Synthesis, molecular modeling and biological evaluation of N-benzylidene-2-((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetohydrazide derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2013.11.004

A series of new 1,3,4-oxadiazole derivatives (6a-6x) containing pyridine and acylhydrazone moieties were synthesized and developed as potential telomerase inhibitors. The bioassay tests demonstrated that compounds 6n, 6o, 6q, 6s and 6t exhibited significant broad-spectrum anticancer activity with IC₅₀ range from 0.76 to 9.59 μM against the four cancer cell lines (HEPG2, MCF7, SW1116 and BGC823). Moreover, all the title compounds were assayed for telomerase inhibition using the TRAP-PCR-ELISA assay. Compound 6s showed the highest anticancer activity with IC₅₀ of 0.76-1.54 μM against the tested cancer cell lines and exhibited the most potent telomerase inhibitory activity with IC₅₀ of 1.18 ± 0.14 μM. The docking simulation was carried out to investigate a possible binding mode of compound 6s into the active site of telomerase (pdb. 3DU6) while the QSAR model was built to check the previous work as well as to introduce new directions.

Full text of DOI:10.1016/j.bmc.2013.11.004

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular modeling and biological evaluation of
N-benzylidene-2-((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)
acetohydrazide derivatives as potential anticancer agents
⇑
Fei Zhang , Xiao-Liang Wang , Jing Shi, She-Feng Wang, Yong Yin, Yu-Shun Yang, Wei-Ming Zhang ,
Hai-Liang Zhu
⇑
Nanjing Institute for the Comprehensive Utilization of Wild Plant, Nanjing 210042, People’s Republic of China
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:
A series of new 1,3,4-oxadiazole derivatives (6a–6x) containing pyridine and acylhydrazone moieties
were synthesized and developed as potential telomerase inhibitors. The bioassay tests demonstrated that
compounds 6n, 6o, 6q, 6s and 6t exhibited significant broad-spectrum anticancer activity with IC50 range
Received 23 August 2013
Revised 3 November 2013
Accepted 4 November 2013
Available online xxxx
from 0.76 to 9.59
all the title compounds were assayed for telomerase inhibition using the TRAP-PCR-ELISA assay. Com-
pound 6s showed the highest anticancer activity with IC50 of 0.76–1.54 M against the tested cancer cell
lines and exhibited the most potent telomerase inhibitory activity with IC50 of 1.18 ± 0.14 M. The dock-
lM against the four cancer cell lines (HEPG2, MCF7, SW1116 and BGC823). Moreover,
l
Keywords:
Telomerase inhibitor
Anticancer activity
l
ing simulation was carried out to investigate a possible binding mode of compound 6s into the active site
of telomerase (pdb. 3DU6) while the QSAR model was built to check the previous work as well as to intro-
duce new directions.
1
,3,4-Oxadiazole
Pyridine
Acylhydrazone
Molecular docking
Ó 2013 Published by Elsevier Ltd.
3
D-QSAR
1
. Introduction
to telomerase-targeting drugs and reduce the probability of toxic-
1
2
ity to normal tissue. Finally, telomerase is encoded by non-
redundant (unique) genes so that tumors are less likely to develop
resistance to telomerase-based therapies than they are to other
cancer targets that are members of a family of genes such as
Telomerase, a reverse transcriptase that maintains telomere
length, is highly activated in tumor cells and practically absent in
somatic cells and hence considered as a potential marker for
tumorigenesis.
1
–4
13
A connection between telomerase activity and
growth factor receptors or signal transduction enzymes. All of
5–7
resistance to apoptosis has been established.
Therefore, telome-
these factors suggest that anti-cancer drugs inhibiting telomerase
might have a broad therapeutic window.
rase has been proposed as a novel and potential selective target for
cancer therapy. The key advantages of targeting telomerase in
comparison with most other cancer targets are its relative univer-
sality, criticality and specificity for cancer cells. Telomerase is ex-
pressed in most tumors (80–90%) from virtually all types of
cancers and is required for long-term maintenance of telomeres,
1,3,4-Oxadiazoles are an important class of heterocyclic com-
1
4–18
pounds with a wide range of biological activities.
spread use of them as scaffold in medicinal chemistry
establishes this moiety as a member of the privileged structures
The wide-
a
1
9,20
class.
oles have exhibited potent antitumor activities
inhibitors of telomerase, which were reported in the previous stud-
Among these, a few different substituted 1,3,4-oxadiaz-
2
1–25
which in turn is crucial for the long-term survival of tumor
including
cells.8
–10
No other tumor-associated gene is as widely expressed
1
1
24,25
in cancers. Moreover, the low or transient expression of telome-
rase in normal tissues and the generally longer telomeres in nor-
mal cells versus tumor cells provide a degree of tumor specificity
ies of our laboratory,
such as compounds A and B in Figure 1. In
addition, oxazole, a similar structure with 1,3,4-oxadiazole, is con-
tained in telomestatin (Fig. 2A), which is a natural product isolated
from Streptomyces anulatus 3533-SV4 and has been shown to be a
2
6,27
very potent telomerase inhibitor.
Besides, pyridine derivatives
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail addresses: botanyzh@163.com (W.-M. Zhang), zhuhl@nju.edu.cn (H.-L.
have attracted significant interest in pharmaceutical and agro-
chemical fields, due to their therapeutic and pharmacological
Zhu).
These two authors equally contributed to this paper.
2
8,29
properties.
Furthermore, pyridine ring is also found in
0
968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.11.004
Please cite this article in press as: Zhang, F.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.004

2
F. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Figure 3. The skeleton of target compounds.
Figure 1. Telomerase inhibitors (containing 1,3,4-oxadiazole) reported in previous
studies.
small-molecule telomerase inhibitors, such as compound B in
2. Results and discussion
2
4
Figure 2. Acylhydrazones, a class of organic compounds with a
structure of –CO–NH–N@CH–, have aroused considerable attention
due to their particular physical, chemical, and biological activities
based on their unique structures. Acylhydrazones have been dem-
onstrated to possess varied biological activities and constituted an
2.1. Chemistry
In this study, twenty-four N-benzylidene-2-((5-(pyridin-4-yl)-
1,3,4-oxadiazol-2-yl)thio)acetohydrazide derivatives (6a–6x) were
synthesized. The synthetic route of compounds 6a–6x was shown
in Scheme 1. They were synthesized from 2-((5-(pyridin-4-yl)-
1,3,4-oxadiazol-2-yl)thio)acetohydrazide (4) and different kinds
3
0–32
important class of compounds for new drug development.
Among them, a few acylhydrazone derivatives have exhibited
potent antitumor activities.3
3–35
3
8
To our knowledge, few reports have been dedicated to the syn-
thesis and telomerase inhibitory activity of 1,3,4-oxadiazole deriv-
atives containing pyridine and acylhydrazone moieties (Fig. 3). We
have performed a virtual screening of these substituted oxadiazole
derivatives by using autodock tools (Discovery Studio). The results
revealed that all compounds have potent telomerase inhibitory
activity, with an estimated binding free energy ranging from about
of aldehyde. The key intermediate 4 was prepared in three steps.
Treatment of the isoniazid 1 with carbon disulfide in the presence
of KOH and 95% ethanol under reflux provided the intermediate 2.
2 3
Refluxing 2 with ethyl bromoacetate in the presence of K CO in
acetone generated 3. The hydrazide 4 was obtained by reacting 3
with 85% hydrazine monohydrate in ethanol. The synthesis of com-
pounds 6a–6x was accomplished by condensing 4 with different
kinds of aldehydes in ethanol. All of the synthetic compounds gave
satisfactory analytical and spectroscopic data, which were in full
accordance with their depicted structures.
ꢀ
10 to ꢀ28 kcal/mol. Virtual screening results also predicted that
compounds with hydroxyl group(s) at the benzene ring have better
telomerase inhibitory activity. Additionally, we also used the re-
sults of molecular orbital calculations (3D-QSAR model) and
molecular modeling to study the structure–activity relationship
and guide the further study.
2.2. Biological activity
In view of the mentioned facts above and an attempt to achieve
new potent antitumor agents with telomerase inhibitory activity, a
series of new 1,3,4-oxadiazole derivatives containing pyridine and
acylhydrazone moieties (Fig. 3) were synthesized and evaluated
2.2.1. Antiproliferation assay
All the synthesized 1,3,4-oxadiazole derivatives 6a–6x were
evaluated for their antiproliferative activity against the HEPG2
(human liver cancer cell), MCF7 (human breast cancer cell),
SW1116 (human colorectal carcinoma cell) and BGC823 (human
gastric cancer) by MTT assay. The results were summarized in
Table 1. The results revealed that most of the synthetic com-
pounds exhibited significant antitumor activities. 5-Fluorouracil
with anti-proliferative activity and telomerase inhibitory activity,
while compounds 6a, 6c, and 6g were reported before.3
6,37
The
SAR (structure–activity relationships) and possible enzyme bind-
ing mode were also illustrated.
Figure 2. Known small-molecule telomerase inhibitors.
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F. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
2 2 2 5 2 3
Schemel 1. General synthesis of compounds 6a–6x. Reagents and conditions: (i): (a) CS /KOH, ethanol (95%), reflux 24 h, (b) HCl, pH 5–6; (ii) BrCH COOC H , acetone, K CO ,
reflux 4 h; (iii) NH
2
NH
2 2
ꢁH O, ethanol; 90 °C; (iv) RCHO, ethanol, water, acetic acid, rt.
Table 1
BGC823,respectively, which was superior to 5-fluorouracil, com-
pound A and compound B.
Antiproliferative activity of the synthesized compounds (6a–6x)
Compounds M) ± SD
SW1116
IC50 (l
Subsequently structure–activity relationships (SAR) studies
were performed to determine how the substituent on benzene ring
affected the anticancer activity. Compound 6a without any substi-
tuent group on benzene ring showed moderate anti-proliferative
activity. Then, we evaluated the effect of introducing different sub-
stituent group(s) on benzene ring of 6a. Firstly, introducing hydro-
xyl group(s) on the benzene ring of 6a to form a class of
compounds (6f, 6n–6t) increased the anticancer activity especially
for 6s with 3,4-dihydroxybenzene moiety whose IC50 values (0.76–
HEPG2
MCF7
BGC823
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
s
t
u
v
w
x
17.31 ± 0.65
12.30 ± 0.43
15.65 ± 0.82
18.59 ± 0.29
17.86 ± 0.60
5.51 ± 0.48
15.84 ± 0.71
10.02 ± 0.18
19.45 ± 0.35
6.52 ± 0.41
9.17 ± 0.61
16.01 ± 0.58
24.70 ± 0.64
3.51 ± 1.03
3.97 ± 0.85
6.49 ± 0.31
3.26 ± 0.42
6.54 ± 0.75
0.76 ± 1.32
2.92 ± 0.69
18.58 ± 0.20
12.47 ± 0.17
14.90 ± 0.52
7.42 ± 0.26
7.43 ± 0.76
14.59 ± 0.67
13.54 ± 0.31
15.28 ± 0.46
19.10 ± 0.79
31.78 ± 0.80
11.68 ± 0.52
17.63 ± 0.84
14.72 ± 0.15
24.89 ± 0.91
14.62 ± 0.38
13.82 ± 0.63
21.80 ± 1.98
35.58 ± 0.78
4.75 ± 0.65
5.83 ± 0.39
7.77 ± 0.63
5.92 ± 1.24
9.01 ± 0.64
0.91 ± 0.13
3.78 ± 0.20
27.87 ± 0.53
16.62 ± 0.78
23.54 ± 2.36
10.83 ± 1.02
9.79 ± 0.49
16.67 ± 0.83
15.71 ± 0.58
17.02 ± 1.05
21.32 ± 2.74
25.82 ± 0.60
12.21 ± 0.87
20.32 ± 0.48
16.82 ± 1.05
26.70 ± 1.85
14.92 ± 0.78
16.52 ± 0.54
21.52 ± 0.86
30.71 ± 1.54
7.38 ± 1.76
5.24 ± 0.73
8.15 ± 1.69
5.03 ± 0.47
8.57 ± 1.09
1.54 ± 0.94
3.59 ± 0.83
28.94 ± 0.70
18.83 ± 0.48
24.91 ± 0.72
14.90 ± 0.59
5.26 ± 0.26
22.05 ± 1.01
17.84 ± 0.54
20.52 ± 0.86
24.51 ± 0.27
31.50 ± 1.62
7.94 ± 0.89
19.79 ± 1.24
21.82 ± 1.76
27.54 ± 2.04
13.03 ± 0.57
15.84 ± 0.83
24.03 ± 0.25
41.63 ± 0.76
6.16 ± 0.41
5.47 ± 0.50
9.59 ± 1.24
6.34 ± 0.89
9.61 ± 0.38
1.26 ± 0.61
4.32 ± 1.74
35.60 ± 0.72
25.75 ± 1.40
26.67 ± 0.98
10.58 ± 0.47
7.48 ± 1.24
1
.54 lM) was superior to that of other compounds and the positive
control. The results indicated that the potent inhibitory activity or-
der of compounds with hydroxyl group(s) on the benzene ring was
3,4-dihydroxy (6s, IC50 = 0.76–1.54
6t, IC50 = 2.92–4.32 M) > 2-hydroxy (6n, IC50 = 3.51–7.38
-hydroxy (6f, IC50 = 5.51–12.21 M). Interestingly, 3,4-dihydroxy-
lM) > 4-hydroxy-3-methoxy
(
l
l
M) >
4
l
benzene moiety is also found in many known small-molecule
telomerase inhibitors (such as compounds C, D and E), which are
24
displayed in Figure 2. Compounds 6n–6r condensed by a series
of substituted salicylaldehyde and hydrazide 4 showed potent
anticancer activities with IC50 values of 3.26–10.57 lM. The
difference in the anticancer activities between compounds (6o,
6p) with a halogen atom on the 5-position of salicylaldehyde and
compounds (6q, 6r) with two halogen atoms on the 3-position
and 5-position of salicylaldehyde is not obvious, but when the sub-
stitute happened on the same position of benzene ring, the potency
-Fluorouracila
a
Used as a positive control.
order was Cl > Br: 6o (3.97–5.83
(3.26–6.34 M) > 6r (6.54–10.57 M). Secondly, the introduction
of the electron-withdrawing groups (halogen groups or –NO ) at
lM) > 6p (6.49–9.59 lM); 6q
was used as the positive control under identical conditions for
comparison. As shown in Table 1, the results revealed that com-
pounds 6n, 6o, 6q, 6s and 6t exhibited significant antitumor
l
l
2
the position of benzene ring weakened the anticancer effects.
activities with IC50 concentration range of 0.76–9.59
rable to 5-fluorouracil (5.26–9.79 M), while antitumor activities
of compound A with IC50 (1.61 M) and compound B with IC50
range of 18.97–23.88 M reported in the previous studies of
Among these compounds, compound 6s
displayed the most potent activity with IC50 values of 0.76,
.91, 1.54 and 1.26 M against HEPG2, MCF7, SW1116 and
l
M compa-
Compounds 6b–6e, 6i, 6l and 6m exhibited poor activities with
IC50 values of 12.30–41.63 lM. The potent inhibitory activity order
of compounds with electron-withdrawing groups on the benzene
ring (e.g., 6b, 6i and 6l) was para > ortho > meta. When the
compounds were para-position-substituted derivatives (e.g., 6b,
l
l
l
2
4,25
our laboratory.
6c, 6d and 6e), the potency order was F > Cl > Br > NO
2
. However,
0
l
the introduction of the electron-donating groups (here it
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4
F. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3 3
was –CH or –OCH ) at the position of benzene ring slightly
strengthened the anticancer effects of compounds 6g, 6h and 6k
with IC50 values ranging from 9.17 to 21.82 M. Finally, the ben-
l
zene ring of 6a was isosterically replaced with furan ring or thio-
phene ring, respectively (6v and 6w), which resulted in no
obvious changes in anticancer activities. Besides, compound 6x
exhibited better anticancer activity than 6a, which indicated that
the introduction of (E)-styryl group improved the anti-proliferative
activity.
2
.2.2. Telomerase inhibitory assay
Then, the telomerase inhibitory activity of the 1,3,4-oxadiazole
derivatives for SW1116 was studied via TRAP-PCR-ELISA assay.
Due to its excellent selective inhibition of telomerase, stauro-
sporine be chosen as the positive control.39 As ethidium bromide
2
4
had been used in the previous studies of our laboratory, it
also be chosen as the positive control. The results were
presented as mean ± SD. As shown in Table 2, most of the tested
compounds displayed good telomerase inhibitory. Among them
compound 6s displayed the most potent inhibitory effect with
Figure 4. The linear regression results of cell lines versus kinases to explore the
correlation between pIC50’s for the inhibition of telomerase and SW1116 cellular
proliferation,R2 = 0.84.
IC50 of 1.18 ± 0.14
control staurosporine (4.18 ± 0.05
mide(2.71 ± 0.18 M). Then, an analysis between telomerase
lM which is lower than that of the positive
l
M) and ethidium bro-
l
inhibition and inhibition of SW1116 cellular proliferation of the
top 10 compounds (6f, 6k, 6n–6t and 6x) indicated that there
was a moderate correlation between telomerase inhibition and
inhibition of SW1116 cellular proliferation with a R square value
of 0.84, as evidenced in Figure 4. The results of telomerase inhibi-
tory activity of the tested compounds correlated with structure–
activity relationships (SAR) of their inhibitory effects on the cell
proliferation assay. This demonstrated that the potent inhibitory
effects of the synthetic compounds on the cell proliferation assay
were causally related to their telomerase inhibitory activities.
These results suggested that compound 6s was an excellent candi-
date as anticancer agents with potent telomerase inhibitory.
2
.2.3. Cytotoxicity test
One of the major obstacles to the clinical use of many com-
pounds with potent antibacterial activities, however, is their ability
to lesion mammalian cell that causes high cytotoxic activity to the
host organism. Then we tested their cytotoxic activity on a mouse
embryonic fibroblast cell line (NIH-3T3) using the MTT assay to
prove the potency of the compounds. The pharmacological re-
sults of these compounds were summarized in Table 2. Based on
the dates, it demonstrated that the compounds were low toxic.
4
0
2
.3. Binding model of compound 6s into telomerase structure
In an effort to elucidate the mechanism by which the title com-
pounds can induce anticancer activity and to establish an SAR
based on our experimental studies, molecular docking of the po-
tent inhibitor 6s into the ATP binding site of telomerase was per-
formed to simulate a binding model derived from telomerase
structure (3DU6. pdb). The binding model of compound 6s and tel-
omerase are depicted in Figure 5. In the binding model, compound
Table 2
Telomerase inhibitory activity and cytotoxicity of compounds 6a–6x
Compounds
Telomerase inhibitation
IC50 M) ± SD
Cytotoxicity
IC50 M) ± SD
(l
(l
6
p
s is nicely bound to the telomerase via four hydrogen bonds and a
–cation interaction. The 3-postion nitrogen atom of 1,3,4-oxadi-
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
9.72 ± 0.02
10.18 ± 0.10
11.88 ± 0.07
12.40 ± 0.05
16.69 ± 0.25
5.36 ± 0.14
9.04 ± 0.04
8.51 ± 0.01
15.73 ± 0.34
5.18 ± 0.06
7.54 ± 0.14
14.55 ± 0.23
18.15 ± 0.27
3.97 ± 0.03
3.13 ± 0.09
4.28 ± 0.18
4.80 ± 0.05
7.12 ± 0.13
1.18 ± 0.14
2.89 ± 0.28
17.82 ± 0.30
9.63 ± 0.03
13.47 ± 0.25
6.57 ± 0.08
4.18 ± 0.05
2.71 ± 0.18
167.43 ± 1.28
170.28 ± 3.03
181.09 ± 2.91
169.15 ± 1.88
195.36 ± 3.74
158.46 ± 2.05
131.99 ± 0.50
184.73 ± 1.13
205.35 ± 2.61
168.66 ± 1.37
178.52 ± 3.15
141.73 ± 0.37
213.91 ± 3.77
154.68 ± 2.63
165.78 ± 1.89
155.96 ± 1.18
171.08 ± 2.58
189.90 ± 3.88
153.77 ± 0.85
147.82 ± 1.59
220.61 ± 3.08
175.02 ± 1.41
188.73 ± 2.45
159.61 ± 2.03
140.12 ± 1.97
134.87 ± 1.08
azole ring forms a hydrogen bond (Nꢁ ꢁ ꢁH-N: 2.41 Å, 159.242°) with
the amino hydrogen atom of Asp254 and the oxadiazole ring forms
a p–cation interaction with the end amino cation (distance: 3.65 Å)
of Lys189, which suggests that the 1,3,4-oxadiazole group plays an
impotant part in the combination of the receptor and ligand. Be-
sides, the nitrogen atom of pyridine ring forms a hydrogen bond
(
Nꢁ ꢁ ꢁH–N: 2.49 Å, 157.766°) with the amino hydrogen atom of
Ala195; the amino hydrogen atom of acylhydrazone group forms
a hydrogen bond (N–Hꢁ ꢁ ꢁO: 2.25 Å, 153.828°) with the carbonyl
oxygen atom of Asp343; the meta-hydroxyl oxygen atom of 3,4-
dihydroxyphenyl moiety forms
2
a
hydrogen bond (Oꢁ ꢁ ꢁH–N:
.25 Å, 114.16°) with the amino hydrogen atom of Arg194. The re-
sults also indicated that the introduction of pyridine, acylhydraz-
one and 3,4-dihydroxyphenyl groups might reinforce the
combination of compound 6s and the receptor, which might en-
hance the binding affinity, resulting in the increased anticancer
activity of this compound. In addition, the receptor surface model
was showed in Figure 6, which revealed that the molecules were
well embedded into the active site lined by Arg253, Ala255,
Tyr256 and Ile196. Moreover, the docking calculation of all the
compounds was also depicted in Table 3. The CDocker Energy
s
t
u
v
w
x
a
Staurosporine
Ethidium bromidea
a
Used as a positive control.
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F. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Figure 5. (A) 2D molecular docking modeling of compound 6s with 3DU6. The four H-bonds (green lines) are displayed as dotted lines and the
p
–cation interaction is shown
as yellow line; the purple circles show the amino acids which participate in hydrogen bonding, electrostatic or polar interactions and the green circles show the amino acids
which participate in the Van der Waals interactions. (B) 3D model of the interaction between compound 6s and 3DU6 bonding site.
Figure 6. The receptor surface model with 6s which revealed that the molecules were well embedded into the active site.
Table 3
ues with corresponding residual errors of 24 compounds by this
QSAR model had been given in Table 4. The graphical relationship
of the observed pIC50 values versus the predicted values had been
illustrated in Figure 7. The linear R value was 0.86, which meant
the dependability of linear fit.
Also the molecules aligned with the iso-surfaces of the 3D-QSAR
model coefficients on Van der Waals grids (Fig. 8C) and electro-
static potential grids (Fig. 8D) were listed. Electrostatic map indi-
cated red contours around regions where high electron density
CDOCKER_ENERGY of compounds 6a–6x and 3DU6
Compounds
-CDOCKER energy
Gb (kcal/mol)
Compounds
-CDOCKER energy
Gb (kcal/mol)
2
D
D
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
g
h
i
16.04
17.52
16.11
15.99
13.24
18.51
16.59
17.51
14.91
18.07
17.82
15.59
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
9.87
22.47
23.14
21.24
19.99
22.89
27.71
24.74
13.11
18.63
15.59
18.86
(
negative charge) was expected to increase activity, and blue con-
tours represent areas where low electron density (partial positive
charge) was expected to increase activity. Similarly, steric map
indicated areas where steric bulk was predicted to increase (green)
or decrease (yellow) activity. It was widely acceptable that a better
inhibitor based on the 3D-QSAR model should have strong Van der
Waals attraction in the green areas and a polar group in the blue
electrostatic potential areas (which were dominant close to the
skeleton).
j
k
l
(
energy of the ligand—receptor complexes) agreed with the telo-
merase inhibitory trend for all the synthesized compounds, which
further confirmed the correlation between the anticancer activities
and telomerase inhibitory activities of our compounds.
As shown in Figure 8, the potent compounds with electron-
donating atom and large substituent not only circumvented the
red subregion but also got more close to the favorable green
spaces. This figure displayed that these bulkier group substituted
in the aromatic ring could have good interaction with the protein
for the nice molecule electrostatic and steric features. So, this QSAR
study could predict that electron-donating atom and large substi-
tuent locating at the benzene ring might enhance binding affinity
of 1,3,4-oxadiazole derivatives. The data summarized above dem-
onstrated that compounds 6s, the most potent Telomerase inhibi-
2
.4. 3D-QSAR
For further exploration of structure and activity relationships of
these compounds, the 3D-QSAR study was performed by means of
the DS 3.5 software (Discovery Studio 3.5, Accelrys, Co. Ltd). The
3
(
D-QSAR model was created with corresponding pIC50 values
ꢀlogIC50) which were converted from the obtained IC50 M) val-
ues of telomerase inhibition. The observed and predicted pIC50 val-
(l
tor (IC50 = 1.18
lM) with suitable substituent possessed an
outstanding activity. The 3D QSAR models fitted the inhibitory
Please cite this article in press as: Zhang, F.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.004

6
F. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 4
Experimental, predicted inhibitory activity of all synthesized compounds against
telomerase by 3D-QSAR models based on active conformations selected fromthe
preliminary molecular docking study
Compounds
Telomerase inhibition
Actual pIC50b
Predicted pIC50
Residual error
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
5.01
4.99
4.93
4.91
4.78
5.27
5.04
5.07
4.80
5.29
5.12
4.84
4.74
5.40
5.50
5.37
5.32
5.15
5.93
5.54
4.75
5.02
4.87
5.18
4.86
4.89
5.06
5.02
4.93
5.32
5.11
5.10
4.83
5.41
4.97
4.90
4.91
5.43
5.37
5.36
5.31
5.22
5.78
5.59
4.80
4.89
5.01
5.05
0.15
0.10
ꢀ0.13
ꢀ0.11
ꢀ0.15
ꢀ0.05
ꢀ0.07
ꢀ0.03
ꢀ0.03
ꢀ0.12
0.15
ꢀ0.06
ꢀ0.17
ꢀ0.03
0.13
d
a
e
f
a
g
h
i
j
k
l
a
a
m
n
a
o
p
q
r
s
t
u
v
w
x
0.01
0.01
Figure 7. Linear fit curve of the observed pIC50 values versus the predicted values
of the training set part and the test set part,R2 = 0.86.
ꢀ0.07
0.15
ꢀ0.05
ꢀ0.05
0.13
4
. Experimental protocols
ꢀ0.14
0.13
4.1. Materials and measurements
a
Compounds were selected as the test sets while the rest ones were in the
training sets.
The IC50 values of the compounds against telomerase (Table 2) were converted
into pIC50 values by using the online calculator (http://www.sanjeevslab.org/tools-
IC50.html).
All chemicals and reagents used in current study were of analyt-
ical grade. The reactions were monitored by thin layer chromatog-
raphy (TLC) on Merck pre-coated silica GF254 plates. Melting
points (uncorrected) were determined on a XT4MP apparatus
b
(
Tai ke Corp., Beijing, China). ESI mass spectra were obtained on
1
a Mariner System 5304 mass spectrometer, and H NMR spectra
were collected on a Bruker DPX500 spectrometer at room temper-
ature with TMS and solvent signals allotted as internal standards.
Chemical shifts are reported in ppm (d). Elemental analyzes were
performed on a CHN-O-Rapid instrument, and were within ±0.4%
of the theoretical values.
activity well thus provide us the direction of further modification.
It could be predict that electron-donating substitute (OH and
OCH ) for the R group and the increasing steric bulk, such as (E)-
3
styryl replacing benzene ring, could benefit the enhancement of
the telomerase inhibitory activity.
4
.2. General procedure for synthesis of the target compounds
3
. Conclusions
(6a–6x)
In conclusion, a series of new 1,3,4-oxadiazole derivatives con-
To a stirred solution of isoniazide (1) (15 mmol) and KOH
taining pyridine and acylhydrazone moieties were synthesized.
Anti-proliferative assay results indicated that compounds 6s
exhibited the most potent activity against four different original
cancer cells (HEPG2, MCF7, SW1116 and BGC823). We further
analyzed their inhibitory effects on telomerase expressed in can-
cer cells which was more active in cancer cells than other normal
ones and responsible for cell immortality. It was shown that com-
pound 6s could be more potent to inhibit telomerase activity than
(12 mmol) in 95% ethanol (30 mL), carbon disulfide (CS2)
(16 mmol) was slowly added. Then, the resulting mixture was stir-
red at 80 °C for 24 h. Then dropwise add 37% HCl to the reaction
until PH 6–7. The 2 precipitated from the solvent, collected using
suction filtration and dried, and followed by recrystallization in
ethanol.
Treatment of 2 (12 mmol) with ethyl bromoacetate (13 mmol)
in acetone at 60 °C for 4 h. Meanwhile, K CO (6 mmol) was added
2
3
other compounds with the IC50 value of 1.18 ± 0.14
lM. Docking
to the mixture. Then the mixture was filtered and the filtrate for
column chromatography. Column chromatography was performed
using silica gel (200–300 mesh) eluting with ethyl acetate and
petroleum ether to give the 3.
Equimolar quantities (10 mmol) of 3 and the 85% hydrazine
monohydrate were dissolved in anhydrous ethanol (25 mL) and
vigorously stirred under ice-bath for 12 h. The 4 was precipitated
from the solvent, collected using suction filtration and dried, and
followed by recrystallization in ethanol.
To a stirred solution of hydrazide 4 (1 mmol) and compound 5
(1 mmol) in ethanol (15 mL), water (1 mL) was added followed
by dropwise addition of glacial acetic acid (0.2 mL). The resulting
mixture was stirred at room temperature until the target product
precipitated from the solvent, which was collected using suction
filtration and dried, followed by recrystallization in ethanol.
simulation was performed to position compound 6s into the ac-
tive site of telomerase (3DU6) to determine the probable binding
model. The enzyme assay data and the molecular docking results
suggested that compound 6s is potential inhibitor of telomerase.
This study reports compounds with oxadiazole, acylhydrazone
and pyridine frameworks as new class of anti-tumor agents and
it may serve as model compounds for design and development
of anticancer agents. Simultaneously, a new 3D QSAR model
was built with the activity data and binding conformations to
check the previous work as well as to introduce new directions.
The introduction of electron-donating substituents (OH and
3
OCH ) for the R group and the increasing steric bulk did reinforce
the anti-tumor activity.
Please cite this article in press as: Zhang, F.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.004

F. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
Figure 8. 3D-QSAR of 1,3,4-oxadiazole derivatives containing pyridine and acylhydrazone moieties for Telomerase (pdb:3DU6). (C) Isosurface of the 3D-QSAR model
coefficients on electrostatic potential grids. The blue triangle mesh represents positive electrostatic potential and the red area represents negative electrostatic potential. (D)
Isosurface of the 3D-QSAR model coefficients on Van der Waals grids. The green triangle mesh representation indicates positive coefficients; the yellow triangle mesh
indicates negative coefficients.
0
4
.2.1. (E)-N -Benzylidene-2-((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-
J = 9.1 Hz, 2H), 8.15 (s, 1H), 8.24–8.34 (m, 2H), 8.78–8.82 (m, 2H),
1
3
yl)thio)acetohydrazide (6a)
6
12.06 (s, 1H). C NMR (100 MHz, DMSO-d , d ppm):171.2, 168.3,
White powder, yield 82%, mp 184–185 °C; 1H NMR (300 MHz,
164.4, 148.2(2), 145.5, 144.6, 141.6, 157.2, 125.2(2), 124.3(2),
+
DMSO-d
6
, d ppm): 4.70 (s, 2H), 7.43 (m, 3H), 7.69–7.72 (m, 2H),
122.4(2), 40.6. MS (ESI): 385.06 (C16
13 6 4
H N O S, [M+H] ). Anal. Calcd
7
.87–7.91 (m, 2H), 8.05 (s, 1H), 8.78–8.81 (m, 2H), 11.81 (s, 1H).
for C16 S: C, 50.00; H, 3.15; N, 21.86%. Found: C, 50.14; H,
3.15; N, 21.80.
12 6 4
H N O
1
3
C
NMR (100 MHz, DMSO-d
6
,
d
ppm):172.1, 169.4, 164.7,
1
4
H
48.4(2),144.6, 144.3, 134.8, 130.2, 129.4(2), 127.3(2), 122.4(2),
+
0
0.2. MS (ESI): 340.08 (C16
14
H N
5
O
2
S, [M+H] ). Anal. Calcd for C16-
4.2.6. (E)-N -(4-Hydroxybenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
oxadiazol-2-yl)thio) acetohydrazide (6f)
13
N
5
O
2
S: C, 56.63; H, 3.86; N, 20.64%. Found: C, 56.50; H, 3.85;
N, 20.70.
White powder, yield 84%, mp 204–205 °C; 1H NMR (300 MHz,
DMSO-d
6
, d ppm): 4.66 (s, 2H), 6.81 (d, J = 5.1, 2H), 7.53 (d,
0
4
.2.2. (E)-N -(4-Fluorobenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
J = 5.2, 2H), 7.88 (m, 2H), 7.94 (s, 1H), 8.78–8.81 (m, 2H), 9.91 (s,
1H), 11.59 (s, 1H). C NMR (100 MHz, DMSO-d
168.3, 164.3, 148.8(2), 145.6, 144.6, 128.6, 167.2, 129.2(2),
114.3(2), 122.2(2), 40.3. MS (ESI): 356.07 (C16
Anal. Calcd for C16 S: C, 54.00; H, 3.69; N, 19.69%. Found:
C, 54.04; H, 3.64; N, 19.79.
1
3
oxadiazol-2-yl)thio)acetohydrazide (6b)
6
, d ppm):171.2,
White powder, yield 80%, mp 192–192 °C; 1H NMR (300 MHz,
+
DMSO-d
.89 (m, 2H), 8.04 (s, 1H), 8.79 (m, 2H), 11.80 (s, 1H). C NMR
100 MHz, DMSO-d
ppm):171.7,13 168.4, 164.2, 148.6(2),
45.6, 144.5, 124.5, 167.2, 131.4(2), 116.3(2), 122.1(2), 40.4. MS
6
, d ppm): 4.69 (s, 2H), 7.26–7.29 (m, 2H), 7.77 (m, 2H),
14 5 3
H N O S, [M+H] ).
1
3
7
(
1
(
13 5 3
H N O
6
,
d
+
0
ESI): 358.07 (C16
5
H13FN O
2
S, [M+H] ). Anal. Calcd for C16
H
12FN
5
O
2-
4.2.7. (E)-N -(4-Methoxybenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
oxadiazol-2-yl)thio) acetohydrazide (6g)
S: C, 53.77; H, 3.88; N, 19.60%. Found: C, 53.87; H, 3.90; N, 19.67.
White powder, yield 84%, mp 173–174 °C; 1H NMR (300 MHz,
0
4
.2.3. (E)-N -(4-Chlorobenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
DMSO-d
6
, d ppm):3.79 (s, 3H), 4.67 (s, 2H), 6.97–7.06 (m, 2H),
oxadiazol-2-yl)thio) acetohydrazide (6c)
7.62–7.66 (m, 2H), 7.87–7.91 (m, 2H), 7.98 (s, 1H), 8.78–8.81 (m,
White powder, yield 84%, mp 205–206 °C; 1H NMR (300 MHz,
2H), 11.66 (s, 1H). C NMR (100 MHz, DMSO-d
168.2, 164.4, 148.6(2), 145.9, 144.8, 128.4, 162.2, 129.8(2),
114.7(2), 122.4(2), 40.7, 55.1. MS (ESI): 370.09 (C17 S,
S: C, 55.27; H, 4.09; N,
13
6
, d ppm):171.3,
6
DMSO-d , d ppm): 5.22 (s, 2H), 7.93 (d, J = 5.0, 2H), 8.24 (d,
J = 4.7, 2H), 8.36 (d, J = 2.9, 2H), 8.59 (s, 1H), 9.26 (d, J = 2.8, 2H),
16 5 3
H N O
1
3
+
1
1
1
1.86 (s, 1H). C NMR (100 MHz, DMSO-d
6
, d ppm):171.6, 168.6,
15 5 3
[M+H] ). Anal. Calcd for C17H N O
64.4, 148.8(2), 145.3, 144.7, 130.7, 137.2, 131.2(2), 119.3(2),
18.96%. Found: C, 55.04; H, 4.10; N, 18.89.
+
22.2(2), 40.7. MS (ESI): 374.04 (C16
12ClN S: C, 51.41; H, 3.24; N, 18.73%. Found: C,
1.55; H, 3.25; N, 18.75.
5 2
H13ClN O S, [M+H] ). Anal.
0
Calcd for C16
5
H
O
5 2
4.2.8. (E)-N -(4-Methylbenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
oxadiazol-2-yl)thio)acetohydrazide (6h)
White powder, yield 86%, mp 179–180 °C; 1H NMR (300 MHz,
0
4
.2.4. (E)-N -(4-Bromobenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
DMSO-d
6
, d ppm):2.34 (s, 3H), 4.68 (s, 2H), 6.98–7.06 (m, 2H),
oxadiazol-2-yl)thio) acetohydrazide (6d)
7.62–7.65 (m, 2H), 7.89–7.92 (m, 2H), 7.99 (s, 1H), 8.79–8.82 (m,
White powder, yield 86%, mp 206–207 °C; 1H NMR (300 MHz,
2H), 11.68 (s, 1H). C NMR (100 MHz, DMSO-d
168.4, 164.2, 148.7(2), 145.7, 144.6, 129.4, 142.2, 126.8(2),
124.7(2), 122.3(2), 40.6, 21.8. MS (ESI): 354.09 (C17 S,
S: C, 57.78; H, 4.28; N,
13
6
, d ppm):171.6,
6
DMSO-d , d ppm): 4.69 (s, 2H), 7.63 (m, 4H), 7.87–7.90 (m, 2H),
13
8
.02 (s, 1H), 8.80 (m, 2H), 11.85 (s, 1H). C NMR (100 MHz,
16 5 2
H N O
+
DMSO-d
6
, d ppm):171.6, 168.4, 164.3, 148.5(2), 145.4, 144.5,
15 5 3
[M+H] ). Anal. Calcd for C17H N O
1
31.6, 127.2, 129.2(2), 131.3(2), 122.3(2), 40.4. MS (ESI): 417.99
19.82%. Found: C, 57.64; H, 4.27; N, 19.89.
+
(
C
16
5 2 16 5 2
H13BrN O S, [M+H] ). Anal. Calcd for C H12BrN O S: C,
0
4
5.94; H, 2.89; N, 16.74%. Found: C, 45.90; H, 2.93; N, 16.70.
4.2.9. (E)-N -(3-Fluorobenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
oxadiazol-2-yl)thio) acetohydrazide (6i)
0
White powder, yield 84%, mp 210–211 °C; 1H NMR (300 MHz,
4
.2.5. (E)-N -(4-Nitrobenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
oxadiazol-2-yl)thio) acetohydrazide (6e)
DMSO-d
3H), 7.88 (m, 2H), 8.03 (s, 1H), 8.78 (m, 2H), 11.88 (s, 1H).
NMR (100 MHz, DMSO-d , d ppm):171.4, 168.3, 164.4, 148.8(2),
6
, d ppm): 4.70 (s, 2H), 7.23–7.26 (m, 1H), 7.44–7.56 (m,
White powder, yield 81%, mp 231–232 °C; 1H NMR (300 MHz,
13
C
DMSO-d
6
, d ppm): 4.72 (s, 2H), 7.86–7.90 (m, 2H), 7.97 (d,
6
Please cite this article in press as: Zhang, F.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.004

8
F. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
1
4
45.5, 144.3, 134.4, 116.2, 124.8, 114.7, 131.2, 166.7, 122.3(2),
129.4, 160.1, 124.2, 118.2, 122.3(2), 40.8. MS (ESI): 390.03 (C16H
13-
ClN O S, [M+H] ). Anal. Calcd for C16H12ClN O S: C, 49.30; H, 3.10;
5 3 5 3
+
+
0.6. MS (ESI): 358.07 (C16
5
H13FN O
2
S, [M+H] ). Anal. Calcd for C16-
5
H12FN O
2
S: C, 53.77; H, 3.38; N, 19.60%. Found: C, 53.61; H, 3.38;
N, 17.97%. Found: C, 49.39; H, 3.11; N, 17.94.
N, 19.64.
0
4
.2.16. (E)-N -(5-Bromo-2-hydroxybenzylidene)-2-((5-(pyridin-
0
4
.2.10. (E)-2-((5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N -(3-
4-yl)-1,3,4-oxadiazol-2-yl)thio)acetohydrazide (6p)
(
trifluoromethyl)benzylidene)acetohydrazide (6j)
White powder, yield 86%, mp 213–214 °C; 1H NMR (300 MHz,
White powder, yield 83%, mp 178–179 °C; 1H NMR (300 MHz,
DMSO-d
6
, d ppm): 4.71 (s, 2H), 6.85–6.88 (t, J = 5.3, 1H), 7.37–
DMSO-d
6
, d ppm): 4.73 (s, 2H), 7.65–7.68 (m, 1H), 7.77 (m, 1H),
7.42 (m, 1H), 7.84 (s, 1H), 7.88 (m, 2H), 8.27 (s, 1H), 8.79 (m,
1
3
7
2
1
1
.86–7.90 (m, 2H), 8.00–8.05 (m, 2H), 8.13 (s, 1H), 8.78–8.81 (m,
2H), 10.39 (s, 1H), 11.79 (s, 1H). C NMR (100 MHz, DMSO-d
ppm):171.6, 168.3, 164.4, 148.5(2), 145.4, 144.3, 119.4, 134.8,
133.4, 160.8, 114.1, 118.9, 122.4(2), 40.8. MS (ESI): 433.98 (C16
6
, d
1
3
H), 11.96 (s, 1H). C NMR (100 MHz, DMSO-d
68.5, 164.6, 148.7(2), 145.4, 144.5, 134.8., 126.2, 149.8, 125.7,
30.2, 132.7, 122.4(2), 40.5,125.2. MS (ESI): 408.07 (C17
6
, d ppm):171.3,
H
13-
+
H
13
F
3
N
5
O
2-
5 3 5 3
BrN O S, [M+H] ). Anal. Calcd for C16H12BrN O S: C, 44.25; H, 2.79;
+
S, [M+H] ). Anal. Calcd for C17
H
12
F
3
N
5
O
2
S: C, 50.12; H, 2.97; N,
N, 16.13%. Found: C, 44.39; H, 2.80; N, 16.18.
1
7.19%. Found: C, 50.20; H, 3.00; N, 17.25.
0
4
.2.17. (E)-N -(3,5-Dichloro-2-hydroxybenzylidene)-2-((5-
0
4
1
.2.11. (E)-N -(3-Methoxybenzylidene)-2-((5-(pyridin-4-yl)-
,3,4-oxadiazol-2-yl)thio)acetohydrazide (6k)
(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetohydrazide (6q)
1
White powder, yield 86%, mp 115–117 °C; H NMR (300 MHz,
DMSO-d , d ppm): 4.68 (s, 2H), 6.82–6.92 (m, 2H), 7.21–7.31 (m,
1H), 7.70 (d, J = 7.7, 1H), 7.87–7.91 (m, 1H), 8.35 (s, 1H), 8.78–
8.81 (m, 1H), 10.04 (s, 1H), 11.72 (s, 1H). C NMR (100 MHz,
DMSO-d ppm):171.8, 168.3, 164.5, 148.5(2),145.3, 144.4,
122.2, 134.9, 129.1, 159.0, 124.4, 128.4, 122.6(2), 40.7. MS (ESI):
White powder, yield 83%, mp 180–181 °C; 1H NMR (300 MHz,
6
DMSO-d
6
, d ppm):3.81 (s, 3H), 4.66 (s, 2H), 6.98–7.05 (m, 2H),
1
3
7
2
1
1
.63–7.67 (m, 2H), 7.88–7.91 (m, 2H), 7.97 (s, 1H), 8.76–8.80 (m,
1
3
H), 11.68 (s, 1H). C NMR (100 MHz, DMSO-d
68.4, 164.6, 148.9(2), 145.6, 144.2, 137.4, 115.2, 123.6, 112.7,
30.2, 163.2, 122.5(2), 40.7,55.6. MS (ESI): 370.09 (C17 S,
S: C, 55.27; H, 4.09; N,
6
, d ppm):171.5,
6
, d
+
H
16
N
5
O
3
2 5 3 2 5 3
424.00 (C16H12Cl N O S, [M+H] ). Anal. Calcd for C16H11Cl N O S:
C, 45.30; H, 2.61; N, 16.71%. Found: C, 45.39; H, 2.60; N, 16.68.
+
[
M+H] ). Anal. Calcd for C17
15
H N
5
O
3
1
8.96%. Found: C, 55.07; H, 4.10; N, 18.87.
0
4
.2.18. (E)-N -(3,5-Dibromo-2-hydroxybenzylidene)-2-((5-
0
4
.2.12. (E)-N -(2-Fluorobenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetohydrazide (6r)
1
oxadiazol-2-yl)thio) acetohydrazide (6l)
White powder, yield 86%, mp 217–219 °C; H NMR (300 MHz,
DMSO-d , d ppm): 4.75 (s, 2H), 7.80–7.83 (m, 2H), 7.88 (m, 2H),
8.36 (s, 1H), 8.79 (s, 2H), 12.06 (s, 1H), 12.48 (s, 1H). C NMR
(100 MHz, DMSO-d , d ppm):171.6, 168.5, 164.3, 148.6(2), 145.8,
144.7, 121.2, 138.9, 129.9, 159.8, 114.2, 118.2, 122.4(2), 40.8. MS
(ESI): 511.89 (C16
S: C, 37.45; H, 2.16; N, 13.65%. Found: C, 37.55; H, 2.17; N,
13.68
White powder, yield 85%, mp 197–198 °C; 1H NMR (300 MHz,
6
13
DMSO-d
6
, d ppm): 4.69 (s, 2H), 7.26 (d, J = 4.2, 2H), 7.48 (d,
J = 2.9, 1H), 7.87 (s, 3H), 8.24 (s, 1H), 8.79 (s, 2H), 11.89 (s, 1H).
6
1
3
C
NMR (100 MHz, DMSO-d
6
, d ppm):171.3, 168.4, 164.7,
+
1
1
48.8(2), 145.3, 144.5, 117.2, 135.1, 131.4, 162.1, 123.2, 113.2,
2 5 3
H12Br N O S, [M+H] ). Anal. Calcd for C16H11Br2-
+
22.4(2), 40.5. MS (ESI): 358.07 (C16
12FN S: C, 53.77; H, 3.38; N, 19.60%. Found: C,
3.62; H, 3.38; N, 19.63.
H13FN
5
O
2
S, [M+H] ). Anal.
5 3
N O
Calcd for C16
5
H
5 2
O
0
4
.2.19. (E)-N -(3,4-Dihydroxybenzylidene)-2-((5-(pyridin-4-yl)-
0
4
.2.13. (E)-N -(2-Nitrobenzylidene)-2-((5-(pyridin-4-yl)-1,3,4-
1,3,4-oxadiazol-2-yl) thio)acetohydrazide (6s)
oxadiazol-2-yl)thio) acetohydrazide (6m)
White powder, yield 87%, mp 245–247 °C; 1H NMR (300 MHz,
White powder, yield 82%, mp 222–223 °C; 1H NMR (300 MHz,
DMSO-d
6
, d ppm): 4.65 (s, 2H), 6.76 (d, J = 5.9, 1H), 6.90 (d,
DMSO-d
6
, d ppm): 4.68 (s, 2H), 7.27 (m, 2H), 7.46 (d, J = 2.9, 1H),
J = 4.9, 1H), 7.17 (d, J = 5.9, 1H), 7.85–8.00 (m, 3H), 8.78 (s, 2H),
13
13
7
.88 (m, 3H), 8.23 (s, 1H), 8.77 (s, 2H), 11.86 (s, 1H). C NMR
9.21 (s, 1H), 9.40 (d, J = 7.5, 1H), 11.55 (s, 1H).
(100 MHz, DMSO-d , d ppm):171.6, 168.7, 164.5, 148.4(2), 145.8,
144.5, 132.2, 148.9, 124.1, 119.2, 117.2, 148.0, 122.3(2), 40.8. MS
C NMR
(
100 MHz, DMSO-d
6
, d ppm):171.7, 168.6, 164.3, 148.6(2), 145.2,
6
1
44.7, 127.3, 130.2, 131.3, 148.1, 133.2, 123.2, 122.6(2), 40.6. MS
+
+
(
ESI): 385.06 (C16
H
13
N
6
O
4
S, [M+H] ). Anal. Calcd for C16
12
H N
6
O
4
S:
14 5 4 13 5 4
(ESI): 372.07 (C16H N O S, [M+H] ). Anal. Calcd for C16H N O S:
C, 50.00; H, 3.15; N, 21.86%. Found: C, 50.16; H, 3.16; N, 21.80.
C, 51.75; H, 3.53; N, 18.86%. Found: C, 51.84; H, 3.54; N, 18.88.
0
0
4
1
.2.14. (E)-N -(2-Hydroxybenzylidene)-2-((5-(pyridin-4-yl)-
4.2.20. (E)-N -(4-Hydroxy-3-methoxybenzylidene)-2-((5-
,3,4-oxadiazol-2-yl)thio) acetohydrazide (6n)
(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetohydrazide (6t)
White powder, yield 86%, mp 192–193 °C; 1H NMR (300 MHz,
White powder, yield 85%, mp 165–167 °C; H NMR (300 MHz,
1
DMSO-d
1
8
6
, d ppm): 4.68 (s, 2H), 6.82–6.92 (m, 2H), 7.21–7.31 (m,
H), 7.70 (d, J = 7.7, 1H), 7.87–7.91 (m, 2H), 8.35 (s, 1H), 8.78–
.81 (m, 2H), 10.04 (s, 1H), 11.72 (s, 1H). 13C NMR (100 MHz,
DMSO-d
6
, d ppm): 3.79 (s, 3H), 4.66 (s, 2H), 6.99–7.06 (m, 2H),
7.62–7.65 (m, 2H), 7.87–7.90 (m, 2H), 7.98 (s, 1H), 8.80 (m, 2H),
11.67 (s, 1H). C NMR (100 MHz, DMSO-d
13
6
, d ppm):171.8, 168.6,
DMSO-d
6
, d ppm):171.9, 168.7, 164.8, 148.9(2), 145.6, 144.8,
164.4, 148.7(2), 145.6, 144.5, 131.1, 152.9, 124.2, 113.1, 117.5,
+
1
3
5
17.9, 133.1, 128.4, 159.1, 121.2, 116.2, 122.6(2), 40.7. MS (ESI):
56.07 (C16
4.00; H, 3.69; N, 19.69%. Found: C, 54.04; H, 3.64; N, 19.79.
148.9, 122.5(2), 40.6. MS (ESI): 386.08 (C17
16 5 4
H N O S, [M+H] ).
+
14
H N
5
O
3
S, [M+H] ). Anal. Calcd for C16
H
13
N
5
3
O S: C,
Anal. Calcd for C17 S: C, 52.98; H, 3.92; N, 18.17%. Found:
15 5 4
H N O
C, 52.84; H, 3.94; N, 18.18.
0
0
4
4
.2.15. (E)-N -(5-Chloro-2-hydroxybenzylidene)-2-((5-(pyridin-
-yl)-1,3,4-oxadiazol-2-yl)thio)acetohydrazide (6o)
4.2.21. (E)-N -(2,4-Dichlorobenzylidene)-2-((5-(pyridin-4-yl)-
1,3,4-oxadiazol-2-yl)thio)acetohydrazide (6u)
White powder, yield 86%, mp 225–226 °C; 1H NMR (300 MHz,
White powder, yield 83%, mp 223–224 °C; 1H NMR (300 MHz,
DMSO-d
7
2
6
, d ppm): 4.71 (s, 2H), 6.85–6.88 (t, J = 5.3, 1H), 7.37–
.42 (m, 1H), 7.84 (s, 1H), 7.88 (m, 2H), 8.27 (s, 1H), 8.79 (m,
H), 10.39 (s, 1H), 11.79 (s, 1H). 13C NMR (100 MHz, DMSO-d
, d
DMSO-d
6
, d ppm): 4.69 (s, 2H), 7.48 (m, 1H), 7.70 (d, J = 6.2, 1H),
7.85 (d, J = 3.5, 2H), 7.98 (d, J = 5.0, 1H), 8.36 (s, 1H), 8.79 (s, 2H),
11.99 (s, 1H). C NMR (100 MHz, DMSO-d
13
6
6
, d ppm):171.8, 168.7,
ppm):171.6, 168.4, 164.6, 148.8(2), 145.5, 144.6, 119.9, 133.8,
164.7, 148.5(2), 145.3, 144.7, 131.9, 132.0, 129.8, 126.9, 127.8,
Please cite this article in press as: Zhang, F.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.004

F. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
1
28.9, 122.5(2), 40.6. MS (ESI): 408.00 (C16
H
12Cl
2
N
5
O
2
S, [M+H]+).
supplemented with 10% fetal bovine serum (GIBCO, New York,
USA), streptomycin (0.1 mg/mL) and penicillin (100 IU/mL) at
Anal. Calcd for C16 11Cl S: C, 47.14; H, 2.72; N, 17.15%. Found:
H
2 5 2
N O
C, 47.26; H, 2.72; N, 17.18.
2
37 °C in a humidified atmosphere containing 5% CO . After trypsin-
4
ization, 5 ꢂ 10 cultured cells in logarithmic growth were seeded
into T25 flasks (Corning, New York, USA) and cultured to allow to
adherence. The cells were then incubated with Staurosporine (San-
ta Cruz, Santa Cruz, USA), Ethidium bromide and the drugs with a
series of concentration as 60, 20, 6.67, 2.22, 0.74, 0.25 and
0
4
.2.22. (E)-N -(Furan-2-ylmethylene)-2-((5-(pyridin-4-yl)-1,3,4-
oxadiazol-2-yl)thio)acetohydrazide (6v)
White powder, yield 82%, mp 193–194 °C; 1H NMR (300 MHz,
DMSO-d
.91 (m, 1H), 8.06 (s, 1H), 8.79–8.82 (m, 2H), 11.81 (s, 1H).
NMR (100 MHz, DMSO-d , d ppm):171.2, 168.5, 164.6, 148.4(2),
45.2, 141.6, 148.9, 119.2, 114.8, 146.9, 122.5(2), 40.6. MS (ESI):
6
, d ppm): 4.71 (s, 2H), 7.44 (m, 2H), 7.70 (m, 2H), 7.88–
1
3
7
C
0.082
vested by cell scraper orderly following by washed once with PBS.
The cells were lysed in 150 L RIPA cell lysis buffer (Santa Cruz,
lg/mL, respectively. After 24 h treatment, the cells were har-
6
1
3
5
l
+
30.06 (C14
H
12
N
5
O
3
S, [M+H] ). Anal. Calcd for C14
11
H N
5
O
3
S: C,
Santa Cruz, USA), and incubated on ice for 30 min. The cellular
supernatants were obtained via centrifugation at 12,000g for
1.06; H, 3.37; N, 21.27%. Found: C, 51.21; H, 3.38; N, 21.30.
2
0 min at 4 °C and stored at ꢀ80 °C.
0
4
.2.23. (E)-2-((5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N -
The TRAP-PCR-ELISA assay was performed using a telomerase
(
thiophen-2-ylmethylene)acetohydrazide (6w)
detection kit (Roche, Basel, Switzerland) according to the manu-
facturer’s protocol. In brief, 2 L of cell extracts were mixed with
48 L TRAP reaction mixtures. PCR was then initiated at 94 °C,
White powder, yield 83%, mp 201–203 °C; 1H NMR (300 MHz,
l
DMSO-d
6
,d ppm): 4.70 (s, 2H), 7.43 (m, 2H), 7.69–7.72 (m, 2H),
l
7
.87–7.91 (m, 2H), 8.05 (s, 1H), 8.79–8.82 (m, 1H), 11.80 (s, 1H).
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
1
3
C
NMR (100 MHz, DMSO-d
6
,
d
ppm):171.5, 168.7, 164.8,
1
48.7(2), 145.4, 131.6, 146.1, 129.2, 124.8, 126.9, 122.4(2), 40.5.
20 ll of PCR products were hybridized to a digoxigenin (DIG)-la-
+
MS (ESI): 346.04 (C14
: C, 48.68; H, 3.21; N, 20.28%. Found: C, 48.78; H, 3.22; N,
0.30.
H
12
N
5
O
S
2 2
, [M+H] ). Anal. Calcd for C14
H
11
N
5-
beled telomeric repeat specific detection probe. And the PCR
products were immobilized via the biotin-labeled primer to a
streptavidin-coated microtiter plate subsequently. The immobi-
lized DNA fragment were detected with a peroxidase-conjugated
anti-DIG antibody and visualized following addition of the stop
regent. The microtitre plate was assessed on TECAN Infinite
M200 microplate reader (Männedorf, Switzerland) at a wave-
length of 490 nm, and the final value were presented as
mean ± SD.
2 2
O S
2
0
4
1
.2.24. (E)-N -((E)-3-Phenylallylidene)-2-((5-(pyridin-4-yl)-
,3,4-oxadiazol-2-yl)thio)acetohydrazide (6x)
White powder, yield 82%, mp 177–178 °C; 1H NMR (300 MHz,
DMSO-d
6
, d ppm): 5.1 (s, 2H), 7.38–7.51 (m, 2H), 7.79–7.86 (m,
4
H), 8.03 (d, J = 4.4, 2H), 8.38 (s, 1H), 9.25 (m, 3H), 11.00 (s, 1H).
1
3
C
NMR (100 MHz, DMSO-d
6
, d ppm):171.7, 168.4, 164.7,
1
1
48.8(2), 145.4, 137.6, 126.1, 129.9, 134.8, 127.9, 130.2(2),
4.5. Cytotoxicity test
+
27.8(2), 122.6(2), 40.3. MS (ESI): 366.09 (C18
16 5 2
H N O S, [M+H] ).
Anal. Calcd for C18
C, 59.26; H, 4.13; N, 19.20.
H
15
N
5
O
2
S: C, 59.16; H, 4.14; N, 19.17%. Found:
The cytotoxic activity in vitro was measured against mamma-
lian cells, human macrophage using the MTT assay. The cell was
grown in DMEM medium supplemented with 10% PBS and 1ꢂ
antimycotic and antibacterial solution (sigma USA) at 37 °C, in
humidified atmosphere having 5% CO . 100 ll of the confluent
2
fibroblast stock suspension was dispensed in 96-well tissue cul-
4
.3. Anti-proliferation assay
The anti-proliferative activity of the title compounds 6a–6x
against the four cell lines HEPG2, MCF7, SW1116 and BGC823,
were evaluated using a standard MTT-based colorimetric assay.
Ten thousand corresponding cells per well were seeded into
ture plate. The original medium from the wells was replaced with
100
lL serum free DMEM when the cells reached 90% confluence
after 5 h incubation in a CO
2
incubator. Various concentrations of
9
3
6-well plates (Corning, New York, USA) and incubated at
the compounds were added to the growing cells and incubated
for 24 h. The absorbance was measured at a wavelength of
570 nm (OD570 nm) on an ELISA microplate reader. Three repli-
cate wells were used for each concentration and each assay was
measured three times, after which the average of IC50 was calcu-
lated. The cytotoxicity of each compound was expressed as the
concentration of compound that reduced cell viability to 50%
(IC50). The results were summarized in Table 2. The morphology
of the cells was observed using Giemsa stain under Phase contrast
microscope.
7 °C, 5% CO for 24 h. And then 100 L a series of concentration
2
l
of drug-containing medium were dispensed into wells to main-
tain the final concentration as 60, 20, 6.67, 2.22, 0.74, 0.25 and
0
.082 lg/mL. Each concentration was in triplicate, and 5-fluoro-
uracil (5-FU) (Sigma–Aldrich, St. Louis, USA) was used as the po-
sitive control. After 48 h incubation, cell survival was determined
by the addition of 25 lL MTT (Sigma–Aldrich, St. Louis, USA)
work solution (5 mg/mL MTT dissolved in Phosphate Buffer Solu-
tion (PBS)). After post-incubation at 37 °C for 4 h, the medium
was discarded following by adding 100 lL DMSO (Sigma–Aldrich,
St. Louis, USA). The plates were then votexed for 10 min for
4.6. Experimental protocol of docking study
complete dissolution. The optical absorbance was measured at
5
70 nm. The data represented the mean of three independent
Molecular docking of compound 6s into the three-dimensional
telomerase complex structure was carried out using the Discovery
Stutio (version 3.1) as implemented through the graphical user
interface DS- CDocker protocal.
experiments in triplicate and were expressed as mean ± SD.
The IC50 value was defined as the concentration at which 50%
of the cells could survive.
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem 3D ultra 12.0 software
4
.4. Telomerase inhibitory assay
[
Chemical Structure Drawing Standard; Cambridge Soft corpora-
Compounds 6a–6x were tested in a search for small molecule
tion, USA (2010)], then they were energetically minimized by using
MMFF94 with 5000 iterations and minimum RMS gradient of 0.10.
The crystal structures of telomerase were retrieved from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb/home/home.-do). All
inhibitors of telomerase activity by using the TRAP-PCR-ELISA as-
say. In detail, the SW116 cells were firstly maintained in Dulbecco
Modified Eagle Medium (DMEM medium (GIBCO, New York, USA))
Please cite this article in press as: Zhang, F.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.004

1
0
F. Zhang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
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Please cite this article in press as: Zhang, F.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.004