Novel coumarin-dihydropyrazole thio-ethanone derivatives: Design, synthesis and anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2013.06.014

A series novel 1-(3-substituted-5-phenyl-4,5-dihydropyrazol-1-yl)-2-thio- ethanone derivatives as potential telomerase inhibitors were designed and synthesized. The bioassays demonstrated that compounds 4a, 4f, 4j and 7b, 7d occupied high antiproliferative activity against SGC-7901, MGC-803, Bcap-37 and HEPG-2 cell lines. By a modified TRAP assay, some title compounds were tested against telomerase, and compound 4f showed the most potent inhibitory activity with IC₅₀ value at 0.92 ± 0.09 μM. The mechanism of antitumor action indicated that title compounds 4f and 7b could suppress cell proliferation through inducing cell cycle arrest in G0/G1 phase.

Full text of DOI:10.1016/j.ejmech.2013.06.014

Accepted Manuscript
Novel coumarin-dihydropyrazole thio-ethanone derivatives: design、synthesis and
anticancer activity
Xiao-Qin Wu, Cheng Huang, Ying-Ming Jia, Bao-An Song, Jun Li, Xin-Hua Liu
PII:
S0223-5234(13)00382-6
10.1016/j.ejmech.2013.06.014
EJMECH 6242
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 February 2013
Revised Date: 3 June 2013
Accepted Date: 10 June 2013
Please cite this article as: X.-Q. Wu, C. Huang, Y.-M. Jia, B.-A. Song, J. Li, X.-H. Liu, Novel coumarin-
dihydropyrazole thio-ethanone derivatives: design、synthesis and anticancer activity, European Journal
of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.06.014.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Novel 4,5-dihydropyrazol-1-yl-2-thio-ethanones as potential telomerase inhibitors
were synthesized. The mechanism of the antitumor action indicated that title
compounds could inhibit the growth of MGC-803 cells via the induction of tumor cell
apoptosis.

ACCEPTED MANUSCRIPT
Novel coumarin-dihydropyrazole thio-ethanone derivatives: designꢀsynthesis and
anticancer activity
Xiao-Qin Wu a, Cheng Huang a, Ying-Ming Jia a, Bao-An Song b, Jun Li *a,
Xin-Hua Liu* a,c
a School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China
b Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of
Education, Guizhou University, Guiyang 550025 , P. R. China
c State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, P. R. China
Abstract:
A series novel 1-(3-substituted-5-phenyl-4,5-dihydropyrazol-1-yl)-2-thio-
ethanone derivatives as potential telomerase inhibitors were designed and synthesized. The
bioassays demonstrated that compounds 4a, 4f, 4j and 7b, 7d occupied high
antiproliferative activity against SGC-7901, MGC-803, Bcap-37 and HEPG-2 cell lines.
By a modified TRAP assay, some title compounds were tested against telomerase, and
compound 4f showed the most potent inhibitory activity with IC50 value at 0.92±0.09 µM.
The mechanism of antitumor action indicated that title compounds 4f and 7b could
suppress cell proliferation through inducing cell cycle arrest in G0/G1 phase.
Keywords: Coumarin; Dihydropyrazole; Thio-ethanone; Anticancer activity
_________________
*Corresponding author.
Jun Li Tel.: +86 0551-5161001; fax: +86 0551-5161001. E-mail: lijun@ahmu.edu.cn.
Xin-Hua Liu Tel.: +86 551 5161115; fax: +86 551 5161115. E-mail: xhliuhx@163.com.
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1. Introduction
Today, about 80% to 90% of cancer cells from a large range of different cancer types have
detectable telomerase activity. Telomerase represents one of the promising targets in drug
discovery [1-4]. Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme
that is required for telomerase activity (TA) and cancer progression [5]. There are two
general strategies of telomerase targeting in cancer treatment. One is targeting telomerase
directly by inhibiting the activity of its catalytic subunit (hTERT) or its RNA template
(hTER), leading to inhibition of telomerase activity. Another strategy is targeting the
telomerase subunit indirectly with G-quadruplex stabilizers, tankyrase or HSP90 inhibitors
[6-7]. Currently, except for BIBR1532 which is the promising hTERT inhibitor [8-10],
there have been only a few successful hTERT inhibitors developed.
Many 4,5-dihydropyrazole-based derivatives showed several biological activity [11-17]. In
our recent publication [18], several dihydropyrazole derivatives were described, which had
potent anticancer activity as potential telomerase inhibitors. Needs to be pointed out, when
we introduced the coumarin moiety in the dihydropyrazole skeleton, compounds with high
antitumor activity were obtained [19]. Importantly, we have identified unoccupied space
above the plane of the coumarin ring lined with region of high electron density due to the
amide backbone of the hTERT (3DU6). Fluorinated compounds in general are widely used
in modern medicinal chemistry, trifluoromethyl-substituted compounds have been reported
to possess activity as fungicides, analgesic agents, antipyretic agents [20-21]. Therefore,
the structure moiety of dihydropyrazole-coumarin- trifluoromethyl substituted group was
first considered in this study.
Our group recently disclosed the first X-ray crystal structure of a small molecule inhibitor
bound to active site of hTERT (3DU6). Since structural information of the binding site was
available to us, we initiated an effort to leverage molecular modeling in combination with
it to provide guidance for future target selection [22]. To this end, computer-generated
molecular models of dihydropyrazole-coumarin thio-ethanone moiety docked into the
allosteric site of hTERT (3DU6) were analyzed. Is that what we expected, when
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trifluoromethyl was introduced, which would formed a halogen bond, the phenyl
dihydropyrazole ring projected into a more stable hydrophobic region, to achieve a better
binding. Our analysis then focus on hydrophilic interactions near the active site, when the
key location of the S group (thio-ethanone) exposed to the binding site, the hydrophobic
aromatic ring could be well combined with the hydrophobic cavity, so, the compounds
with phenyl substituted thio-ethanone were considered for design.
Furthermore, structure-activity relationship of my lab showed that amination transformed
could enhance antitumor activity [23]. Because -S- and –NH2- is a classic isostere, and S
atom in the ether bond has loneelectronic energy to attract protons, having a hydrophilic,
also the next C atom has lipophilic, this kind of moiety (-C-S-C-) could directionally
arrange in the fat-water interface, and this structure is in favor of molecule transiting the
cell membrane, so we at last focus on the molecule design of coumarin-dihydropyrazole
thio-ethanone.
Based on all above, as a continuation of our research for finding novel, efficient lead
compound with anticancer activity, we designed and synthesized a series of
dihydropyrazole-coumarin- trifluoromethylphenyl thio-ethanones. In order to understand
whether the reduced cell proliferation was due to cell cycle arrest, we used flow cytometric
analyses to measure the effect of titled compounds on induction of cell cycle.
2. Results and discussion
2.1 Chemistry
The synthesis of compounds 4 (Schemes 1, 2) started from salicylaldehyde and catalyzed
o
by piperazine at 20~30 C was added ethyl acetoacetate. Claisen-Schmidt condensation
3-acetyl-2H-chromen-2-one and benzaldehyde using mild catalyst piperazine, the butanol
used as solvent, proved to be an efficient method for the synthesis of α, β unsaturated
ketone 2. Using chloride, proved to be an efficient alternative method for the synthesis of
title compounds 3. In addition, in order to facilitate the synthesis of title compounds 4,
catalytic amount of KI was added. Compounds 1~3, 5~6 were prepared according to a
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previously published report [18]. The structure of compounds 4a, 4b, 4c and 7a were
determined by X-ray crystallography. Their molecular structures were shown in Figure 1.
(Figure 1)
2.2 In vitro anticancer activity
In the screening assay studies, all title compounds were evaluated for their cytotoxic
activity against SGC-7901, MGC-803, Bcap-37 and HEPG-2 cell lines. Also included was
the activity 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 4a, 4f, 4j and 7b, 7d exhibited high
activity against SGC-7901 and MGC-803 with IC50 values of 4.42±0.70, 2.91±0.11,
4.39±0.80, 2.30±0.35, 3.30±0.45 and 3.14±0.35, 2.35±0.27, 2.89±0.09, 2.97±0.08,
2.27±0.31 µM respectively, surpassing that of the positive control 5-Fluorouracil.
Compounds 4f, 4j, 7b, 7d showed anticancer activity against Bcap-37 with IC50 values of
3.55±1.00, 3.77±0.21, 3.24±0.29, 3.70±0.15 µM respectively, surpassing that of the
positive control 5-Fluorouracil. Compounds 7b, 7d showed anticancer activity against
HEPG-2 with IC50 value of 4.00±0.31, 2.95±0.40 µM, surpassing that of positive control
5-Fluorouracil. From the data presented in Table 1, it can be concluded that among all the
synthesized compounds, two classifications can be made, one containing straight chain
substituent on the S moiety which include compounds 4a, 4f and 7b, 7d, and the other
containing ring substitute on the S moiety (see Scheme 1, 2). We can see that compounds
4a, 4f and 7b, 7d showed obvious anticancer activity against the gastric SGC-7901,
MGC-803, Bcap-37 and HEPG-2 cell lines, while most of the other compounds containing
ring substitute on the S moiety only show moderate anticancer activity against above cell
lines.
(Table 1)
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2.3 Flow cytometry (FCM) analysis
Proliferation of MGC-803 cells was evaluated against selected compounds 4f and 7b (with
high activity against MGC-803 cell). The results of MTT assay showed that two
compounds could inhibit proliferation of MGC-803 cell. In order to understand whether
the reduced cell proliferation was due to cell cycle arrest, we used flow cytometric
analyses to measure the effect of compound 4f on induction of cell cycle. As shown in
Figure 2, the results of assays indicated that compound 4f could inhibit its proliferation.
Also the fraction of MGC-803 cells staying in the G0/G1 phase was distinctly increased,
whereas that in the S and G2/M phase decreased by stimulated of compounds 4f and 7b.
So this result showed that some title compounds could inhibit proliferation of MGC-803, at
least in part, via influencing cell cycle arrest.
(Figure 2)
2.4 Telomerase activity
Some purified title compounds were assayed for telomerase inhibition, using a MGC-803
cell extract, also included the activity of reference compounds Ethidium bromide and
BIBR1532. The results (Table 2) suggested that compounds 4f and 7b showed strong
telomerase inhibitory activity with IC50 values of 0.92±0.09, 1.04±0.10 µM respectively,
which surpassing that of the positive control ethidium bromide, comparable to that of
positive control BIBR1532, furthermore, there was a good correlation between
antiproliferative activity and telomerase activity of compounds 4f and 7b (Tables 1 and 2),
indicating that inhibition of the telomerase by the synthesized derivatives caused inhibition
of cancer cell growth. Among them, trifluoromethylphenyl- coumarin-dihydropyrazole
derivative 4f showed higher inhibitory activity than the other. Structure containing a
trifluoromethyl moiety may be matched with the dipole moment of binding sites, so that
the dipole moment is parallel and in the opposite direction, resulting in higher activity. This
also pointed out the direction for us to further optimize the structure of dihydropyrazoles
with anticancer activity as potential telomerase inhibitors.
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(Table 2)
2.5 Taq polymerase activity
By a modified TRAP assay, telomerase activity was measured first in vitro screening
potential telomerase inhibitors, and several compounds showed high inhibitory activity
(Table 2). In order to avoid false-positive, several compounds with higher inhibitory
activity against telomerase were selected to assay the inhibitory activity against Taq
polymerase. The bioassays demonstrated that compounds 4a, 4e, 4f, 4h, 7b and 7d
occupied no obvious effect on Taq polymerase (Figure 3). Therefore, it can initially
determine that the telomerase is the primary target of our tested compounds.
(Figure 3)
2.6 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 compound 4f into binding site of
telomerase was performed to simulate a binding model derived from telomerase TERT
(3DU6. pdb). The binding model of compound 4f with telomerase TERT was depicted in
Figure 4. Visual inspection of the pose of 4f into the ATP-site revealed that optimal
intramolecular hydrogen bond was observed (N–H…..O, with amino hydrogen group of Val
342). Also the dihydropyrazole ring projects into a hydrophobic region, which is
comprised of the side chains of Tyr 217, Leu 220, Thr 226, Ser 227, that is important for
the potent inhibitory activity of compound 4f. 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,
hydrophobic - hydrophobic interaction was formed by straight chain thioether moiety,
which made the 3D structure more stable and may be mainly due to higher activity of
compounds 4f, 7b. Furthermore, the introduction of fluorine atom affects the distribution
of the electrons in the molecule, strengthened dipole - dipole interaction, which should
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improve the activity of title compounds.
(Figure 4)
3. Conclusions
In summary, we designed and synthesized some novel 1-(3-substituted-5-phenyl-4,5-
dihydropyrazol-1-yl)-2-thio-ethanones as potential telomerase inhibitors, followed by
chemical synthesis and biological evaluated for their antiproliferative activity against
several cell lines including MGC-803, Bcap-37, SGC-7901 and HEPG-2. Among them,
compound 4f occupied high antiproliferative activity against above cell lines and showed
the most potent telomerase inhibitory activity with IC50 value at 0.92±0.09 µM. In addition,
the mechanism of antitumor action indicated that some title compounds could suppress cell
proliferation through inducing cell cycle arrest in G0/G1 phase. In brief, based on
reasonable molecular design, compared with the results of our previous studies, the
advantages of this study was that we found more efficient telomerase inhibitors. These
results are of help in the rational design of more efficient telomerase inhibitors in further.
4. Experimental section
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
1
(Taike Corp., Beijing, China). H 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 (ꢁ). Elemental analyses were performed on a CHN–O-Rapid
instrument, and were within ±0.4% of the theoretical values.
4.2 General procedure for the synthesis of title compound 4
To a chloroform (20 ml) solution of 3-(1-(2-bromoacetyl)-5-(2 or 4-trifluoromethyl)
phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one 3 (10 mmol) was added
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substituted-thiol (10 mmol), KHCO3 (8 mmol) and catalytic KI, the reaction mixture was
allowed to stand at 40~50 ˚C for 5 h. The mixture was cooled, washed with water. The
product was collected by filtration and the crude residue was purified by chromatography
on SiO2 (dichloromethane / methanol, v:v= 68:1) to give title compounds 4a~4l (Scheme 1)
as colorless solids.
(Scheme 1)
4a: (R)-3-(1-(2-(butylthio)acetyl)-5-(2-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-
yl)-2H-chromen-2-one, colorless crystals, yield, 70%; mp 171-172 oC; 1H NMR (400 MHz,
CDCl3) δ (ppm): 0.85 (t, 3H, J=8.0 Hz), 1.33 (m, 2H), 1.53 (m, 2H), 2.60 (t, 2H, J=4.0 Hz),
3.17 (dd, 1H, J1 = 20.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 3.71 (m, 2H), 3.99 (dd, 1H, J1 =
12.0 Hz, J2 = 20.0 Hz, pyrazole 4-Hb), 5.78 (dd, 1H, J1 = 12.0 Hz, J2 = 4.0 Hz, pyrazole
5-H), 7.27-7.87 (m, 8H, ArH), 8.61 (s, 1H, C4-H). Anal. calcd for C25H23F3N2O3S: C,
61.46; H, 4.75; N, 5.73%. Found: C, 61.27; H, 5.03; N, 6.01%.
4b: (R)-3-(1-(2-(furan-2-ylmethylthio)acetyl)-5-(2-(trifluoromethyl)phenyl)-4,5-dihydro-
o
1
1H-pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 65%; mp 141-142 C; H
NMR (400 MHz, CDCl3) δ (ppm): 3.21 (dd, 1H, J1 = 4.0 Hz, J2 = 20.0 Hz, pyrazole 4-Ha),
3.70-3.90 (m, 4H), 4.03 (dd, 1H, J1 = 12.0 Hz, J2 = 20.0 Hz, pyrazole 4-Hb), 5.83 (dd, 1H,
J1 = 12.0 Hz, J2 = 4.0 Hz, pyrazole 5-H), 6.34-6.45 (m, 2H), 7.32-7.90 (m, 9H), 8.64 (s, 1H,
C4-H). Anal. calcd for C26H19F3N2O4S: C, 60.93; H, 3.74; N, 5.47%. Found: C, 60.69; H,
3.49; N, 5.70%.
4c: (R)-3-(1-(2-(4-bromophenylthio)acetyl)-5-(2-(trifluoromethyl)phenyl)-4,5-dihydro-1H-
o
pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 72%; mp 143-145 C; 1H NMR
(400 MHz, CDCl3) δ (ppm): 3.15 (dd, 1H, J1 = 18.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 3.98
(dd, 1H, J1 = 20.0 Hz, J2 = 12.0 Hz, pyrazole 4-Hb), 4.16-4.49 (m, 2H), 5.76 (dd, 1H, J1 =
12.0 Hz, J2 = 4.0 Hz, pyrazole 5-H), 7.13 (d, 1H, J = 8.0 Hz), 7.38-7.76 (m, 10H), 7.86 (d,
1H, J = 8.0 Hz), 8.61 (s, 1H, C4-H). Anal. calcd for C27H18BrF3N2O3S: C, 55.21; H, 3.09;
N, 4.77%. Found: C, 55.01; H, 3.34; N, 5.05%.
4d: (R)-3-(1-(2-(4-nitrophenylthio)acetyl)-5-(2-(trifluoromethyl)phenyl)-4,5-dihydro-1H-
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o
pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 62%; mp 150-151 C; 1H NMR
(400 MHz, CDCl3) δ (ppm): 3.21 (dd, 1H, J1 = 18.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 4.02
(dd, 1H, J1 = 18.0 Hz, J2 = 12.0 Hz, pyrazole 4-Hb), 4.05-4.40 (m, 2H), 5.66 (dd, 1H, J1 =
12.0 Hz, J2 = 4.0 Hz, pyrazole 5-H), 7.19-7.81 (m, 11H), 8.04 (d, 1H, J = 8.0 Hz), 8.65 (s,
1H, C4-H). Anal. calcd for C27H18F3N3O5S: C, 58.59; H, 3.28; N, 7.59%. Found: C, 58.47;
H, 3.55; N, 7.84%.
4e: (R)-3-(1-(2-(pyrimidin-2-ylthio)acetyl)-5-(2-(trifluoromethyl)phenyl)-4,5-dihydro-1H-
o
pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 60%; mp 161-162 C; 1H NMR
(400 MHz, CDCl3) δ (ppm): 3.27 (dd, 1H, J1 = 18.7 Hz, J2 = 4.9 Hz, pyrazole 4-Ha), 4.03
(dd, 1H, J1 = 18.7 Hz, J2 = 12.0 Hz, pyrazole 4-Hb), 4.38-4.74 (m, 2H), 5.98 (dd, 1H, J1 =
12.0 Hz, J2 = 5.4 Hz, pyrazole 5-H), 7.21-7.89 (m, 9H), 8.64 (d, 3H, containing C4-H and
pyrimidine H). 13C NMR (75 MHz, CDCl3) δ (ppm): 33.94, 44.17, 60.53, 116.75, 116.78,
118.70, 119.16, 125.13, 125.93, 126.11, 128.97, 133.21, 141.64, 148.97, 151.36, 154.23,
157.22, 160.14, 166.18, 171.23. Anal. calcd for C25H17F3N4O3S: C, 58.82; H, 3.36; N,
10.98%. Found: C, 59.11; H, 3.10; N, 11.15%.
4f: (R)-3-(1-(2-(butylthio)acetyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-
yl)-2H-chromen-2-one, colorless crystals, yield, 61%; mp 117-118 oC; 1H NMR (400 MHz,
CDCl3) δ (ppm): 0.88 (t, 3H, J = 8.0 Hz), 1.38 (m, 2H), 1.59 (m, 2H), 2.62 (m, 2H), 3.44
(dd, 1H, J1 = 18.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 3.56-3.77 (m, 2H), 4.02 (dd, 1H, J1 =
20.0 Hz, J2 = 12.0 Hz, pyrazole 4-Hb), 5.63 (dd, 1H, J1 = 12.0 Hz, J2 = 4.0 Hz, pyrazole
5-H), 7.35-7.65 (m, 8H), 8.47 (s, 1H, C4-H). Anal. calcd for C25H23F3N2O3S: C, 61.46; H,
4.75; N, 5.73%. Found: C, 61.24; H, 5.02; N, 6.00%.
4g: (R)-3-(1-(2-(furan-2-ylmethylthio)acetyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-
o
1
1H-pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 61%; mp 125-126 C; H
NMR (400 MHz, CDCl3) δ (ppm): 3.21 (dd, 1H, J1 = 4.0 Hz, J2 = 20.0 Hz, pyrazole 4-Ha),
3.68-3.89 (m, 4H), 3.99 (dd, 1H, J1 = 12.0 Hz, J2 = 20.0 Hz, pyrazole 4-Hb), 5.67 (dd, 1H,
J1 = 12.0 Hz, J2 = 4.0 Hz, pyrazole 5-H), 6.28-6.40 (m, 2H), 7.39-7.74 (m, 8H), 7.85 (d,
1H, J = 8.0 Hz), 8.58 (s, 1H, C4-H). Anal. calcd for C26H19F3N2O4S: C, 60.93; H, 3.74; N,
5.47%. Found: C, 61.04; H, 3.99; N, 5.35%.
4h. (R)-3-(1-(2-(4-bromophenylthio)acetyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-
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o
pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 57%; mp 191-193 C; 1H NMR
(400 MHz, CDCl3) δ (ppm): 3.25 (dd, 1H, J1 = 16.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 3.97
(dd, 1H, J1 = 20.0 Hz, J2 = 12.0 Hz, pyrazole 4-Hb), 4.18-4.40 (m, 2H), 5.65 (dd, 1H, J1 =
12.0 Hz, J2 = 4.0 Hz, pyrazole 5-H), 7.35-7.72 (m, 11H), 7.86 (d, 1H, J = 8.0 Hz), 8.59 (s,
1H, C4-H). Anal. calcd for: C27H18BrF3N2O3S: C, 55.21; H, 3.09; N, 4.77%. Found: C,
55.15; H, 2.77; N, 5.10%.
4i. (R)-3-(1-(2-(4-nitrophenylthio)acetyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-
o
pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 67%; mp 164-165 C; 1H NMR
(400 MHz, CDCl3) δ (ppm): 3.25 (dd, 1H, J1 = 18.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 3.91
(dd, 1H, J1 = 18.0 Hz, J2 = 12.0 Hz, pyrazole 4-Hb), 4.00-4.36 (m, 2H), 5.75 (dd, 1H, J1 =
12.0 Hz, J2 = 4.0 Hz, pyrazole 5-H), 7.10-7.70 (m, 11H), 8.08 (d, 1H, J = 8.0 Hz), 8.69 (s,
1H, C4-H). Anal. calcd for: C27H18F3N3O5S: C, 58.59; H, 3.28; N, 7.59%. Found: C, 58.94;
H, 3.55; N, 7.33%.
4j. (R)-3-(1-(2-(pyrimidin-2-ylthio)acetyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-
o
pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 63%; mp 170-171 C; 1H NMR
(400 MHz, CDCl3) δ (ppm): 3.26 (dd, 1H, J1 = 18.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 4.08
(dd, 1H, J1 = 18.0 Hz, J2 = 12.0 Hz, pyrazole 4-Hb), 4.30-4.76 (m, 2H), 5.71 (dd, 1H, J1 =
12.0 Hz, J2 = 4.0 Hz, pyrazole 5-H), 7.23 (d, 1H, J = 8.0 Hz), 7.42-7.74 (m, 7H), 7.88 (d,
1H, J = 8.0 Hz), 8.65 (d, 3H, J = 8.0 Hz containing C4-H and pyrimidine H); Anal. calcd
for: C25H17F3N4O3S: C, 58.82; H, 3.36; N, 10.98%. Found: C, 59.04; H, 3.58; N, 11.17%.
4k: (R)-3-(1-(2-(butylthio)acetyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2
-one, colorless crystals, yield, 72%; mp 178-179 oC; 1H NMR (400 MHz, CDCl3) δ (ppm):
0.91 (t, 3H, J=8.0 Hz), 1.36 (m, 2H), 1.55 (m, 2H), 2.69 (t, 2H, J=4.0 Hz), 3.12 (dd, 1H, J1
= 20.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 3.74 (m, 2H), 3.87 (dd, 1H, J1 = 12.0 Hz, J2 = 20.0
Hz, pyrazole 4-Hb), 5.75 (dd, 1H, J1 = 12.0 Hz, J2 = 4.0 Hz, pyrazole 5-H), 7.15-7.37 (m,
9H, ArH), 8.66 (s, 1H, C4-H). Anal. calcd for C24H24N2O3S: C, 68.55; H, 5.75; N, 6.66%.
Found: C, 68.18; H, 6.01; N, 7.03%.
4l: (R)-3-(1-(2-(4-bromophenylthio)acetyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-2H-
chromen-2-one, colorless crystals, yield, 75%; mp 147-148 oC; 1H NMR (400 MHz, CDCl3)
δ (ppm): 3.10 (dd, 1H, J1 = 18.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 3.95 (dd, 1H, J1 = 20.0
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Hz, J2 = 12.0 Hz, pyrazole 4-Hb), 4.12-4.43 (m, 2H), 5.68 (dd, 1H, J1 = 12.0 Hz, J2 = 4.0
Hz, pyrazole 5-H), 6.97-7.46 (m, 13H), 8.63 (s, 1H, C4-H). Anal. calcd for C26H19BrN2O3S:
C, 60.12; H, 3.69; N, 5.39%. Found: C, 60.44; H, 3.95; N, 5.41%.
4m: (R)-3-(5-phenyl-1-(2-(phenylthio)acetyl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-
2-one, colorless crystals, yield, 70%; mp 155-156 oC; 1H NMR (400 MHz, CDCl3) δ (ppm):
3.14 (dd, 1H, J1 = 18.0 Hz, J2 = 4.0 Hz, pyrazole 4-Ha), 3.88 (dd, 1H, J1 = 20.0 Hz, J2 =
12.0 Hz, pyrazole 4-Hb), 4.17-4.40 (m, 2H), 5.75 (dd, 1H, J1 = 12.0 Hz, J2 = 4.0 Hz,
pyrazole 5-H), 6.95-7.29 (m, 14H), 8.65 (s, 1H, C4-H). Anal. calcd for C26H20N2O3S: C,
70.89; H, 4.58; N, 6.36%. Found: C, 71.05; H, 4.93; N, 6.35%.
4.3 General procedure for the synthesis of title compound 7
To
a
chloroform
(20
ml)
solution
of
2-bromo-1-(5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)ethanone
6 (10 mmol) was added substituted-thiol (10 mmol), DMAP (12 mmol) and catalytic KI,
the reaction mixture was allowed to stand at 40~50 ˚C for 3 h. The mixture was cooled,
washed with water. The product was collected by filtration and the crude residue was
purified by chromatography on SiO2 (dichloromethane / methanol, v:v= 4:1) to give title
compounds 7a~7f (Scheme 2) as colorless solids.
(Scheme 2)
7a. (R)-2-(4-bromophenylthio)-1-(5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydro-
pyrazol-1-yl)ethanone, colorless crystals, yield, 69%; mp 176-177 oC; 1H NMR (400 MHz,
CDCl3) δ (ppm): 2.09 (s, 3H, -CH3), 2.75 (dd, 1H, J1 = 18.6 Hz, J2 = 3.8 Hz, pyrazole
4-Ha), 3.34 (dd, 1H, J1 = 18.2 Hz, J2 = 11.2 Hz, pyrazole 4-Hb), 3.89-4.06 (m, 2H), 5.63
(dd, 1H, J1 = 11.4 Hz, J2 = 3.9 Hz, pyrazole 5-H), 6.97-7.49 (m, 6H), 7.99 (s, 1H). 13C
NMR (75 MHz, CDCl3) δ (ppm): 16.04, 36.26, 44.45, 54.40, 112.84, 113.04, 120.96,
128.01, 129.69, 131.90, 132.03, 134.17, 134.27, 149.86, 158.40, 167.00. Anal. calcd for:
C18H15Br3N2O2S: C, 38.39; H, 2.68; N, 4.97%. Found: C, 38.01; H, 3.03; N, 4.65%.
7b. (R)-1-(5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-2-
11

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o
(propylthio)ethanone, colorless crystals, yield, 65%; mp 163-164 C; 1H NMR (400 MHz,
CDCl3) δ (ppm): 0.99 (t, 3H, -CH3), 1.64-2.33 (m, 7H), 2.93 (dd, 1H, J1 = 18.0 Hz, J2 = 3.5
Hz, pyrazole 4-Ha), 3.30 (dd, 1H, J1 = 18.0 Hz, J2 = 11.0 Hz, pyrazole 4-Hb), 3.42 (m, 2H),
5.68 (dd, 1H, J1 = 11.0 Hz, J2 = 3.5 Hz, pyrazole 5-H), 7.03-7.28 (m, 2H); Anal. calcd for:
C15H18Br2N2O2S: C, 40.02; H, 4.03; N, 6.22%. Found: C, 39.84; H, 4.27; N, 6.40%.
7c. (R)-2-(benzo[d]thiazol-2-ylthio)-1-(5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-
o
dihydropyrazol-1-yl)ethanone, colorless crystals, yield, 72%; mp 180-182 C; 1H NMR
(400 MHz, CDCl3) δ (ppm): 2.01 (s, 3H, -CH3), 2.64 (dd, 1H, J1 = 18.4 Hz, J2 = 4.6 Hz,
pyrazole 4-Ha), 3.47 (dd, 1H, J1 = 18.4 Hz, J2 = 11.7 Hz, pyrazole 4-Hb), 4.50-4.78 (m, 2H),
5.51 (dd, 1H, J1 = 11.6 Hz, J2 = 4.8 Hz, pyrazole 5-H), 7.03-7.98 (m, 6H), 9.72 (s, 1H).
13C NMR (75 MHz, CDCl3) δ (ppm): 15.84, 36.46, 44.94, 55.56, 111.75, 112.96, 121.45,
122.0, 124.68, 126.56, 127.91, 133.22, 133.38, 134.92, 150.24, 152.86, 159.04, 163.65,
166.30. Anal. calcd for: C19H15Br2N3O2S2: C, 42.16; H, 2.79; N, 7.76%. Found: C, 42.44;
H, 2.98; N, 8.03%.
7d. (R)-methyl2-(2-(5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)
-2-oxoethylthio)acetate, colorless crystals, yield, 70%; mp 160-161 oC; 1H NMR (400 MHz,
CDCl3) δ (ppm): 2.15 (s, 3H, Me), 2.59-2.80 (m, 4H), 2.97 (dd, 1H, J1 = 18.0 Hz, J2 = 3.5
Hz, pyrazole 4-Ha), 3.31 (dd, 1H, J1 = 18.0 Hz, J2 = 11.0 Hz, pyrazole 4-Hb), 3.45 (m, 2H),
3.78 (s, 3H, OMe), 5.65 (dd, 1H, J1 = 11.0 Hz, J2 = 3.5 Hz, pyrazole 5-H), 7.00-7.22 (m,
2H); Anal. calcd for: C15H16Br2N2O4S: C, 37.52; H, 3.36; N, 5.83%. Found: C, 37.71; H,
3.15; N, 6.08%.
7e. (R)-1-(5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-2-(pyridin
o
-2-ylthio)ethanone, colorless crystals, yield, 60%; mp 175-176 C; 1H NMR (400 MHz,
CDCl3) δ (ppm): 2.10 (s, 3H, Me), 3.02 (dd, 1H, J1 = 18.2 Hz, J2 = 3.6 Hz, pyrazole 4-Ha),
3.38 (dd, 1H, J1 = 18.2 Hz, J2 = 11.0 Hz, pyrazole 4-Hb), 3.87 (m, 2H), 5.62 (dd, 1H, J1 =
11.0 Hz, J2 = 3.6 Hz, pyrazole 5-H), 7.09-8.51 (m, 6H); Anal. calcd for: C17H15Br2N3O2S:
C, 42.08; H, 3.12; N, 8.66%. Found: C, 42.21; H, 2.96; N, 8.87%.
7f. (R)-1-(5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-2-
o
(phenylthio)ethanone, colorless crystals, yield, 65%; mp 172-173 C; 1H NMR (400 MHz,
CDCl3) δ (ppm): 2.12 (s, 3H, Me), 3.00 (dd, 1H, J1 = 18.0 Hz, J2 = 3.0 Hz, pyrazole 4-Ha),
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3.32 (dd, 1H, J1 = 18.0 Hz, J2 = 11.0 Hz, pyrazole 4-Hb), 3.67 (m, 2H), 5.67 (dd, 1H, J1 =
11.0 Hz, J2 = 3.5 Hz, pyrazole 5-H), 7.00-7.32 (m, 7H); Anal. calcd for: C18H16Br2N2O2S:
C, 44.65; H, 3.33; N, 5.79%. Found: C, 45.02; H, 3.00; N, 6.04%.
4.4 Crystallographic Studies
X-ray single-crystal diffraction data for compounds 4a, 4b, 4c and 7a were collected on a
Bruker SMART APEX CCD diffractometer at 296(2) K using MoKα radiation (λ =
0.71073 Å) by the ω scan mode. The 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 [23]. All non-hydrogen atoms of compounds 4a, 4b, 4c, 7a were refined with
anisotropic thermal parameters. All hydrogen atoms were generated theoretically onto the
parent atoms and refined isotropically with fixed thermal factors.
4.5 Crystal structure analysis
Crystal data of 4a: Colorless crystals, yield, 70%; mp 171-172 oC; C25 H23 F3 N2 O3 S, M =
488.5, Triclinic, space group P-1; a = 9.985(10), b = 11.076(11), c = 11.792(12) (Å);
α =
84.947(9), = 65.299(8),
β
γ
= 80.938(8) (°), V = 1170(2) nm3, T =293(2) K, Z =2, Dc =
1.387 g/cm3, F(000)= 508, Reflections collected / unique = 8269 / 4246, Data / restraints /
parameters = 4246 / 48 / 309, Goodness of fit on F2 =1.040, Fine, R1= 0.0658, wR(F2) =
o
0.1649. Crystal data of 4b: Colorless crystals, yield, 65%; mp 141-142 C; C26 H19 F3 N2
O4 S, M = 512.49, Monoclinic, space group P21/n; a = 7.308(2), b = 8.219(2), c =
38.718(11) (Å);
α = 90, β = 95.217(3), γ
= 90 (°), V = 2316.1(12) nm3, T =293(2) K, Z =4,
Dc = 1.470 g/cm3, F(000)= 1056, Reflections collected / unique = 16328 / 4290, Data /
restraints / parameters = 4290 / 72 / 325, Goodness of fit on F2 =1.040, Fine, R1= 0.0624,
o
wR(F2) = 0.1520. Crystal data of 4c: Colorless crystals, yield, 72%; mp 143-145 C; C27
H18 Br F3 N2 O3 S, M = 587.40, Triclinic, space group P-1; a = 8.883(7), b = 10.944(9), c =
13.139(11) (Å);
α = 91.551(9), β = 107.298(8), γ
= 91.465(9) (°), V = 1218.3(17) nm3, T
=293(2) K, Z =2, Dc = 1.601 g/cm3, F(000)= 592, Reflections collected / unique = 8304 /
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4320, Data / restraints / parameters = 4320 / 0 / 334, Goodness of fit on F2 =0.979, Fine,
R1= 0.0672, wR(F2) = 0.0952. Crystal data of 7a: Colorless crystals, yield, 69%; mp
o
176-177 C; C18 H15 Br3 N2 O2 S, M = 563.11, Monoclinic, space group P2(1)/c; a =
11.048(9), b = 14.875(11), c = 12.436(10) (Å);
α = 90, β = 102.912(7), γ = 90 (°), V =
1170(2) nm3, T =293(2) K, Z =4, Dc = 1.878 g/cm3, F(000)= 1096, Reflections collected /
unique = 12138 / 3518, Data / restraints / parameters = 3518 / 36 / 238, Goodness of fit on
F2 = 1.011, Fine, R1= 0.0652, wR(F2) = 0.1233.
4.6 Anticancer assay
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 3×104 cells mL-1 with the
complete medium, 100 ꢀL 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 ꢀL of PBS containing 2.5 mg mL-1 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 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.
4.7 Cell cycle analysis
For cell cycle analysis, we performed cell cycle kit (Beyotime, China). Cells were washed
three times by cold PBS, and then cells were fixed in 70% ethanol at -20 ˚C for 12 h. After
fixation, cells were washed with cold PBS and stained with 0.5 mL of propidium iodide (PI)
◦
staining buffer, which contain 200 mg/mL RNase A, 50 ꢀg/mL PI, at 37 C for 30 min in
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the dark. Analyses were performed on FACScan flow cytometer. The experiments were
repeated three times.
4.8 Telomerase activity assay
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
bovineserum (GIBCO, NewYork, USA), streptomycin (0.1 mg/mL) and penicillin (100
IU/mL) at 37 °C in a humidified atmosphere containing 5% CO2. After trypsinization,
5×104 cultured cells in logarithmic growth were seeded into T25 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 concentration
as 60, 20, 6.67, 2.22, 0.75, 0.25 and 0.082l g/mL, respectively. After 24 h treatment, the
cells were harvested by cell scraper orderly following by washed once with PBS. The cells
were lysed in 150 ꢀL RIPA cell lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated
on ice for 30 min. The cellular supernatants were obtained via centrifugation at 12,000 g
for 20 min at 4 °C and stored at -80 °C. The TRAP-PCR-ELISA assay was performed
using a telomerase detection kit (Roche, Basel, Switzerland) according to the
manufacturer’s protocol. In brief, 2 ꢀL of cell extracts were mixed with 48 ꢀL 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 ꢀL of PCR products were hybridized to a digoxigenin (DIG)-labeled telomeric
repeat specific detection probe. And the PCR products were immobilized via the
biotin-labeled primer to a streptavidin-coated microtiter plate subsequently. The
immobilized 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.
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4.9 Taq polymerase activity
Take 25 ꢀl reactions, add 0.5 ꢀl of pEGFP plasmid containing the fluorescent proteion
gene fragment, 0.25 ꢀl of determinand or positive control, 2 U Taq polymerase (Promega)
and 20 ꢀl of paraffin oil at room temperature for 15 min. The protocol for PCR
amplification consists of denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94
°C for 30 seconds, and extenson at 72 °C for 30 seconds. PCR products were separated by
2% agarose gel electrophoresis and hybridization with ethidium bromide and
photographed.
4.10 General procedure for molecular docking
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-annealingbased 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.
5. Acknowledgments
The authors wish to thank the National Natural Science Foundation of China (No.
21272008), the governor fund of Guizhou province (20120037), China Postdoctoral
Science Foundation funded project (2012M511948) and the Research fundation of doctor,
Anhui Medical University (XJ201120).
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6. Supporting information
Crystallographic data (excluding structure factors) for the structure had been deposited
with the Cambridge Crystallographic Data Center as supplementary publication No.
CCDC-886613, CCDC-886612, CCDC-886614 and CCDC-887778. 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: (+44) 1223 336033;
e-mail: deposit@ccdc.cam.ac.uk)
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Figure Captions
Table 1. In vitro anticancer activity of the synthesized compounds against SGC-7901,
MGC-803, Bcap-37 and HEPG-2 cell lines
Table 2. Inhibitory effects of slected compounds against telomerase
Scheme 1. Synthesis of title compounds 4
Scheme 2. Synthesis of title compounds 7
Figure 1. ORTEP drawing of compounds 4a, 4b, 4c, 7a
Figure 2. Flow cytometry (FCM) analysis of 4f and 7b
Figure 3. Effect of compounds 4a, 4e, 4f, 4h, 7b, 7c and 7d on Taq polymerase activity. 1:
positive control (Staurosporine).
Figure 4. Molecular docking modeling of compound 4f (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 4f in the ATP
binding site of 3DU6.
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Table 1. In vitro anticancer activity of the synthesized compounds against SGC-7901,
MGC-803, Bcap-37 and HEPG-2 cell lines a
Compound
IC50 (µM) b
SGC-7901
4.42±0.70
10.25±1.02
14.66±0.98
9.77±0.68
4.97±0.87
2.91±0.11
9.74±1.21
12.07±0.84
8.51±0.29
4.39±0.80
7.30±0.65
14.30±0.50
20.31±1.33
8.21±0.62
2.30±0.35
12.30±1.12
3.30±0.45
10.30±1.18
24.14±0.99
7.37±0.21
MGC-803
3.14±0.35
15.74±1.29
21.33±1.29
7.58±0.41
4.00±1.03
2.35±0.27
15.20±1.11
10.79±0.49
10.21±0.29
2.89±0.09
12.85±0.54
16.81±1.02
22.75±2.01
12.80±0.29
2.97±0.08
7.80±1.02
2.27±0.31
12.29±1.09
12.65±0.79
3.68±0.11
Bcap-37
9.73±0.99
10.30±1.68
10.35±1.22
10.81±0.98
4.09±0.69
3.55±1.00
12.00±1.33
7.99±1.15
16.50±0.89
3.77±0.21
9.70±0.25
13.00±1.20
14.85±0.91
3.20±0.11
3.24±0.29
9.70±0.77
3.70±0.15
16.70±1.20
13.07±0.85
5.58±0.20
HEPG-2
5.01±0.85
18.00±2.01
17.89±1.55
7.70±0.89
6.54±0.94
8.68±1.17
11.22±1.56
12.70±1.55
7.76±2.00
4.75±0.47
6.64±0.40
14.52±0.77
16.32±0.87
6.87±0.45
4.00±0.31
8.70±0.65
2.95±0.40
24.70±0.89
10.35±0.97
4.35±0.20
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
7a
7b
7c
7d
7e
7f
5-Fluorouracil c
a
b
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.
c Used as a positive control.
Negative control DMSO, no activity.
Table 2. Inhibitory effects of slected compounds against telomerase
Compound
IC50 (µM) telomerase d
4a
4e
4f
4h
7b
7c
7d
2.00±0.41
8.89±0.65
0.92±0.09
25.11±1.50
1.04±0.10
4.65±0.50
1.19±0.26
Ethidium bromide e 2.10±0.19
BIBR1532e
0.28±0.10
d Telomerase supercoiling activity
e Ethidium bromide and BIBR1532 are reported as a control. The inhibition constant of ethidium toward
telomerase has been reported previously.
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O
O
O
O
O
(A)
CH3
H
(B)
H3C
O
CH3
O
OH
1
R1
O
O
O
O
(D)
H
(C)
R1
N
N
Br
O
O
2
3
4f: R1=4-CF3, R2=
CH3
4a: R1=2-CF3, R2=
CH3
R1
O
4g: R1=4-CF3, R2=
4h: R1=4-CF3, R2=
O
O
4b: R1=2-CF3, R2=
4c: R1=2-CF3, R2=
O
H
R2
N
N
Br
NO2
S
4
Br
4i: R1=4-CF3, R2=
4j: R1=4-CF3, R2=
O
4d: R1=2-CF3, R2=
4e: R1=2-CF3, R2=
NO2
N
N
N
N
4m: R1= H, R2=
4k: R1= H, R2=
4l: R1= H, R2=
CH3
Br
Scheme 1. Synthesis of title compounds 4
Reagent and conditions: (A) piperazine, 20~30 oC, 1 h. (B) benzaldehyde, piperazine,
butanol, reflux, 12 h. (C). NH2-NH2.H2O, CHCl3, BrCH2COCl, 30 oC, 2 h. (D) substituted
thiol, KHCO3, KI, 40~50 oC, 3 h.
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O
O
H3C
HO
Br
Br
Br
(E)
CH3
H
(F)
N
N
H
OH
5
OH
Br
O
Br
Br
Br
6
O
O
H3C
7a: R3=
7b: R3=
7c: R3=
7d: R3=
7e: R3=
H3C
Br
HO
Br
CH3
(G)
N
N
N
H
S
7f: R3=
S
R3
O
N
Br
7
Scheme 2. Synthesis of title compounds 7
o
Reagent and conditions: (E) CH3COCH3, NaOH, C2H5OH, 20~25 C, 15 h. (F)
o
NH2-NH2.H2O, CHCl3, BrCH2COCl, 20~30 C, 2 h. (G) substituted thiol, DMAP, KI,
40~50 oC, 3 h.
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Figure 1. ORTEP drawing of compounds 4a, 4b, 4c, 7a
4a
4b
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4c
7a
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7a
Figure 2. Flow cytometry (FCM) analysis showed cell cycle of MGC-803 cells after 48 h
of incubation.10,000 cells were scored for the analysis (n=3). (5) Compound 4f (0.92 ꢀM);
(6) Compound 7b (1.04 ꢀM); (9) Control group; (10) LY294002 (25 ꢀM).
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Figure 3.
1
4a
4e
4f
4h
7b
7c
7d
Effect of compounds 4a, 4e, 4f, 4h, 7b, 7c and 7d on Taq polymerase activity. 1: positive
control (Staurosporine 0.28 ꢀM).
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Figure 4. Molecular docking modeling of compound 4f (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.
hydrophobic - hydrophobic
G
H
l
u
i
s
3
3
3
9
,
1
8
,
A
T
l
a
h
r
S
O
N
3
2
2
3
4
1
,
A
s
N
p
F
3
2
5
,
F
I
l
e
F
3
3
O
O
8
,
Val 342
B) Schematic representation of the binding mode of 4f in the ATP binding site of 3DU6.
(
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Highlights
► Novel dihydropyrazole as potential telomerase inhibitors were designed.
► Compound 4f showed the most potent inhibitory activity.
► Compounds could induce tumor cell apoptosis.