Design and synthesis of N-phenylacetyl (sulfonyl) 4,5-dihydropyrazole derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2011.03.066

A series of novel N-phenylacetyl (sulfonyl) 4,5-dihydropyrazole derivatives as potential telomerase inhibitors were synthesized. The bioassay tests show that compound 4a exhibited high activity against human gastric cancer cell SGC-7901, liver cancer Hep-G2 and human prostate PC-3 cell lines with IC ₅₀ values of 21.23 ± 0.99, 29.43 ± 0.32 and 30.89 ± 1.07 μM, respectively. All title compounds were assayed for telomerase inhibition by a modified TRAP assay, the results show that compound 4a can inhibit telomerase with IC₅₀ value of 4.0 ± 0.32 μM. Docking simulation was performed to position compound 4a into the telomerase (3DU6) active site to determine the probable binding model.

Full text of DOI:10.1016/j.bmcl.2011.03.066

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2916–2920
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of N-phenylacetyl (sulfonyl) 4,5-dihydropyrazole
derivatives as potential antitumor agents
Xin-Hua Liu a,b, , Ban-Feng Ruan a, , Jing-Xin Liu a, Bao-An Song c, Ling-Hong Jing c, Jun Li b, , Yang Yang a,
⇑
d
Hai-Liang Zhu a, , Xing-Bao Qi
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b School of Pharmacy, Anhui Medical University, Hefei, 230032, PR China
c Education Ministry Key Laboratory of Green Pesticide and Agriculture Bioengineering, 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 N-phenylacetyl (sulfonyl) 4,5-dihydropyrazole derivatives as potential telomerase inhib-
itors were synthesized. The bioassay tests show that compound 4a exhibited high activity against human
gastric cancer cell SGC-7901, liver cancer Hep-G2 and human prostate PC-3 cell lines with IC50 values of
Received 27 January 2011
Revised 16 March 2011
Accepted 17 March 2011
Available online 23 March 2011
21.23 0.99, 29.43 0.32 and 30.89 1.07
merase inhibition by a modified TRAP assay, the results show that compound 4a can inhibit telomerase
with IC50 value of 4.0 0.32 M. Docking simulation was performed to position compound 4a into the
telomerase (3DU6) active site to determine the probable binding model.
lM, respectively. All title compounds were assayed for telo-
l
Keywords:
Synthesis
Dihydropyrazole
Antitumor agent
Telomerase inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
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 cancer.3
Dihydropyrazole, a small bioactive molecule, is a prominent
structural motif found in numerous pharmaceutically active com-
pounds. Many 3,4-dihydropyrazole-based derivatives have shown
several biological activities as seen in cannabinoid receptor 1
antagonist, monoamine oxidase inhibitors, and tumor necrosis
inhibitors,4,5 Many of them are currently being tested and/or clin-
ically evaluated for new drug discovery. Of late, the dihydropyraz-
ole derivatives used as potent and selective inhibitors capable of
causing cancer cell death have also been reported.6–10
In an effort to synthesize novel 3,4-dihydropyrazole systems
with potential anticancer activity, our group has recently reported
on the anticancer activity of some acetyl 4,5-dihydropyrazole
derivatives,11 and a 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 projects into a hydrophobic region, which is comprised of the
side chains of 200–250, that is, important for the potent inhibitory
activity. In current molecular design, when acetyl replaced by
N-phenylacetyl, the 4,5-dihydropyrazole ring projects into a more
stable hydrophobic region, which comprised of the side chains of
220–227, should be enhance the anticancer activity. Since there
are only a very few systematic reports on the synthetic methodol-
ogy and evaluation of anticancer activities of these compounds,
herein, in continuation to extend our research on anticancer com-
pounds, we prepared a series novel N-phenylacetyl 4,5-dihydropy-
razole derivatives and tested their activities against human gastric
cancer cell SGC-7901, liver cancer Hep-G2 and human prostate
cancer PC-3 cell lines. In order to compare the activity of
4,5-dihydropyrazole moiety with carbonyl or sulfone, some sulfo-
nyl-4,5-dihydropyrazole derivatives were synthesized.
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, enzymelinked immunosorbentassay; TRAP, telomere repeat ampli-
fication protocol; DMAP, 4-dimethylaminopyridine.
⇑
Corresponding authors. Tel./fax: +86 0551 5161001 (J.L.); tel./fax: +86 25
83592672 (H.-L.Z.).
The synthesis of compound 2 (Scheme 1) started from substi-
tuted-salicylaldehyde and catalyzed by NaOH at 20 °C was added
E-mail addresses: lijun@ahmu.edu.cn (J. Li), zhuhl@nju.edu.cn (H.-L. Zhu).
These authors contributed equally to this paper.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.066

X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2916–2920
2917
O
O
H
(A)
CH3
(B)
R
R
CH3COCH3
OH
OH
2
1
H3C
HO
N
(C)
R
N
H3C
R1
O
HO
4
N
R
N
H
H3C
HO
(D)
N
R
3
N
S
O
O
R2
5
Scheme 1. Synthesis of title compounds. Reagent and conditions: (A) CH3COCH3, NaOH, C2H5OH, 20 °C, 15 h. (B) N2H4ÁH2O, 98% CH3CH2OH, reflux, 2 h. (C) carboxylic acid,
tosyl chloride, DMAP, reflux, 3 h. (D) substituted-benzenesulfonyl chloride, CHCl3, reflux. R = H, R1 = phenyl (4a); R = H, R1 = 4-methylphenyl (4b); R = H, R1 = 2-methylphenyl
(4c); R = H, R1 = 4-nitrophenylethyl (4d); R = H, R1 = chloromethyl (4e); R = H, R1 = 2-chlorophenyl) (4f); R = 3-methyl, R1 = phenyl (4g); R = H, R2 = phenyl (5h); R = H, R2 = p-
methylphenyl (5i); R = H, R2 = 4-nitrophenylethyl (5j).
Figure 1. ORTEP drawing of 4a.
Figure 2. ORTEP drawing of 4e.
acetone. Compound 3 was prepared according to a previously pub-
lished report.9 Using catalysts tosyl chloride and DMAP, proved to
be an efficient alternative for the synthesis of title compounds 4.
The general synthetic procedure process and spectral data of com-
pounds 4 and 5 can be found in the Supplementary data.12
In order to facilitate molecular docking, we trained a single-crys-
tal of compounds 4a, 4e and the single-crystal structure of com-
pounds 4a and 4e were determined by X-ray crystallography.
Compound 4a: Colorless crystals, yield, 70%; mp 202–203 °C; crystal
data: C17H16N2O2, M = 280.3, monoclinic, space group P2(1)/c;
a = 8.666(3), b = 16.043(5), c = 10.585(4) (Å);
a
= 90, b = 99.551,
c
= 90(°), V = 1451.2(8) nm3, T = 296(2) K, Z = 4, Dc = 1.283 g/cm3,
Table 1
Cytotoxic activity of the synthesized compounds against SGC-7901, Hep-G2 and PC-3 cell linesa
IC50
(l
M)b
Hep-G2
IC50
(
l
M)b
Hep-G2
Compound
SGC-7901
PC-3
30.89 1.07
115.53 0.85
106.32 3.06
132.15 1.74
20.10 1.27
15.69 1.92
Compound
SGC-7901
PC-3
4a
4b
4c
4d
21.23 0.99
51.48 0.68
103.77 1.49
97.26 0.85
26.83 0.79
31.46 4.38
29.43 0.32
78.87 0.92
95.17 1.29
107.28 2.51
47.92 3.60
18.85 0.85
4f
4g
5h
5i
103.02 2.16
149.81 2.96
66.40 2.84
73.64 0.67
52.82 1.30
31.46 4.38
95.55 2.86
177.40 1.87
57.24 2.05
66.77 1.76
83.04 1.66
18.85 0.85
84.21 2.35
154.29 2.07
50.60 2.43
85.47 2.03
38.77 0.83
15.69 1.92
4e
5j
5-Fluorouracilc
5-Fluorouracilc
a
b
c
The data represented the mean of three experiments in triplicate and were expressed as means SD; only descriptive statistics were done in the text.
The IC50 value was defined as the concentration at which 50% survival of cells was observed. The results are listed in the table.
Used as a positive control.

2918
X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2916–2920
Table 2
clinic, space group P2(1)/c; a = 9.646(3), b = 9.558(3), c = 13.364(4)
(Å);
a = 90, b = 90.536(4), c
= 90(°), V = 1232.1(7) nm3, T = 296(2) K,
Inhibitory effects of the title compounds against telomerase
Z = 4, Dc = 1.362 g/cm3, F(0 0 0) = 528. Reflections collected/unique:
Compound
IC50
polymerase
(
l
M) Taq
IC50
telomerase
(
l
M)
Selectivity
indexa (SI)
5246/2374, Fine, R1 = 0.0427, wR(F2) = 0.1045.
The molecular structures of compounds 4a and 4e are shown in
Figures 1 and 2. Crystallographic data (excluding structure factors)
for the structures have been deposited with the Cambridge
Crystallographic Data Center as Supplementary publication No.
CCDC-808895 and CCDC-808896.14
In the screening assay studies, compounds 4 and 5 were evalu-
ated for their cytotoxic activity against SGC-7901, Hep-G2 and PC-
3 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
4a exhibited high activity against the human gastric cell
4a
4b
4c
4d
4e
4f
4g
5h
8.0 2.0
no
50.5 1.80
no
20.4 2.0
36.0 2.2
no
31.0 2.0
30.0 1.0
23.5 0.29
4.0 0.32
31.5 1.1
24.5 0.87
no
9.5 0.46
no
2
—
2.1
—
2.1
—
no
—
17.3 0.21
16.8 0.38
12.5 0.25
2.6 0.7
1.8
1.9
1.9
5i
5j
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.
SGC-7901 with the IC50 value of 21.23 0.99
the positive control 5-fluorouracil. Also compound 4a can inhibit
telomerase with IC50 value of 4.0 0.32 M.
lM, comparable to
a
b
l
From the data presented in Table 1, it can be concluded that
phenylsulfonyl-4,5-dihydropyrazole derivatives showed moderate
inhibition against SGC-7901, Hep-G2 and PC-3 cell lines, 2-hydro-
xy-substitutedphenyl-4,5-dihydropyrazole (4g) displayed poor
activity against above cell lines, also poor activity against telome-
rase, whereas 2-hydroxy-phenyl-4,5-dihydropyrazole derivatives,
F(0 0 0) = 592. Reflections collected/unique: 6878/2818, Fine,
R1 = 0.0450, wR(F2) = 0.1070. Compound 4e: Colorless crystals, yield,
91%; mp 174–175 °C; crystal data: C12H13ClN2O2, M = 252.7, mono-
A
,
5
2
2
s
A
l
y
H
L
a
,
i
N
4
s
3
2
OH
3
2
7
2
2
3
2
r
,
9
2
N
O
y
A
,
r
T
y
T
e
s
,
p
S
0
r
,
3
2
6
3
4
2
5
2
2
2
u
5
,
e
r
I
L
l
e
h
T
3
3
8
2.54 A
,
H
N
Ary 340
B
Figure 3. Molecular docking modeling of compound 4a (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 4a in the ATP binding site of 3DU6.

X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2916–2920
2919
5. Dmytro, H.; Borys, Z.; Olexandr, V.; Lucjusz, Z.; Andrzej, G.; Roman, L. Eur. J.
Med. Chem. 2009, 44, 1396.
6. Manna, F.; Chimenti, F.; Fioravanti, R.; Bolasco, A.; Secci, D.; Chimenti, P.;
Ferlinib, C.; Scambia, G. Bioorg. Med. Chem. Lett. 2005, 15, 4632.
7. Havrylyuk, D.; Zimenkovsky, B.; Vasylenko, O.; Zaprutko, L.; Gzella, A.; Lesyk, R.
Eur. J. Med. Chem. 2009, 44, 1396.
8. Insuasty, B.; Tigreros, A.; Orozco, F.; Quiroga, J.; Abonía, R.; Nogueras, M.;
Sanchez, A.; Cobo, J. Bioorg. Med. Chem. 2010, 18, 4965.
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45, 114.
11. Liu, X. H.; Liu, H. F.; Song, B. A.; Zhu, H. L.; Bai, L. S.; Pan, C. X.; Liu, J. X.; Yang, Y.;
Qi, X. B. Bioorg. Med. Chem. Lett. 2010, 20, 5705.
12. General synthetic procedure process for compounds 4: To a chloroform (20 ml)
solution of carboxylic acid (12 mmol), tosyl chloride (12 mmol) and DMAP
in general, showed relatively higher activity against the above
cells, so, some compounds in this series deserve further
investigation.
All purified title compounds were assayed for telomerase inhi-
bition by
a modified telomere repeat amplification protocol
(TRAP)17 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 Telomerase activity was detected by a modified
version of the TRAP protocol. Telomerase products were resolved
by 10% polyacrylamide gel electrophoresis and visualized by stain-
ing with SYBER Green. As a source of telomerase, the total cell ly-
sates derived from SGC-7901 cell line was used. Protein
concentration of the lysates was assayed using Bio-Rad protein as-
say kit using BSA standards.
(15 mmol) was added 2-(3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenol
3
(10 mmol), then the reaction mixture was refluxed for 3 h. The mixture was
cooled, washed with water, and allowed to stand at 0 °C over night. The
product was collected by filtration and the crude residue was purified by
chromatography on SiO2 (acetone/petroleum, v:v = 3:1) to give title
compounds 4 (Scheme 1) as colorless solids.13
To avoid false positive results due to drug interference with the
amplification step, Taq polymerase inhibition was additionally
monitored. The results are summarized in Table 2, where a selec-
tivity index (SI, ratio between IC50 for Taq polymerase vs telome-
rase inhibition) is also included. The results suggested that the
compound 4a showed strong telomerase inhibitory ability with
Compound
4a.
(5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)
(phenyl)methanone: colorless crystals, yield, 60%; mp 202–204 °C; 1H NMR
(CDCl3, 300 MHz): d 2.20 (3H, s, –Me), 3.09 (1H, dd, J = 18.6 and 3.9 Hz,
pyrazole, 4-Ha), 3.40 (1H, dd, J = 11.4 and 18.6 Hz, pyrazole, 4-Hb), 5.91 (1H, dd,
J = 11.4 and 3.9 Hz, pyrazole, 5-H), 6.89–7.90 (9H, m, ArH), 9.31 (1H, br s, –OH);
13C NMR (CDCl3, 125 MHz): d 16.7, 44.1, 55.0, 118.1, 122.4, 127.2, 129.1, 129.6,
130.7, 132.2, 132.9, 135.1, 144.2, 150.9, 156.8; ESI-MS: 280.9 (C17H16N2O2,
[M+H]+); Anal. Calcd for C17H16N2O2: C, 72.84; H, 5.75; N, 9.99. Found: C, 73.08;
H, 6.00; N, 9.62.
IC50 value of 4.0 0.32
bromide.
lM, which comparable to the ethidium
In an effort to elucidate the mechanism by which the title com-
pound can induce anticancer activity in the human gastric cell
SGC-7901, molecular docking of the potent inhibitors 4a into ATP
binding site of telomerase was performed to simulate a binding
model derived from telomerase structure (PDB: 3DU6).19 See Fig-
ure 3. Visual inspection of the pose of 4a into the ATP-site revealed
that optimal intramolecular hydrogen bond is observed (N–HÁ Á ÁO:
2.54 Å, with amino hydrogen group of Ary 340). Also the 4,5-
dihydropyrazole ring projects into a hydrophobic region, which is
comprised of the side chains of Ala 220, Tyr 224, Lys 225, Thr
226, Ser 227, that is, important for the potent inhibitory activity
of 4a. These residues influenced the accessibility of the hydropho-
bic 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 2-hydroxyphenyl interacted with
the residue His 339, which made the 3D structure more stable.
In summary, we prepared a series of novel N-phenylacetyl 4,5-
dihydropyrazole derivatives as potential telomerase inhibitors. The
result showed that compound 4a had high activity against SGC-
7901, Hep-G2 and PC-3 cell lines. Compounds 5h–5j showed mod-
erate inhibition against above cell lines. Docking simulation result
shows compound 4a can bind well with the telomerase active site
and act as potential telomerase inhibitor.
Compound 4b. (5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl) (p-
tolyl)methanone: colorless crystals, yield, 54%; mp 200–201 °C; 1H NMR
(CDCl3, 300 MHz):
d 2.18 (3H, s, –Me), 2.39 (3H, s, –Me), 3.12 (1H, dd,
J = 18.6 and 3.9 Hz, pyrazole, 4-Ha), 3.45 (1H, dd, J = 11.4 and 18.6 Hz, pyrazole,
4-Hb), 5.86 (1H, dd, J = 11.4 and 3.9 Hz, pyrazole, 5-H), 6.82–7.96 (8H, m, ArH),
9.27 (1H, br s, –OH); 13C NMR (CDCl3, 125 MHz): d 16.9, 25.5, 43.8, 54.7, 117.7,
122.8, 128.4, 129.7, 130.0, 131.8, 132.4, 133.9, 141.5, 143.9, 152.7, 156.0; ESI-
MS: 293.8 (C18H18N2O2, [M+H]+); Anal. Calcd for C18H18N2O2: C, 73.45; H, 6.16;
N, 9.52. Found: C, 73.17; H, 6.32; N, 9.88.
Compound 4c. (5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl) (o-
tolyl)methanone: colorless crystals, yield, 56%; mp 198–199 °C; 1H NMR
(CDCl3, 300 MHz):
d 2.10 (3H, s, –Me), 2.42 (3H, s, –Me), 3.06 (1H, dd,
J = 18.5 and 3.9 Hz, pyrazole, 4-Ha), 3.40 (1H, dd, J = 11.4 and 18.5 Hz, pyrazole,
4-Hb), 5.95 (1H, dd, J = 11.4 and 3.9 Hz, pyrazole, 5-H), 6.89–8.04 (8H, m, ArH),
9.15 (1H, br s, –OH); 13C NMR (CDCl3, 125 MHz): d 16.2, 18.9, 44.2, 55.1, 117.5,
121.9, 126.3, 127.2, 129.0, 129.5, 130.5, 131.6, 132.1, 136.7, 137.0, 144.1, 152.4,
157.8; ESI-MS: 294.5 (C18H18N2O2, [M+H]+); Anal. Calcd for C18H18N2O2: C,
73.45; H, 6.16; N, 9.52. Found: C, 73.61; H, 6.04; N, 9.27.
Compound 4d. 1-(5-(2-Hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-2-(4-
nitrophenyl)ethanone: colorless crystals, yield, 72%; mp 210–212 °C; 1H NMR
(CDCl3, 300 MHz): d 2.17 (3H, s, –Me), 3.05 (1H, dd, J = 18.6 and 3.9 Hz,
pyrazole, 4-Ha), 3.27 (1H, dd, J = 11.4 and 18.6 Hz, pyrazole, 4-Hb), 3.51 (2H, s, –
CH2–), 5.84 (1H, dd, J = 11.4 and 3.9 Hz, pyrazole, 5-H), 6.85–8.18 (8H, m, ArH),
9.26 (1H, br s, –OH); 13C NMR (CDCl3, 125 MHz): d 16.2, 40.8, 43.7, 54.5, 117.1,
122.4, 123.0, 129.1, 129.6, 130.2, 130.8, 143.1, 148.6, 149.1, 154.0, 158.9; ESI-
MS: 340.2 (C18H17N3O4, [M+H]+); Anal. Calcd for C18H17N3O4: C, 63.71; H, 5.05;
N, 12.38. Found: C, 63.45; H, 4.91; N, 12.57.
Compound 4e. 2-Chloro-1-(5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-
yl)ethanone: colorless crystals, yield, 91%; mp 174–175 °C; 1H NMR (CDCl3,
300 MHz): d 2.19 (3H, s, –Me), 3.05 (1H, dd, J = 18.6 and 3.6 Hz, pyrazole, 4-Ha),
3.39 (1H, dd, J = 11.1 and 18.6 Hz, pyrazole, 4-Hb), 4.40 (2H, s, –CH2–), 5.71 (1H,
dd, J = 11.1 and 3.6 Hz, pyrazole, 5-H), 6.86–7.26 (4H, m, ArH), 8.53 (1H, br s, –
OH); 13C NMR (CDCl3, 125 MHz): d 16.8, 43.2, 44.5, 54.3, 116.8, 122.3, 128.6,
129.0, 130.5, 144.0, 152.0, 158.9; ESI-MS: 253.3 (C12H13ClN2O2, [M+H]+); Anal.
Calcd for C12H13ClN2O2: C, 57.04; H, 5.19; N, 11.09. Found: C, 57.33; H, 5.42; N,
11.34.
Supplementary data: CCDC-808895 and CCDC-808896 contain
the Supplementary crystallographic data for this paper. These data
can be obtained free of charge via the URL http://www.ccdc.cam.a-
c.uk/conts/retrieving.html (or from the CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk).
Compound
4f.
(2-Chlorophenyl)
(5-(2-hydroxyphenyl)-3-methyl-4,5-
dihydropyrazol-1-yl)methanone: colorless crystals, yield, 62%; mp 207–209 °C;
1H NMR (CDCl3, 300 MHz): d 2.10 (3H, s, –Me), 3.08 (1H, dd, J = 18.6 and 3.3 Hz,
pyrazole, 4-Ha), 3.45 (1H, dd, J = 11.1 and 18.6 Hz, pyrazole, 4-Hb), 5.90 (1H, dd,
J = 11.1 and 3.3 Hz, pyrazole, 5-H), 6.74–7.40 (8H, m, ArH), 8.97 (1H, br s, –OH);
13C NMR (CDCl3, 125 MHz): d 16.2, 44.2, 54.0, 117.0, 121.2, 126.0, 126.6, 128.7,
129.6, 129.9, 130.7, 132.3, 132.4, 133.4, 155.0, 160.3, 165.9; ESI-MS: 316.1
(C17H15ClN2O2, [M+H]+); Anal. Calcd for C17H15ClN2O2: C, 64.87; H, 4.80; N,
8.90. Found: C, 65.06; H, 4.92; N, 9.11.
Acknowledgment
The authors thank the National Natural Science Foundation of
China (No. 20902003).
Compound 4g. (5-(2-Hydroxy-3-methylphenyl)-3-methyl-4,5-dihydropyrazol-1-
yl) (phenyl)methanone: colorless crystals, yield, 54%; mp 195–196 °C; 1H
NMR (CDCl3, 300 MHz): d 2.09 (3H, s, –Me), 2.32 (3H, s, –Me), 3.17 (1H, dd,
J = 18.6 and 3.6 Hz, pyrazole, 4-Ha), 3.37 (1H, dd, J = 11.4 and 18.6 Hz, pyrazole,
4-Hb), 5.95 (1H, dd, J = 11.4 and 3.6 Hz, pyrazole, 5-H), 6.77–7.99 (8H, m, ArH),
9.04 (1H, br s, –OH); 13C NMR (CDCl3, 125 MHz): d 14.9, 16.2, 43.3, 55.1, 121.7,
125.8, 126.3, 127.8, 128.9, 129.3, 130.7, 132.9, 134.5, 143.6, 152.2, 156.8; ESI-
MS: 293.2 (C18H18N2O2, [M+H]+); Anal. Calcd for C18H18N2O2: C, 73.45; H, 6.16;
N, 9.52. Found: C, 73.66; H, 6.08; N, 9.35.
References and notes
1. Wright, W. E.; Shay, J. W. Curr. Opin. Genet. Dev. 2001, 11, 98.
2. Bodnar, A. G.; Ouellette, M.; Frolkis, M.; Holt, S. E.; Chiu, C. P.; Morin, G. B.;
Harley, C. B.; Shay, J. W.; Lichtsteiner, S.; Wright, W. E. Science 1998, 279, 349.
3. Andrew, J. G.; Anthony, P. S.; Emmanuel, S. Nature 2008, 455, 633.
4. Need, A. B.; Davis, R. J.; Alexander-Chacko, J. T.; Eastwood, B.; Chernet, E.;
Phebus, L. A.; Sindelar, D. K.; Nomikos, G. G. Psychopharmacology 2006, 184, 26.
General synthetic procedure process for compounds 5: To a chloroform (20 ml)

2920
X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2916–2920
(296 K) using graphite monochromated MoK
solution of 2-(3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenol 3 (10 mmol) was
a
radiation (k = 0.71073 Å) and an
added tosyl chloride (10 mmol) and DMAP (12 mmol), the reaction mixture
was refluxed for 1 h. The mixture was cooled, washed with water, and allowed
to stand at 0–5 °C 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 5 (Scheme 1) as colorless solids.13
Enraf-Nonius CAD-4 four-circle diffractometer. The systematic absences and
intensity symmetries indicated the Monoclinic Cc space group. The corrections
for LP factors were applied. The structure was solved by direct methods and
refined by full-matrix least-squares techniques on F2 with anisotropic thermal
parameters for all non-hydrogen atoms. The calculations were performed with
SHELXL-97 program.
Compound
5h.
2-(3-Methyl-1-(phenylsulfonyl)-4,5-dihydro-1H-pyrazol-5-
yl)phenol: colorless crystals, yield, 78%; mp 218–219 °C; 1H NMR (CDCl3,
300 MHz): d 2.02 (3H, s, –Me), 2.89 (1H, dd, J = 4 and 12 Hz, pyrazole, 4-Ha),
3.08 (1H, dd, J = 12 and 18 Hz, pyrazole, 4-Hb), 4.92 (1H, dd, J = 4 and 12 Hz,
pyrazole, 5-H), 5.94 (1H, s, –OH), 6.82–7.81 (9H, m, ArH); 13C NMR (CDCl3,
125 MHz): d 16.4, 45.4, 60.5, 117.1, 122.0, 126.8, 128.0, 129.8, 130.5, 131.7,
138.9, 144.0, 152.9, 161.4; ESI-MS: 317.1 (C16H16N2O3S, [M+H]+); Anal. Calcd
for C16H16N2O3S: C, 60.74; H, 5.10; N, 8.85. Found: C, 61.03; H, 5.29; N, 9.04.
Compound 5i. 2-(3-Methyl-1-tosyl-4,5-dihydro-1H-pyrazol-5-yl)phenol: colorless
crystals, yield, 82%; mp 221–222 °C; 1H NMR (CDCl3, 300 MHz): d 2.00 (3H, s, –
Me), 2.82 (1H, dd, J = 18 and 3.6 Hz, pyrazole, 4-Ha), 2.44 (3H, s, –Me), 3.03 (1H,
dd, J = 12 and 18 Hz, pyrazole, 4-Hb), 4.89 (1H, dd, J = 12 and 3.6 Hz, pyrazole,
5-H), 5.99 (1H, s, –OH), 6.85–7.78 (8H, m, ArH); 13C NMR (CDCl3, 125 MHz): d
16.2, 21.8, 45.9, 61.2, 117.7, 121.1, 126.1, 128.3, 129.4, 129.6, 131.6, 138.1,
144.7, 153.6, 161.0; ESI-MS: 331.0 (C17H18N2O3S, [M+H]+); Anal. Calcd for
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 six 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.
C17H18N2O3S: C, 61.80; H, 5.49; N, 8.48. Found: C, 61.51; H, 5.68; N, 8.60.
Compound 5j. 2-(3-Methyl-1-(4-nitrophenylsulfonyl)-4,5-dihydro-1H-pyrazol-5-
yl)phenol: colorless crystals, yield, 87%; mp 232–233 °C; 1H NMR (CDCl3,
300 MHz): d 2.02 (3H, s, –Me), 2.81 (1H, dd, J = 3.6 and 12 Hz, pyrazole, 4-Ha),
3.16 (1H, dd, J = 10.5 and 17.7 Hz, pyrazole, 4-Hb), 4.89 (1H, dd, J = 3.6 and
10.5 Hz, pyrazole, 5-H), 6.74–8.36 (9H, m, ArH); 13C NMR (CDCl3, 125 MHz): d
16.7, 44.9, 60.7, 116.5, 121.0, 121.6, 128.7, 128.9, 129.2, 131.3, 145.2, 148.7,
152.5, 156.7; ESI-MS: 362.0 (C16H15N3O5S, [M+H]+); Anal. Calcd for
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 4 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.
C16H15N3O5S: C, 53.18; H, 4.18; N, 11.63. Found: C, 53.42; H, 4.01; N, 12.00.
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). Elemental analysis was performed by a Vario-III CHN analyzer and
was within 0.4% of the theoretical values.
14. Crystal structure determination
A sample of size 0.26 Â 0.23 Â 0.15 mm3 was selected for the crystallographic
study. The diffraction measurement was performed at room temperature