Synthesis, biological evaluation, 3D-QSAR studies of novel aryl-2H-pyrazole derivatives as telomerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.12.010

A series of novel aryl-2H-pyrazole derivatives bearing 1,4-benzodioxan or 1,3-benzodioxole moiety were designed as potential telomerase inhibitors to enhance the ability of aryl-2H-pyrazole derivatives to inhibit telomerase, a target of anticancer. The telomerase inhibition tests showed that compound 16A displayed the most potent inhibitory activity with IC₅₀ value of 0.9 μM for telomerase. The antiproliferative tests showed that compound 16A exhibited high activity against human gastric cancer cell SGC-7901 and human melanoma cell B16-F10 with IC₅₀ values of 18.07 and 5.34 μM, respectively. Docking simulation showed that compound 16A could bind well with the telomerase active site and act as telomerase inhibitor. 3D-QSAR model was also built to provide more pharmacophore understanding that could be used to design new agents with more potent telomerase inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2012.12.010

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1091–1095
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation, 3D-QSAR studies of novel
aryl-2H-pyrazole derivatives as telomerase inhibitors
Yin Luo a, , Shuai Zhang a, , Ke-Ming Qiu a, Zhi-Jun Liu a, Yu-Shun Yang a, Jie Fu c, Wei-Qing Zhong b,
,
⇑
Hai-Liang Zhu a,
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P.R. China
b School of Pharmacy, Second Military Medical University, Shanghai 200433, P.R. China
c State Key Laboratory of Pollution Control & Resource Reuse, Nanjing University, Nanjing 210046, P.R. 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 novel aryl-2H-pyrazole derivatives bearing 1,4-benzodioxan or 1,3-benzodioxole moiety were
designed as potential telomerase inhibitors to enhance the ability of aryl-2H-pyrazole derivatives to
inhibit telomerase, a target of anticancer. The telomerase inhibition tests showed that compound 16A
Received 4 November 2012
Revised 6 December 2012
Accepted 7 December 2012
Available online 20 December 2012
displayed the most potent inhibitory activity with IC50 value of 0.9
ative tests showed that compound 16A exhibited high activity against human gastric cancer cell SGC-
7901 and human melanoma cell B16-F10 with IC50 values of 18.07 and 5.34 M, respectively. Docking
lM for telomerase. The antiprolifer-
l
Keywords:
simulation showed that compound 16A could bind well with the telomerase active site and act as telo-
merase inhibitor. 3D-QSAR model was also built to provide more pharmacophore understanding that
could be used to design new agents with more potent telomerase inhibitory activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Aryl-2H-pyrazole
Telomerase inhibitor
Antiproliferative
3D-QSAR
In the early stage of life, telomerase is active and maintains telo-
mere length and the chromosomal integrity of frequently dividing
cells. During adulthood, telomerase keeps dormant in most so-
matic cells.1 However, telomerase gets reactivated first and then
works tirelessly to keep the short length of telomeres during the
dividing stage of cancer cells, which leads to the long life of cancer
cells.2 The essential role of telomerase makes it an important target
for the development of therapies to treat cancer and other age-
associated disorders.3
Several applications were reported about 2H-pyrazole derivatives
in pharmaceutical fields. According to the research papers, it showed
that 2H-pyrazole derivatives have strong bioactivity, such as antican-
cer activity.4–7 In 2005, Manna et al. reported a series of replacing aryl
group 2H-pyrazole, which could directly combine and inhibit p-gly-
coprotein mediated resistance to drugs.8 In order to discover some
potential anticancer leading structure, Insuasty et al. synthesized a
series of 3-aryl-2H-pyrazole derivatives in 2009.9 And then, US
National Cancer Institute screened some compounds of those deriva-
tives inhibiting about 60 variety of human tumor cell lines.10
Oxygen-bearing heterocycles especially 1,3-benzodioxole and
1,4-benzodioxan have caught significant attention in the medicinal
chemical research field.11 The benzodioxole group is widely used in
synthetic medicinal chemistry as a component of enzyme inhibi-
tors; it is present in piperine, a known enzyme inhibitor. Besides,
more and more chemical compounds bearing 1,4-benzodioxan
moiety as potential anticancer agents have been confirmed.12–14
Relevant research indicated that 1,3,4-oxadiazole derivatives bear-
ing 1,4-benzodioxan moiety show low toxicity to human liver cells
and have a good inhibitory effect on cancer cells.15
Andrew et al. revealed the telomerase key active site with X-ray in
2008, which was three-dimensional structure of TERT protein catalysis
subunit.3 At present, our group designed and synthesized several
kinds of telomerase inhibitors.16–18 All of these compounds had the
similar skeleton structure: 2H-pyrazole. So we deduced that 2H-
pyrazole might be used as key group to show telomerase inhibitory
activity.
Above all, we continued our previous study and designed new
2H-pyrazoles bearing oxygen-bearing heterocycles targeting
telomerase. In this Letter, we described the synthesis and the struc-
ture–activity relationships of series of aryl-2H-pyrazole derivatives
bearing 1,5-benzdioxepine or 1,4-benzodioxan or 1,3-benzodioxole
moiety as potential antitumor agents, which was based on molecu-
lar modeling. Biological evaluation was also carried out for screening
potential telomerase inhibitors among the synthesized compounds.
Docking simulations were performed using the X-ray crystallo-
graphic structure of the telomerase in complex with an inhibitor
to explore the binding mode of the compound at the active site.
Based on the activity data, QSAR model was also built to study the
structure–activity relationship and guide the further study.
A series of novel aryl-2H-pyrazole derivatives were synthesized
by the routes outlined in Scheme 1. Compounds 1–3 were synthe-
sized from 3,4-dihydroxybenzal-dehyde and dibromomethane or
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contribute to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.010

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Y. Luo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1091–1095
OHC
OH
OH
OHC
O
(I)
+
n
Br
n=1,2,3
OHC
Br
n
O
1-3
O
O
O
O
O
O
(II)
+
n
n
R
R
R
1-3
4-10
11-31
O
O
N
N
O
O
(III)
R
O
O
n
n
11-31
11A-31A
Scheme 1. Synthetic pathway of compounds 11A–31A. Reagents and conditions: (I) acetone, K2CO3, reflux, 24 h; (II) EtOH, KOH (40% aq), rt 2 h; (III) hydrazine hydrate, acetic
acid, reflux, 2 h.
dibromoethane or dibromopropane with acetone as solvent in the
presence of potassium carbonate. The synthesized compounds
were then reacted with substituted acetophenones (4–10) to pre-
pare the corresponding chalcone derivatives 11–31. After that
solution of compounds 11–31 in acetic acid were reacted with
hydrazine hydrate to synthesize corresponding 2H-pyrazoles
11A–31A. Compounds 17A–31A reported synthesized for the first
time. These compounds gave satisfactory elementary analyses. 1H
NMR and ESI MS spectra were consistent with the assigned
structures.
Table 1
Inhibitory effects of the title compounds against telomerase and cancer cell lines
O
N
N
R
O
O
n
Compounds
n
R
IC50
B16-F10
(
l
M)
IC50
SCG-7901
(
lM)
IC50 (lM)
Telomerase
Compounds 11A–31A were evaluated for telomerase inhibitory
activities by a modified telomere repeat amplification protocol
(TRAP) assay.20 Modified TRAP assay was proved to be a useful
technology to give some information about small molecules inhib-
iting telomere elongation qualitatively and quantitatively.20 As
summarized in Table 1, most of the synthesized 2H-pyrazole deriv-
atives displayed good telomerase inhibitory activities and among
them compound 16A displayed the best inhibitory activity with
11A
12A
13A
14A
15A
16A
17A
18A
19A
20A
21A
22A
23A
24A
25A
26A
1
1
1
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
3
3
3
H
F
Cl
Br
CH3
OCH3
NO2
H
F
Cl
13.96
22.17
20.83
18.93
12.78
5.34
24.74
39.52
53.11
47.34
44.06
37.93
31.67
57.8
No
No
No
96.95
89.71
85.26
No
25.67
40.85
35.33
30.09
21.24
18.07
41.93
50.17
68.39
59.46
52.31
49.22
45.83
70.05
No
No
No
No
No
94.01
No
46.35
3.4
7.3
6.7
6.2
1.3
0.9
8.7
18.5
25.7
23.8
22.9
13.4
10.6
37.4
68.3
83.6
82.6
76.4
54.9
46.7
93.2
IC50 of 0.9 lM for telomerase which could be comparable to the
reference compound ethidium bromide.
Br
CH3
OCH3
NO2
H
F
Cl
According to the data presented in Table 1, it could be con-
cluded that aryl-2H-pyrazole derivatives bearing 1,3-benzodioxole
group (compounds 11A–17A) displayed better telomerase inhibi-
tory activities, whereas aryl-2H-pyrazole derivatives bearing
1,4-benzodioxan and 1,5-benzdioxepine group, in general, showed
relatively worse activities. And the results suggested that the
compounds with 1,3-benzodioxole group (11A–17A) and elec-
tron-donating groups in R position (15A, 16A, 22A, 23A, 29A and
30A) showed better telomerase inhibitory activities than com-
pounds with electron-withdrawing substituents. Some compounds
in this series deserve further investigation.
Based on above results, compounds 11A–31A were also chosen
to finish the screening assay studies. These compounds were eval-
uated for their anticancer activities against gastric SGC-7901 (hu-
man gastric cancer) and B16-F10 (mouse melanoma) cell lines.
From the results summarized in Table 1, it was obvious that com-
pound 16A exhibited best activity against B16-F10 cells with the
27A
28A
29A
30A
Br
CH3
OCH3
No2
31A
5-Fluorouracil
21.41
Ethidium bromidea
2.6
No, not observed in the tested concentration range 0–100 lM.
Ethidium bromide is reported as a control. The inhibition constant of ethidium
toward telomerase has been reported previously.
a
in contrast, all the synthesized compounds exhibited worse activ-
ity against SGC-7901 cells. The results were similar with previous
paper.21
Molecular docking is an application wherein molecular
modeling techniques are used to predict how a protein (enzyme)
interacts with small molecules (ligands).22 In order to explore
probable interaction model of inhibitors (ligands) and enzyme
active site, molecular docking of the most potent enzyme inhibitor
16A into active binding site of telomerase was performed to
IC50 value of 5.34
rouracil with the IC50 value of 21.41
l
M, much better than the reference drug 5-fluo-
M. Most of the compounds
l
bearing 1,3-benzodioxole (11A–17A) exhibited better activity
against B16-F10 compared with reference drug. And also it was
obvious that compounds bearing 1,3-benzodioxole (11A–17A)
group showed better anticancer activity than those bearing
1,4-benzodioxan and 1,5-benzdioxepine groups. At the same time,

Y. Luo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1091–1095
1093
Figure 1. (a) Ligand interaction diagram of compound 16A with TERT protein catalytic subunit (PDB ID: 3DU6) using Discovery Studio program with the essential amino acid
residues at the binding site are tagged in circles. 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) The 3D model structure of compound 16A binding model with telomerase
complex.
Table 2
Experimental, predicted inhibitory activity of compounds 11A–31A by 3D-QSAR
models based upon active conformation achieved by molecular docking
Compound
Experimental pIC50
Predicted pIC50
Residual error
11A
12A
13A
14A
15A
16A
17A
18A
19A
20A
21A
22A
23A
24A
25A
26A
27A
28A
29A
30A
31A
5.469
5.137
5.174
5.208
5.886
6.046
5.060
4.733
4.590
4.623
4.640
4.873
4.975
4.427
4.166
4.078
4.083
4.117
4.260
4.331
4.031
5.200
5.506
4.991
5.523
5.702
5.876
4.702
5.070
4.368
5.027
4.621
4.707
4.704
4.711
4.549
4.283
4.202
4.372
4.447
4.212
4.075
0.269
ꢀ0.369
0.183
ꢀ0.315
0.184
0.169
0.359
ꢀ0.337
0.222
ꢀ0.403
0.020
0.166
0.270
ꢀ0.283
ꢀ0.383
ꢀ0.205
ꢀ0.119
ꢀ0.255
ꢀ0.187
0.119
Figure 2. Plot of experimental versus predicted telomerase inhibitory activities of
training set and test set.
Based on the preliminary biological results, a 3D-QSAR model of
these 2H-pyrazole derivates was initiated by using a novel 3D-
QSAR protocol of Discovery Studio 3.1. The calculated pIC50 values
ranged from 4.08 to 6.04.
ꢀ0.044
simulate a binding model derived from TERT protein catalytic sub-
unit (3DU6.pdb).
Twenty one compounds (Table 1) with IC50s ranging from 0.9 to
82.6 lM for inhibiting telomerase were selected as the model data-
All docking runs were applied CDOCKER protocol of Discovery
Studio 3.1. The 2D and 3D binding model of compound 16A and
telomerase was depicted in Figure 1. In the binding model, com-
pound 16A was nicely bound to the TERT protein catalytic subunit
with four interaction bonds with binding interaction energy of
ꢀ35.76 KJ molꢀ1. Visual inspection of the pose of 16A into the ac-
tive site revealed that two optimal intermolecular hydrogen bonds
were observed (LYS249: N–Hꢁ ꢁ ꢁO: 2.3 Å, angle: 137.6°; LYS406: N–
set. The IC50 values were converted into corresponding pIC50 values
by the formula in Eq. (1):
pIC50 ¼ ꢀlog10 IC50
ð1Þ
Because molecular alignment was considered as a crucial step
for 3D-QSAR study,23 we applied CDOCKER protocol to explore
each molecule with lowest energy before alignment conformation.
4,5-Dihydro-1H-pyrazole was selected as substructure to build
alignment conformation before building the QSAR model. The ini-
tial set of compounds has been randomly divided into training set
and test set by the Molecules method in Discovery Studio.
Hꢁ ꢁ ꢁO: 2.1 Å, angle: 137.6°). Also the two benzene rings formed
p-
cation interactions with LYS189 and LYS372, respectively. These
residues influenced the accessibility of the hydrophobic pocket
that flanks the active binding site, and their size could be impor-
tant in controlling telomerase selectivity. In the other end of the
binding pocket, the oxygen atom of 2H-pyrazole acetyl interacted
with the residue GLN308, which might make the 3D structure
more stable.
The correlation coefficient r2 between observed and predicted
activity of training set was found to be 0.829, while that of test
set was found to be 0.966, which proved this QSAR model was
acceptable. Predicted pIC50 values and residual errors of 21 com-
pounds by this QSAR model had been given in Table 2. The plot

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Y. Luo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1091–1095
Figure 3. (a) 3D-QSAR model coefficients on van der Waals grids. Green represents positive coefficients; yellow represents negative coefficients. (b) 3D-QSAR model
coefficients on electrostatic potential grids. Blue represents positive coefficients; red represents negative coefficients.
As a result, data summarized above demonstrated that com-
pounds 16A, the most potent telomerase inhibitor (IC50 = 0.9 lM),
containing suitable substituents had an outstanding activity.
In all, a series of novel aryl-2H-pyrazole derivatives bearing
oxygen-bearing heterocycle group were prepared as potential
telomerase inhibitors. The bioactivity assay results showed that
compound 16A showed most potent inhibition activity for telo-
merase and good activity against human melanoma cell B16-F10
better than reference drug. Docking simulation was performed
to position compound 16A into 3DU6 active site, the result
showed compound 16A could bind well with the telomerase ac-
tive site and act as telomerase inhibitor. QSAR model was also
built by the activity data and binding conformations to provide
a reliable tool for rational design of novel telomerase inhibitors.
The above results provided theoretical basis for further struc-
tural optimization of 2H-pyrazole derivatives as telomerase
inhibitors.
Figure 4. 3D-QSAR model coefficients on van der Waals grids from another
perspective.
Acknowledgment
This work was supported by the National Natural Science Foun-
dation of China (Grant Nos. 20371050, 21273283 and J1103512).
of the observed pIC50 versus the predicted data was shown in Fig-
ure 2. Besides, we have built a random model by adding two re-
ported compounds in previous paper (4a and 4b in reference18).
The results showed that r2 for training set was 0.984 and that of
test set was 0.567 while that of test set was 0.996 in statistical
model. This model could lead to increase residual errors. We can
assure that the statistical model is much more reliable than ran-
dom model.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.010.
Also the molecules aligned with the iso-surfaces of the 3D-QSAR
model coefficients on van der Waals grids (Fig. 3a) and electrostatic
potential grids (Fig. 3b) are listed. Electrostatic map indicates red
contours around regions where high electron density (negative
charge) is expected to increase activity, and blue contours repre-
sent areas where low electron density (partial positive charge) is
expected to increase activity. Similarly, steric map indicates areas
where steric bulk is predicted to increase (green) or decrease (yel-
low) activity.
According to the maps, it suggested the compound with high
positive charged and small oxygen-bearing heterocycle group
would show higher activity, validating that 1,3-benzodioxole
group being a better choice than 1,4-benzodioxan and 1,5-benzdi-
oxepine. Whereas, a high negative charged R group would help
obtain good activity, validating the methoxy substituent could be
more effective. As shown in Figure 4, it was clear that R groups
were surrounded by several green and yellow grids, which sug-
gested that molecular size did not influence the activity obviously.
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