Synthesis and molecular docking study of novel coumarin derivatives containing 4,5-dihydropyrazole moiety as potential antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2010.08.017

A series of novel coumarin derivatives containing 4,5-dihydropyrazole moiety as potential telomerase inhibitors were synthesized. The bioassay tests show that compound 3d exhibited potentially high activity against human gastric cancer cell SGC-7901 with IC₅₀ value of 2.69 ± 0.60 μg/mL. All title compounds were assayed for telomerase inhibition by a modified TRAP assay, the results show that compounds 3d and 3f can strongly inhibit telomerase with IC₅₀ values of 2.0 ± 0.07 and 1.8 ± 0.35 μM, respectively. Docking simulation was performed to position compound 3d into the telomerase (3DU6) active site to determine the probable binding model.

Full text of DOI:10.1016/j.bmcl.2010.08.017

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5705–5708
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and molecular docking study of novel coumarin derivatives
containing 4,5-dihydropyrazole moiety as potential antitumor agents
Xin-Hua Liu a,b, Hui-Feng Liu a, Jin Chen b, Yang Yang a, Bao-An Song c, Lin-Shan Bai a, Jing-Xin Liu a,
d
Hai-Liang Zhu a,b, , Xing-Bao Qi
⇑
a School of Chemistry and Chemical Engineering, Anhui University of Technology, Maanshan 243002, PR China
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
c Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China
d 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 coumarin derivatives containing 4,5-dihydropyrazole moiety as potential telomerase
Received 17 May 2010
Revised 21 July 2010
Accepted 4 August 2010
Available online 10 August 2010
inhibitors were synthesized. The bioassay tests show that compound 3d exhibited potentially high activ-
ity against human gastric cancer cell SGC-7901 with IC50 value of 2.69 0.60
were assayed for telomerase inhibition by a modified TRAP assay, the results show that compounds 3d
and 3f can strongly inhibit telomerase with IC50 values of 2.0 0.07 and 1.8 0.35 M, respectively.
lg/mL. All title compounds
l
Docking simulation was performed to position compound 3d into the telomerase (3DU6) active site to
determine the probable binding model.
Keywords:
Synthesis
Coumarin
Ó 2010 Elsevier Ltd. All rights reserved.
Dihydropyrazole
Antitumor agent
Molecular docking
Telomerase remains active in the early stages of life maintain-
ing 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 rap-
idly dividing cells, leading to their immortality.2 The essential role
of telomerase in cancer and aging makes it an important target for
the development of therapies to treat cancer and other age-associ-
ated disorders. Telomere and telomerase are closely related to the
occurrence and development of human gastric cancer.3
Coumarins are present in natural and synthetic compounds
possessing biological activity. They are acting at different stages
of cancer formation. Some of them have cytostatic properties and
the others have cytotoxic activity.4 Two naturally occurring cou-
marins have been found to exhibit cytotoxicity against a panel of
mammalian cancer cell lines.5 In view of their importance as drugs,
biologically active natural products, and in other related applica-
tions, extensive studies have been carried out on the synthesis of
coumarin compounds in recent years. On the other hand, many lit-
eratures discussed the antitumor activity of pyrazole derivatives.6,7
Furthermore, 3,4-dihydropyrazole, a small bioactive molecule, is a
prominent structural motif found in numerous pharmaceutically
active compounds. Many 3,4-dihydropyrazole-based derivatives
have shown several biological activities as seen in CB1 antagonist,8
and tumor necrosis inhibitor.9 Many of them are currently being
tested and/or clinically evaluated for new drug discovery.
In an effort to synthesize novel 3,4-dihydropyrazole heterocy-
clic systems with potential biological activity, our group has re-
cently reported on the activity of some 3,4-dihydropyrazole
derivatives.10,11 At present, we focus on screening of novel precur-
sor structure with anti-gastric cancer activity and found some
2-chloro-pyridine derivatives containing phenylpropanoid (flavone,
chrome) or dihydropyrazole unit used as potential telomerase
inhibitors.12 Based on above reports, we considered the possibility
of introducing heterocyclic 4,5-dihydropyrazole moiety into the
parent coumarin unit (phenylpropanoids) to design novel struc-
tures with enhanced anticancer activities. Since there are only a
very few systematic reports on the synthetic methodology and
evaluation of anticancer activities of these compounds, Herein, in
continuation to extend our research on antiproliferative activity
Abbreviations: MTT, 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium
bromide; DMSO, dimethyl sulfoxide; MH, Mueller-Hinton; PBS, phosphate buffered
saline; ELISA, enzyme linked immunosorbent assay; TRAP, telomere repeat
amplification protocol.
⇑
Corresponding author. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
against cancer cells, we prepared
a series novel coumarin
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.017

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X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5705–5708
Table 1
(phenylpropanoids) derivatives containing 4,5-dihydropyrazole
moiety and tested their activities against SGC-7901 (human gastric
cancer), PC-3 (human prostate cancer) cell, and A431 (human epi-
dermoid carcinoma) cell. In order to elucidate the potential mech-
anism by which the title compounds induce anticancer activity,
docking simulation was performed to position selected compounds
into the active site of telomerase 3DU6.
The synthesis of compound 1 (Scheme 1) started from salicylal-
dehyde and catalyzed by piperazine at 30 °C was added acetoace-
tate. Claisen–Schmidt condensation 3-acetyl-2H-chromen-2-one
and substituted-benzaldehyde using mild catalyst piperidine, by
following a reported method,10 proved to be an efficient alternative
Cytotoxic activity of test compounds against SGC-7901 cell linea negative control
DMSO, no activity
b
b
b
Compound
IC50
(l
g/mL)
IC50
(lg/mL)
IC50 (lg/mL)
SGC-7901
PC-3
A431
d
d
d
2a
4
3a
3b
3c
3d
3e
3f
—
—
—
—
—
d
d
65.10 1.22
d
48.74 2.40
35.93 0.77
29.93 0.09
2.69 0.60
39.93 0.49
28.93 0.77
22.35 1.66
7.38 0.98
—
68.74 0.15
72.97 2.11
50.65 2.00
51.74 0.09
68.74 1.12
48.12 1.40
68.35 0.56
2.17 0.20
58.74 0.40
55.08 1.95
40.16 2.31
46.74 2.00
58.09 1.08
38.77 0.98
2.48 0.11
3g
for the synthesis of a,b unsaturated ketone 2. The general synthetic
5-Fluorouracilc
procedure process and spectral data of compounds 3 can be found
in Ref. 14. Compound 4 was prepared according to a previously
published Letter.11
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
Compound 2a: (E)-3-(3-(4-methoxyphenyl)acryloyl)-2H-chro-
men-2-one, colorless crystals, yield, 60%; mp 153–154 °C; 1H
NMR (CDCl3, 300 MHz): d 3.85 (3H, s, –OMe), 6.93 (2H, d, J =
8.8 Hz, ArH), 7.32 (2H, d, J = 7.5 Hz, ArH), 7.37–8.37 (6H, m, C5–8-H
and –CH@CH–), 8.57 (1H, s, C4-H); 13C NMR (CDCl3, 125 MHz):
d 56.3, 113.7, 122.9, 124.7, 124.9, 126.6, 126.9, 128.0, 128.2, 128.7,
134.3, 148.1, 152.0, 154.1, 159.4, 160.3, 184.2; ESI-MS: 306.3
(C19H14O4, [M+H]+); Anal. Calcd for C19H14O4: C, 74.5; H, 4.6. Found:
C, 74.9; H, 4.2.
was observed. The results are listed in the table.
c
Used as a positive control.
No significant activity.
d
Table 2
Biological properties of test compounds
Compound
IC50
(
lM) Taq
IC50
(
l
M)
Selectivity
indexa (SI)
polymerase
telomerase
2a
No
No
—
Compound 4: 5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyraz-
ole-1-carbaldehyde, colorless crystals, yield, 90%; mp 155–156 °C;
1H NMR (CDCl3, 300 MHz): d 2.19 (3H, s, –Me), 3.10 (1H, dd,
J = 18.7 and 3.7 Hz, pyrazole, 4-Ha), 3.39 (1H, dd, J = 11.2 and
18.6 Hz, pyrazole, 4-Hb), 5.67 (1H, dd, J = 11.6 and 3.5 Hz, pyrazole,
5-H), 6.89–7.26 (5H, m, ArH and –OH), 8.63 (1H, s, –COH); 13C NMR
(CDCl3, 125 MHz): d 21.2, 43.1, 48.5, 115.4, 121.9, 129.6, 129.9,
131.3, 149.7, 155.0, 160.6; ESI-MS: 205.1 (C11H12N2O2, [M+H]+);
Anal. Calcd for C11H12N2O2: C, 64.7; H, 5.9; N, 13.7. Found: C,
64.5; H, 6.1; N, 14.0.
4
3a
3b
3c
3d
3e
3f
3g
No
No
No
—
—
36.7 1.1
29.1 1.03
6.5 0.16
2.0 0.16
9.6 0.09
1.8 0.35
4.0 0.25
2.5 0.8
58.5 1.8
16.8 2.0
6.0 2.2
20.7 1.9
4.0 2.0
10.0 2.0
2.0
2.6
3.0
2.1
2.2
2.5
Ethidium
bromideb
No, not observed in the tested concentration range 0–60 lM.
Ratio between the drug concentrations at which 50% Taq polymerase/telome-
rase inhibition was observed.
Ethidium bromide is reported as a control. The inhibition constant of ethidium
toward telomerase has been reported previously.
a
In the screening assay studies, compounds 2a and 4 and all the
compounds 3 were evaluated for their cytotoxic activity against
gastric SGC-7901, PC-3 (human prostate cancer), and A431 (human
b
O
O
O
O
CH3
H
R1
R1
(I)
(II)
O
H3C
CH3
O
O
OH
1
CH3
O
O
O
N
N
O
R2
(III)
R2
R1
R1
O
O
2
3
H3C
O
HO
O
N
H
(IV)
CH3
(V)
N
CH3COCH3
OH
OH
H
O
4
R1=H, R2 = 4-OMe (a); R1=H, R2 =4-OH (b); R1=H, R2= 2-Cl (c); R1=H, R2 =H (d);
R1=H, R2 =2,4-2Cl (e); R1=5-Br (f), R2=H; 5-Br, R2=4-OH (g)
Scheme 1. Synthesis of title compounds. Reagent and conditions: (I) piperzine, 25–30 °C, 1 h; (II) substituted-benzaldehyde, piperzine, ethanol, reflux, 6 h; (III) 80% NH2–
NH2ÁH2O, 98% CH3COOH, reflux, 2 h; (IV) NaOH, CH3COCH3, HCl, 20 °C, 15 h; (V) 80% NH2–NH2ÁH2O, HCOOH, ethanol, reflux, 1 h.

X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5705–5708
5707
A
B
GLy 182
O
Phe 200
N
H
O
N
H
O
O
N
N
O
H3C
Figure 1. Molecular docking modeling of compound 3d (A) with telomerase; the small molecule and the critical interaction of 3DU6 are represented by sticks. Panel is a view
into the active site cavity. (B) Schematic representation of the binding mode of 3d in the ATP binding site of 3DU6.
epidermoid carcinoma) cell lines.15 The cells were 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 compound 3d exhibited high activity against the hu-
ATP binding site of telomerase was performed to simulate a
binding model derived from telomerase structure (3DU6.pdb)
(see Fig. 1).19 Visual inspection of the pose of 3d into the ATP-site
revealed that two more optimal intramolecular hydrogen bonds
are observed (N–HÁ Á ÁO: 3.06 Å, with amino hydrogen group of
Gly 182; N–HÁ Á ÁO: 3.07 Å, with amino hydrogen group of Phe
200). Also the 4,5-dihydropyrazole ring projects into a hydropho-
bic region, which is comprised of the side chains of Pro 201, Asp
202, Ser 203, Ala 204, that is, important for the potent inhibitory
activity of 3d. These residues influenced the accessibility of the
hydrophobic pocket that flanks the ATP binding site, and their size
can be a key factor in controlling telomerase selectivity. In the
other end of the ATP-binding pocket, the O of dihydropyrazole
acetyl interacted with the residue Ile 199, which made the 3D
structure more stable.
In summary, we prepared a series of novel coumarin derivatives
containing 4,5-dihydropyrazole moiety as potential telomerase
inhibitors. The result showed that compound 3d had strongly high
activity against human gastric cancer cell SGC-7901. Docking sim-
ulation was performed to position compounds 3d into the telome-
rase 3DU6 active site, the result shows compound 3d can bind well
with the telomerase active site and act as telomerase inhibitor.
man gastric cell SGC-7901 with the IC50 value of 2.69 0.60 lg/mL,
which was even better than that of the 5-fluorouracil, while all the
synthesized compounds exhibited poor activity against PC-3 cell
(human prostate cancer) and A431 (human epidermoid carcinoma)
cell.
From the data presented in Table 1, it can be concluded that
coumarin
a,b unsaturated ketone (2a) and 4,5-dihydropyrazole
(4), respectively, displayed poor activity against human gastric cell
SGC-7901, whereas coumarin derivatives containing 4,5-dihydro-
pyrazole moiety, in general, showed relatively higher activity
against the gastric human SGC-7901 cell, so, some compounds in
this series deserve further investigation.
All purified title compounds were assayed for telomerase inhi-
bition by a modified TRAP17 assay, using a SGC-7901 cell extract.
Modified TRAP is a powerful technique and could give us some
information about small molecules inhibiting telomere elongation
qualitatively and quantitatively.18 To avoid false positive results
due to drug interference with the amplification step, Taq polymer-
ase inhibition was additionally monitored. The results are summa-
rized in Table 2, where a selectivity index (SI, ratio between IC50 for
Taq polymerase vs telomerase inhibition) is also included. The re-
sults suggested that the telomerase inhibitory abilities of certain
coumarin derivatives containing 4,5-dihydropyrazole moiety were
strong. Especially compounds 3d and 3f with IC50 values of
Acknowledgment
The authors wish to thank the National Natural Science Founda-
tion of China (No. 20902003); the Key Research Projects of Univer-
sity Natural Science, Anhui Province (No. KJ2009A011Z).
2.0 0.16 and 1.8 0.35 lM, respectively, which were even better
than that of the ethidium bromide.
References and notes
In an effort to elucidate the mechanism by which the title
compound can induce anticancer activity in the human gastric
cell SGC-7901, molecular docking of the potent inhibitors 3d into
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and 4.8 Hz, pyrazole, 5-H), 7.12–7.95 (9H, m, C5–8-H and ArH), 8.37 (1H, s, C4-
H); 13C NMR (CDCl3, 125 MHz): d 24.0, 39.8, 58.6, 122.0, 123.1, 124.0, 126.7,
126.9, 127.5, 127.8, 128.9, 129.2, 134.3, 144.4, 151.7, 157.2, 161.7, 169.8; ESI-
MS: 333.0 (C20H16N2O3, [M+H]+); Anal. Calcd for C20H16N2O3: C, 72.3; H, 4.9; N,
8.4. Found: C, 72.8; H, 5.3; N, 8.0.
6. Mohammad, A.; Harish, K.; Suroor, A. K. Bioorg. Med. Chem. Lett. 2008, 18, 918.
7. Sayed, M. R.; Thoraya, A. F.; Magda, A. A.; Mohamed, M. A. Eur. J. Med. Chem.
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9. Dmytro, H.; Borys, Z.; Olexandr, V.; Lucjusz, Z.; Andrzej, G.; Roman, L. Eur. J.
Med. Chem. 2009, 44, 1396.
Compound 3e: 3-(1-acetyl-5-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)-
2H-chromen-2-one, colorless crystals, yield, 68%; mp 197–199 °C; 1H NMR
(CDCl3, 300 MHz): d 2.40 (3H, s, –Me), 3.22 (1H, dd, J = 19.0 and 5.5 Hz,
pyrazole, 4-Ha), 3.97 (1H, dd, J = 12.2 and 19.2 Hz, pyrazole, 4-Hb), 5.76 (1H, dd,
J = 12.1 and 5.5 Hz, pyrazole, 5-H), 6.89–7.94 (7H, m, C5–8-H and ArH), 8.35 (1H,
s, C4-H); 13C NMR (CDCl3, 125 MHz): d 24.6, 38.2, 53.1, 122.0, 123.1, 124.5,
126.2, 127.7, 127.8, 128.9, 131.2, 131.5, 133.3, 133.8, 140.0, 142.8, 150.3, 156.7,
160.5, 169.4; ESI-MS: 401.9 (C20H14Cl2N2O3, [M+H]+); Anal. calcd for
10. Liu, X. H.; Zhu, J.; Zhou, A. N.; Song, B. A.; Zhu, H. L.; Bai, L. S.; Bhadury, P. S.;
Pan, C. X. Bioorg. Med. Chem. 2009, 17, 1207.
C
20H14Cl2N2O3: C, 59.9; H, 3.5; N, 7.0. Found: C, 60.3; H, 3.8; N, 6.6.
11. Liu, X. H.; Cui, P.; Song, B. A.; Bhadury, P. S.; Zhu, H. L.; Wang, S. F. Bioorg. Med.
Chem. 2008, 16, 4075.
12. Liu, X. H.; Liu, H. F.; Xu, S.; Song, B. A.; Bhadury, P. S.; Zhu, H. L.; Liu, J. X.; Qi, X.
B. Bioorg. Med. Chem. Lett. 2010, 20, 4163.
13. 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). ESI mass spectra were
obtained on a Mariner System 5304 mass spectrometer, and 1H NMR spectra
were collected on PX300 spectrometer at room temperature with TMS and
solvent signals allotted as internal standards. Chemical shifts are reported in
ppm (d).
Compound 3f: 3-(1-acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-6-bromo-2H-
chromen-2-one, colorless crystals, yield, 57%; mp 158–159 °C; 1H NMR (CDCl3,
300 MHz): d 2.30 (3H, s, –Me), 3.38 (1H, dd, J = 19.0 and 4.5 Hz, pyrazole, 4-Ha),
3.98 (1H, dd, J = 11.8 and 19.0 Hz, pyrazole, 4-Hb), 5.29 (1H, dd, J = 11.8 and
4.5 Hz, pyrazole, 5-H), 6.99 (H, d, J = 8.2 Hz, C8-H), 7.08–7.36 (6H, m, ArH and
C7-H), 7.67 (1H, s, C5-H), 8.28 (1H, s, C4-H); 13C NMR (CDCl3, 125 MHz): d 25.1,
39.3, 56.2, 121.5, 123.3, 124.0, 125.2, 126.7, 128.0, 129.4, 130.9, 131.4, 134.5,
144.9, 150.3, 156.7, 160.8, 169.9; ESI-MS: 410.8 (C20H15BrN2O3, [M+H]+); Anal.
Calcd for C20H15BrN2O3: C, 58.4; H, 3.7; N, 6.8. Found: C, 58.6; H, 4.0; N, 6.5.
Compound 3g: 3-(1-acetyl-5-(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-
bromo-2H-chromen-2-one, colorless crystals, yield, 63%; mp 183–185 °C; 1H
NMR (CDCl3, 300 MHz): d 2.35 (3H, s, –Me), 3.32 (1H, dd, J = 19.0 and 4.5 Hz,
pyrazole, 4-Ha), 3.85 (1H, dd, J = 11.8 and 19.0 Hz, pyrazole, 4-Hb), 5.35 (1H, dd,
J = 11.8 and 4.5 Hz, pyrazole, 5-H), 5.75 (1H, s, –OH), 6.74 (2H, d, J = 8.4 Hz,
ArH), 6.98–7.32 (4H, m, ArH and C7, C8-H), 7.60 (1H, s, C5-H), 8.21 (1H, s, C4-H);
13C NMR (CDCl3, 125 MHz): d 24.5, 40.0, 57.4, 116.2, 120.4, 123.0, 124.2, 124.7,
129.1, 130.8, 131.6, 134.2, 138.5, 150.6, 156.9, 157.1, 160.7, 169.2; ESI-MS:
426.4 (C20H15BrN2O4, [M+H]+); Anal. Calcd for C20H15BrN2O4: C, 56.2; H, 3.5; N,
6.6. Found: C, 55.9; H, 3.2; N, 6.5.
14. General synthetic procedure process for compounds 3: To a solution of
a,b
unsaturated ketone 2 (10 mmol) in acetic acid (20 mL) was added hydrazine
monohydrate (40 mmol) and the reaction mixture was refluxed for 2 h. The
mixture was cooled, adjusted pH to 7 with 10% Na2CO3 solution, poured into
crush ice, and allowed to stand at room temperature over night. The product
was collected by filtration and the crude residue was purified by
chromatography on SiO2 (acetone/petroleum, V:V = 2:1) to give title
compounds 3 (Scheme 1) as colorless solids.13
Compound 3a: 3-(1-acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)-
2H-chromen-2-one, colorless crystals, yield, 64%; mp 180–182 °C; 1H NMR
(CDCl3, 300 MHz): d 2.34 (3H, s, –Me), 3.34 (1H, dd, J = 19.0 and 4.7 Hz,
pyrazole, 4-Ha), 3.70 (3H, s, –OMe), 3.86 (1H, dd, J = 12.0 and 19.0 Hz, pyrazole,
4-Hb), 5.48 (1H, dd, J = 11.7 and 4.6 Hz, pyrazole, 5-H), 6.77 (2H, d, J = 8.6 Hz,
ArH), 7.08 (2H, d, J = 8.6 Hz, ArH), 7.20–7.95 (4H, m, C5–8-H), 8.35 (1H, s, C4-H);
13C NMR (CDCl3, 125 MHz): d 24.2, 40.0, 54.9, 56.3, 115.0, 121.9, 123.4, 123.7,
125.0, 127.3, 128.5, 128.8, 134.2, 136.8, 152.1, 158.2, 159.0, 160.5, 169.2; ESI-
MS: 362.0 (C21H18N2O4, [M+H]+); Anal. Calcd for C21H18N2O4: C, 69.6; H, 5.0; N,
7.7. Found: C, 70.0; H, 4.6; N, 8.0.
Compound 3b: 3-(1-acetyl-5-(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)-
2H-chromen-2-one, colorless crystals, yield, 61%; mp 167–168 °C; 1H NMR
(CDCl3, 300 MHz): d 2.44 (3H, s, –Me), 3.45 (1H, dd, J = 19.0 and 4.6 Hz,
pyrazole, 4-Ha), 3.95 (1H, dd, J = 11.9 and 19.0 Hz, pyrazole, 4-Hb), 5.41 (1H, s, –
OH), 5.55 (1H, dd, J = 11.9 and 4.6 Hz, pyrazole, 5-H), 6.71 (2H, d, J = 8.4 Hz,
ArH), 6.99 (2H, d, J = 8.4 Hz, ArH), 7.27–8.09 (4H, m, C5–8-H), 8.44 (1H, s, C4-H);
13C NMR (CDCl3, 125 MHz): d 24.7, 38.9, 55.4, 114.7, 121.0, 122.9, 124.2, 126.1,
127.7, 128.9, 129.0, 134.1, 137.3, 150.9, 156.1, 158.8, 160.0, 169.4; ESI-MS:
349.1 (C20H16N2O4, [M+H]+); Anal. Calcd for C20H16N2O4: C, 69.0; H, 4.6; N, 8.0.
Found: C, 68.6; H, 4.9; N, 7.8.
Compound 3c: 3-(1-acetyl-5-(2-chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-
chromen-2-one, colorless crystals, yield, 62%; mp 184–186 °C; 1H NMR (CDCl3,
300 MHz): d 2.41 (3H, s, –Me), 3.23 (1H, dd, J = 19.0 and 4.2 Hz, pyrazole, 4-Ha),
3.98 (1H, dd, J = 12.2 and 19.0 Hz, pyrazole, 4-Hb), 5.83 (1H, dd, J = 12.0 and
4.9 Hz, pyrazole, 5-H), 6.95–7.55 (8H, m, C5–8-H and ArH), 8.34 (1H, s, C4-H);
13C NMR (CDCl3, 125 MHz): d 24.2, 39.3, 54.7, 122.1, 123.3, 124.5, 126.1, 127.0,
127.2, 128.8, 128.9, 129.1, 129.5, 133.5, 133.9, 144.7, 151.7, 157.8, 160.3,
169.0; ESI-MS: 367.9 (C20H15ClN2O3, [M+H]+); Anal. Calcd for C20H15ClN2O3: C,
65.5; H, 4.1; N, 7.6. Found: C, 65.2; H, 3.8; N, 7.5.
Compound 3d: 3-(1-acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-
2-one, colorless crystals, yield, 71%; mp 199–200 °C; 1H NMR (CDCl3,
300 MHz): d 2.36 (3H, s, –Me), 3.34 (1H, dd, J = 19.0 and 4.8 Hz, pyrazole,
4-Ha), 3.89 (1H, dd, J = 12.0 and 19.0 Hz, pyrazole, 4-Hb), 5.52 (1H, dd, J = 12.0
15. The cytotoxicity evaluation was conducted by using a modified procedure as
described in the literature.16 Briefly, target tumor cells were grown to log
phase in RPMI 1640 medium supplemented with 10% fetal bovine serum. After
diluting to 3 Â 104 cells mLÀ1 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 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-assayed as a
positive reference. After 48 h exposure period, 25
2.5 mg mLÀ1 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.
lL of PBS containing
l
The absorbance at 570 nm of each well was measured on an ELISA plate reader.
The data represented 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.
16. Chen, X. Y.; Plasencia, C.; Hou, Y.; Neamati, N. J. Med. Chem. 2005, 48, 1098.
17. Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L.;
Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Science 1994, 266, 2011.
18. Han, H.; Hurly, L. H.; Salazar, M. Nucleic Acid Res. 1999, 27, 537.
19. 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 C-DOCKER, a molecular dynamics (MD) simulated-annealing-
based algorithm module from DS 2.1. Random substrate conformations are
generated using high-temperature MD. Candidate poses are then created using
random rigid-body rotations followed by simulated annealing. The structure of
protein, substrate were subjected to energy minimization using CHARMm
forcefield as implemented in DS 2.1. A full potential final minimization was
then used to refine the substrate poses. Based on C-DOCKER, energy docked
conformation of the substrate was retrieved for postdocking analysis.