Synthesis, biological evaluation, and molecular docking studies of pyrazolyl-acylhydrazone derivatives as novel anticancer agents

Article abstract of DOI:10.1007/s00044-014-0909-0

A series of pyrazolyl-acylhydrazone derivatives (1e-20e) have been designed and synthesized and their biologic activities were also evaluated for telomerase inhibition and tumor cell antiproliferation. Among all the compounds, 12e showed the most potent activity in vitro, which inhibited the growth of MCF-7 and B16-F10 cell lines with IC₅₀ values of 0.57 ± 0.03 and 0.49 ± 0.07 μM, respectively. Compound 12e also exhibited significant telomerase inhibitory activity (IC₅₀ = 1.9 ± 0.43 μM). The result of flow cytometry demonstrated that compound 12e induced cell apoptosis. Docking simulation was performed to insert compound 12e into the crystal structure of telomerase at ATP binding site to determine the probable binding model. Based on the preliminary results, compound 12e with potent inhibitory activity in tumor growth may be a potential anticancer agent.

Full text of DOI:10.1007/s00044-014-0909-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0909-0
ORIGINAL RESEARCH
Synthesis, biological evaluation, and molecular docking studies
of pyrazolyl-acylhydrazone derivatives as novel anticancer agents
•
Man Xing Ting-Ting Zhao Yu-Jia Ren Na-Na Peng Xian-Hui Yang
•
•
•
•
•
•
•
Xi Li Hui Zhang Gao-Qi Liu Li-Rong Zhang Hai-Liang Zhu
•
Received: 24 February 2013 / Accepted: 6 January 2014
Ó Springer Science+Business Media New York 2014
Abstract A series of pyrazolyl-acylhydrazone derivatives
(1e–20e) have been designed and synthesized and their bio-
logic activities were also evaluated for telomerase inhibition
and tumor cell antiproliferation. Among all the compounds,
12e showed the most potent activity in vitro, which inhibited
the growth of MCF-7 and B16-F10 cell lines with IC50 values
of 0.57 0.03 and 0.49 0.07 lM, respectively. Com-
pound 12e also exhibited significant telomerase inhibitory
activity (IC50 = 1.9 0.43 lM). The result of flow cytom-
etry demonstrated that compound 12e induced cell apoptosis.
Docking simulation was performed to insert compound 12e
into the crystal structure of telomerase at ATP binding site to
determine the probable binding model. Based on the pre-
liminary results, compound12e with potent inhibitoryactivity
in tumor growth may be a potential anticancer agent.
of frequently dividing cells, but becomes dormant in most
somatic cells during adulthood (Wright and Shay, 2001;
Zhang et al., 2011). 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 (Bodnar et al., 1998;
Masutomi and Harn, 2003). These findings have prompted
us to expect that the suppression of telomerase activity in
tumor cells can be an encouraging strategy of anticancer
therapy (Saretzki, 2003; Argyle and Nasir, 2003).
The X-ray crystallography revealed that the key active
site of telomerase, a unique ribonucleoprotein enzyme, is
composed of two major components, i.e. the human telo-
merase RNA (hTR) and the human telomerase reverse
transcriptase (hTERT) catalytic subunit, as well as many
associated proteins that are critical for proper function
(Francois et al., 2000). These distinct parts, especially the
hTERT, have been an attractive target for the discovery of
telomerase inhibitors (Olaussen et al., 2006; Gillis et al.,
2008). Till date, several structurally distinct compounds
targeting telomerase have been reported (Fig. 1) (Luo et al.,
2012; Subhabrata et al., 2006; Elzbieta et al., 2009; Pascolo
et al., 2002; Sun et al., 1997; Tian et al., 2010; Moorthy
et al., 2012).
Keywords Pyrazolyl-acylhydrazone derivatives ꢀ
Anticancer ꢀ Telomerase ꢀ Molecular docking ꢀ 3D-QSAR
Introduction
Telomerase keeps its activity in the early stages of life cycle
maintaining telomere length and the chromosomal integrity
Pyrazole derivatives are a very encouraging class of
heterocyclic compounds that have remarkable pharmaco-
logical activities as antibacterial–antifungal, tumor necrosis
inhibitor, and antiangiogenic agent (Cabri et al., 2009;
Aggarwal et al., 2006; Kumar et al., 2005; Kane et al.,
2003; Liu et al., 2011). Some telomerase inhibitors
(Fig. 1a–c), carrying the pyrazole skeleton, exhibit telo-
merase inhibitory. For instance, VER49009 (Fig. 1b) tar-
gets the telomerase subunit indirectly with HSP90
inhibitors, thus inhibiting binding of telomerase-associated
proteins leading to telomere uncapping and cell apoptosis
Man Xing and Ting-Ting Zhao equally contributed to this paper.
M. Xing ꢀ T.-T. Zhao ꢀ Y.-J. Ren ꢀ N.-N. Peng ꢀ X.-H. Yang ꢀ
X. Li ꢀ H. Zhang ꢀ L.-R. Zhang ꢀ H.-L. Zhu (&)
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing
University, Nanjing 210093, People’s Republic of China
e-mail: zhuhl@nju.edu.cn
G.-Q. Liu
School of Materials Science and Engineering, Zhengzhou
University, Zhengzhou 450001, People’s Republic of China
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Med Chem Res
C
B
A
E
D
F
G
Fig. 1 Several reported anticancer agents especially telomerase inhibitors share some similar pharmacodynamic scaffolds with the title
compounds
(Ruden and Puri, 2012). Consequently, pyrazole frame-
work plays an essential role and was chosen as a template
for medicinal chemistry.
Ni2?, leading to blocking the physiologic reaction which is
catalyzed by ions or promoting the absorption, transportation,
distribution, and metabolism of drugs (Bernhardtet al., 2007).
Furthermore, acylhydrazone group might form hydrophobic
interaction and hydrogen bond with targeting protein or DNA
resulting in, probably, inhibiting cell proliferation (Tian et al.,
2010).
Much literature discussed the anticancer activity of amide
derivatives. It is a prominent structural motif found in
numerous pharmaceutically active compounds. BIBR1532,
(E)-2-(3-(naphthalen-2-yl)but-2-enamido)benzoic
acid,
(Fig. 1d) is one of the most promising hTERT specific
active site inhibitors to date (Tian et al., 2010). It acts as a
non-competitive inhibitor of telomerase, which specifically
blocks the elongation of DNA (Pascolo et al., 2002).
Treatment of different human cancer cell lines in vitro
resulted in reduced telomere length, inhibition of cell pro-
liferation, and cellular senescence (Philippi et al., 2010).
Due to the unique structure, hydrazone derivatives, in
particular, acylhydrazone derivatives, exhibit potent coordi-
nation in multiple forms. First of all, acylhydrazone deriva-
tives could form complexes with metal ions, such as Fe3? and
Given the above research and hypothesis of the phar-
macological function of acylhydrazone derivatives, hydra-
zone group was introduced into the pyrazole-amide motif,
expecting that they might exhibit synergistic effect in anti-
cancer activities by the inhibition against telomerase.
Telomerase has been elevated in over 90 % of breast
carcinomas MCF-7 cells (Anshu et al., 2004) and besides
B16-F10 cells have also played a key role in telomerase-
targeting cancer therapeutics (Hiroyuki et al., 2003),
therefore the two cancer cell lines, MCF-7 and B16-F10,
have been chosen in this study.
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Med Chem Res
derivatives was started from the interaction of substituted 1,3-
diphenyl-1H-pyrazole-4-carbaldehyde (compounds b) and
substituted 2-((hydrazinyloxy)carbonyl)phenol (compounds
d). And these starting materials were prepared in two ways,
respectively. Firstly, substituted acetophenone was inter-
acted with phenylhydrazine hydrochloride coupled with
sodium acetate in anhydrous ethanol to form compound a,
which was then added to a cold solution of DMF and
POCl3 to give the desired compound b. Secondly, substi-
tuted salylic acid was interacted with methanol, using
thionyl chloride as catalyst to yield compounds c. and then
substituted compounds c reacted with 80 % N2H4ꢀH2O to
gain compounds d.
Table 1 Structure of compounds 1e–20e
R2
H
O
C3'
C2'
O
C11
N
C1'
B
4'
C
H
C
N
C6'
5'
C
C10
R3
H
C
C9
C8
C7
C2
C3
C4
C6"
C1
N
5"
C
A
N
C1"
C2"
R1
C6
C4"
C5
Among these compounds, 2e–6e, 8e–10e, 12e–14e, 16e–
18e, and 20e are reported for the first time. All the synthetic
compounds gave satisfactory analytical and spectroscopic
C3"
1
data. H NMR and ESI–MS spectra were consistent with
Compound 1e 2e 3e
4e
5e
6e
7e 8e 9e 10e
the assigned structures.
R1
R2
R3
F
F
F
F
Cl
H
Cl
H
Cl Cl
H
H
H
H
H
H
H
H
Me
H
H
H
Me
Telomerase inhibitory assay
Cl Br
Cl
Br
H
Cl Br
Compound 11e 12e 13e 14e 15e 16e 17e 18e 19e 20e
To examine whether the compounds inhibit the telomerase,
we screen compounds 1e–20e against the telomerase. As
shown in Table 2. Most of the tested compounds displayed
potent telomerase inhibitory activities. Among them,
compound 12e (IC50 = 1.9 0.43 lM for telomerase)
displayed the most potent telomerase inhibitory, which is
much better than that of the ethidium bromide
(IC50 = 2.5 0.23 lM for telomerase).
R1
R2
R3
H
H
H
H
OMe OMe OMe OMe Me Me Me Me
Me
H
H
H
H
H
Me
H
H
H
H
H
Me
H
Cl
Br
Cl Br
Since there are only a very few systematic reports on the
synthetic methodology and evaluation of anticancer activ-
ity of this moiety, herein, as a continuation to extend our
research on telomerase inhibitors, we report the synthesis
and bioactivities of a series of novel (Z)-N0-((1,3-diphenyl-
From the results listed in Table 2, we can arrive at the
conclusion that the telomerase inhibitory activity of the
tested compounds may be correlated to structure variation
and modification. A general review of the relationships of
structure–activity can draw a conclusion, that is, com-
pounds with either substituent (3e, 7e, 9e–12e, 15e, and
19e) showed more potent telomerase inhibitory activity
than those with each substituent (such as, 1e, 2e, and 4e) in
both A-ring and B-ring (Table 1), and the compound
without substituent (11e) was just in between. A pre-
sumption might be drawn that both stereospecific blockade
and liposolubility of the substituent might influence telo-
merase inhibitory activity. Compared among the pare-
substituent on the A-ring, electron-withdrawing and small
group have improved telomerase inhibitory activity, with
the potency order: F [ Cl. As to the B-ring with constant
A-ring substituent, the compounds with electron-donating
(–Me) substituent on B-ring are of better telomerase
inhibitory activity comparing to those with electron-with-
drawing substituent, quite different with ring A, and the
potency of substituent on B-ring is ordered as:
Me [ H [ Br [ Cl. Among all the compounds, 12e with F
group on A-ring and Me group on B-ring respectively, led
to a noteworthy activity.
1H-pyrazol-4-yl)methylene)-2-hydroxybenzo
hydrazide
derivatives. Biologic evaluation was carried out for
screening potential telomerase inhibitors among the syn-
thesized compounds. Docking simulations were performed
using the X-ray crystallographic 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, quantitative structure–activity relationship (QSAR)
model was also built to study the structure–activity rela-
tionship and guide the further study.
Results and discussion
Chemistry
In this study, twenty (Z)-N0-((1,3-diphenyl-1H-pyrazol-4-
yl)methylene)-2-hydroxyl benzo hydrazide derivatives 1e–
20e (Table 1) were synthesized, and 15 of them were
reported for the first time.
The synthesis of compounds (1e–20e) followed the
general pathway outlined in Scheme 1, the synthesis of these
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Med Chem Res
A
B
C
Scheme 1 a General synthesis of 1,3-diphenyl-1H-pyrazole-4-carbal-
dehyde derivatives (compounds b). Reagents and conditions: (i) etha-
nol, 50–60 °C, 3 h; (ii) POCl3, DMF, 50–60 °C, 5 h. b General
(compounds d). Reagents and conditions: (iii) SOCl2, 0 °C, 0.5 h;
RT, 2 h; reflux, 5 h; (iv) 80 % N2H4ꢀH2O, 80–90 °C, overnight.
c General synthesis of compounds 1e–20e. Reagents and conditions:
(v) ethanol, 50–60 °C, 4 h
synthesis
of
2-((hydrazinyloxy)carbonyl)phenol
derivatives
Antiproliferative activities
Apoptosis assay
The synthesized compounds were evaluated to test the anti-
proliferative activities against MCF-7 and B16-F10 cells. The
results were summarized in Table 3. As shown in Table 3,
these (Z)-N0-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)-2-
hydroxybenzohydrazide derivatives bearing the acylhydraz-
one moiety and pyrazole moiety exhibited remarkable anti-
proliferative effects. Among them, compound 12e displayed
the most potent inhibitory activity (IC50 = 0.57 0.03 lM
for MCF-7 and IC50 = 0.49 0.07 lM for B16-F10),
comparable to the positive control 5-fluorouracil (5-FU)
(IC50 = 2.75 0.07 lM for MCF-7 and IC50 = 0.46
0.14 lM for B16-F10, respectively). Apart from the above
presumption summarized from A-ring, F group exhibited
better antiproliferative activity than Cl group, which might be
due to the high lipophilicity and metabolic stability of fluorine
substituent, inother words, thepresenceoffluorine often leads
to increased lipid solubility, thereby enhancing rates of
absorption and transport of drugs (Filler and Saha, 2009; Zhao
et al., 2012).
As representative of these (Z)-N0-((1,3-diphenyl-1H-pyrazol-
4-yl)methylene)-2-hydroxybenzohydrazide derivatives, com-
pound 12e has been under investigations in vitro experiment.
We detected the mechanism of compound 12e inhibition
effect by flow cytometry (FCM) (Fig. 2), and found that the
compound could induce the apoptosis of activated MCF-7
cells in a dose dependent manner. As shown in Fig. 2, MCF-7
cells were treated with 0, 0.3, 0.6, 0.9 lM of compound 12e
for 24 h. The compoundincreased the percentage ofapoptosis
by Annexin V-FITC/PI. The result indicated that compound
12e could induce apoptosis of MCF-7 cells.
Molecular modeling
To gain better understanding on the potency of the studied
compounds and guide further SAR studies, we proceeded
to examine the interaction of compound 12e with telome-
rase (PDB code: 3DU6). The molecular docking was per-
formed by simulation of compound 12e into the ATP
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Med Chem Res
binding site of telomerase. All docking runs were applied
LigandFit Dock protocol of Discovery Studio 3.1. The
binding modes of compound 12e and telomerase were
depicted in Fig. 3. All the amino acid residues which had
interactions with telomerase were exhibited in Fig. 3b. In
the binding mode, compound 12e was nicely bound to the
ATP binding site of telomerase via hydrophobic interac-
tions and binding is stabilized by two hydrogen bonds,
three Pi–Cation interactions, and one Pi–Sigma interaction.
The nitrogen atom of the pyrazole ring formed one
hydrogen bond with the amino hydrogen of Arg A: 340
Table 2 Telomerase inhibitory activity of compounds 1e–20e
Compound
Telomerase (IC50 SD, lM)
1e
7.1 0.78
7.5 0.63
2.6 0.18
6.1 0.79
9.2 0.93
9.0 0.81
3.8 0.27
7.1 0.63
3.6 0.18
3.2 0.05
2.4 0.21
1.9 0.43
13.8 0.93
15.3 0.97
4.1 0.44
6.2 0.70
16.1 1.07
13.3 1.02
4.5 0.04
6.9 0.53
2.5 0.23
2e
3e
4e
5e
6e
7e
8e
˚
(bond length: 2.44 A; bond angle: 93.8°), and the hydrogen
atom of the amide bond formed another with the carboxylic
9e
10e
˚
oxygen of Ser A: 227 (bond length: 2.02 A; bond angle:
11e
147.8°), respectively. Overall, these results of the molec-
ular docking study revealed that the molecule was well
embedded in the active pocket, suggested that compound
12e is a potential inhibitor of telomerase.
12e
13e
14e
15e
16e
QSAR model
17e
18e
In order to give a systematic evaluation on pyrazolyl-acy-
lhydrazone derivatives as telomerase inhibitors and to explore
more potent inhibitors, a 3D-QSAR model was initiated by
using a novel 3D-QSAR protocol of Discovery Studio 3.1.
The calculated pIC50 values ranged from 4.79 to 5.72.
Twenty compounds with definite IC50 values were
selected as the model dataset. By convention, we use the
pIC50 scale (-lgIC50), in which higher value indicates
exponentially greater potency, to measure the inhibitory
activity. The training set and test set were chosen by the
Diverse Molecules method in Discovery Studio 3.1.
To insure a good alignment, we chose the alignment
conformation of each molecule with lowest energy in the
docked results of CDOCKER protocol. And, before
building the QSAR model, we apply the alignment by the
substructure (Z)-N0-((1,3-diphenyl-1H-pyrazol-4-yl)methy-
lene)-2-hydroxybenzohydrazide.
The correlation coefficient r2 between observed activity
of testing set and training set was found to be 0.813, which
proved that the QSAR model built by us is acceptable.
Further, the molecules aligned with the iso-surfaces of
the 3D QSAR model coefficients on electrostatic potential
grids (Fig. 4a) and Van der Waals grids (Fig. 4b) are listed.
Electrostatic map indicates red contours around regions
where high electron density (negative charge) is expected
to increase activity, and blue contours represent areas
where low electron density (partial positive charge) is
expected to increase activity. Similarly, steric map indi-
cates areas where steric bulk is predicted to increase
(green) or decrease (yellow) activity.
19e
20e
Ethidium bromide
Table 3 Inhibition (IC50) of MCF-7 and B16-F10 cells proliferation
Compound
IC50 SD (lM)
MCF-7a
B16-F10a
1e
1.44 0.09
1.44 0.05
0.70 0.07
1.12 0.13
1.49 0.28
1.48 0.08
0.86 0.07
1.26 0.21
0.93 0.09
0.89 0.08
0.63 0.16
0.57 0.03
2.07 0.07
1.74 0.14
1.00 0.12
1.28 0.13
2.42 0.11
2.23 0.09
1.10 0.05
1.42 0.25
2.75 0.07
1.48 0.08
1.47 0.24
0.87 0.13
1.20 0.11
1.64 0.17
1.53 0.08
1.06 0.15
1.42 0.10
1.10 0.02
1.08 0.09
0.80 0.02
0.49 0.07
1.75 0.12
1.73 0.12
1.11 0.10
1.43 0.05
3.02 0.14
1.84 0.06
1.13 0.18
1.43 0.07
0.46 0.14
2e
3e
4e
5e
6e
7e
8e
9e
10e
11e
12e
13e
14e
15e
16e
17e
18e
19e
20e
According to the maps, on A-ring, it suggested the
compound with low negative charged and small R1 group
5-Fluorouracil
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Med Chem Res
Fig. 2 MCF-7 cells were cultured with various concentrations of compound 12e for 24 h. Cells were stained by Annexin V-FITC/PI, and
apoptosis was analyzed by flow cytometry. Inhibition included early and late apoptosis
on the para-position would bring higher activity, proving
that H-substituent was a better choice than F- or Cl-sub-
stituent. As for substituent on B-ring, a bulky and low
negative charged R2 group would enhance the bioactivity
while small and low negative charged R3 group might
show even better activity. From the data summarized
above, compound 12e was demonstrated to be the most
potent telomerase inhibitor (IC50 = 1.9 0.43 lM).
Above all, the 3D QSAR model agrees with the inhibitory
activity well and provides us the direction of further
modification.
Experimental
Telomerase inhibitory assay
Compounds 1e–20e were tested in a search for small
molecule inhibitors of telomerase activity by the TRAP-
PCR-ELISA assay (Zhang et al., 2011), using a MCF-7 cell
extract. The TRAP-PCR-ELISA assay was performed
using a telomerase detection kit (Roche, Basel, Switzer-
land) according to the manufacturer’s protocol. In brief,
2 lL of cell extracts were mixed with 48 lL TRAP reac-
tion mixtures. PCR was then initiated at 94 °C, 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 digox-
igenin (DIG)-labeled telomeric repeat specific detection
probe, and the PCR products were immobilized via the
Fig. 3 a Compound 12e (colored by atom: carbons, gray; hydrogen,
white; oxygen, red; nitrogen, blue) is bond into telomerase (entry 3DU6
plate subsequently. The immobilized DNA fragment were
in the Protein Data Bank). The dotted lines show the hydrogen bond and
biotin-labeled primer to a streptavidin-coated microtiter
detected with a peroxidase-conjugated anti-DIG antibody
the yellow lines show the Pi–Cation interaction and Pi–Sigma interac-
tion. b 2D ligand interaction diagram of compound 12e with telomerase
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 (Color figure online)
and visualized following addition of the stop regent. The
microtitre plate was assessed on TECAN Infinite M200
¨
microplate reader (Mannedorf, Switzerland) at a wave-
length of 490 nm, and the final value were presented as
mean SD.
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Med Chem Res
Fig. 4 a 3D-QSAR model
coefficients on electrostatic
potential grids. Blue represents
positive coefficients; red
represents negative coefficients.
b 3D-QSAR model coefficients
on van der Waals grids. Green
represents positive coefficients;
yellow represents negative
coefficients (Color figure
online)
Antiproliferation assay
rcsb.org/pdb/home/home.do). The molecular docking pro-
cedure was performed by using LigandFit protocol within
Discovery Studio 3.1. For ligand preparation, the 3D struc-
tures of 12e were generated and minimized using Discovery
Studio 3.1. For protein preparation, the hydrogen atoms
were added, and the water and impurities were removed.
The molecular docking was performed by inserting com-
pound 12e into the ATP binding site of telomerase crystal
structure. Types of interactions of the docked protein with
ligand were analyzed after the end of molecular docking.
The antiproliferative activities of the prepared compounds
against MCF-7 and B16-F10 cells were evaluated as
described in the literature (Boumendjel et al., 2008) with
some modifications. Target tumor cell lines were grown to
log phase in RPMI 1640 medium supplemented with 10 %
fetal bovine serum. After diluting to 2 9 104 cells mL-1
with the complete medium, 100 lL of the obtained cell
suspension was added to each well of 96-well culture plates.
The subsequent incubation was permitted at 37 °C, 5 %
CO2 atmosphere for 24 h before the cytotoxicity assess-
ments. Tested samples at pre-set concentrations were added
to six wells with 5-FU as positive references. After 48 h
exposure period, 40 lL of PBS containing 2.5 mg mL-1 of
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)) was added to each well. Four hours later, 100 lL
extraction solution (10 % SDS–5 % isobutyl alcohol–
0.01 M HCl) was added. After an overnight incubation at
37 °C, the optical density was measured at a wavelength of
570 nm on an ELISA microplate reader. In all experiments,
three replicate wells were used for each drug concentration.
Each assay was carried out for at least three times. The
results were summarized in Table 3.
QSAR model
Among all the 20 compounds, 75 % (i.e., 15) were utilized
as a training set for QSAR modeling. The remaining 25 %
(i.e., 5) were chosen as an external test subset for validating
the reliability of the QSAR model by the Diverse Mole-
cules protocol in Discovery Studio 3.1. The selected test
compounds were: 5e, 8e, 10e, 13e, and 15e.
The inhibitory activity of the compounds in literature
[IC50 (mol/L)] was initially changed into the minus loga-
rithmic scale [pIC50 (mol/L)] and then used for subsequent
QSAR analysis as the response variable.
In Discovery Studio, the CHARMm force field is applied
and the electrostatic potential together with the Van der
Waals potential are treated as separate terms. As the elec-
trostatic potential probe, A?le point change is used while
distance-dependent dielectric constant is used to mimic the
solvent effect. As for the Van der Waals potential, a carbon
Apoptosis assay
Apoptosis induced by telomerase inhibitors in MCF-7
cells. Confluenced cells were treated with a series of con-
centrations for 24 h and stained with Annexin V-FITC on
ice in dark for 30 min. After PI added, labeled cells were
analyzed on BD FCM. The data were calculated using cell
quest soft.
˚
atom with a radius of 1.73 A is used as a probe.
A partial least-squares model is built using energy grids
as descriptors. QSAR models were built by using the
Create 3D QSAR Model protocol in Discovery Studio 3.1.
Chemistry
Docking simulations
All chemicals and reagents used in the current study were
of analytical grade. Melting points (uncorrected) were
The crystal structure of telomerase (PDB code: 3DU6) was
obtained from the RCSB Protein Data Bank (http://www.
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Med Chem Res
determined on an XT4 MP apparatus (Taike Corp., Beijing,
China). All the 1H NMR spectra were recorded on a Bruker
DPX300 model Spectrometer in CDCl3 and chemical shifts
were reported in ppm (d). ESI–MS spectra were recorded
on a Mariner System 5304 Mass spectrometer. Elemental
analyses were performed on a CHN–O-Rapid instrument.
TLC was performed on the glassbacked silica gel sheets
(Silica Gel 60 GF254) and visualized in UV light
(254 nm).
ethyl acetate. The extract was washed successively with
saturated NaCl solution, and then dried over anhydrous
Na2SO4, filtered and evaporated. Finally, the precipitate
was filtered and recrystallized from ethanol to yield 1e–
20e.
(Z)-5-Chloro-N0-((3-(4-fluorophenyl)-1-phenyl-1H-
pyrazol-4-yl)methylene)-2-hydroxybenzohydrazide (1e)
White solid, yield 65 %, mp: 114–119 °C. 1H NMR
(CDCl3, 300 MHz): 3.71–3.78 (m, 1H), 7.01 (d,
J = 8.97 Hz, 1H), 7.16–7.24 (m, 3H), 7.62–7.67 (m, 3H),
7.80 (t, J = 5.90 Hz, 3H), 7.86–7.90 (m, 1H), 8.27 (s, 1H),
8.62 (s, 1H), 9.53 (s, 1H), 11.71 (s,1H), 13C NMR (CDCl3,
100.6 MHz): d = 163.11 (C-11), 162.12 (C-4), 156.51 (C-
200), 150.61 (C-7), 143.11 (C-10), 139.14 (C-100), 134.9 (C-
4 ), 130.41 (C-2), 130.38 (C-6), 130.10 (C-9), 129.49 (C-
300), 129.29 (C-500), 128.59 (C-60), 128.20 (C-1), 127.12 (C-
50), 126.22 (C-400), 121.13 (C-10), 119.87 (C-600), 119.83
(C-200), 118.57 (C-30), 115.92 (C-3), 115.73 (C-5), 113.23
(C-8); ESI–MS: 434.65 (C23H17ClFN4O2 [M?H]?). Anal.
Calcd for C23H16ClFN4O2: C, 63.53; H, 3.71; N, 12.88.
Found: C, 63.61; H, 3.72; N, 12.84.
General synthetic procedure of 1,3-diphenyl-1H-
pyrazole-4-carbaldehyde derivatives (compounds b)
The starting material substituted 1,3-diphenyl-1H-pyr-
azole-4-acrylic acid was synthesized as following: para-
substituted acetophenone (20 mmol) interact with phen-
ylhydrazine hydrochloride (25 mmol) coupled with sodium
acetate (40 mmol) in anhydrous ethanol to form 1-phenyl-
2-(1-phenylethylidene)hydrazine, which was then added to
a cold solution of DMF (10 mL), then POCl3 (8 mL) was
added and the resulting mixture was stirred at 50–60 °C for
6 h. The mixture was poured into ice-cold water. A satu-
rated solution of sodium hydroxide was added to neutralize
the mixture. And the solid precipitate was filtered, washed
with water, dried, and recrystallized from ethanol to give
the desired compound b (Li et al., 2012).
(Z)-5-Bromo-N0-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-
4-yl)methylene)-2-hydroxybenzohydrazide (2e)
General synthetic procedure of 2-((hydrazinyloxy)
White solid, yield 74 %, mp: 127–130 °C. 1H NMR
(CDCl3, 300 MHz): 3.71–3.78 (m, 1H), 6.95 (t, J =
15.72 Hz, 2H), 7.11–7.28 (m, 2H), 7.43 (t, J = 12.99 Hz,
1H), 7.65–7.69 (m, 3H), 7.87 (d, J = 5.49 Hz, 2H), 8.27 (s,
1H), 8.56 (s, 1H), 8.69 (s, 1H), 9.38 (s, 1H), 10.06 (s,1H);
13C NMR (CDCl3, 100.6 MHz): d = 163.42 (C-11), 162.81
(C-4), 158.29 (C-20), 150.27 (C-7), 143.18 (C-10), 139.61
(C-100), 136.19 (C-40), 134.77 (C-60), 130.52 (C-2), 130.49
(C-6), 130.21 (C-9), 129.27 (C-300), 129.19 (C-500), 128.43
(C-1), 126.03 (C-400), 122.11 (C-10), 119.72 (C-600), 119.69
(C-200), 119.21 (C-30), 116.10 (C-5), 116.02 (C-3), 115.73
(C-50), 113.13 (C-8); ESI–MS: 479.34 (C23H17BrFN4O2
[M?H]?). Anal. Calcd for C23H16BrFN4O2: C, 57.64; H,
3.36; N, 11.69. Found: C, 57.87; H, 3.37; N, 11.66.
carbonyl)phenol derivatives (compounds d)
Thionyl chloride (6 mmol) was dropped slowly into the
mixture of substituted salylic acid (3 mmol) and methanol
(30 mL). And the reaction was run in an ice bath for 1 h,
and then at RT for 2 h, and finally was refluxed for 5 h.
The reaction was monitored by TLC. The product was
extracted with ethyl acetate and saturated NaHCO3, filtered
and recrystallized from ethanol to gain substituted com-
pound c. Then a mixture of substituted compound c, 80 %
N2H4ꢀH2O (6 mmol) was dissolved in absolute ethanol and
stirred at 80–90 °C overnight. The reaction was monitored
by TLC. The solvent was evaporated until precipitate was
partially separated out, and to the residue, water was added.
And our target product (compound d) was washed with
water and collected by filtration (Samia et al., 2009).
(Z)-N0-((3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-
4-yl)methylene)-2-hydroxybenzohydrazide (3e)
General synthetic procedure of target compounds
White solid, yield 68 %, mp: 98–102 °C. 1H NMR (CDCl3,
300 MHz): 3.73 (d, J = 6.39 Hz, 1H), 6.88 (t,
J = 15.36 Hz, 1H), 7.06 (d, J = 8.58 Hz, 1H), 7.13–7.24
(m, 2H), 7.36 (t, J = 15.00 Hz, 1H), 7.42–7.54 (m, 3H),
7.60–7.65 (m, 2H), 7.75–7.86 (m, 2H), 8.24 (s, 1H), 8.58
(d, J = 20.49 Hz, 1H), 10.05 (s, 1H), 11.80 (s, 1H); 13C
NMR (CDCl3, 100.6 MHz): d = 163.43 (C-11), 163.16
(C-4), 158.11 (C-20), 153.92 (C-7), 149.50 (C-10), 139.31
(1e–20e)
A mixture of substituted compound b (1 mmol) and
substituted compound d (1.2 mmol) was dissolved in
absolute ethanol and refluxed for 4 h. The reaction was
monitored by TLC. The solvent was evaporated until no
longer liquid outflow. The products were extracted with
123

Med Chem Res
(C-100), 136.53 (C-40), 132.15 (C-9), 127.33 (C-2), 127.01
(C-6), 125.88 (C-300), 125.35 (C-500),124.91 (C-60), 124.35
(C-400), 123.34 (C-1), 120.45 (C-50), 115.42 (C-200), 114.70
(C-600), 112.23 (C-3), 112.10 (C-5), 111.43 (C-30), 110.85
(C-10). 107.39 (C-8); ESI–MS: 400.14 (C23H18FN4O2
[M?H]?). Anal. Calcd for C23H17FN4O2: C, 68.99; H,
4.28; N, 13.99. Found: C, 68.71; H, 4.29; N, 14.03.
(CDCl3, 100.6 MHz): d = 162.99 (C-11), 159.31 (C-20),
150.44 (C-7), 143.28 (C-10), 139.81 (C-100), 136.55 (C-40),
134.83 (C-60), 134.28 (C-4), 133.17 (C-1), 130.11 (C-9),
129.28 (C-500), 129.26 (C-300), 129.17 (C-5), 129.09 (C-3),
128.74 (C-6), 128.70 (C-2), 126.01 (C-400), 122.08 (C-10),
119.81 (C-600), 119.77 (C-200), 119.39 (C-30), 115.70 (C-50),
113.02 (C-8); ESI–MS: 496.08 (C23H17BrClN4O2
[M?H]?). Anal. Calcd for C23H16BrClN4O2: C, 55.72; H,
3.25; N, 11.30. Found: C, 55.94; H, 3.24; N, 11.26.
(Z)-N0-((3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-
4-yl)methylene)-2-hydroxy-3-methylbenzohydrazide (4e)
(Z)-N0-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-
Light yellow solid, yield 67 %, mp: 135–138 °C. 1H NMR
(CDCl3, 300 MHz): 2.28 (s, 3H), 2.31 (s, 1H), 6.80 (t,
J = 15.36 Hz, 1H), 7.16–7.24 (m, 1H), 7.48–7.57 (m, 2H),
7.63–7.67 (m, 1H), 7.79 (d, J = 7.86 Hz, 4H), 7.86–7.91
(m, 1H), 8.22 (s, 1H), 8.55 (s, 1H), 8.66 (s, 1H), 9.41 (s,
1H), 12.12 (s, 1H); 13C NMR (CDCl3, 100.6 MHz):
d = 166.99 (C-11), 164.29 (C-4), 161.81 (C-20), 155.51
(C-7), 152.23 (C-10), 142.32 (C-100), 139.14 (C-40), 135.84
(C-9), 130.38 (C-2), 130.30 (C-6), 129.61 (C-1), 128.15
(C-300), 128.11 (C-500), 127.41 (C-30), 127.22 (C-400),
126.78 (C-60), 123.09 (C-50), 119.20 (C-200), 118.42 (C-600),
115.89 (C-10), 115.79 (C-3), 115.67 (C-5), 112.00 (C-8),
12.89 (CH3); ESI–MS: 414.24 (C24H20FN4O2 [M?H]?).
Anal. Calcd for C24H19FN4O2: C, 69.55; H, 4.62; N, 13.52.
Found: C, 69.82; H, 4.63; N, 13.55.
4-yl)methylene)-2-hydroxybenzohydrazide (7e)
Light yellow solid, yield 67 %, mp: 123–125 °C. 1H NMR
(CDCl3, 300 MHz): 3.71–3.78 (m, 1H), 6.91 (t,
J = 13.71 Hz, 1H), 7.07 (d, J = 8.58 Hz, 2H), 7.46–7.54
(m, 3H), 7.60 (d, J = 12.06 Hz, 2H), 7.79–7.87 (m, 4H),
8.25 (s, 1H), 8.67 (s, 1H), 9.46 (s, 1H), 11.80 (s, 1H); 13C
NMR (CDCl3, 100.6 MHz): d = 162.91 (C-11), 159.28
(C-20), 150.33 (C-7), 143.00 (C-10), 139.79 (C-100), 134.43
(C-4), 133.66 (C-40), 133.01 (C-1), 130.50 (C-9), 129.38
(C-500), 129.31 (C-300), 129.22 (C-5), 129.19 (C-3),129.11
(C-2), 129.06 (C-6), 128.81 (C-60), 126.28 (C-400), 121.47
(C-50), 119.77 (C-600), 119.72 (C-200), 117.82 (C-30), 117.52
(C-10), 114.00 (C-8); ESI–MS: 416.74 (C23H18ClN4O2
[M?H]?). Anal. Calcd for C23H17ClN4O2: C, 66.27; H,
4.11; N, 13.44. Found: C, 66.53; H, 4.10; N, 13.47.
(Z)-5-Chloro-N0-((3-(4-chlorophenyl)-1-phenyl-1H-
pyrazol-4-yl)methylene)-2-hydroxybenzohydrazide (5e)
(Z)-N0-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-
yl)methylene)-2-hydroxy-3-methylbenzohydrazide (8e)
Light yellow solid, yield 81 %, mp: 164–168 °C. 1H NMR
(CDCl3, 300 MHz): 3.71–3.78 (m, 1H), 7.02 (d,
J = 9.00 Hz, 1H), 7.39 (t, J = 6.21 Hz, 3H), 7.50 (s, 1H),
7.63 (d, J = 8.22 Hz, 2H), 7.79–7.87 (m, 4H), 8.28 (s, 1H),
8.67 (s, 1H), 9.47 (s, 1H), 11.71 (s, 1H); 13C NMR (CDCl3,
100.6 MHz): d = 164.09 (C-11), 157.63 (C-20), 150.33 (C-
7), 142.99 (C-10), 139.77 (C100), 134.55 (C-40), 134.28 (C-
4), 133.01 (C-1), 130.38 (C-9), 129.33 (C-300), 129.29 (C-
500), 128.99 (C-5), 128.94 (C-3), 128.71 (C-60), 128.69 (C-
2), 128.65 (C-6), 126.88 (C-50), 126.11 (C-400), 121.01 (C-
10), 119.91 (C-600), 119.87 (C-200), 118,55 (C-30), 113.28
(C-8); ESI–MS: 451.69 (C23H17Cl2N4O2 [M?H]?). Anal.
Calcd for C23H16Cl2N4O2: C, 61.21; H, 3.57; N, 12.41.
Found: C, 60.96; H, 3.58; N, 12.45.
White solid, yield 59 %, mp: 127–130 °C. 1H NMR
(CDCl3, 300 MHz): 2.29 (s, 3H), 2.31 (s, 1H), 6.80 (t,
J = 15.36 Hz, 1H), 7.33–7.39 (m, 3H), 7.47 (s, 1H), 7.61
(t, J = 8.61 Hz, 3H), 7.77–7.83 (m, 3H), 8.22 (s,1H), 8.55
(s, 1H), 9.44 (s, 1H), 12.11 (s, 1H); 13C NMR (CDCl3,
100.6 MHz): d = 166.88 (C-11), 161.70 (C-20), 155.40 (C-
7), 152.12 (C-10), 142.21 (C-100), 139.30 (C-40), 136.11 (C-
4), 135.73 (C-9), 130.27 (C-2), 130.19 (C-6), 129.50 (C-1),
128.04 (C-300), 128.00 (C-500), 127.30 (C-30), 127.11 (C-
400), 126.67 (C-60), 123.11 (C-50), 119.01 (C-600), 118.21
(C-200), 115.78 (C-10), 115.68 (C-3), 115.56 (C-5), 112.02
(C-8), 12.78 (CH3); ESI–MS: 430.68 (C24H20ClN4O2
[M?H]?). Anal. Calcd for C24H19ClN4O2: C, 66.90; H,
4.44; N, 13.00. Found: C,66.63; H, 4.43; N, 13.04.
(Z)-5-Bromo-N0-((3-(4-chlorophenyl)-1-phenyl-1H-
pyrazol-4-yl)methylene)-2-hydroxybenzohydrazide (6e)
(Z)-5-Chloro-N0-((1,3-diphenyl-1H-pyrazol-4-
yl)methylene)-2-hydroxybenzohydrazide (9e)
Yellow solid, yield 72 %, mp: 167–170 °C. 1H NMR
(CDCl3, 300 MHz): 3.71–3.78 (m, 1H), 6.98 (d,
J = 8.97 Hz, 2H), 7.43–7.55 (m, 3H), 7.65 (d,
J = 8.61 Hz, 2H), 7.80–7.87 (m, 2H), 8.28 (s, 1H), 8.56 (s,
2H), 8.70 (s, 1H), 10.07 (s, 1H), 11.75 (s, 1H); 13C NMR
White solid, yield 72 %, mp: 132–139 °C. 1H NMR (CDCl3,
300 MHz): 3.72–3.76 (m, 1H), 6.99 (d, J = 5.31 Hz, 1H),
7.37–7.39 (m, 3H), 7.43–7.47 (m, 3H), 7.71 (d, J = 39.54 Hz,
2H), 7.79–7.84 (m, 3H), 8.30(s, 1H), 8.61(s, 1H), 9.69(s, 1H),
123

Med Chem Res
11.32 (s, 1H); 13C NMR (CDCl3, 100.6 MHz): d = 164.10
(C-11), 157.28 (C-20), 149.88 (C-7), 143.11 (C-10), 139.62
(C-100), 134.55 (C-40), 133.01 (C-1), 129.77 (C-9), 129.40 (C-
500), 129.32 (C-300), 129.20 (C-5), 129.14 (C-3), 128.77 (C-60),
128.62 (C-4), 127.64 (C-6), 127.52 (C-2), 126.91 (C-50),
126.01 (C-400), 121.33 (C-10), 119.99 (C-600), 119.81 (C-200),
118.72(C-30), 112.77(C-8);ESI–MS:416.94(C23H18ClN4O2
[M?H]?). Anal. Calcd for C23H17ClN4O2: C, 66.27; H, 4.11;
N, 13.44. Found: C, 66.54; H, 4.12; N, 13.47.
7.66 (d, J = 4.11 Hz, 2H), 7.80–7.85 (m, 3H), 8.24 (s, 1H),
8.68 (s, 1H), 9.40 (s, 1H), 12.13 (s, 1H); 13C NMR (CDCl3,
100.6 MHz): d = 164.21 (C-11), 159.23 (C-20), 150.33 (C-
7), 143.19 (C-10), 139.80 (C-100), 135.27 (C-40), 133.27 (C-
1), 130.52 (C-9), 129.27 (C-300), 129.23 (C-500), 129.11 (C-
5), 129.09 (C-3), 128.77 (C-4), 127.70 (C-6), 127.63 (C-2),
126.22 (C-400), 126.14 (C-30), 125.77 (C-60), 121.21 (C-50),
119.98 (C-600), 119.89 (C-200), 119.67 (C-10), 113.42 (C-8),
15.33 (CH3); ESI–MS: 396.13 (C24H21N4O2 [M?H]?).
Anal. Calcd for C24H20N4O2: C, 72.71; H, 5.08; N, 14.13.
Found: C, 72.42; H, 5.10; N, 13.17.
(Z)-5-Bromo-N0-((1,3-diphenyl-1H-pyrazol-4-
yl)methylene)-2-hydroxybenzohydrazide (10e)
(Z)-5-Chloro-2-hydroxy-N0-((3-(4-methoxyphenyl)-1-
White solid, yield 72 %, mp: 138–148 °C. 1H NMR (CDCl3,
300 MHz): 3.71–3.78 (m, 1H), 6.96 (d, J = 8.97 Hz, 1H),
7.30 (s, 1H), 7.37 (d, J = 7.50 Hz, 2H), 7.50 (t,
J = 15.36 Hz, 3H), 7.72 (s, 2H), 7.77 (t, J = 18.12 Hz, 3H),
8.30 (s, 1H), 8.62 (s, 1H), 9.66 (s, 1H), 11.80 (s, 1H); 13C
NMR (CDCl3, 100.6 MHz): d = 164.01 (C-11), 157.93 (C-
20), 150.55 (C-7), 144.00 (C-10), 139.88 (C-100), 136.51 (C-
40), 135.21 (C-60), 133.12 (C-1), 130.57 (C-9), 129.04 (C-500),
129.00 (C-300), 128.92 (C-5), 128.88 (C-3), 128.41(C-4),
127.42 (C-6), 127.39 (C-2), 126.01 (C-400), 122.04 (C-10),
120.01 (C-600), 119.99 (C-200), 118.97 (C-30), 115.71 (C-50),
114.01 (C-8); ESI–MS: 461.23 (C23H18BrN4O2 [M?H]?).
Anal. Calcd for C23H17BrN4O2: C, 59.88; H, 3.71; N, 12.15.
Found: C, 59.64; H, 3.72; N, 12.19.
phenyl-1H-pyrazol-4-yl)methylene)benzohydrazide (13e)
1
Light yellow solid, yield 79 %, mp: 123–125 °C. H NMR
(CDCl3, 300 MHz): 2.31 (s, 3H); 2.43 (s, 1H), 6.10 (d,
J = 8.97 Hz, 1H), 7.27 (s, 1H), 7.31–7.40 (m, 3H),
7.46–7.53 (m, 4H), 7.73 (t, J = 14.32 Hz, 2H), 8.31 (s, 1H),
8.59 (s, 1H), 9.75 (s, 1H), 10.11 (s, 1H); 13C NMR (CDCl3,
100.6 MHz): d = 165.68 (C-11), 162.43 (C-4), 160.65 (C-
20), 154.83 (C-7), 146.34 (C-10), 141.07 (C-100), 138.42 (C-
40), 134.13 (C-9), 129.56 (C-2), 129.24 (C-6), 128.35 (C-1),
127.48 (C-300), 127.17 (C-500), 126.65 (C-60), 125.21 (C-50),
122.32 (C-400), 118.56 (C-10), 117.23 (C-200), 117.20 (C-600),
115.21 (C-30), 114.01 (C-3), 113.81 (C-5), 111.87 (C-8),
55.81 (OCH3); ESI–MS: 446.54 (C24H20ClN4O3 [M?H]?).
Anal. Calcd for C24H19ClN4O3: C, 64.50; H, 4.29; N, 12.54.
Found: C, 64.76; H, 4.30; N, 12.50.
(Z)-N0-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-2-
hydroxybenzohydrazide (11e)
(Z)-5-Bromo-2-hydroxy-N0-((3-(4-methoxyphenyl)-1-
White solid, yield 68 %, mp: 118–122 °C. 1H NMR (CDCl3,
300 MHz): 3.71–3.76 (m, 1H), 6.89 (t, J = 8.97 Hz, 1H),
7.05 (t, J = 19.32 Hz, 1H), 7.28 (s, 2H), 7.35 (t,
J = 8.79 Hz, 1H), 7.45 (t, J = 7.89 Hz, 3H), 7.64 (d,
J = 4.29 Hz, 2H), 7.77–7.84 (m, 3H), 8.26 (s, 1H), 8.63 (s,
1H), 9.57 (s, 1H), 11.83 (s, 1H); 13C NMR (CDCl3,
100.6 MHz): d = 163.41 (C-11), 159.57 (C-20), 150.77 (C-
7), 144.02 (C-10), 140.21 (C-100), 133.51 (C-40), 133.21 (C-
1), 131.22 (C-9), 129.52 (C-500), 129.44 (C-300), 129.14 (C-5),
129.10 (C-3), 128.77 (C-60), 128.41 (C-4), 127.32 (C-6),
127.29 (C-2), 126.55 (C-400), 121.83 (C-50), 120.11 (C-600),
120.01 (C-200), 117.84 (C-30), 117.66 (C-10), 112.83 (C-8);
ESI–MS: 382.19 (C23H19N4O2 [M?H]?). Anal. Calcd for
C23H18N4O2: C, 72.24; H, 4.74; N, 14.65. Found: C, 72.53;
H, 4.75; N, 14.62.
phenyl-1H-pyrazol-4-yl)methylene)benzohydrazide (14e)
White solid, yield 83 %, mp: 111–118 °C. 1H NMR
(CDCl3, 300 MHz): 2.37 (s, 3H), 2.41 (s, 1H), 6.08 (d,
J = 8.79 Hz, 1H), 7.15 (s, 1H), 7.25–7.34 (m, 3H),
7.36–7.45 (m, 4H), 7.73 (t, J = 13.31 Hz, 2H), 8.11 (s, 1H),
8.28 (s, 1H), 9.34 (s, 1H), 10.10 (s, 1H); 13C NMR (CDCl3,
100.6 MHz): d = 163.27 (C-11), 160.68 (C-4), 158.41 (C-
20), 150.49 (C-7), 143.39 (C-10), 140.01 (C-100), 136.57 (C-
40), 135.01 (C-60), 128.97 (C-9), 139.33 (C-500), 139.30 (C-
300), 128.47 (C-6), 128.45 (C-2), 125.79 (C-400), 125.04 (C-
1), 121.83 (C-10), 119.99 (C-600), 119.95 (C-200), 119.27 (C-
30), 115.91 (C-50), 114.88 (C-5), 114.85 (C-3), 113.02 (C-8),
55.81(OCH3); ESI–MS: 491.23 (C24H20BrN4O3 [M?H]?).
Anal. Calcd for C24H19BrN4O3: C, 58.67; H, 3.90; N, 11.40.
Found: C, 58.90; H, 4.01; N, 11.36.
(Z)-N0-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-2-
hydroxy-3-methylbenzohydrazide (12e)
(Z)-2-Hydroxy-N0-((3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)methylene)benzohydrazide (15e)
Light yellow solid, yield 73 %, mp: 126–131 °C. 1H NMR
(CDCl3, 300 MHz): 2.27 (s, 3H), 2.31 (s, 1H), 6.79 (t,
J = 9.15 Hz, 1H), 7.32–7.38 (m, 3H), 7.41–7.46 (m, 3H),
White solid, yield 69 %, mp: 106–109 °C. 1H NMR (CDCl3,
300 MHz): 2.29 (s, 3H), 2.33 (s, 1H), 6.81 (t, J = 9.15 Hz,
123

Med Chem Res
(Z)-5-Bromo-2-hydroxy-N0-((1-phenyl-3-(p-tolyl)-1H-
1H), 7.33–7.39 (m, 3H), 7.44–7.48 (m, 3H), 7.67 (d,
J = 4.21 Hz, 2H), 7.81–7.86 (m, 3H), 8.27 (s, 1H), 8.69 (s,
1H), 9.43 (s, 1H), 12.12 (s, 1H); 13C NMR (CDCl3,
100.6 MHz): d = 165.21 (C-11), 160.81 (C-4), 159.71 (C-
20), 149.92 (C-7), 143.56 (C-10), 140.20 (C-100), 133.58 (C-
40), 131.00 (C-9), 129.51 (C-500), 129.49 (C-300), 128.97 (C-
60), 128.44 (C-6), 128.42 (C-2), 126.22 (C-400), 125.30 (C-1),
121.49 (C-50), 119.70 (C-600), 119.67 (C-200), 117.88 (C-30),
117.61 (C-10), 114.88 (C-5), 114.83 (C-3), 113.57 (C-8),
55.56 (OCH3); ESI–MS: 412.68 (C24H21N4O3 [M?H]?).
Anal. Calcd for C24H20N4O3: C, 69.89; H, 4.89; N, 13.58.
Found: C, 70.17; H, 4.91; N, 13.54.
pyrazol-4-yl)methylene)benzohydrazide (18e)
White solid, yield 77 %, mp: 145–151 °C. 1H NMR
(CDCl3, 300 MHz): 2.38 (s, 3H), 2.43 (s, 1H), 6.08 (d,
J = 8.97 Hz, 1H), 7.15 (s, 1H), 7.24–7.31 (m, 3H),
7.38–7.45 (m, 4H), 7.75 (t, J = 14.32 Hz, 2H), 8.14 (s, 1H),
8.29 (s, 1H), 9.37 (s, 1H), 10.11 (s, 1H); 13C NMR (CDCl3,
100.6 MHz): d = 165.11 (C-11), 158.51 (C-20), 151.03 (C-
7), 143.38 (C-10), 140.01 (C-100), 136.44 (C-40), 134.82 (C-
60), 131.77 (C-4), 130.39 (C-9), 129.98 (C-1), 129.61 (C-5),
129.56 (C-3), 129.31 (C-500), 129.28 (C-300), 128.49 (C-6),
128.45 (C-2), 126.41 (C-400), 121.97 (C-10), 118.77 (C600),
118.69 (C-200), 118.09 (C-30), 116.01 (C-50), 113.29 (C-8),
21.59(CH3); ESI–MS: 475.13 (C24H20BrN4O2 [M ? H]?).
Anal. Calcd for C24H19BrN4O2: C, 60.64; H, 4.03; N, 11.79.
Found: C, 60.88; H,3.74; N, 11.82.
(Z)-2-Hydroxy-N0-((3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)methylene)-3-methylbenzohydrazide (16e)
White solid, yield 75 %, mp: 120–125 °C. 1H NMR
(CDCl3, 300 MHz): 2.39 (s, 3H), 2.42 (s, 3H), 2.43 (s, 1H),
6.79 (t, J = 7.79 Hz, 1H), 7.37 (s, 2H), 7.50–7.65 (m, 4H),
7.73 (d, J = 8.23 Hz, 1H), 7.80 (d, J = 8.25 Hz, 3H), 8.31
(s, 1H), 8.53 (s, 1H), 8.75 (s, 1H), 10.12 (s, 1H); 13C NMR
(CDCl3, 100.6 MHz): d = 166.12 (C-11), 160.57 (C-4),
159.22 (C-20), 151.00 (C-7), 144.28 (C-10), 139.82 (C-100),
135.41 (C-40), 130.33 (C-9), 129.30 (C-500), 129.26 (C-300),
128.57 (C-6), 128.52 (C-2), 126.27 (C-30),126.24 (C-400),
125.91 (C-60), 125.30 (C-1), 121.40 (C-50), 119.97 (C-600),
119.92 (C-200), 119.72 (C-10), 114.81 (C-5), 114.79 (C-3),
113.32 (C-8), 55.81(OCH3), 15.18(CH3); ESI–MS: 426.13
(C25H23N4O3 [M?H]?). Anal. Calcd for C25H22N4O3: C,
70.41; H, 5.20; N, 13.14. Found: C, 70.69; H, 5.19; N,
13.11.
(Z)-2-Hydroxy-N0-((1-phenyl-3-(p-tolyl)-1H-pyrazol-
4-yl)methylene)benzohydrazide (19e)
White solid, yield 75 %, mp: 132–137 °C. 1H NMR
(CDCl3, 300 MHz): 2.37 (s, 3H), 2.45 (s, 1H), 6.90 (t,
J = 14.46 Hz, 1H), 7.06 (d, J = 8.43 Hz, 2H), 7.33 (d,
J = 7.14 Hz, 2H), 7.48–7.58 (m, 2H), 7.74 (d, J = 8.07 Hz,
3H), 7.82 (t, J = 7.50 Hz, 2H), 8.24 (s, 1H), 8.55 (s, 1H),
8.71 (s, 1H), 10.07 (s, 1H); 13C NMR (CDCl3, 100.6 MHz):
d = 164.21 (C-11), 160.01 (C-20), 150.49 (C-7), 143.34 (C-
10), 139.71 (C-100), 133.55 (C-40), 131.80 (C-4), 130.29 (C-
9), 129.91 (C-1), 129.82 (C-5), 129.79 (C-3), 129.41 (C-500),
129.38 (C-300), 129.01 (C-60), 128.21 (C-6), 128.19 (C-2),
126.53 (C-400), 121.31 (C-50), 119.91 (C-600), 119.88 (C-200),
117.74 (C-30), 117.62 (C-10), 114,01 (C-8), 21.21(CH3);
ESI–MS: 396.58 (C24H21N4O2 [M?H]?). Anal. Calcd for
C24H20N4O2: C, 72.71; H, 5.08; N, 14.13. Found: C, 73.00;
H, 5.10; N, 14.18.
(Z)-5-Chloro-2-hydroxy-N0-((1-phenyl-3-(p-tolyl)-1H-
pyrazol-4-yl)methylene)benzohydrazide (17e)
White solid, yield 79 %, mp: 137–140 °C. 1H NMR
(CDCl3, 300 MHz): 2.40 (s, 3H), 2.45 (s, 1H), 6.10 (d,
J = 8.97 Hz, 1H), 7.25 (s, 1H), 7.32–7.41 (m, 3H),
7.48–7.55 (m, 4H), 7.75 (t, J = 21.96 Hz, 2H), 8.29 (s,
1H), 8.58 (s, 1H), 9.72 (s, 1H), 10.01 (s, 1H); 13C NMR
(CDCl3, 100.6 MHz): d = 163.88 (C-11), 157.48 (C-20),
149.91 (C-7), 143.39 (C-10), 140.01 (C-100), 134.73 (C-40),
131.78 (C-4), 130.38 (C-9), 130.01 (C-1), 129.53 (C-5),
129.49 (C-3), 129.30 (C-500), 129.29 (C-300), 128.99 (C-60),
128.47 (C-2), 128.43 (C-6), 127.09 (C-50), 125.88 (C-400),
121.29 (C-10),119.94 (C-600), 119.91 (C-200), 118.60 (C-30),
112.91 (C-8), 21.28 (CH3); ESI–MS: 430.94
(C24H20ClN4O2 [M?H]?). Anal. Calcd for C24H19ClN4O2:
C, 66.90; H, 4.44; N, 13.00. Found: C, 67.12; H, 4.46; N,
12.96.
(Z)-2-Hydroxy-3-methyl-N0-((1-phenyl-3-(p-tolyl)-1H-
pyrazol-4-yl)methylene)benzohydrazide (20e)
White solid, yield 66 %, mp: 139–143 °C. 1H
NMR(CDCl3, 300 MHz): 2.37 (s, 3H), 2.44 (s, 3H), 2.45
(s, 1H), 6.80 (t, J = 15.36 Hz, 1H), 7.34 (s, 2H), 7.53–7.62
(m, 4H), 7.74 (d, J = 8.10 Hz, 1H), 7.82 (d, J = 8.25 Hz,
3H), 8.24 (s, 1H), 8.55 (s, 1H), 8.73 (s, 1H), 10.07 (s, 1H);
13C NMR (CDCl3, 100.6 MHz): d = 162.33 (C-11),
159.28 (C-20), 150.51 (C-7), 143.41 (C-10), 139.71 (C-100),
135.44 (C-40), 131.82 (C-4), 130.33 (C-9), 129.90 (C-1),
129.61 (C-5), 129.57 (C-3), 129.20 (C-500), 129.17 (C-300),
128.47 (C-6), 128.44 (C-2), 126.39 (C-400), 126.23 (C-30),
125.91 (C-60), 121.58 (C-50), 119.81 (C-600), 119.79 (C-200),
119.51 (C-10), 112.88 (C-8), 21.77(CH3), 15.21(CH3);
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Med Chem Res
ESI–MS: 410.21 (C25H23N4O2 [M?H]?). Anal. Calcd for
C25H22N4O2: C, 73.16; H, 5.38; N, 13.67. Found: C, 73.45;
H, 5.40; N, 13.63.
Elzbieta B, Magdalena M, Ingo PL, Peter M, Urszula K, Marek R
(2009) Synthesis and X-ray structure of platinum(II), palla-
dium(II) and copper(II) complexes with pyridine–pyrazole
ligands: influence of ligands’ structure on cytotoxic activity.
Polyhedron 28:637–645
Filler R, Saha R (2009) Fluorine in medicinal chemistry: a century of
progress and a 60-year retrospective of selected highlights.
Future Med Chem 1:777–791
Conclusions
Francois L, Jean FR, Abdelazize L, Patrick M (2000) Telomerase: a
therapeutic target for the third millennium. Crit Rev Oncol
Hematol 34:111–126
Gillis AJ, Schuller AP, Skordalakes E (2008) Structure of the
Tribolium castaneum telomerase catalytic subunit TERT. Nature
455:633–636 (luoyin 3)
Hiroyuki K, Katsuyo S, Kenji F, Barrett JC, Oshimura M (2003)
Human chromosome 5 carries a putative telomerase repressor
gene. Genes Chromosomes Cancer 36:37–47
Kane JL, Hirth BH, Laing O, Gourlie BB, Nahill S, Barsomiam G
(2003) Ureas of 5-aminopyrazole and 2-aminothiazole inhibit
growth of gram-positive bacteria. Bioorg Med Chem Lett
13:4463–4466
Kumar V, Aggarwal R, Tyagi P, Singh SP (2005) Synthesis and
antibacterial activity of some new 1-heteroaryl-5-amino-4-
phenyl-3-trifluoromethylpyrazoles. Eur J Med Chem 40:922–927
Li X, Liu JL, Yang XH, Lu X, Zhao TT, Gong HB, Zhu HL (2012)
Synthesis, biological evaluation and molecular docking studies
of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide deriv-
atives as inhibitors of HDAC activity. Bioorg Med Chem
20:4430–4436
Liu XH, Ruan BF, Li J (2011) Synthesis and biological activity of
chiral dihydropyrazole: potential lead for drug design. Mini Rev
Med Chem 11:771–821
Luo Y, Zhang S, Qiu KM, Liu ZJ, Yang YS, Fu J, Zhu HL (2012)
Synthesis, biological evaluation, 3D-QSAR studies of novel
aryl-2H-pyrazole derivatives as telomerase inhibitors. Bioorg
Med Chem Lett 23:1091–1095
In our present work, a series of novel telomerase inhibitors
(1e–20e) have been synthesized and evaluated. These
compounds exhibited potent antiproliferative activities
against MCF-7 and B16-F10 cells and telomerase inhibi-
tory activity. Among them, compound 12e demonstrated
the most potent activity which inhibited the growth of
MCF-7 and B16-F10 cells with IC50 values of 0.57 0.03
and 0.49 0.07 lM, and inhibited the activity of telo-
merase with IC50 of 1.9 0.43 lM. The result of FCM
demonstrated that compound 12e induced cell apoptosis.
Molecular docking showed that compound 12e bound to
the ATP binding site by two hydrogen bonds, three Pi–
Cation interactions and one Pi–Sigma interaction which
might play a crucial role in tumor cell antiproliferation and
telomerase inhibition. 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. This study might be helpful for the
design and synthesis of telomerase inhibitors with stronger
activities.
Acknowledgments This work was financed by National Natural
Science Foundation of China (No. J1103512).
Masutomi K, Harn WC (2003) Telomerase and tumorigenesis. Cancer
Lett 194:163–172
Moorthy NSHN, Nuno SC, Maria JR, Pedro AF (2012) QSAR
analysis of 2-benzoxazolyl hydrazone derivatives for anticancer
activity and its possible target prediction. Med Chem Res
21:133–144
References
Aggarwal R, Kumar V, Tyagi P, Singh SP (2006) Synthesis and
antibacterial activity of some new 1-heteroaryl-5-amino-3H/
methyl-4-phenylpyrazoles. Bioorg Med Chem 14:1785–1791
Anshu M, Mitchell SP, Rebecca CW, Trygve OT, Santosh KK (2004)
EGCG down-regulates telomerase in human breast carcinoma
MCF-7 cells, leading to suppression of cell viability and
induction of apoptosis. Int J Oncol 24:703–710
Olaussen KA, Dubrana K, Dornont J (2006) Telomeres and telome-
rase as targets for anticancer drug development. Crit Rev Oncol
Hematol 57:191–214
Pascolo E, Wenz C, Lingner J, Hauel N, Priepke H, Kauffmann I
(2002) Mechanism of human telomerase inhibition by
BIBR1532, a synthetic, non-nucleosidic drug candidate. J Biol
Chem 277:15566–15572
Argyle DJ, Nasir L (2003) Telomerase: a potential diagnostic and
therapeutic tool in canine oncology. Vet Pathol 40:1–7
Bernhardt PV, Chin P, Sharpe PC, Richardson DR (2007) Hydrazone
chelators for the treatment of iron overload disorders: iron
coordination chemistry and biological activity. Dalton Trans
30:3232–3244
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB,
Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998)
Extension of life-span by introduction of telomerase into normal
human cells. Science 279:349–352
Philippi C, Loretz B, Schaefer UF, Lehr CM (2010) Telomerase as
emerging target to fight cancer—opportunities and challenges for
nanomedicine. J Control Release 146:228–240
Ruden M, Puri N (2012) Novel anticancer therapeutics targeting
telomerase. Cancer Treat Rev 06:007
Samia MR, Manal NSS, Amal MY, Madiha AH (2009) Synthesis and
biological evaluation of the pyrazole class of cyclooxy-genase-2-
inhibitors. Lett Org Chem 6:282–288
Saretzki G (2003) Telomerase inhibition as cancer therapy. Cancer
Lett 194:209–219
Boumendjel A, Boccard J, Carrupt PA, Nicolle E, Blanc M, Geze A,
Choisnard L, Wouessidjewe D, Matera EL, Dumontet C (2008)
Antimitotic and antiproliferative activities of chalcones: forward
structure–activity relationship. J Med Chem 51:2307–2310
Cabri W, Vesci L, Pisano C (2009) N-Hydroxy-(4-oxime)-cinnamide:
a versatile scaffold for the synthesis of novel histone deacetilase
(HDAC) inhibitors. Bioorg Med Chem Lett 19:3694
Subhabrata C, Timothy RW, Brian SJB (2006) Hsp90 as a target for
drug development. ChemMedChem 1:1331–1340
Sun D, Thompson B, Cathers BE (1997) Inhibition of human
telomerase by a G-quadruplex-interactive compound. J Med
Chem 40:2113–2116
Tian FF, Jiang FL, Han XL, Xiang C, Ge YS, Li JH, Zhang Y, Li R,
Ding XL, Liu YJ (2010) Synthesis of a novel hydrazone
123

Med Chem Res
derivative and biophysical studies of its interactions with bovine
serum albumin by spectroscopic, electrochemical, and molecular
docking methods. Phys Chem B 114:14842–14853
docking studies of 1,3,4-oxadiazole derivatives possessing 1,4-
benzodioxan moiety as potential anticancer agents. Bioorg Med
Chem 19:6518–6524
Wright WE, Shay JW (2001) Cellular senescence as a tumor-
protection mechanism: the essential role of counting. Curr Opin
Genet Dev 11:98–103
Zhang XM, Qiu M, Sun J, Zhang YB, Yang YS, Wang XL, Tang JF,
Zhu HL (2011) Synthesis, biological evaluation, and molecular
Zhao TT, Lu X, Yang XH, Wang LM, Li X, Wang ZC, Gong HB,
Zhu HL (2012) Synthesis, biological evaluation, and molecular
docking studies of 2,6-dinitro-4-(trifluoromethyl)pheno-
xysalicylaldoxime derivatives as novel antitubulin agents. Bio-
org Med Chem 20:3233–3241
123