz, 1H), 8.29
(d, J ¼ 8.5 Hz,1H), 8.35 (d, J ¼ 8.5 Hz,1H). 13C NMR (300 MHz, CDCl3)
4.1.1.8. 3-(((3-Chlorophenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4h). Light yellow powder, yield: 70%, mp:
171e173 ꢁC. 1H NMR (300 MHz, CDCl3)
d: 5.28 (brs, 1H), 5.60 (d,
J ¼ 7.3 Hz, 2H), 6.78e6.86 (m, 2H), 6.97 (t, J ¼ 2.0 Hz, 1H), 7.13 (t,
J ¼ 8.1 Hz, 1H), 7.66 (t, J ¼ 7.0 Hz, 1H), 7.82 (t, J ¼ 7.0 Hz, 1H), 7.88 (d,
J ¼ 8.2 Hz, 1H), 8.01 (d, J ¼ 8.6 Hz, 1H), 8.26 (d, J ¼ 8.6 Hz, 1H), 8.34
d
: 176.4, 163.3, 148.2, 147.9, 137.1, 135.4, 133.5, 128.1, 127.3, 127.0,
125.7, 125.1, 123.9, 123.2, 116.4, 115.3, 114.2, 58.8. MS (ESI): 352.19,
228.29, 170.27, 128.07, 124.11, 76.26. (C18H14FN4OS, [M þ H]þ). Anal.
Calcd for C18H13FN4OS: C, 61.35; H, 3.72; N, 15.90; Found: C, 61.45;
H, 3.77; N, 15.81.
(d, J ¼ 8.6 Hz, 1H). 13C NMR (300 MHz, CDCl3)
d: 177.3, 152.2, 150.6,
149.5, 149.3, 136.4, 135.4, 133.1, 130.8, 130.4, 129.6, 129.3, 128.9,
122.8, 121.1, 117.5, 115.7, 60.4. MS (ESI): 368.74, 228.25, 170.18,

J. Sun et al. / European Journal of Medicinal Chemistry 60 (2013) 23e28
27
140.50, 128.29, 76.24. (C18H14ClN4OS, [M þ H]þ). Anal. Calcd for
C18H13ClN4OS: C, 58.61; H, 3.55; N, 15.19; Found: C, 58.55; H, 3.49;
N, 15.28.
4.1.1.14. 3-(Morpholinomethyl)-5-(quinolin-2-yl)-1,3,4-oxadiazole-
2(3H)-thione (4n). White crystals, yield: 85%, mp: 175e176 ꢁC. 1H
NMR (500 MHz, CDCl3)
d
: 2.94 (t, J ¼ 4.5 Hz, 4H), 3.75 (t, J ¼ 4.5 Hz,
4H), 5.18 (s, 2H), 7.70 (t, J ¼ 7.5 Hz, 1H), 7.85 (t, J ¼ 7.5 Hz, 1H), 7.93
4.1.1.9. 3-(((4-Chlorophenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
(d, J ¼ 7.5 Hz, 1H), 8.09 (d, J ¼ 8.0 Hz, 1H), 8.30 (d, J ¼ 8.0 Hz, 1H),
oxadiazole-2(3H)-thione (4i). Light yellow crystals, yield: 75%, mp:
8.38 (d, J ¼ 8.0 Hz, 1H). 13C NMR (300 MHz, CDCl3)
d: 177.7, 159.8,
205e208 ꢁC. 1H NMR (300 MHz, CDCl3)
d
: 5.25 (brs, 1H), 5.60 (d,
150.6, 148.5, 136.4, 135.1, 130.8, 130.1, 129.4, 128.9, 115.6, 79.0, 67.4,
67.1, 50.7, 50.3. MS (ESI): 328.19, 228.15, 170.09, 128.23, 100.04,
44.26. (C16H17N4O2S, [M þ H]þ)þ. Anal. Calcd for C16H16N4O2S: C,
58.52; H, 4.91; N, 17.06; Found: C, 58.36; H, 4.89; N, 17.12.
J ¼ 7.5 Hz, 2H), 6.89 (d, J ¼ 8.9 Hz, 2H), 7.17 (d, J ¼ 8.9 Hz, 2H), 7.66 (t,
J ¼ 8.0 Hz,1H), 7.82 (t, J ¼ 8.0 Hz,1H), 7.89 (d, J ¼ 8.0 Hz,1H), 8.01 (d,
J ¼ 8.6 Hz, 1H), 8.26 (d, J ¼ 8.0 Hz, 1H), 8.33 (d, J ¼ 8.6 Hz, 1H). 13C
NMR (300 MHz, CDCl3) d: 173.8, 149.4, 148.6, 147.3, 116.2, 134.4,
133.4, 128.8, 128.3, 127.6, 126.9, 125.8, 124.7, 123.3, 123.5, 122.8,
113.7, 58.3. MS (ESI): 368.58, 228.13, 170.22, 140.48, 128.32, 76.11.
(C18H14ClN4OS, [M þ H]þ). Anal. Calcd for C18H13ClN4OS: C, 58.61;
H, 3.55; N, 15.19; Found: C, 58.73; H, 3.51; N, 15.09.
4.1.1.15. 3-((4-Phenylpiperazin-1-yl)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4o). White powder, yield: 88%, mp: 177e
178 ꢁC. 1H NMR (300 MHz, CDCl3)
d
: 3.08 (t, J ¼ 4.2 Hz, 4H), 3.22 (t,
J ¼ 4.2 Hz, 4H), 5.24 (s, 2H), 6.82e6.92(m, 3H), 7.22e7.24 (m, 2H), 7.67
(t, J ¼ 7.1 Hz,1H), 7.82 (t, J ¼ 7.1 Hz,1H), 7.89 (d, J ¼ 8.2 Hz,1H), 8.06 (d,
J ¼ 8.6 Hz,1H), 8.28 (d, J ¼ 8.6 Hz,1H), 8.35 (d, J ¼ 8.6 Hz,1H).13C NMR
4.1.1.10. 3-(((2-Bromophenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4j). White powder, yield: 80%, mp: 188e
(300 MHz, CDCl3) d: 176.3,152.6,151.7,149.7,147.3,134.4,133.3,131.8,
191 ꢁC. 1H NMR (500 MHz, CDCl3)
d
: 5.71 (d, J ¼ 8.0 Hz, 2H), 5.82
131.4,129.7,129.1,128.9,126.9,121.3,117.5,117.1,114.7, 70.1, 55.3, 54.9,
49.5, 49.1. MS (ESI): 403.28, 228.21,175.19,170.18,128.20, 84.11, 76.23.
(C22H22N5OS, [Mþ H]þ). Anal. Calcd for C22H21N5OS: C, 65.49;H, 5.25;
N, 17.36; Found: C, 65.62; H, 5.22; N, 17.28.
(t, J ¼ 8.0 Hz, 1H), 6.72 (t, J ¼ 7.0 Hz, 1H), 7.25e7.31 (m, 2H), 7.47 (d,
J ¼ 7.0 Hz, 1H), 7.69 (t, J ¼ 8.0 Hz, 1H), 7.85 (t, J ¼ 8.0 Hz, 1H), 7.91 (d,
J ¼ 8.5 Hz, 1H), 8.04 (d, J ¼ 8.5 Hz, 1H), 8.29 (d, J ¼ 8.5 Hz, 1H), 8.36
(d, J ¼ 8.5 Hz, 1H). 13C NMR (300 MHz, CDCl3)
d: 174.3, 150.3, 147.6,
146.3, 135.7, 135.3, 134.3, 132.7, 128.5, 127.7, 127.4, 126.9, 125.9,
125.4, 125.1, 124.9, 114.7, 56.9. MS (ESI): 413.18, 228.12, 185.17,
170.21, 128.30, 76.31. (C18H14BrN4OS, [M þ H]þ). Anal. Calcd for
C18H13BrN4OS: C, 52.31; H, 3.17; N, 13.56; Found: C, 52.19; H, 3.21;
N, 13.65.
4.2. Cell proliferation assay
The in vitro anticancer activities of the prepared compounds 4ae
4o against HepG2, SGC-7901 and MCF-7 cell lines were evaluated as
described in the literature [28] with some modifications. Target
tumor cells were grown to log phase in RPMI 1640 medium sup-
plemented with 10% fetal bovine serum. After reaching a dilution
4.1.1.11. 3-((Methyl(phenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4k). White crystals, yield: 81%, mp: 158e
of 3 ꢂ 104 cells mLꢃ1 with the medium, 100
mL of the obtained cell
159 ꢁC. 1H NMR (300 MHz, CDCl3)
d
: 3.34 (s, 3H), 5.77 (s, 2H), 6.86 (t,
suspension was added to each well of 96-well culture plates.
Subsequently, incubation was performed at 37 ꢁC in 5% CO2 atmo-
sphere for 48 h before the cytotoxicity assessment. Tested samples
at pre-set concentrations were added to 6 wells with 5-fluorouracil
being employed as a positive reference. After 72 h exposure period,
J ¼ 7.3 Hz, 1H), 7.09 (d, J ¼ 7.3 Hz, 2H), 7.28e7.33 (m, 2H), 7.65 (t,
J ¼ 7.0 Hz, 1H), 7.80 (t, J ¼ 7.0 Hz, 1H), 7.87 (d, J ¼ 8.4 Hz, 1H), 7.99 (d,
J ¼ 8.6 Hz, 1H), 8.24 (d, J ¼ 8.4 Hz, 1H), 8.29 (d, J ¼ 8.4 Hz, 1H). 13C
NMR (300 MHz, CDCl3) d: 175.3, 159.8, 151.2, 149.6, 148.3, 136.4,
135.4, 132.7, 131.5, 131.0, 129.9, 129.4, 128.6, 128.1, 116.8, 116.0, 115.7,
72.1, 36.2. MS (ESI): 348.28, 228.23, 170.17, 128.34, 120.16, 76.22.
(C19H17N4OS, [M þ H]þ). Anal. Calcd for C19H16N4OS: C, 65.50; H,
4.63; N, 16.08; Found: C, 65.38; H, 4.58; N, 16.12.
25
After 4 h, the medium was replaced by 150
m
L of PBS containing 2.5 mg mLꢃ1 of MTT was added to each well.
m
L DMSO to dissolve the
purple formazan crystals produced. The absorbance at 570 nm of
each well was measured with an ELISA plate reader. The data rep-
resented the mean of three independent experiments in triplicate
and were expressed as means ꢀ SD. The IC50 value was defined as
the concentration at which 50% of the cells could survive.
4.1.1.12. 3-((Ethyl(phenyl)amino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4l). Light yellow powder, yield: 76%, mp:
157e158 ꢁC. 1H NMR (300 MHz, CDCl3)
d
: 1.32 (t, J ¼ 7.1 Hz, 3H),
3.79 (q, J ¼ 7.1 Hz, 2H), 5.74 (s, 2H), 6.85 (t, J ¼ 7.3 Hz, 1H), 7.10 (d,
J ¼ 8.0 Hz, 2H), 7.27e7.33 (m, 2H), 7.65 (t, J ¼ 7.0 Hz, 1H), 7.81 (t,
J ¼ 7.0 Hz, 1H), 7.87 (d, J ¼ 8.2 Hz, 1H), 8.01 (d, J ¼ 8.6 Hz, 1H), 8.23e
4.3. Telomerase inhibitory assay
Fifteen oxadiazole derivatives containing quinoline moiety were
tested in a search for small molecule inhibitors of telomerase using
the TRAP-PCR-ELISA assay. In detail, the HepG2 cells were firstly
maintained in DMEM medium (GIBCO, New York, USA) supple-
mented with 10% fetal bovine serum (GIBCO, New York, USA),
streptomycin (0.1 mg/mL) and 420 penicillin (100 IU/mL) at 37 ꢁC in
a humidified atmosphere containing 5% CO2. After trypsinization,
5 ꢂ 104 cultured cells in logarithmic growth were seeded into T25
flasks (Corning, New York, USA) and cultured to allow for adher-
ence. The cells were then incubated with Staurosporine (Santa Cruz,
Santa Cruz, USA) and the drugs with a series of concentration as 60,
8.31 (m, 2H). 13C NMR (300 MHz, CDCl3)
d: 176.3, 160.9, 152.2, 150.6,
149.3, 137.4, 136.6, 131.9, 131.5, 131.1, 130.8, 130.2, 129.6, 128.7, 121.4,
120.8, 116.7, 70.8, 48.6, 14.9. MS (ESI): 362, 39, 228.18, 170.26,
134.29, 128.44, 76.21. (C20H19N4OS, [M þ H]þ). Anal. Calcd for
C20H18N4OS: C, 66.28; H, 5.01; N, 15.46; Found: C, 66.41; H, 4.98; N,
15.55.
4.1.1.13. 3-((Pyridin-2-ylamino)methyl)-5-(quinolin-2-yl)-1,3,4-
oxadiazole-2(3H)-thione (4m). Yellow powder, yield: 79%, mp:
154e155 ꢁC. 1H NMR (300 MHz, CDCl3)
d: 5.80e5.87 (m, 3H), 6.71e
6.81 (m, 2H), 7.51 (t, J ¼ 8.0 Hz, 1H), 7.65 (t, J ¼ 7.0 Hz, 1H), 7.81 (t,
J ¼ 7.1 Hz,1H), 7.88 (d, J ¼ 8.0 Hz,1H), 8.04 (d, J ¼ 8.6 Hz,1H), 8.18 (d,
J ¼ 5.0 Hz, 1H), 8.26 (d, J ¼ 8.6 Hz, 1H), 8.32 (d, J ¼ 8.6 Hz, 1H). 13C
20, 6.67, 2.22, 0.74, 0.25 and 0.082
treatment, the cells were harvested by cell scraper orderly following
by washed once with PBS. The cells were lysed in 150 L RIPA cell
mg/mL, respectively. After 48 h
m
NMR (300 MHz, CDCl3)
d
: 176.1, 173.2, 152.3, 149.4, 148.2, 144.5,
lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated on ice for
30 min. The cellular supernatants were obtained via centrifugation
430 at 12,000 g for 20 min at 4 ꢁC and stored at ꢃ80 ꢁC.
140.0, 137.1, 135.7, 131.5, 130.7, 130.2, 128.9, 117.0, 114.7, 111.6, 57.1.
MS (ESI): 335.18, 228.06, 170.11, 128.14, 107.24, 77.06. (C17H14N5OS,
[M þ H]þ). Anal. Calcd for C17H13N5OS: C, 60.88; H, 3.91; N, 20.88;
Found: C, 61.02; H, 3.90; N, 20.82.
The TRAP-PCR-ELISA assay was performed using a telomerase
detection kit (Roche, Basel, Switzerland) according to the

28
J. Sun et al. / European Journal of Medicinal Chemistry 60 (2013) 23e28
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M.J. Edwards, Cancer 116 (2010) 2307e2315.
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with 48
120 s for pre-denaturation and performed using 35 cycles each
consisting of 94 ꢁC for 30 s, 50 ꢁC for 30 s, 72 ꢁC for 90 s. Then 20
m
L TRAP reaction mixtures. PCR was then initiated at 94 ꢁC,
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mL
of PCR products were hybridized to a digoxigenin (DIG)-labeled
telomeric repeat specific detection probe. And the PCR products
were immobilized via the biotin-labeled primer to a streptavidin-
coated 440 microtiter plate subsequently. The immobilized DNA
fragment was detected with a peroxidase-conjugated anti-DIG
antibody and visualized following addition of the stop regent. The
microtitre plate was assessed on TECAN Infinite M 200 microplate
reader (Männedorf, Switzerland) at a wavelength of 570 nm, and
the final value was presented as mean ꢀ SD.
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Automated docking studies were carried out using Discovery
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The three-dimensional structures of the aforementioned
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