Design, synthesis and biological evaluation of heterocyclic azoles derivatives containing pyrazine moiety as potential telomerase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.08.059

Three series of novel heterocyclic azoles derivatives containing pyrazine (5a-5k, 8a-8k and 11a-11k) have been designed, synthesized, structurally determined, and their biological activities were evaluated as potential telomerase inhibitors. Among the oxadiazole derivatives, compound 5c showed the most potent biological activity against SW1116 cancer cell line (IC₅₀ = 2.46 μM against SW1116 and IC₅₀ = 3.55 μM for telomerase). Compound 8h performed the best in the thiadiazole derivatives (IC₅₀ = 0.78 μM against HEPG2 and IC₅₀ = 1.24 μM for telomerase), which was comparable to the positive control. While compound 11f showed the most potent biological activity (IC₅₀ = 4.12 μM against SW1116 and IC₅₀ = 15.03 μM for telomerase) among the triazole derivatives. Docking simulation by positioning compounds 5c, 8h and 11f into the telomerase structure active site was performed to explore the possible binding model. The results of apoptosis demonstrated that compound 8h possessed good antitumor activity against HEPG2 cancer cell line. Therefore, compound 8h with potent inhibitory activity in tumor growth inhibition may be a potential antitumor agent against HEPG2 cancer cell. Therefore, the introduction of oxadiazole, thiadiazole and triazole structures reinforced the combination of our compounds and the receptor, resulting in progress of bioactivity.

Full text of DOI:10.1016/j.bmc.2012.08.059

Bioorganic & Medicinal Chemistry 20 (2012) 6356–6365
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of heterocyclic azoles
derivatives containing pyrazine moiety as potential telomerase inhibitors
⇑
Yan-Bin Zhang, Xiao-Liang Wang, Wen Liu, Yu-Shun Yang, Jian-Feng Tang, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of novel heterocyclic azoles derivatives containing pyrazine (5a–5k, 8a–8k and 11a–11k)
have been designed, synthesized, structurally determined, and their biological activities were evaluated
as potential telomerase inhibitors. Among the oxadiazole derivatives, compound 5c showed the most
Received 2 August 2012
Revised 30 August 2012
Accepted 31 August 2012
Available online 11 September 2012
potent biological activity against SW1116 cancer cell line (IC50 = 2.46
IC50 = 3.55 for telomerase). Compound 8h performed the best in the thiadiazole derivatives
(IC50 = 0.78 M against HEPG2 and IC50 = 1.24 M for telomerase), which was comparable to the positive
control. While compound 11f showed the most potent biological activity (IC50 = 4.12 M against SW1116
and IC50 = 15.03 M for telomerase) among the triazole derivatives. Docking simulation by positioning
lM against SW1116 and
l
M
l
l
Keywords:
Telomerase
Oxadiazole
l
l
compounds 5c, 8h and 11f into the telomerase structure active site was performed to explore the possible
binding model. The results of apoptosis demonstrated that compound 8h possessed good antitumor
activity against HEPG2 cancer cell line. Therefore, compound 8h with potent inhibitory activity in tumor
growth inhibition may be a potential antitumor agent against HEPG2 cancer cell. Therefore, the introduc-
tion of oxadiazole, thiadiazole and triazole structures reinforced the combination of our compounds and
the receptor, resulting in progress of bioactivity.
Thiadiazole
Triazole
Anti-tumor activity
Pyrazine
Molecular docking
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
indicated that a smaller moiety with higher electronic density
might improve the bioactivity.
Telomerase is a ribonucleoprotein for telomere maintenance
which utilizes its RNA component as the template to extend telo-
meric DNA length.1 The activity of telomerase could be detected in
about 85–90% of tumor cells, whereas it is low or not present in most
somatic cells.2 This is not surprising since one of the hallmarks of
tumor is the indefinite growth managed by preventing telomere
shortening and senescence.3 Thus, the maintenance of telomere
length is considered as a biological marker for determining the
proliferation of cancer cells.4 The essential role of telomerase in
cancer and ageing makes it an important target for the development
of therapies to treat cancer and other age-associated disorders.5
Pyrazine is the heterocyclic compound having two nitrogen
atoms in the para-position of the six-membered ring. Ligands con-
1,3,4-Oxadiazoles are an important class of heterocyclic
compounds with a wide range of biological such as antiviral,10
antimicrobial,11 fungicidal,12 anticancer,13,14 tyrosinase inhibi-
tory15 activities. Further, 1,3,4-oxadiazole heterocycles are very
good bioisosteres of amides and esters, which can contribute sub-
stantially in increasing pharmacological activity by participating in
hydrogen bonding interactions with the receptors.16 1,3,4-Thiadia-
zole nucleus constitutes the active part of several biologically
active compounds, including anticancer,17,18 antibacterial19 and
anti-inflammatory agents.20 Moreover, the introduction of differ-
ent heterocyclic backbone to the thiadiazole nucleus will affect
its biological activity, which has drawn widespread attention.21
The chemistry of N-bridged heterocycles derived from 1,2,4-tria-
zole has received considerable attention in recent years due to
their usefulness in different areas of biological activities and as
industrial intermediates.22 The derivatives of 1,2,4-triazole possess
a wide range of antimicrobial23,24 and antitumour25,26 activities.
Recently, the compounds containing 1,2,4-triazole were discovered
as a novel class of potent tubulin polymerization inhibitors.27,28
1,3,4-Thiadiazole and 1,2,4-triazole were introduced to see if
1,3,4-oxadiazole could be replaced by a similar heterocyclic one,
thus, to seek for more potent telomerase inhibitors.
taining a pyrazine ring are widely studied and their p-donor prop-
erties are interesting.6 Pyrazine has been paid great attention,
because the diazine rings form an important class of compounds
presented in several natural and synthetic compounds.7 Pyrazine
derivatives have been widely used in the fields of medicinal chem-
istry for the skeleton of biologically active sites.8,9 Pyrazine was
used to replace benzo[b]dioxin mainly according to the result of
CADD (computer assistant drug design) method. The result
Some 1,3,4-oxadiazole derivatives have been reported as poten-
tial telomerase inhibitors.29,30 However, to our knowledge, few
reports have been dedicated to the synthesis and telomerase
⇑
Corresponding author. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.059

Y.-B. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 6356–6365
6357
structure inhibitory activity of heterocyclic azoles derivatives
containing pyrazine. Herein, in continuation to extend our research
on antitumor compounds with telomerase structure inhibitory
activity, in the present work we sought to synthesis three series
of heterocyclic azoles derivatives containing pyrazine as antitumor
agents, besides, we discussed the differences of biological activities
among the three series, oxadiazoles, thiadiazoles, and triazoles.
Biological evaluation indicated that some of the synthesized com-
pounds were potent inhibitors of telomerase structure.
9 was stirred under refluxing for 30 min, yielding the desired com-
pound 10. Then, eleven 1,2,4-triazole derivatives (11a–11k) were
prepared by refluxing in anhydrous acetonitrile of 10 with halo-
gen-substituted benzyl bromide compounds.
All of the synthetic compounds gave satisfactory analytical and
spectroscopic data, which were in full accordance with their
depicted structures. Additionally, the structure of compounds 5k,
8k, 11e and 11i were further confirmed by X-ray diffraction. The
crystal data are presented in Table 1 and Figure 1, which give a
perspective view of these compounds together with the atomic
labeling system.
2. Results and discussion
2.1. Chemistry
2.2. Biological activity
2.2.1. Anti-proliferation assay
In this study, thirty-three heterocyclic azoles derivatives con-
taining pyrazine were synthesized. The synthetic routes of com-
pounds 5a–5k, 8a–8k and 11a–11k were shown in Scheme 1.
It was prepared in several steps. Firstly, esterification of the
2-pyrazine carboxylic acid 1 with methanol and concentrated
sulfuric acid afforded the corresponding ester 2. The pyrazine-
2-carbohydrazide 3 was obtained by reacting 2 with 85% hydrazine
monohydrate in ethanol. Then oxadiazole, thiadiazole and triazole
derivatives containing pyrazine were systhesized from the
pyrazine-2-carbohydrazide 3. Treatment of the hydrazide 3 with
carbon disulfide in the presence of KOH and 95% ethanol under re-
flux gave the intermediate 4. The synthesis of compounds 5a–5k
was accomplished by refluxing 4 with halogen-substituted benzyl
bromide in the presence of K2CO3 in acetone. The hydrazide 3 was
stirred with KOH in absolute methanol, and carbon disulfide was
slowly added, then compound 6 was precipitated. Dried compound
6 was stirred in concentrated sulfuric acid at 0 °C, the mixture was
then poured into ice-water, obtaining the intermediate 7. The syn-
thesis of compounds 8a–8k was accomplished by refluxing 7 with
halogen-substituted benzyl bromide in the presence of K2CO3 in
acetone. A mixture of the hydrazide 3 and phenyl isothiocyanate
was refluxed in ethanol for 30 min. The solution was cooled and
compound 9 was precipitated. A solution of NaOH (2 N) containing
All the synthesized heterocyclic azoles derivatives were evalu-
ated for their anti-proliferative activity against the HEPG2 (human
liver cancer cell), SW1116 (human colorectal carcinoma cell), HELA
(human cervical cancer cell) and BGC823 (human gastric cancer).
The results were summarized in Table 2.
As illustrated in Table 2, the active analogs showed a distinctive
potential pattern of selectivity as well as broad-spectrum antitu-
mor activity. With regard to selectivity against individual cell lines,
most of the compounds showed better activities against cell lines
HEPG2 and SW1116 than HELA and BGC823 with lower IC50 values.
Especially, these compounds showed effectiveness against cell line
human hepatocellular liver carcinoma HEPG2 with IC50 values
range of 0.78–22.57
Among these compounds, compound 8h showed the most potent
biological activity (IC50 = 0.78 M). Overall, series 8a–8k mostly
lM comparative to staurosporine (1.30 lM).
l
showed better activities against the cell lines than series 5a–5k
and 11a–11k, but not absolutely. When regarding HEPG2 cell line,
IC50 values of compounds 8e, 8g, 8h and 8j ranged of 0.78–
6.90
11i (10.07–10.64
with compounds 8e, 8g, 8h and 8i with IC50 values range of 1.47–
lM, compared with 5c, 5d, 5e (4.22–7.50
lM) and 11e, 11f,
lM). Also, the same regular pattern was observed
5.26
l
M, comparable with 5c, 5d, 5e (2.46–7.88
lM) and 11d, 11f,
N
N
COOH
R4
1
N
N
R1
a
b
N
O
N
O
C
SH
R2
R3
N
N
R3
d
Br
S
O
N
N
N
N
CH3
O
+
R1
R2
R4
4
5
2
R4
O
C
N
O
C
N
R1
H
N
N
N
N
N
NH2
SH
N
N
SK
N
N
R2
R3
N
H
R3
g
S
N
H
f
Br
S
N
S
+
S
S
R1
R2
R4
3
R4
8
6
7
N
N
N
O
C
R1
H
N
H
N
N
SH
N
N
N
N
N
R2
R3
j
i
R3
N
N
H
S
Br
N
N
+
N
R1
R2
R4
11
9
10
5a/8a/11a R1=F, R2=R3=R4=H
5b/8b/11b R1=Cl, R2=R3=R4=H
5e/8e/11e
5h/8h/11h R3=Br, R1=R2=R4=H
5k/8k/11k R1=R2=R3=R4=H
5c/8c/11c R1=Br, R2=R3=R4=H
5d/8d/11d R2=Cl, R1=R3=R4=H
5g/8g/11g R3=Cl, R1=R2=R4=H
R2=Br, R1=R3=R4=H
5f/8f/11f
5i/8i/11i
R3=F, R1=R2=R4=H
R1=R3=F, R2=R4=H
5j/8j/11j
R1=R4=F, R2=R3=H
Scheme 1. General synthesis of compounds (5a–5k, 8a–8k and 11a–11k). Reagents and conditions: (a) methanol, concentrated sulfuric acid; reflux, 8–12 h; (b) NH2NH2ÁH2O
(85%), ethanol; reflux, 8–12 h; (c) (1) CS2/KOH, ethanol (95%), reflux, 24 h, (2) HCl, pH 5–6; (d) K2CO3, acetone, reflux, 1 h; (e) methanol, KOH, CS2, rt; (f) concentrated sulfuric
acid, 0 °C; (g) K2CO3, acetone, reflux, 1 h; (h) phenyl isothiocyanate, ethanol, reflux, 30 min; (i) NaOH (2 N), reflux, 30 min; (j) reflux, acetonitrile.

6358
Y.-B. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 6356–6365
Table 1
Crystallographical and experimental data for compounds 5k, 8k, 11e and 11i
Crystal parameters
Compound 5k
13H10N4OS
270.32
Monoclinic
P21/n
Compound 8k
Compound 11e
Compound 11i
Formula
C
C13H10N4S2
286.39
Monoclinic
P21/n
C19H14BrN5S
424.32
Monoclinic
P21/c
C19H14F2N5S
381.41
Monoclinic
P21/c
Formula weight
Crystal system
Space group
a
(°)
b (°)
(°)
90
90
90
90
110.054(7)
90
101.575(5)
90
107.906(18)
90
90.468(10)
90
c
a (Å)
b (Å)
c (Å)
V (Å)
7.607(5)
17.209(11)
10.265(7)
1262.3(14)
4
5.808(4)
29.459(18)
7.882(5)
1321.2(15)
4
14.27(3)
13.49(3)
9.847(19)
1804(7)
4
16.022(18)
10.288(12)
10.868(12)
1789(4)
4
Z
Calculated density
1.422
1.440
1.562
1.416
(Mg/cm3)
Mu(MoKa) (mmÀ1
)
0.253
0.756
0.393
0.767
2.407
9.983
0.215
0.760
Absorption coefficient
(mmÀ1
)
F (000)
560.0
592
856
784
h range (°)
2.37–28.14
2.73–28.33
2.65–26.50
2.35–29.05
hkl limits
À9 6 h 6 10, À22 6 k 6 18,
À7 6 h 6 7, À39 6 k 6 38,
À17 6 h 6 16, À15 6 k 6 16,
À20 6 h 6 21, À13 6 k 6 13,
À13 6 l 6 8
À9 6 l 6 10
À12 6 l 6 12
À14 6 i 6 11
Reflections collected/
unique
6361/2861 [Rint = 0.052]
11246/3216 [Rint = 0.039]
9979/3669 [Rint = 0.083]
13649/4427 [Rint = 0.060]
Data/restraints/
parameters
2861/0/172
3216/0/172
3669/0/235
4427/0/244
R1/wR2 [I > 2
R1/wR2 (all data)
GOF
r
(I)]
0.0640/0.1339
0.1255/0.1682
1.069
0.0525/0.1156
0.0721/0.1241
1.065
0.1695/0.1863
0.0629/0.1419
0.994
0.1867/0.4226
0.1323/0.3748
1.488
Figure 1. Crystal structure diagrams of compounds 5k, 8k, 11e and 11i.
11i (4.12–6.61
proved to be sensitive toward 11e, 11f and 11h with IC50
concentration range of 7.30–9.58 M comparative with 5c, 5d, 5k
l
M) against SW1116 cell line. HELA cell line was
(8.89–21.12
was seemed to be less effective to compounds 8d, 8g and 8i with
IC50 concentration range of 13.45–14.65 together with
lM) and 8e, 8g, 8h (5.43–18.87 lM). BGC823 cell line
l
lM

Y.-B. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 6356–6365
6359
Table 2
which presented as F = Br > Cl (e.g., 8a, 8b, 8c). However, the potent
inhibitory activity order of 1,2,4-triazole derivatives 11a–11k with
halogen atom substituent was quite different from the compounds
mentioned before. Among the meta-position halogen-substituted
compounds, the potent inhibitory action order could be summa-
rized as Br > Cl (e.g., 11d, 11e); while when the substitute hap-
pened on the para-position, the potency order was F > Br > Cl
(e.g., 11f, 11g, 11h), but the potency was not very obvious when
happened on ortho-position. Overall, most Br-substituted com-
pounds showed better activities than other halogen-substituted
compounds, like compounds 5c, 5h, 8c, 8e, 8h, 11e and 11h with
Antiproliferative activity of the synthesized compounds (5a–5k, 8a–8k and 11a–11k)
Compounds
IC50(lM)
HEPG2
SW1116
HELA
BGC823
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
12.47
12.29
4.22
7.50
5.79
16.46
11.47
13.21
14.41
17.13
11.21
9.52
10.78
9.81
7.03
16.78
15.08
2.46
7.88
5.06
22.66
17.29
14.43
19.28
23.76
34.00
6.77
7.53
6.95
12.09
5.26
17.13
4.77
25.33
23.01
8.89
14.63
30.31
41.64
24.61
21.57
26.72
>50
21.12
32.67
28.86
21.30
29.73
5.43
18.18
27.73
17.55
15.75
11.48
>50
30.01
25.68
33.04
>50
25.04
18.43
20.33
18.86
14.65
16.72
29.33
13.43
25.18
14.45
18.59
21.20
>50
34.66
36.24
20.26
18.29
27.15
27.67
19.76
17.82
>50
IC50 values ranged of 0.78–25.68 lM. The same halogen atom
substituent on different positions of benzene ring also presented
quite different orders in different skeletons. With regard to the
Br-substituted compounds, no similarities were found as follows:
ortho > meta > para (e.g., 5c, 5e, 5h) in 1,3,4-oxadiazole derivatives,
para > meta > ortho (e.g., 8c, 8e, 8h) in 1,3,4-thiadiazole derivatives
and meta > para = ortho (e.g., 11c, 11e, 11h) in 1,2,4-triazole deriv-
atives. While the potent inhibitory activity orders of compounds
with Cl-substituted on the benzene ring were meta > para = ortho
(e.g., 5b, 5d, 5g) in 1,3,4-oxadiazole derivatives, para > meta > ortho
(e.g., 8b, 8d, 8g) which was similar to the Br-substituted com-
pounds in 1,3,4-thiadiazole derivatives, and para > ortho = meta
(e.g., 11b, 11d, 11g) in triazole derivatives. However, the potent
inhibitory activity orders were uncertain of F-substituted
compounds, the potency order of fluorine substituted 1,2,4-triazole
derivatives was summarized as ortho > 2,4-disubstituded > para =
2,6-disubstituded. The results showed that Br-substituted on ben-
zene ring could enhance the activity of compounds, but the
position of bromine atom was not absolutely. It could be a promis-
ing lead for the further development of novel telomerase inhibition
agents.
1.79
10.12
6.23
0.78
9.63
>50
18.87
12.36
20.90
29.17
34.40
44.10
>50
>50
13.52
9.58
7.30
>50
8.63
12.53
>50
1.47
5.18
6.90
16.83
7.88
18.84
18.13
19.39
6.61
5.03
4.12
12.54
6.21
4.65
11.35
19.27
18.83
11.63
19.62
10.64
10.07
15.34
11.38
10.41
22.57
17.14
1.30
24.11
15.42
2.41
11k
5-Fluorouracil
Staurosporine
35.55
3.25
9.12
>50
1.52
6.83
6.73
4.95
2.2.2. Telomerase inhibition assay
In addition, we also selected 15 compounds which had better or
worse anti-proliferative activity to test their telomerase inhibitory
activity against HEPG2 cell line. The results were summarized in
Table 3. Most of the tested compounds displayed potent telome-
rase inhibitory. Among them, compound 8h showed the most
compounds 5c, 5d, 5e (11.48–17.55
and 11i (17.82–19.76 M). It was concluded that compounds 5c,
5d, 8e, 8g, 8h, 11e, 11f, 11h and 11i showed broad-spectrum anti-
lM) and compounds 11e, 11h,
l
tumor activity with IC50 concentration range of 0.78–27.15
against the mentioned four cancer cell lines.
l
M
potent inhibitory activity with IC50 of 1.24 lM. Then, an analysis
between telomerase inhibition and inhibition of HEPG2 cellular
proliferation of the selected 15 compounds indicated that there
was a moderate correlation between telomerase inhibition and
inhibition of HEPG2 cellular proliferation with R square value of
0.839. The results of telomerase inhibitory activity of the tested
compounds were corresponding to the structure relationships of
their antitumor activities. This demonstrated that the potent
Subsequently preliminary SAR (structure–activity relationship)
studies were performed to deduce how the structure variation
(oxadiazole, thiadiazole, and triazole) and different halogen atom
substituent could affect the anticancer activity. Through investi-
gating on the selectivity of the tested compounds over the four cell
lines, it was clear that the tested compounds showed stronger
activities against HEPG2 than other three cancer cell lines. Among
the tested compounds, compound 8h showed the most potent
biological activity against HEPG2 cancer cell line with IC50 value
Table 3
Telomerase inhibitory activity of selected compounds
of 0.78 lM. Some rules were summarized, respectively, in different
Compounds
Telomerase inhibitation (IC50, lM)
skeletons (oxadiazole, thiadiazole, and triazole) with different hal-
ogen atom substituent on benzene ring against the four cell lines,
the rules were quite different, but not absolutely, which might lead
to different antitumor activity. With regard to the 1,3,4-oxadiazole
derivatives 5a–5k, comparisons of antitumor activities with differ-
ent halogen atoms substituent on benzene ring were demonstrated
as follows: when the compounds were ortho-position halogen-
substituted derivatives, the potency order was Br > Cl = F (e.g., 5a,
5b, 5c) and among the meta-position halogen-substituted com-
pounds the potent inhibitory action order could be summarized
as Br > Cl (e.g., 5d, 5e); while when the substitute happened on
the para-position of benzene ring, the potency order was Cl > Br > F
(e.g., 5f, 5g, 5h). The rules were quite similar to the 1,3,4-
thiadiazole derivatives 8a–8k, possibly because their structures
were very alike. The difference was just on the ortho-position,
5c
5d
5e
5f
8c
8d
8e
8f
8g
8h
11a
11f
11g
11h
11j
3.55
6.38
3.59
17.12
3.97
3.12
1.54
12.09
7.88
1.24
17.65
15.03
13.44
7.13
24.16
4.14
Staurosporine

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Y.-B. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 6356–6365
antitumor activities of the synthetic compounds were probably
correlated to their telomerase inhibitory activities.
oxadiazole group and the amino hydrogen atom of Gln 308 form
another hydrogen bond interaction, which means the oxadiazole
group plays an impotant part in the combination of the receptor
and ligand. Besides, the two amino hydrogen atoms of Lys 189,
respectively, from two hydrogen bonds with the nitrogen atom
of pyrazine group. Compound 8h is also nicely bound to the
telomerase with four interactions. The amino hydrogen atom of
Arg 194 forms two hydrogen bonds with the two nitrogen atoms
of thiadiazole group at the same time. Also the amino hydrogen
atom of Gly 309 forms a hydrogen bond with the nitrogen atom
of pyrazine group. Besides, the benzene ring of compound 8h forms
2.2.3. Apoptosis assay
Apoptosis is an essential mechanism used to eliminate acti-
vated HEPG2 cells during the shutdown process of excess immune
responses and maintain proper immune homeostasis, while defi-
cient apoptosis of activated HEPG2 cells is associated with a wide
variety of immune disorders. We detected the mechanism of
compound 8h (Fig. 2) inhibition effects by flow cytometry (FCM),
and found that the compound could induce the apoptosis of
activated HEPG2 cells in a dose dependent manner. The result
indicated that compound 8h induced apoptosis of antitumor stim-
ulated HEPG2 cells.
a p-cation interaction with Lys 189. This ensures the binding affin-
ity and results in an increased telomerase inhibitory activity. In
addition, Compound 11f is nicely bound to the telomerase with
five interactions. The amino hydrogen atom of Arg 194 also forms
two hydrogen bonds with the two nitrogen atoms of thiadiazole
group at the same time. Besides, another amino hydrogen atom
2.3. Binding models of compounds 5c, 8h and 11f into
telomerase
of Arg 194 forms a
p-cation interaction with the pyrazine ring.
To gain better understanding on the potency of the synthesized
compounds and guide further structure–activity relationships
(SARs) studies. The molecular docking was performed by potent
inhibitor 5c (Fig. 3A), 8h (Fig. 3B) and 11f (Fig. 3C) into binding site
of telomerase. All docking runs were applied Discovery Studio 3.1
(DS 3.1). In the binding model, compound 5c is nicely bound to the
telomerase with five hydrogen bonds. The amino hydrogen atom of
Arg 194 forms two hydrogen bonds with the two nitrogen atoms of
oxadiazole group at the same time. Also, the oxygen atom of the
The sulfur atom between the benzene ring and triazole ring forms
a hydrogen bond with the nitrogen atom of Gln 308. The amino
hydrogen atom of Lys 189 forms a hydrogen bond with the fluorine
atom of the benene ring. In summary, the choosed heterocyclic
azoles derivatives containing pyrazine are nicely combined to the
telomerase, Arg 194 and Lys 189 play an important part in the
combination of the receptor and ligand. These results, along with
the data of inhibitory activity assay indicated that compounds 5c,
8h and 11f would be a kind of potential inhibitor.
Figure 2. HEPG2 cells isolated from naïve mice were cultured with anticancer and various concentrations of 8h for 24 h. Cells were stained by Annexin VeFITC/PI and
apoptosis was analyzed by flow cytometry.

Y.-B. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 6356–6365
6361
Figure 3A. Left: 3D model of the interaction between compound 5c and the 3DU6 binding site. The H-bond (green lines) is displayed as dotted lines. Right: 2D molecular
docking modeling of compound 5c with 3DU6. The H-bond (blue arrows) is displayed as dotted arrows.
Figure 3B. Left: 3D model of the interaction between compound 8h and the 3DU6 binding site. The H-bond (green lines) is displayed as dotted lines, and the
interactions are shown as orange lines. Right: 2D molecular docking modeling of compound 8h with 3DU6. The H-bond (blue or green arrows) is displayed as dotted arrows,
and the -cation interactions are shown as orange lines.
p-cation
p
Figure 3C. Left: 3D model of the interaction between compound 11f and the 3DU6 binding site. The H-bond (green lines) is displayed as dotted lines, and the
interactions are shown as orange lines. Right: 2D molecular docking modeling of compound 11f with 3DU6. The H-bond (blue arrows) is displayed as dotted arrows, and the
-cation interactions are shown as orange lines.
p-cation
p

6362
Y.-B. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 6356–6365
289.1 (C13H10FN4OS, [M+H]+). Anal. Calcd for C13H9FN4OS: C,
3. Conclusions
54.16; H, 3.15; N, 19.43. Found: C, 54.31; H, 3.16; N, 19.49.
Three series of heterocyclic azoles derivatives containing pyra-
zine have been synthesized and evaluated for their antitumor
activities. Compound 5c demonstrated potent inhibitory activity
4.1.1.2. 2-((2-Chlorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-oxadiaz-
ole (5b).
(500 MHz, CDCl3): 4.69 (s, 2H); 7.22–7.28 (m, 2H); 7.42 (d,
J = 7.45 Hz, 1H); 7.65 (d, J = 7.20 Hz, 1H); 8.73 (s, 2H); 9.41 (s,
1H). ESI-MS: 305.0 (C13H10ClN4OS, [M+H]+). Anal. Calcd for
White solid, yield 93%; mp: 75–76 °C. 1H NMR
that inhibited the growth of HEPG2 cells with IC50 of 2.46
and inhibited the growth of SW1116 cells with IC50 of 4.22
lM
l
M.
While compound 11f demonstrated potent inhibitory activity that
inhibited the growth of HELA cells with IC50 of 7.30 M and inhib-
ited the growth of SW1116 cells with IC50 of 4.12 M. Compound
8h demonstrated the most potent inhibitory activity that inhibited
C13H9ClN4OS: C, 51.23; H, 2.98; N, 18.38. Found: C, 51.38; H,
l
l
2.97; N, 18.44.
4.1.1.3. 2-((2-Bromobenzyl)oxy)-5-(pyrazin-2-yl)-1,3,4-oxadiaz-
ole (5c).
the growth of HEPG2 cells with IC50 of 0.78
growth of SW1116 cells with IC50 of 1.47
l
l
M and inhibited the
M, it also inhibited
White solid, yield 91%; mp: 86–88 °C. 1H NMR
(500 MHz, CDCl3): 4.71 (s, 2H); 7.16–7.22 (m, 1H); 7.30 (t,
J = 7.50 Hz, 1H); 7.61 (d, J = 7.86 Hz, 1H); 7.68 (dd, J1 = 1.65 Hz,
J2 = 6.03 Hz, 1H); 8.74 (s, 2H); 9.42 (s, 1H). ESI-MS: 349.0
(C13H10BrN4OS, [M+H]+). Anal. Calcd for C13H9BrN4OS: C, 44.71;
H, 2.60; N, 16.04. Found: C, 44.84; H, 2.59; N, 16.09.
the activity of telomerase with IC50 of 1.24
rable to the positive control staurosporine.
lM, which was compa-
In order to gain deeper understanding of the structure–activity
relationships observed at the telomerase, molecular docking of the
potent inhibitors 5c, 8h and 11f into the binding site of telomerase
was performed on the binding model based on the telomerase
complex structure. Analysis of the three compounds binding
conformation demonstrated that these compounds were stabilized
by hydrogen bonding interaction with Arg194. Apoptosis assay
showed the compounds were potential antitumor agents. The re-
sult provided valuable information for the design of telomerase
inhibitors as antitumor agents.
4.1.1.4. 2-((3-Chlorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-oxadiaz-
ole (5d).
White solid, yield 88%; mp: 76–77 °C. 1H NMR
(500 MHz, CDCl3): 4.53 (s, 2H); 7.26–7.29 (m, 2H); 7.37 (s, 1H);
7.48 (s, 1H); 8.73 (s, 2H); 9.42 (s, 1H). ESI-MS: 305.0
(C13H10ClN4OS, [M+H]+). Anal. Calcd for C13H9ClN4OS: C, 51.23; H,
2.98; N, 18.38. Found: C, 51.11; H, 2.99; N, 18.45.
4.1.1.5. 2-((3-Bromobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-oxadiaz-
ole (5e).
(500 MHz, CDCl3): 4.54 (s, 2H); 7.20–7.27 (m, 1H); 7.30 (t,
J = 7.68 Hz, 2H); 7.65 (s, 1H); 8.72–8.75 (m, 2H); 9.43 (s, 1H). ESI-
MS: 349.0 (C13H10BrN4OS, [M+H]+). Anal. Calcd for C13H9BrN4OS:
C, 44.71; H, 2.60; N, 16.04. Found: C, 44.87; H, 2.59; N, 16.11.
4. Experimental section
4.1. Methods of synthesis
White solid, yield 92%; mp: 90–91 °C. 1H NMR
All the NMR spectra were recorded on a Bruker DPX 500 model
Spectrometer in CDCl3. Chemical shifts (d) for 1H NMR spectra were
reported in parts per million to residual solvent protons. Melting
points were measured on a Boetius micro melting point apparatus.
The ESI-MS spectra were recorded on a Mariner System 5304 Mass
spectrometer. All chemicals and reagents used in current study
were of analytical grade. TLC was run on the silica gel coated
aluminum sheets (Silica Gel 60 GF254, E. Merk, Germany) and
visualized in UV light (254 nm).
4.1.1.6. 2-((4-Fluorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-oxadiaz-
ole (5f).
White solid, yield 90%; mp: 121–123 °C. 1H NMR
(500 MHz, CDCl3): 4.55 (s, 2H); 7.15 (d, J = 7.90 Hz, 2H); 7.35 (d,
J = 7.80 Hz, 2H); 8.72 (s, 2H); 9.42 (s, 1H). ESI-MS: 289.1
(C13H10FN4OS, [M+H]+). Anal. Calcd for C13H9FN4OS: C, 54.16; H,
3.15; N, 19.43. Found: C, 54.32; H, 3.16; N, 19.48.
4.1.1.7. 2-((4-Chlorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-oxadiaz-
ole (5g).
4.1.1. General procedure for synthesis of the target compounds
(5a–5k), (8a–8k) and (11a–11k)
White solid, yield 83%; mp: 118–119 °C. 1H NMR
(500 MHz, CDCl3): 4.53 (s, 2H); 7.31 (d, J = 8.61 Hz, 2H); 7.42 (d,
J = 8.40 Hz, 2H); 8.73 (s, 2H); 9.42 (s, 1H). ESI-MS: 305.0
(C13H10ClN4OS, [M+H]+). Anal. Calcd for C13H9ClN4OS: C, 51.23; H,
2.98; N, 18.38. Found: C, 51.08; H, 2.99; N, 18.44.
To a solution of compound 4 (1 mmol) in acetone, the corre-
sponding benzyl bromide compounds (1 mmol) were added and
the mixture was stirred under reflux for 1–2 h in the presence of
K2CO3 (1 mmol). Then, the solvent was removed under reduced
pressure and a solid obtained. The solid was recrystallized from
ethylalcohol to afford compounds 5a–5k.
To a solution of compound 7 (1 mmol) in acetone, the corre-
sponding benzyl bromide compounds (1 mmol) were added and
the mixture was stirred under reflux for 1–2 h in the presence of
K2CO3 (1 mmol). Then, the solvent was removed under reduced
pressure and a solid obtained. The solid was recrystallized from
ethylalcohol to afford compounds 8a–8k.
To a solution of compound 10 (1 mmol) in acetonitrile, the cor-
responding benzyl bromide compounds (1 mmol) were added and
the mixture was stirred under reflux for 4–8 h in the presence of
NaOH (2 mmol). Then, the solvent was removed under reduced
pressure and a solid obtained. The solid was recrystallized from
acetonitrile to afford compounds 11a–11k.
4.1.1.8. 2-((2-Bromobenzyl)oxy)-5-(pyrazin-2-yl)-1,3,4-oxadiaz-
ole (5h).
White solid, yield 91%; mp: 117–118 °C. 1H NMR
(500 MHz, CDCl3): 4.51 (s, 2H); 7.36 (d, J = 8.25 Hz, 2H); 7.47 (d,
J = 8.35 Hz, 2H); 8.73 (d, J = 6.70 Hz, 2H); 9.42 (s, 1H). ESI-MS:
349.0 (C13H10BrN4OS, [M+H]+). Anal. Calcd for C13H9BrN4OS: C,
44.71; H, 2.60; N, 16.04. Found: C, 44.85; H, 2.61; N, 16.10.
4.1.1.9. 2-((2,4-Difluorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-oxa-
diazole (5i).
White solid, yield 85%; mp: 85–87 °C. 1H NMR
(500 MHz, CDCl3): 4.58 (s, 2H); 6.83–6.92 (m, 2H); 7.55–7.62 (m,
1H); 8.75 (s, 2H); 9.43 (s, 1H). ESI-MS: 307.0 (C13H9F2N4OS,
[M+H]+). Anal. Calcd for C13H8F2N4OS: C, 50.98; H, 2.63; N, 18.29.
Found: C, 51.11; H, 2.64; N, 18.33.
4.1.1.10.
oxadiazole (5j).
2-((2,6-Difluorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-
White solid, yield 87%; mp: 72–73 °C. 1H
4.1.1.1. 2-((2-Fluorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-oxadiaz-
ole (5a).
(500 MHz, CDCl3): 4.61 (s, 2H); 7.05–7.13 (m, 2H); 7.26–7.32 (m,
1H); 7.56 (t, J = 7.65 Hz, 1H); 8.73 (s, 2H); 9.42 (s, 1H). ESI-MS:
White solid, yield 89%; mp: 142–143 °C. 1H NMR
NMR (500 MHz, CDCl3): 4.67 (s, 2H); 6.92–6.97 (m, 2H); 7.27–
7.34 (m, 1H); 8.75 (s, 2H); 9.45 (s, 1H). ESI-MS: 307.0

Y.-B. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 6356–6365
6363
(C13H9F2N4OS, [M+H]+). Anal. Calcd for C13H8F2N4OS: C, 50.98; H,
2.63; N, 18.29. Found: C, 51.08; H, 2.64; N, 18.23.
MS: 365.0 (C13H10BrN4S2, [M+H]+). Anal. Calcd for C13H9BrN4S2:
C, 42.75; H, 2.48; N, 15.34. Found: C, 42.88; H, 2.47; N, 15.38.
4.1.1.11.
(5k).
2-(Benzylthio)-5-(pyrazin-2-yl)-1,3,4-oxadiazole
4.1.1.20.
thiadiazole (8i).
2-((2,4-Difluorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-
White solid, yield 72%; mp: 140–142 °C. 1H
White solid, yield 88%; mp: 79–80 °C. 1H NMR
(500 MHz, CDCl3): 4.58 (s, 2H); 7.31–7.36 (m, 3H); 7.47 (d,
J = 7.15 Hz, 1H); 8.72 (s, 2H); 9.42 (s, 1H). ESI-MS: 271.0
(C13H11N4OS, [M+H]+). Anal. Calcd for C13H10N4OS: C, 57.76; H,
3.73; N, 20.73. Found: C, 57.61; H, 3.72; N, 20.78.
NMR (500 MHz, CDCl3): 4.69 (s, 2H); 6.83–6.87 (m, 2H); 7.54–
7.59 (m, 1H); 8.60 (s, 1H); 8.66 (s, 1H); 9.54 (s, 1H). ESI-MS:
323.0 (C13H9F2N4S2, [M+H]+). Anal. Calcd for C13H8F2N4S2: C,
48.44; H, 2.50; N, 17.38. Found: C, 48.56; H, 2.51; N, 17.33.
4.1.1.12. 2-((2-Fluorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadi-
azole (8a).
4.1.1.21.
thiadiazole (8j).
2-((2,6-Difluorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-
White solid, yield 68%; mp: 165–166 °C. 1H
White solid, yield 71%; mp: 120–121 °C. 1H NMR
(500 MHz, CDCl3): 4.69 (s, 2H); 7.07–7.13 (m, 2H); 7.24–7.32 (m,
1H); 7.56 (t, J = 7.60 Hz, 1H); 8.59 (s, 1H); 8.66 (s, 1H); 9.54 (s,
1H). ESI-MS: 305.0 (C13H10FN4S2, [M+H]+). Anal. Calcd for
NMR (500 MHz, CDCl3): 4.74 (s, 2H); 6.92–6.95 (m, 2H); 7.28–
7.33 (m, 1H); 8.60 (s, 1H); 8.67 (s, 1H); 9.56 (s, 1H). ESI-MS:
323.0 (C13H9F2N4S2, [M+H]+). Anal. Calcd for C13H8F2N4S2: C,
48.44; H, 2.50; N, 17.38. Found: C, 48.60; H, 2.51; N, 17.34.
C13H9FN4S2: C, 53.10; H, 2.98; N, 18.41. Found: C, 53.28; H, 2.99;
N, 18.47.
4.1.1.22.
(8k).
2-(Benzylthio)-5-(pyrazin-2-yl)-1,3,4-thiadiazole
4.1.1.13. 2-((2-Chlorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadi-
azole (8b).
White solid, yield 66%; mp: 127–128 °C. 1H NMR
White solid, yield 68%; mp: 116–117 °C. 1H NMR
(500 MHz, CDCl3): 4.64 (s, 2H); 7.28–7.36 (m, 3H); 7.46 (d,
J = 7.45 Hz, 2H); 8.58 (s, 1H); 8.64 (s, 1H); 9.53 (s, 1H). ESI-MS:
287.0 (C13H11N4S2, [M+H]+). Anal. Calcd for C13H10N4S2: C, 54.52;
H, 3.52; N, 19.56. Found: C, 54.61; H, 3.51; N, 19.62.
(500 MHz, CDCl3): 4.79 (s, 2H); 7.24–7.27 (m, 2H); 7.43 (d,
J = 9.33 Hz, 1H); 7.63–7.66 (m, 1H); 8.63 (d, J = 17.76 Hz, 2H);
9.55 (s, 1H). ESI-MS: 321.0 (C13H10ClN4S2, [M+H]+). Anal. Calcd
for C13H9ClN4S2: C, 48.67; H, 2.83; N, 17.46. Found: C, 48.78; H,
2.82; N, 17.41.
4.1.1.23. 2-(5-((2-Fluorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-
3-yl)pyrazine (11a).
Yellow solid, yield 74%; mp: 131–132 °C.
4.1.1.14. 2-((2-Bromobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadi-
1H NMR (500 MHz, CDCl3): 4.64 (s, 2H); 7.01–7.10 (m, 2H); 7.16 (d,
J = 7.35 Hz, 2H); 7.24–7.30 (m, 1H); 7.43–7.50 (m, 3H); 7.54 (t,
J = 7.60 Hz, 1H); 8.24 (s, 1H); 8.49 (s, 1H); 9.39 (s, 1H). ESI-MS:
364.1 (C19H15FN5S, [M+H]+). Anal. Calcd for C19H14FN5S: C, 62.79;
H, 3.88; N, 19.27. Found: C, 62.61; H, 3.89; N, 19.33.
azole (8c).
White solid, yield 63%; mp: 133–135 °C. 1H NMR
(500 MHz, CDCl3): 4.80 (s, 2H); 7.15–7.21 (m, 1H); 7.27–7.32 (m,
1H); 7.60–7.68 (m, 2H); 8.61 (s, 1H); 8.66 (s, 1H); 9.55 (s, 1H).
ESI-MS: 365.0 (C13H10BrN4S2, [M+H]+). Anal. Calcd for
C13H9BrN4S2: C, 42.75; H, 2.48; N, 15.34. Found: C, 42.88; H,
2.49; N, 15.38.
4.1.1.24. 2-(5-((2-Chlorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-
3-yl)pyrazine (11b).
Yellow solid, yield 77%; mp: 144–145 °C.
4.1.1.15. 2-((3-Chlorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadi-
1H NMR (500 MHz, CDCl3): 4.69 (s, 2H); 7.13–7.16 (m, 2H); 7.19–
7.26 (m, 2H); 7.35–7.38 (m, 1H); 7.41–7.49 (m, 3H); 7.61–7.64
(m, 1H); 8.24 (s, 1H); 8.49 (s, 1H); 9.38 (s, 1H). ESI-MS: 380.1
(C19H15ClN5S, [M+H]+). Anal. Calcd for C19H14ClN5S: C, 60.07; H,
3.71; N, 18.44. Found: C, 60.21; H, 3.70; N, 18.40.
azole (8d).
Yellow solid, yield 77%; mp: 127–128 °C. 1H NMR
(500 MHz, CDCl3): 4.62 (s, 2H); 7.27–7.36 (m, 2H); 7.38–7.40 (m,
1H); 7.48 (s, 1H); 8.59–8.61 (m, 1H); 8.66 (s, 1H); 9.54 (s, 1H).
ESI-MS: 321.0 (C13H10ClN4S2, [M+H]+). Anal. Calcd for C13H9ClN4S2:
C, 48.67; H, 2.83; N, 17.46. Found: C, 48.54; H, 2.84; N, 17.51.
4.1.1.25. 2-(5-((2-Bromobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-
4.1.1.16. 2-((3-Bromobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadi-
azole (8e).
(500 MHz, CDCl3): 4.61 (s, 2H); 7.20–7.27 (m, 1H); 7.42 (s, 2H);
7.64 (s, 1H); 8.60 (s, 1H); 8.66 (s, 1H); 9.54 (s, 1H). ESI-MS: 365.0
(C13H10BrN4S2, [M+H]+). Anal. Calcd for C13H9BrN4S2: C, 42.75; H,
2.48; N, 15.34. Found: C, 42.60; H, 2.49; N, 15.39.
3-yl)pyrazine (11c).
Yellow solid, yield 65%; mp: 160–162 °C.
White solid, yield 70%; mp: 120–121 °C. 1H NMR
1H NMR (500 MHz, CDCl3): 4.70 (s, 2H); 7.12–7.17 (m, 3H); 7.24–
7.28 (m, 1H); 7.42–7.51 (m, 3H); 7.55 (d, J = 7.50 Hz, 1H); 7.64
(d, J = 7.50 Hz, 1H); 8.24 (s, 1H); 8.49 (s, 1H); 9.38 (s, 1H). ESI-
MS: 424.0 (C19H15BrN5S, [M+H]+). Anal. Calcd for C19H14BrN5S: C,
53.78; H, 3.33; N, 16.50. Found: C, 53.60; H, 3.34; N, 16.44.
4.1.1.17. 2-((4-Fluorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadi-
azole (8f).
(500 MHz, CDCl3): 4.61 (s, 2H); 7.12–7.17 (m, 2H); 7.36 (d,
J = 7.80 Hz, 2H); 8.58 (s, 1H); 8.65 (s, 1H); 9.54 (s, 1H). ESI-MS:
305.0 (C13H10FN4S2, [M+H]+). Anal. Calcd for C13H9FN4S2: C,
53.10; H, 2.98; N, 18.41. Found: C, 53.26; H, 2.99; N, 18.35.
4.1.1.26. 2-(5-((3-Chlorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-
White solid, yield 72%; mp: 136–137 °C. 1H NMR
3-yl)pyrazine (11d).
White solid, yield 78%; mp: 117–118 °C.
1H NMR (500 MHz, CDCl3): 4.55 (s, 2H); 7.15–7.17 (m, 2H); 7.23–
7.27 (m, 2H); 7.38 (s, 1H); 7.45–7.51 (m, 4H); 8.25 (s, 1H); 8.50
(s, 1H); 9.39 (s, 1H). ESI-MS: 380.1 (C19H15ClN5S, [M+H]+). Anal.
Calcd for C19H14ClN5S: C, 60.07; H, 3.71; N, 18.44. Found: C,
59.92; H, 3.70; N, 18.49.
4.1.1.18. 2-((4-Chlorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadi-
azole (8g).
Yellow solid, yield 61%; mp: 153–155 °C. 1H NMR
4.1.1.27. 2-(5-((3-Bromobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-
(500 MHz, CDCl3): 4.60 (s, 2H); 7.31 (d, J = 8.10 Hz, 2H); 7.41 (d,
J = 7.90 Hz, 2H); 8.58 (s, 1H); 8.65 (s, 1H); 9.52 (s, 1H). ESI-MS:
321.0 (C13H10ClN4S2, [M+H]+). Anal. Calcd for C13H9ClN4S2: C,
48.67; H, 2.83; N, 17.46. Found: C, 48.79; H, 2.84; N, 17.41.
3-yl)pyrazine (11e).
Yellow solid, yield 67%; mp: 135–136 °C.
1H NMR (500 MHz, CDCl3): 4.50 (s, 2H); 7.14–7.18 (m, 3H); 7.34 (d,
J = 7.60 Hz, 1H); 7.39 (d, J = 7.90 Hz, 1H); 7.44–7.51 (m, 3H); 7.52
(s, 1H); 8.24 (s, 1H); 8.49 (s, 1H); 9.38 (s, 1H). ESI-MS: 424.0
(C19H15BrN5S, [M+H]+). Anal. Calcd for C19H14BrN5S: C, 53.78; H,
3.33; N, 16.50. Found: C, 53.89; H, 3.34; N, 16.45.
4.1.1.19. 2-((4-Bromobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadi-
azole (8h).
Yellow solid, yield 68%; mp: 158–160 °C. 1H NMR
(500 MHz, CDCl3): 4.59 (s, 2H); 7.37 (d, J = 8.61 Hz, 2H); 7.48 (d,
4.1.1.28. 2-(5-((4-Fluorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-
J = 8.58 Hz, 2H); 8.59–8.61 (m, 1H); 8.66 (s, 1H); 9.54 (s, 1H). ESI-
3-yl)pyrazine (11f).
White solid, yield 76%; mp: 144–145 °C.

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Y.-B. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 6356–6365
1H NMR (500 MHz, CDCl3): 4.59 (s, 2H); 7.10–7.16 (m, 4H); 7.28 (s,
2H); 7.37–7.49 (m, 3H); 8.24 (s, 1H); 8.49 (s, 1H); 9.38 (s, 1H).
ESI-MS: 364.1 (C19H15FN5S, [M+H]+). Anal. Calcd for C19H14FN5S:
C, 62.79; H, 3.88; N, 19.27. Found: C, 62.62; H, 3.89; N, 19.34.
serum (GIBCO, New York, USA), streptomycin (0.1 mg/mL) and
penicillin (100 IU/mL) at 37 °C in
a humidified atmosphere
containing 5% CO2. After trypsinization, 5 Â 104 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 (Santa Cruz, Santa Cruz, USA) and
the drugs with a series of concentration as 30, 10, 3.33, 1.11,
4.1.1.29. 2-(5-((4-Chlorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-
3-yl)pyrazine (11g).
Yellow solid, yield 77%; mp: 169–170 °C.
1H NMR (500 MHz, CDCl3): 4.52 (s, 2H); 7.14 (d, J = 6.78 Hz, 1H);
7.24 (s, 1H); 7.27 (s, 1H); 7.34 (d, J = 8.43 Hz, 2H); 7.44–7.48 (m,
4H); 8.23 (s, 1H); 8.49 (s, 1H); 9.37 (s, 1H). ESI-MS: 380.1
(C19H15ClN5S, [M+H]+). Anal. Calcd for C19H14ClN5S: C, 60.07; H,
3.71; N, 18.44. Found: C, 60.18; H, 3.70; N, 18.39.
0.37 and 0.12
were harvested by cell scraper orderly following by washed once
with PBS. The cells were lysed in 150 L RIPA cell lysis buffer
lg/mL, respectively. After 24 h treatment, the cells
l
(Santa Cruz, Santa Cruz, USA), and incubated on ice for 30 min.
The cellular supernatants were obtained via centrifugation at
12,000 g for 20 min at 4 °C and stored at À80 °C.
4.1.1.30. 2-(5-((4-Bromobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-
The TRAP–PCR–ELISA assay was performed using a telomerase
detection kit (Roche, Basel, Switzerland) according to the manufac-
3-yl)pyrazine (11h).
White solid, yield 72%; mp: 167–169 °C.
1H NMR (500 MHz, CDCl3): 4.52 (s, 2H); 7.16 (d, J = 6.75 Hz, 2H);
7.34 (d, J = 10.05 Hz, 2H); 7.41–7.53 (m, 5H); 8.24 (s, 1H); 8.50 (s,
1H); 9.39 (s, 1H). ESI-MS: 424.0 (C19H15BrN5S, [M+H]+). Anal. Calcd
for C19H14BrN5S: C, 53.82; H, 3.33; N, 16.51. Found: C, 53.69; H,
3.32; N, 16.46.
turer’s protocol. In brief, 2
lL of cell extracts were mixed with
48 L TRAP reaction mixtures. PCR was then initiated at 94 °C,
l
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 lL 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 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 wavelength
of 490 nm, and the final values were presented.
4.1.1.31. 2-(5-((2,4-Difluorobenzyl)thio)-4-phenyl-4H-1,2,4-tria-
zol-3-yl)pyrazine (11i).
Yellow solid, yield 80%; mp: 156–
157 °C. 1H NMR (500 MHz, CDCl3): 4.56 (s, 2H); 6.77–6.84 (m,
2H); 7.17–7.21 (m, 2H); 7.44–7.51 (m, 3H); 7.57 (dd, J1 = 8.40 Hz,
J2 = 14.95 Hz, 1H); 8.24 (s, 1H); 8.49 (s, 1H); 9.39 (s, 1H). ESI-MS:
382.1 (C19H14F2N5S, [M+H]+). Anal. Calcd for C19H13F2N5S: C,
59.83; H, 3.44; N, 18.36. Found: C, 59.67; H, 3.45; N, 18.42.
4.1.1.32. 2-(5-((2,6-Difluorobenzyl)thio)-4-phenyl-4H-1,2,4-tria-
4.4. Apoptosis assay
zol-3-yl)pyrazine (11j).
Yellow solid, yield 78%; mp: 172–
173 °C. 1H NMR (500 MHz, CDCl3): 4.59 (s, 2H); 6.87 (t,
J = 7.80 Hz, 2H); 7.21–7.24 (m, 3H); 7.44–7.48 (m, 3H); 8.24 (s,
1H); 8.49 (s, 1H); 9.40 (s, 1H). ESI-MS: 382.1 (C19H14F2N5S,
[M+H]+). Anal. Calcd for C19H13F2N5S: C, 59.83; H, 3.44; N, 18.36.
Found: C, 59.97; H, 3.45; N, 18.30.
HEPG2 cells were treated with various concentrations of com-
pound 8h for 24 h and then stained with both Annexin V-FITC
(fluorescein isothiocyanate) and propidium iodide (PI). Then
samples were analyzed by FACSCalibur flow cytometer (Becton
Dickinson, SanJose, CA).
4.1.1.33. 2-(5-(Benzylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)pyra-
zine (11k).
4.5. Molecular docking study
Yellow solid, yield 86%; mp: 140–141 °C. 1H NMR
(500 MHz, CDCl3): 4.58 (s, 2H); 7.13 (d, J = 7.65 Hz, 2H); 7.26–7.29
(m, 3H); 7.38 (d, J = 7.50 Hz, 1H); 7.42–7.48 (m, 3H); 8.24 (s, 1H);
8.49 (s, 1H); 9.38 (s, 1H). ESI-MS: 346.1 (C19H16N5S, [M+H]+). Anal.
Calcd for C19H15N5S: C, 66.07; H, 4.38; N, 20.27. Found: C, 66.21; H,
4.39; N, 20.32.
Molecular docking of compounds into the 3D telomerase
complex structure (PDB code:3DU6) was carried out using the Dis-
covery Studio (version 3.1) as implemented through the graphical
user interface DS-CDocker protocal.
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem 3D ultra 12.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
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 complex were retrieved from
the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/
home.do). All bound water and ligands were eliminated from the
protein and the polar hydrogen was added. Types of interactions
of the docked protein with ligand were analyzed after the end of
molecular docking.
4.2. Cell proliferation assay
The antitumor activities of compounds 5a–5k, 8a–8k and 11a–
11k were determined using a standard (MTT)-based colorimetric
assay (Sigma). Seed 104 cells per well into 96-well plates, incubate
at 37 °C, 5% CO2 for 24 h. Then add 100
drug-containing medium into wells to maintain the final concen-
tration of drug as 30, 10, 3.33, 1.11, 0.37 and 0.12 g/mL. One con-
lL a series concentration of
l
centration should be triplicated and staurosporine was used for the
positive control. After 48 h, cell survival was determined by the
addition of an MTT solution (25
lL of 5 mg/mL MTT in PBS). After
4.6. Crystal structure determination
4 h, discard the medium and add 100
lL DMSO; the plates were
votexed for 10 min to make completely dissolution. Optical
absorbance was measured at 490 nm.
Crystal structure determination of compounds 5k, 8k, 11e and
11i were carried out on a Nonius CAD4 diffractometer equipped
with graphitemonochromated Mo K
a (k = 0.71073 Å) radiation
4.3. Telomerase inhibitory assay
(Table 1). The structure was solved by direct methods and refined
on F2 by fullmatrix least-squares methods using SHELX-97.31 All
non-hydrogen atoms of compounds 5k, 8k, 11e and 11i were
refined with anisotropic thermal parameters. All hydrogen atoms
were placed in geometrically idealized positions and constrained
to ride on their parent atoms.
Compounds were tested in a search for small molecule inhibi-
tors of telomerase activity by using the TRAP–PCR–ELISA assay.
In detail, the HEPG2 cells were firstly maintained in DMEM med-
ium (GIBCO, New York, USA) supplemented with 10% fetal bovine

Y.-B. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 6356–6365
6365
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Acknowledgments
The work was financed by National Natural Science Foundation
of China (No. J1103512).
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