Design and synthesis of novel 5-phenyl-N-piperidine ethanone containing 4,5-dihydropyrazole derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2012.02.040

A series of novel 5-phenyl-N-piperidine ethanone-4,5-dihydropyrazole derivatives as potential telomerase inhibitors were synthesized. The bioassays demonstrated that compounds 4d, 4f, 7a and 7b occupied high antiproliferative activities against SGC-7901, MGC-803 and Bcap-37 cell lines. By a modified TRAP assay, some titled compounds were tested against telomerase, and compound 7b showed the most potent inhibitory activity with IC₅₀ value at 2.00 ± 0.40 μM. The active compound 4d was also docked into the telomerase TERT active site to determine the probable binding model. The results indicated that conserved residues Lys189, Asp254 and Gln308 were important for ligand binding via hydrogen bond interactions.

Full text of DOI:10.1016/j.ejmech.2012.02.040

European Journal of Medicinal Chemistry 51 (2012) 294e299
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design and synthesis of novel 5-phenyl-N-piperidine ethanone containing
4,5-dihydropyrazole derivatives as potential antitumor agents
,
b
,
a
a
b
,
c
Xin-Hua Liu a , Jun Li , Jing Bo Shi , Bao-An Song
, Xing-Bao Qi
*
**
a School of Pharmacy, Anhui Medical University, Hefei 230032, PR China
b Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China
c School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR 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 5-phenyl-N-piperidine ethanone-4,5-dihydropyrazole derivatives as potential telome-
rase inhibitors were synthesized. The bioassays demonstrated that compounds 4d, 4f, 7a and 7b occu-
pied high antiproliferative activities against SGC-7901, MGC-803 and Bcap-37 cell lines. By a modified
TRAP assay, some titled compounds were tested against telomerase, and compound 7b showed the most
Received 8 February 2012
Received in revised form
17 February 2012
Accepted 18 February 2012
Available online 25 February 2012
potent inhibitory activity with IC50 value at 2.00 ꢀ 0.40
mM. The active compound 4d was also docked
into the telomerase TERT active site to determine the probable binding model. The results indicated that
conserved residues Lys189, Asp254 and Gln308 were important for ligand binding via hydrogen bond
interactions.
Keywords:
Synthesis
Piperidine
Ó 2012 Elsevier Masson SAS. All rights reserved.
4,5-Dihydropyrazole
Antitumor agents
1. Introduction
In our recent publications [9,10], several dihydropyrazole
derivatives were described, which had potent anticancer activity,
Telomerase remains active in the early stages of life maintaining
telomere length and the chromosomal integrity of frequently
dividing cells. It turns dormant in most somatic cells during adult-
hood [1]. In cancer cells, however, telomerase gets reactivated and
works tirelessly to maintain the short length of telomeres of rapidly
dividing cells, leading to their immortality [2]. 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. Telomere and telomerase are closely related
to the occurrence and development of human gastric cancer [3].
Dihydropyrazole, a small bioactive molecule, is a prominent
structural motif found in numerous pharmaceutically active
compounds [4,5]. The dihydropyrazole structure has been
demonstrated to bear important biological activities such as anti-
cancer, antimicrobial, antimalarial, antinociceptive, antiviral, anti-
tubercular, anti-inflammatory [6e8].
especially against SGC-7901 cell line. According to preliminary
anticancer activity of structureeactivity relationship (SAR), we
found that anticancer activity changed with the transformation of
N-acetyl. The novel docking model based on the protein structure of
telomerase inhibitors was developed using LigandFit module
within Discovery Studio 2.1, we found 4,5-dihydropyrazole ring
project into a hydrophobic region, which was comprised of the side
chains of 220e260, that was important for the potent inhibitory
activity [9]. In current molecular design, when the hydrogen atom
of N-acetyl was replaced by piperidine, the 5-phenyl 4,5-
dihydropyrazole ring projected into a more stable hydrophobic
region, which comprised of the side chains of 252e256, should be
enhance the anticancer activity. So we designed and synthesized
some 5-phenyl-dihydropyrazole derivatives containing piperidine
moiety.
Furthermore, tetracyclic coumarin compounds, have been
demonstrated to bear the best anti-HIV-1 virus activity. Because of
the mechanistic and structural similarity between reverse tran-
scriptases and hTERT, it is supposed that known reverse tran-
scriptase inhibitors may potently inhibit human telomerase [11,12].
Based on this hypothesis, since there are only a very few systematic
reports on the synthetic methodology and evaluation of anticancer
activities of these compounds, we introducted the coumarin
* Corresponding author. School of Pharmacy, Anhui Medical University, Hefei,
230032, PR China. Tel./fax: þ86 551 5161115.
** Corresponding author. Tel.: þ86 851 3620521; fax: þ86 851 3622211.
E-mail addresses: xhliuhx@163.com (X.-H. Liu), songbaoan22@yahoo.com
(B.-A. Song).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.040

X.-H. Liu et al. / European Journal of Medicinal Chemistry 51 (2012) 294e299
295
moiety in the above-mentioned 5-phenyl-dihydropyrazole piperi-
dine skeleton, designed and synthesized of N-substituted-dihy-
dropyrazole-piperidine derivatives containing coumarin moiety.
In order further investigate the detailed structural basis of the
synthesized inhibitor for telomerase TERT, high inhibitory activity
compound was docked into the active site of telomerase TERT
(3DU6).
2. Results and discussion
2.1. Chemistry
The synthesis of compound 5 (Scheme 2) started from salicy-
laldehyde and catalyzed by piperazine at 20e30 ꢁC was added
acetoacetate. ClaiseneSchmidt condensation 3-acetyl-2H-chro-
men-2-one and benzaldehyde using mild catalyst piperazine, by
following a reported method [10], proved to be an efficient method
Fig. 2. ORTEP drawing of compound 7b.
for the synthesis of a,b unsaturated ketone 5. Using catalysts DMAP,
proved to be an efficient alternative method for the synthesis of
title compounds 4, 7. In addition, in order to facilitate the synthesis
of target compound 4, catalytic amount of KI was added. In the
process of synthesis of compound 7, the best choice to control pH
value less than 8.5 with organic base, when the pH value is greater
than 8.5, the coumarin ring will open. Compounds 1e3 were
prepared according to a previously published report [13].
Crystallographic Data Center as supplementary publication No.
CCDC-837387 and CCDC-836554. These data can be obtained free of
charge via the URL http://www.ccdc.cam.ac.uk/conts/retrieving.
html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK;
fax: (þ44) 1223 336033; e-mail: deposit@ccdc.cam.ac.uk).
In order to facilitate molecular docking, the structures of
compounds 4d and 7b were determined by X-ray crystallography.
Crystal data of 4d: Colorless crystals, yield, 64%; mp 159 ꢁC;
C18H25N3O2, M ¼ 315.4, Orthorhombic, space group P212121;
2.2. In vitro anticancer activity
In the screening assay studies, all title compounds were evalu-
ated for their cytotoxic activity against gastric SGC-7901, MGC-803
and Bcap-37 cell lines. Also included were the activities of reference
compound 5-fluorouracil. The cell was allowed to proliferate in
presence of tested material for 48 h, and the results are reported in
terms of IC50 values (Table 1). It is obvious from the data that
compounds 4f, 7b exhibited high activity against SGC-7901 and
MGC-803 with the IC50 values of 3.22 ꢀ 0.40, 2.64 ꢀ 0.31 and
a ¼ 7.571(5), b ¼ 11.028(8), c ¼ 21.407(15) (Å);
a
¼ 90,
b
¼ 90,
g
¼ 90(ꢁ), V ¼ 1787(2) nm3, T ¼ 293(2) K, Z ¼ 4, Dc ¼ 1.172 g/cm3,
F(000) ¼ 680, Reflections collected/unique ¼ 7851/2989, Data/
restraints/parameters ¼ 2989/3/208, Goodness of fit on F2 ¼ 1.027,
Fine, R1 ¼ 0.0258, wR(F2) ¼ 0.2970. Crystal data of 7b: colorless
crystals, yield, 59%; mp 209e211 ꢁC; C26H29N3O4, M ¼ 447.5,
Triclinic, space group Pꢂ1;
a
¼
9.425(4),
¼ 103.263(4),
b
g
¼
10.453(4),
4.70 ꢀ 0.09, 3.01 ꢀ 0.24
mM respectively, surpassing that of the
positive control 5-fluorouracil. Compounds 7a, 7b showed anti-
cancer activity against the Bcap-37 with IC50 values of 7.30 ꢀ 1.01,
c ¼ 13.384(6) (Å);
a
¼ 103.385(4),
b
¼ 95.469(4) (ꢁ),
V ¼ 1233.1(9) nm3, T ¼ 293(2) K, Z ¼ 2, Dc ¼ 1.205 g/cm3,
F(000) ¼ 476, Reflections collected/unique ¼ 9122/4688, Data/
restraints/parameters ¼ 4688/0/298, Goodness of fit on F2 ¼ 1.003,
Fine, R1 ¼ 0.078, wR(F2) ¼ 0.2050.
6.57 ꢀ 0.34
mM respectively, comparable to that of positive control
5-fluorouracil. From the data presented in Table 1, it can be
concluded that N-substituted-dihydropyrazole-piperidine-
The molecular structures of compounds 4d and 7b were shown
in Figs. 1 and 2. Crystallographic data (excluding structure factors)
for the structure had been deposited with the Cambridge
coumarin derivatives, in general, showed relatively higher activity
against SGC-7901, MGC-803 and Bcap-37 cell lines (7aec), so, some
compounds in this series deserve further investigation.
Table 1
Cytotoxic activity of the synthesized compounds against SGC-7901, MGC-803 and
Bcap-37 cell lines.a
Compound
IC50 (m
M)b
SGC-7901
MGC-803
Bcap-37
4a
4b
4c
4d
4e
4f
4g
7a
5.71 ꢀ 0.78
11.22 ꢀ 1.31
12.91 ꢀ 0.28
4.60 ꢀ 0.30
39.22 ꢀ 1.51
3.22 ꢀ 0.40
30.71 ꢀ 2.20
6.00 ꢀ 0.51
4.70 ꢀ 0.09
10.33 ꢀ 0.89
7.11 ꢀ 0.33
4.12 ꢀ 0.87
11.70 ꢀ 1.45
15.74 ꢀ 1.26
3.55 ꢀ 0.19
38.79 ꢀ 2.11
2.64 ꢀ 0.31
28.27 ꢀ 2.00
8.71 ꢀ 0.45
3.01 ꢀ 0.24
7.01 ꢀ 0.29
3.42 ꢀ 15
18.70 ꢀ 2.47
16.33 ꢀ 2.55
10.70 ꢀ 1.40
6.81 ꢀ 0.87
41.05 ꢀ 2.74
8.55 ꢀ 2.01
32.31 ꢀ 2.66
7.30 ꢀ 1.01
6.57 ꢀ 0.34
8.76 ꢀ 0.44
5.33 ꢀ 0.27
7b
7c
5-Fluorouracilc
Negative control DMSO, no activity.
a
The data represented the mean of three experiments in triplicate and were
expressed as means ꢀ SD; Only descriptive statistics were done in the text.
b
The IC50 value was defined as the concentration at which 50% survival of cells
was observed. The results are listed in the table.
c
Fig. 1. ORTEP drawing of compound 4d.
Used as a positive control.

296
X.-H. Liu et al. / European Journal of Medicinal Chemistry 51 (2012) 294e299
Some purified title compounds were assayed for telomerase
inhibition, using a MGC-803 cell extract, also included the activity
of reference compound ethidium bromide. The results were
summarized in Table 2. The results suggested that the compound
7b showed strong telomerase inhibitory ability with IC50 value of
2.00 ꢀ 0.40
mM, which surpassing that of the positive control
ethidium bromide.
2.3. Molecular docking
In an effort to elucidate the mechanism by which the title
compound can induce anticancer activity against above cancer cell
lines and to establish an SAR based on our experimental studies,
molecular docking of the potent inhibitor 4d into binding site of
telomerase was performed to simulate a binding model derived from
telomerase TERT (3DU6 pdb). The binding model of compounds 4d
with telomerase TERT was depicted in Fig. 3. Visual inspection of the
pose of 4d into the ATP-site revealed that three more optimal intra-
molecular hydrogen bonds were observed (NeH/Ne, with amino
hydrogen group of Lys189; NeH/OeH, with amino hydrogen group
of Asp254, NeH/OeC, withamino hydrogen group ofGln308). Inthe
other end of the ATP-binding pocket, the N of peridine interacted with
the residue Phe 193, which made the 3D structure more stable [10].
3. Conclusion
In summary, we designed 5-phenyl-4,5-dihydropyrazole con-
taining piperidine ethanone derivatives as potential telomerase
inhibitors, followed by chemical synthesis and biological evaluated
for their antiproliferative activities against several cell lines
including MGC-803, Bcap-37 and SGC-7901. Compounds 4d, 4f, 7a
and 7b occupied high antiproliferative activities against above cell
lines. Compound 7b showed the most potent inhibitory activity
Fig. 3. Molecular docking modeling of compound 4d (A) with telomerase TERT
(3DU6); the small molecule and the critical interaction of 3DU6 are represented by
sticks. (B) Schematic representation of the binding mode of 4d in the ATP-binding site
of 3DU6.
with IC50 value at 2.00 ꢀ 0.40
mM. In addition, the binding mode of
high active compound 4d on the telomerase TERT indicated that
conserved residuces Lys189, Asp254 and Gln308 are important for
ligand binding via hydrogen bond interactions. These results are of
help in the rational design of more efficient telomerase inhibitors
for chemoprevention and chemotherapy in further.
4.2. General procedure for the synthesis of compound 4
To an acetone (20 mL) solution of 2-bromo-1-(5-(2-
hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)ethanone
3
(10 mmol) was added substituted-piperazine (10 mmol), DMAP
(12 mmol) andcatalytic KI, the reaction mixturewas allowed to stand
at 40e50 ꢁC for 4 h. The mixture was cooled, washed with water. The
productwascollectedbyfiltrationandthe crude residuewas purified
bychromatography on SiO2 (dichloromethane/methanol, v:v ¼ 65:1)
to give title compounds 4aeg (Scheme 1) as colorless solids.
4. Experimental
4.1. Chemistry
The reactions were monitored by thin layer chromatography
(TLC) on Merck pre-coated silica GF254 plates. Melting points
(uncorrected) were determined on a XT4MP apparatus (Taike Corp.,
Beijing, China). 1H NMR, 13C NMR spectra were collected on PX400
spectrometer at room temperature with TMS and solvent signals
allotted as internal standards. Chemical shifts are reported in ppm
4.2.1. 1-(5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-
2-(4-hydroxypiperidin-1-yl) ethanone (4a)
Colorless crystals, yield, 64%; mp 179e181 ꢁC; 1H NMR
(400 MHz, CDCl3)
d (ppm): 1.60e1.67 (m, 2H), 1.83e1.91 (m, 2H),
(d). Elemental analyses were performed on a CHNeO-Rapid
instrument, and were within ꢀ0.4% of the theoretical values.
2.18 (s, 3H), 2.31e2.37 (m, 2H), 2.83e2.86 (m, 2H), 3.05 (dd, 1H,
J1 ¼ 18.8 Hz, J2 ¼ 3.0 Hz, pyrazole 4-Ha), 3.33 (dd, 1H, J1 ¼ 18.4 Hz,
J2 ¼ 11.2 Hz, pyrazole 4-Hb), 3.52 (s, 2H), 3.66e3.70 (m, 1H), 5.65
(dd, 1H, J1 ¼ 11.2 Hz, J2 ¼ 3.2 Hz, pyrazole 5-H), 6.87e6.98 (m, 3H),
7.18e7.22 (m, 1H). Anal. calcd for C17H23N3O3: C, 64.33; H, 7.30; N,
13.24%. Found: C, 64.00; H, 7.66; N, 12.85%.
Table 2
Inhibitory effects of selected compounds against telomerase.
Compound
IC50 (m
M) Telomerasea
4a
4d
4f
4g
7b
7c
9.11 ꢀ 0.77
5.99 ꢀ 0.31
8.81 ꢀ 0.25
42.12 ꢀ 1.87
2.00 ꢀ 0.40
4.11 ꢀ 0.55
2.47 ꢀ 0.29
4.2.2. 1-(5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-
2-(2-methylpiperidin-1-yl) ethanone (4b)
Colorless crystals, yield, 60%; mp 190e191 ꢁC; 1H NMR
Ethidium bromideb
(400 MHz, CDCl3)
d
(ppm): 1.25 (d, 3H, J ¼ 6.0 Hz), 1.46e1.48 (m,
1H), 1.71e1.77 (m, 4H), 1.88e1.96 (m, 1H), 2.12 (s, 3H), 2.92 (dd, 1H,
J1 ¼ 18.4 Hz, J2 ¼ 3.2 Hz, pyrazole 4-Ha), 3.11e3.21 (m, 2H, con-
taining pyrazole 4-Hb), 3.40 (s, 2H), 3.97e4.01 (m, 1H), 4.11e4.17
a
Telomerase supercoiling activity.
b
Ethidium bromide is reported as a control. The inhibition constant of
ethidium toward telomerase has been reported previously.

X.-H. Liu et al. / European Journal of Medicinal Chemistry 51 (2012) 294e299
297
H3C
OH
O
HO
CHO
A
C
B
CH3
N
N
OH
H
2
1
4a: R1=4 -OH
4b: R1=2 -CH3
4e: R1=2 -CO-
NH2
4f: R1=4 -CF3
H3C
HO
HO
R1
(D)
N
N
4c: R1=2 -CH -CH
1
N
N
34g: R =2 -NO2
2
3
Br
N
O
O
4
4d: R1=4 -CH3
Scheme 1. Synthesis of title compound 4. Reagent and conditions: (A) CH3COCH3, C2H5ONa, 20e30 ꢁC, 10 h. (B) NH2eNH2$H2O, C2H5OH, reflux, 3 h. (C) BrCH2COOH, 40e50 ꢁC, 2 h.
(D) substituted piperidine DMAP, 60 ꢁC, 4 h.
(m, 1H), 5.61 (dd, 1H, J1 ¼ 11.2 Hz, J2 ¼ 3.2 Hz, pyrazole 5-H),
6.82e6.86 (m, 1H), 6.90e6.93 (m, 1H), 7.02e7.04 (m, 1H), 7.11e7.15
(m, 1H). Anal. calcd for C18H25N3O2: C, 68.44; H, 7.99; N, 13.32%.
Found: C, 68.71; H, 7.65; N, 12.97%.
4.2.7. 1-(5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-
2-(2-nitropiperidin-1-yl) ethanone (4g)
Colorless crystals, yield, 67%; mp 194e195 ꢁC; 1H NMR
(400 MHz, CDCl3)
d (ppm): 1.66e1.78 (m, 4H), 2.10 (s, 3H),
2.37e1.48 (m, 4H), 3.04 (dd, 1H, J1 ¼18.4 Hz, J2 ¼ 3.3 Hz, pyrazole 4-
Ha), 3.38 (dd, 1H, J1 ¼ 18.3 Hz, J2 ¼ 11.2 Hz, pyrazole 4-Hb), 3.48 (s,
2H), 4.63 (t,1H), 5.68 (dd,1H, J1 ¼11.2 Hz, J2 ¼ 3.3 Hz, pyrazole 5-H),
6.80e6.87 (m, 1H), 6.92e6.95 (m, 1H), 7.00e7.02 (m, 1H), 7.13e7.16
(m, 1H). Anal. calcd for C17H22N4O4: C, 58.95; H, 6.40; N, 16.17%.
Found: C, 59.20; H, 6.31; N, 15.94%.
4.2.3. 2-(2-Ethylpiperidin-1-yl)-1-(5-(2-hydroxyphenyl)-3-methyl-
4,5-dihydropyrazol-1-yl)ethanone (4c)
Colorless crystals, yield, 52%; mp 166e167 ꢁC; 1H NMR
(400 MHz, CDCl3)
d
(ppm): 0.89e0.97 (t, 3H, J ¼ 7.2 Hz), 1.74e2.06
(m, 8H), 2.09 (s, 3H), 2.78 (dd, 1H, J1 ¼18.4 Hz, J2 ¼ 3.4 Hz, pyrazole
4-Ha), 3.35 (dd, 1H, J1 ¼ 18.4 Hz, J2 ¼ 11.2 Hz, pyrazole 4-Hb),
3.40e3.51 (m, 3H), 4.18e4.26 (m, 2H), 5.63 (dd, 1H, J1 ¼ 11.2 Hz,
J2 ¼ 3.4 Hz, pyrazole 5-H), 6.78e6.92 (m, 2H), 7.01e7.10 (m, 1H),
4.3. General procedure for the synthesis of compound 7
7.17e7.19 (m, 1H). 13C NMR (CDCl3, 125 MHz):
d
10.1, 16.3, 21.9, 22.2,
To an acetone (20 mL) solution of 3-(1-(2-bromoacetyl)-5-phenyl-
4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one 6 (10 mmol) was
added substituted-piperazine (10 mmol), DMAP (10 mmol) and
catalytic KI, the reaction mixture was reflux for 6 h. The mixture was
cooled,washedwithwater. Theproductwascollectedbyfiltrationand
the crude residue was purified by chromatography on SiO2
(dichloromethane/methanol, v:v ¼ 70:1) to give title compounds
7ae7c (Scheme 2) as colorless solids.
26.6, 27.4, 45.3, 56.0, 59.1, 64.8, 118.1, 120.8, 126.2, 126.7, 129.6,
153.8, 154.0, 160.9, 161.7; Anal. calcd for C19H27N3O2: C, 69.27; H,
8.26; N, 12.76%. Found: C, 69.00; H, 8.49; N, 13.04%.
4.2.4. 1-(5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-
2-(4-methylpiperidin-1-yl) ethanone (4d)
Colorless crystals, yield, 64%; mp 159e160 ꢁC; 1H NMR
(400 MHz, CDCl3)
d (ppm): 0.93e1.00 (d, 3H), 1.59e1.83 (m, 4H),
2.12 (s, 3H), 2.72e2.77 (m, 2H), 2.84 (dd,1H, J1 ¼18.4 Hz, J2 ¼ 3.3 Hz,
pyrazole 4-Ha), 2.90e2.97 (m, 1H), 3.28e3.37 (m, 2H), 3.41 (dd, 1H,
J1 ¼ 18.4 Hz, J2 ¼ 11.2 Hz, pyrazole 4-Hb), 3.96 (s, 2H), 5.66 (dd, 1H,
J1 ¼11.2 Hz, J2 ¼ 3.3 Hz, pyrazole 5-H), 6.83e6.95 (m, 2H), 7.07e7.15
(m, 2H). Anal. calcd for C18H25N3O2: C, 68.54; H, 7.99; N, 13.32%.
Found: C, 68.81; H, 7.65; N, 13.55%.
4.3.1. 3-(1-(2-(2-Ethylpiperidin-1-yl)acetyl)-3-phenyl-4,5-dihydro-
1H-pyrazol-5-yl)-2H-chromen-2-one (7a)
Colorless crystals, yield, 60%; mp 210e211 ꢁC; 1H NMR
(400 MHz, CDCl3)
d (ppm): 0.98 (t, 3H), 1.43 (m, 2H), 1.50e1.69
(m, 6H), 2.06e2.16 (m, 1H), 2.72e2.77 (m, 2H), 3.34 (dd, 1H,
J1 ¼ 19.2 Hz, J2 ¼ 4.8 Hz, pyrazole 4-Ha), 3.49 (s, 2H), 3.88 (dd, 1H,
J1 ¼ 19.2 Hz, J2 ¼ 12.0 Hz, pyrazole 4-Hb), 5.57 (dd, 1H,
J1 ¼ 12.0 Hz, J2 ¼ 4.8 Hz, pyrazole 5-H), 7.19e7.38 (m, 7H),
7.59e7.75 (m, 1H), 7.76e7.78 (m, 1H), 8.67 (1H, s, C4-H); 13C NMR
4.2.5. 1-(2-(5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-
yl)-2-oxoethyl)piperidine-4-carboxamide (4e)
Colorless crystals, yield, 55%; mp 157e158 ꢁC; 1H NMR
(CDCl3, 125 MHz): d 9.9, 22.7, 24.0, 28.3, 44.5, 54.2, 61.0, 61.1,
(400 MHz, CDCl3)
d
(ppm): 1.74e1.82 (m, 4H), 2.13 (s, 3H),
63.1, 63.2, 116.7, 118.7, 125.3, 125.5, 125.6, 127.7, 129.2, 140.6,
140.7, 142.8, 142.9, 153.7, 154.4, 159.3; Anal. calcd for:
C27H29N3O3: C, 73.11; H, 6.59; N, 9.47%. Found: C, 73.26; H, 6.77;
N, 9.86%.
2.09e2.23 (m, 2H), 2.91 (dd, 1H, J1 ¼18.8 Hz, J2 ¼ 3.2 Hz, pyrazole 4-
Ha), 2.97e3.02 (m, 2H), 3.31 (dd, 1H, J1 ¼ 18.8 Hz, J2 ¼ 11.1 Hz,
pyrazole 4-Hb), 3.54 (s, 2H), 5.64 (dd, 1H, J1 ¼ 11.2 Hz, J2 ¼ 3.2 Hz,
pyrazole 5-H), 5.92 (s, 2H), 6.82e6.90 (m, 4H), 7.10e7.14 (m, 1H).
Anal. calcd for C18H24N4O3: C, 62.77; H, 7.02; N, 16.27%. Found: C,
62.43; H, 7.35; N, 16.12%.
4.3.2. 3-(1-(2-(4-Methylpiperidin-1-yl)acetyl)-3-phenyl-4,5-
dihydro-1H-pyrazol-5-yl)-2H-chromen-2-one (7b)
Colorless crystals, yield, 59%; mp 209e211 ꢁC; 1H NMR
4.2.6. 1-(5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-
2-(4-(trifluoromethyl)piperidin-1-yl) ethanone (4f)
(400 MHz, CDCl3)
d
(ppm): 0.94 (d, 3H, J ¼ 4.8 Hz), 1.60e1.75 (m,
4H), 3.04e3.11 (m, 3H, containing pyrazole 4-Ha), 3.38 (m, 3H), 3.96
(dd, 1H, J1 ¼19.2 Hz, J2 ¼ 12.0 Hz, pyrazole 4-Hb), 4.34 (m, 2H), 5.53
(dd, 1H, J1 ¼ 4.8 Hz, J2 ¼ 12.0 Hz, pyrazole 5-H), 7.19e7.35 (m, 9H),
Colorless crystals, yield, 66%; mp 170e172 ꢁC; 1H NMR
(400 MHz, CDCl3)
d (ppm): 1.68e1.86 (m, 4H), 2.07 (s, 3H),
2.48e2.79 (m, 5H), 2.99 (dd,1H, J1 ¼18.3 Hz, J2 ¼ 3.2 Hz, pyrazole 4-
Ha), 3.48 (dd, 1H, J1 ¼ 18.3 Hz, J2 ¼ 11.2 Hz, pyrazole 4-Hb), 3.61 (s,
2H), 5.75 (dd, 1H, J1 ¼11.2 Hz, J2 ¼ 3.2 Hz, pyrazole 5-H), 6.87e6.92
(m, 2H), 7.02e7.11 (m, 2H). Anal. calcd for C18H22F3N3O2: C, 58.53;
H, 6.00; N, 11.38%. Found: C, 58.51; H, 6.37; N, 10.99%.
8.76 (1H, s, C4-H); 13C NMR (CDCl3,125 MHz):
d 21.2, 28.7, 32.0, 44.6,
53.4, 57.4, 61.1, 116.6, 118.5, 118.8, 125.2, 125.6, 128.2, 129.2, 129.7,
133.5, 140.6, 143.2, 153.7, 154.4, 159.3, 164.7; Anal. calcd for:
C26H27N3O3: C, 72.71; H, 6.34; N, 9.78%. Found: C, 72.90; H, 6.28; N,
10.05%.

298
X.-H. Liu et al. / European Journal of Medicinal Chemistry 51 (2012) 294e299
O
O
O
O
(E)
CH3
H
(F)
H3C
O
CH3
O
N
O
OH
4'
O
O
O
O
(H)
(G)
N
Br
O
O
O
5
6
7a: R2=2 -CH2-CH3
7b: R2=4 -CH3
7c: R2=4 - Br
O
N
N
N
R2
O
7
Scheme 2. Synthesis of title compound 7. Reagent and conditions: (E) piperazine, 20e30 ꢁC, 1 h. (F) benzaldehyde, piperidine, ethanol, reflux, 10 h. (G). NH2eNH2$H2O, C2H5OH,
BrCH2COOH, reflux, 2 h. (H) substituted piperidine DMAP, KI, reflux, 6 h.
4.3.3. 3-(1-(2-(4-Bromopiperidin-1-yl)acetyl)-3-phenyl-4,5-
dihydro-1H-pyrazol-5-yl)-2H-chromen-2-one (7c)
4.6. Telomerase activity assay
Colorless crystals, yield, 55%; mp 200e202 ꢁC; 1H NMR
Compounds 4 and 7 were tested in a search for small molecule
inhibitors of telomerase activity by using the TRAP-PCR-ELISA assay.
In detail, the MGC-803 cells were firstly maintained in DMEM
medium (GIBCO, New York, USA) supplemented with 10% fetal bovine
serum (GIBCO, New York, USA), streptomycin (0.1 mg/mL) and peni-
cillin (100 IU/mL) at 37 ꢁC in a humidified atmosphere containing 5%
CO2. After trypsinization, 5 ꢃ 104 cultured cells in logarithmic growth
were seeded intoT25 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 concen-
tration as 60, 20, 6.67, 2.22, 0.74, 0.25 and 0.0821 g/mL, respectively.
After 24 h treatment, the cells were harvested by cell scraper orderly
(400 MHz, CDCl3)
d (ppm): 1.97e2.16 (m, 4H), 2.28e2.31 (m, 4H),
3.31 (s, 2H), 3.78 (dd, 1H, J1 ¼ 19.2 Hz, J2 ¼ 4.8 Hz, pyrazole 4-Ha),
3.52 (m, 1H), 3.90 (dd, 1H, J1 ¼19.2 Hz, J2 ¼ 12.0 Hz, pyrazole 4-Hb),
5.65 (dd, 1H, J1 ¼ 4.8 Hz, J2 ¼ 12.0 Hz, pyrazole 5-H), 7.11e7.38 (m,
9H), 8.70 (1H, s, C4-H); Anal. calcd for: C25H24BrN3O3: C, 60.74; H,
4.89; N, 8.50%. Found: C, 61.02; H, 5.15; N, 8.67%.
4.4. Crystallographic studies
X-ray single-crystal diffraction data for compounds 4d, 7b were
collected on a Bruker SMART APEX CCD diffractometer at 296(2) K
following by washed once with PBS. The cells were lysed in 150 mL
using MoK
a
radiation (
l
¼ 0.71073 Å) by the
u scan mode. The
RIPA cell lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated on
ice for 30 min. The cellular supernatants were obtained via centrifu-
gation at 12,000 g for 20 min at 4 ꢁC and stored at ꢂ80 ꢁC. The TRAP-
PCR-ELISA assay was performed using a telomerase detection kit
(Roche, Basel, Switzerland) according to the manufacturer’s protocol.
program SAINT was used for integration of the diffraction profiles.
The structures were solved by direct methods using the SHELXS
program of the SHELXTL package and refined by full-matrix least-
squares methods with SHELXL [14]. All non-hydrogen atoms of
compounds 4d, 7b were refined with anisotropic thermal param-
eters. All hydrogen atoms were generated theoretically onto the
parent atoms and refined isotropically with fixed thermal factors.
In brief, 2 mL of cell extracts were mixed with 48 mL TRAP reaction
mixtures. PCR was then initiated at 94 ꢁC, 120 s for predenaturation
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
mL of PCR products were hybridized to
4.5. Anticancer assay
a digoxigenin (DIG)-labeled telomeric repeat specific detectionprobe.
And thePCRproducts 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 (Mannedorf, Switzerland) at a wavelength of
490 nm, and the final value were presented as mean ꢀ SD.
The cytotoxicity evaluation was conducted by using a modified
procedure as described in the literature. Briefly, target tumor cells
were grown to log phase in RPMI 1640 medium supplemented with
10% fetal bovine serum. After diluting to 3 ꢃ 104 cells mLꢂ1 with the
complete medium, 100
mL of the obtained cell suspension was
added to each well of 96-well culture plates. The subsequent
incubation was performed at 37 ꢁC, 5% CO2 atmosphere for 24 h
before subjecting to cytotoxicity assessment. Tested samples at pre-
set concentrations were added to 6 wells with 5-fluorouracil co-
4.7. General procedure for molecular docking
assayed as a positive reference. After 48 h exposure period, 25
of PBS containing 2.5 mg mLꢂ1 of MTT was added to each well. After
4 h, the medium was replaced by 150 L DMSO to dissolve the
purple formazan crystals produced. The absorbance at 570 nm of
each well was measured on an ELISA plate reader. The data repre-
sented the mean of three experiments in triplicate and were
expressed as means ꢀ SD using Student t test. The IC50 value was
defined as the concentration at which 50% of the cells could survive.
mL
Discovery Studio 2.1 (DS 2.1, Accelrys Software Inc., San Diego,
California, USA). Crystal structure of telomerase (PDB entry 3DU6)
was used as template. Hydrogen atoms were added to protein model.
The added hydrogen atoms were minimized to have stable energy
conformation and to also relax the conformation from close contacts.
The active site was defined and sphere of 5 Å was generated around
the active site pocket, with the active site pocket of BSAI model using
m

X.-H. Liu et al. / European Journal of Medicinal Chemistry 51 (2012) 294e299
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