Benzophenone-nucleoside derivatives as telomerase inhibitors: Design, synthesis and anticancer evaluation in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2016.09.011

Based on telomerase, thirteen novel phenstatin moiety linked stavudine derivatives (8a～8e and 11a～11f) were synthesized. The structures were determined by NMR and TOF-HRMS. The screening results showed that some compounds had better anti-cancer activity i

Full text of DOI:10.1016/j.ejmech.2016.09.011

European Journal of Medicinal Chemistry 124 (2016) 729e739
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Benzophenone-nucleoside derivatives as telomerase inhibitors:
Design, synthesis and anticancer evaluation in vitro and in vivo
Jing Bo Shi a, Liu Zeng Chen a, Yang Wang a, Cheng Xiou a, Wen Jian Tang a, Hai Pin Zhou c,
,
c
,
, **
Xin Hua Liu a *, Qi Zheng Yao b
a School of Pharmacy, Anhui Medical University, Hefei, 230032, PR China
b School of Pharmacy, China Pharmaceutical University, NanJing, 210009, PR China
c School of Material Science Chemical Engineering, ChuZhou University, ChuZhou, 239000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2016
Received in revised form
22 August 2016
Accepted 3 September 2016
Available online 5 September 2016
Based on telomerase, thirteen novel phenstatin moiety linked stavudine derivatives (8a~8e and 11a~11f)
were synthesized. The structures were determined by NMR and TOF-HRMS. The screening results
showed that some compounds had better anti-cancer activity in vivo and in vitro. Among them, Com-
pound 8d showed high inhibitory activity against telomerase and showed good antiproliferative activity
against SGC-7901 cell with IC50 value 0.77 mM by inducing cell cycle arrest at G2 phase. It also could
improve SGC-7901 cell apoptosis, mitochondrial membrane potential assay indicated that the dissipation
of MMP might participate in apoptosis induced by title compound. In vivo studies showed that com-
pound 8d displayed potent anticancer activity with inhibition tumor growth.
© 2016 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzophenone-nucleoside derivatives
Anticancer activity
Telomerase
Inhibitor
1. Introduction
its structure-activity relationships (SARs) studies of this compound
have been widely carried out [15e17]. Among them, phenstatin
Telomerase plays important role in early stage of life-
maintaining telomere and chromosomal integrity of frequently
dividing cells [1]. About 90% of cancer cells from a large range of
different cancer types have detectable telomerase activity, telo-
merase is hence believed as a potential anticancer target [2e5]. As
the component of telomerase, expression of telomerase reverse
transcriptase (TERT) is the rate-limiting factor for telomerase ac-
tivity, and most human somatic cells do not show detectable
telomerase activity due to lack of TERT, TERT is hence regarded as
the key role for the drug discovery [6,7]. Target telomerase, a lot of
works have been carried out in recent years [8e10]. Unfortunately,
highly efficient and selective activity molecule has not been
designed so far.
(Fig. 1B) strongly inhibited cancer cellular growth and tubulin
polymerization and caused significant arrest of mitosis.
BIBR1532, a selective TERT inhibitor shows the mechanism of
action similar to inhibitors of HIV-1 reverse transcriptase. Known
to all, stavudine was demonstrated potent anti-HIV-1 activity.
Motivated by the afore-mentioned findings, taking into account
the similarity of their mechanistic and structural similarities be-
tween TERT and HIV-1 reverse transcriptase, we anticipate that the
presence of stavudine in the phenstatin moiety should play an
important role in the telomerase activity. Herein, in continuation
to extend our research for searching effective telomerase in-
hibitors [18e20], some novel compounds of phenstatin moiety
linked stavudine (Fig. 1C) will been designed and optimized in this
study.
Combretastatin A-4 (Fig. 1A) is a natural product, its cis-stilbene
shows potent cytotoxicity against a broad spectrum of human
cancer cell lines [11]. Based on CA-4, large analogues [12e14] and
2. Results and discussion
2.1. Chemistry
* Corresponding author. School of Pharmacy, Anhui Medical University, Hefei,
230032, PR China.
** Corresponding author.
According to known procedure [21], benzophenones, analogues
of phenstatin (5a~5e) were prepared (Scheme 1) from guaiacol 1. 13
novel benzophenone-nucleoside hybrid derivatives were designed
E-mail addresses: xhliuhx@163.com (X.H. Liu), qz_yao@yahoo.com.cn (Q.Z. Yao).
http://dx.doi.org/10.1016/j.ejmech.2016.09.011
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739
Fig. 1. The workflow of the general design strategy in this study.
Scheme 1. Synthesis of compounds 5a~5e Reagents and conditions: (A) ClCH2COCl, 120e130 ꢀC, 5e8 h; (B) MeSO3H/P2O5, 40 ꢀC, 8e10 h, (C) NaOAc, MeOH, reflux, 30 min.
and synthesized (Schemes 2e3). In order to optimize the reaction
conditions, we applied a modified methodology in the second and
third stage. Thus, Eaton's reagent (MeSO3H/P2O5) at 40e60 ꢀC was
used, afforded the precursors 4a~4e with high yields. Deprotection
of chloroacetyl group was achieved with sodium acetate and ob-
tained benzophenone analogues (5a~5e).
11a~11h. All compounds were characterized by HR-MS, 1H NMR
and 13C NMR spectral analysis.
2.2. Anticancer activity
At first, all title compounds were evaluated for their anti-
proliferative activities against cervical cancer cell (Hela), human
hepatocellular carcinoma cell (SMMC-7721), human gastric can-
cer cell (SGC-7901) and human hepatocellular carcinoma
(HepG2) cell lines. Also included was the activity of reference
AMD. The cells were allowed to proliferate in presence of tested
material for 48 h, and the results were reported in terms of IC50
values (Table 1). From Table 1, it is obvious to see that com-
pounds 8a and 8d exhibited high activity against four cell lines.
Among them, compound 8d possessed potent activity against
Title compounds 8a~8e were obtained with steps: First, succinic
anhydride was introduced at the 50-position of stavudine in dry
dichloromethane (DCM) in the presence of triethylamine (TEA) to
obtain compound 7. The compounds were treated with corre-
sponding benzophenones (5a~5e) in dry DCM, in the presence of
dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) to afford compounds 8a~8e, as show in Scheme 2.
The target compounds 11a~11h were synthesized according to
Scheme 3. Treatment of the key benzophenones 5a~5e with ethyl
bromoacetate in dry acetone in the presence of K2CO3 produced the
corresponding benzophenone derivatives 9a~9h. They were con-
verted to corresponding carboxylic acid derivatives 10a~10h in
base. Finally, stavudine 6 was treated with corresponding 10a~10h
in dry DCM, in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
car-bodiimide (EDC$HCl), N-hydroxybenzotriazole monohydrate
(HoBt) and triethylamine (TEA) to afford target compounds
Hela,
SMMC-7721
and
SGC-7901
cell
lines
with
IC50 ¼ 1.58 0.20, 0.82 0.11 and 0.77 0.33
m
M, respectively,
which were even better than that of the commercial AMD. But,
compounds 11a~11f exhibited poor inhibitory activity against all
the tested cells. The preliminary results show that the length of
the linker between phenstatin and stavudine had a great influ-
ence on the activities.

J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739
731
Scheme 2. Synthesis of title compounds 8a~8e Reagents and conditions: (A) Succinic anhydride, Et3N, CH2Cl2, r.t., 12 h; (B) DCC, DMAP, CH2Cl2, r.t., 12 h.
Scheme 3. Synthesis of title compounds 11a~11h Reagents and conditions: (A) Ethyl bromoacetate, K2CO3, Acetone, reflux, 4e5 h; (B) 2 N NaOH, MeOH, r.t., 2e3 h; (C) EDC$HCl,
HoBt, DCM, Et3N, r.t., 12 h.
2.3. Annexin V/propidium iodide assay
The apoptosis ratios of compound 8d measured at different con-
centration points were found to 13.3% (1 M), 38% (2 M) and 59.6%
(4 M), respectively, while that of control was 3.23%. The results
m
m
To determine whether the cells death induced by the compound
8d was due to apoptosis or necrosis, the compound was further
investigated to SGC-7901 cells using an Annexin V-FITC/propidium
iodide (PI) assay. As phosphatidylserine (PS) exposure usually
precedes loss of plasma membrane integrity in apoptosis, the
presence of AnnexinVþ/PI- cells can be considered as an indicator
of apoptosis. The apoptosis ratios induced by compound 8d were
quantitatively determined by flow cytometry. And SGC-7901 cells
not treated with compound 8d were used as control. Four quadrant
images were observed by flow cytometric analysis: the Q1 area
represented damaged cells appearing in the process of cell collec-
tion, the Q2 area represented necrotic cells and later period
apoptotic cells, Q3 area represented early apoptotic cells, and the
normal cells were located in the Q4 area. The results were given in
Fig. 2. After 48 h, apoptosis ratios (including the early and late
apoptosis ratios) were obtained by treatment at the concentration
m
revealed that compound 8d could induce SGC-7901 cells apoptosis.
2.4. Mitochondrial membrane potential (MMP) assay
The loss of mitochondrial membrane potential (Dj) is regarded
as a limiting factor in the apoptotic pathway, which has been
considered as an antitumor target and is of high importance to the
control of the cell apoptosis. To investigate the role of mitochondria
in the apoptosis induced by compound 8d, the effect of compound
on MMP was measured by flow cytometry after SGC-7901 cells
were stained with JC-1, which is capable of selectively entering
mitochondria to form monomers that emit green fluorescence at
low MMP, and form JC-1 aggregates that emit red fluorescence at
high MMP. After treated for 48 h with compound 8d, compared
with the control group, the number of treated cells emitting red
fluorescence significantly decreased while the number of treated
cells emitting green fluorescence obviously increased which
of 1
mM, 2 mM and 4 mM. The apoptosis of SGC-7901 cells treated
with compound 8d increased gradually in a concentration manner.

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J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739
Table 1
suggested the disruption of MMP. The results indicated that dissi-
pation of MMP might participate in apoptosis induced by com-
pound 8d (Figs. 3e4).
In vitro anticancer activity of compounds 8 and 11 against Hela, SMMC-7721, HepG2
and SGC-7901 cell linesb.
Comp
IC50
/m
m
Hela
SMMC-7721
HepG2
SGC-7901
8a
8b
8c
8d
2.65 0.39
17.80 2.35
21.11 2.11
1.58 0.20
6.51 0.88
12.80 1.03
28.60 1.87
36.31 2.01
da
2.86 0.39
22.59 1.93
30.51 1.80
0.82 0.11
3.21 0.98
21.15 2.02
20.11 2.20
40.29 1.28
e
6.85 1.22
20.30 4.00
24.02 1.55
3.15 0.78
11.09 1.40
14.96 1.06
28.57 2.41
37.99 1.87
e
2.27 1.48
8.25 1.81
10.20 1.67
0.77 0.33
4.19 1.62
23.39 1.60
31.42 2.97
29.10 1.95
e
2.5. Cell cycle analysis
To understand whether the reduced cell proliferation was due to
cell cycle, the effect of compound 8d on the cells cycle was inves-
tigated by flow cytometry. Cytometric profiles of the PI-stained
DNA showed cell cycle arrest of SGC-7901 cells treated with com-
pound 8d for 48 h at different concentrations. As shown in Fig. 5,
the treatment of SGC-7901 cells with compound 8d enhanced cell
cycle arrest at the G2 phase at different concentrations, resulting in
concomitant population increase in the G2 phase (30%, 42.6% and
60.9%) compared with the control cells (24.6%). The population of
the G1 phase decrease at a certain extent (32.9%, 28.5% and 15.8%)
compared with the control cells (38.1%). For S phase, the cell per-
centages were decreased obviously from 37.4% to 37.1%, 28.9% and
23.2%. These data indicated that compound 8d significantly
induced cells cycle arrest at G2 phase.
8e
11a
11b
11c
11d
11e
11f
11g
11h
AMDc
50.21 2.89
e
e
e
48.14 2.51
e
e
e
30.21 2.33
45.80 1.65
3.99 0.35
32.33 2.95
40.60 2.51
1.95 0.17
e
e
e
e
2.67 0.31
3.41 0.20
a
Not observed in the tested concentration range.
b
The standard deviation (SD) of three time independent tests. The IC50 value was
defined as the concentration at which 50% survival of cells was observed. Inactive at
50 M (highest concentration tested).
m
c
AMD used as a positive control.
Fig. 2. Apoptosis ratio detected by Annexin V/PI assay on the SGC-7901 cells treated with compound 8d of 1 mM, 2 mM and 4 mM for 48 h, respectively. Analyses were performed at
least 3 times, and a representative experiment was presented.

J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739
733
Fig. 3. MMP assay on the SGC-7901 cells treated with compound 8d of 1
m
M, 2
mM and 4
mM for 48 h, respectively. Analyses were performed at least 3 times, and a representative
experiment was presented.
2.6. Anticancer activity evaluation in vivo
Therefore, the length of the linker between phenstatin and
stavudine played an important role for the telomerase activity. This
preliminary SAR could offer the useful information for further
structural optimization.
Mice sarcoma cells S180 and hepatoma cells HepG2 models are
recognized as the reliable research models for study of antitumor
pharmacodynamics. To further verify the inhibitory against the
growth of tumor cells in vivo, sarcoma cells S180 and hepatoma
cells HepG2 models were selected to evaluate its antitumor effects.
The inhibitory effects of compound 8d against the growth of the
transplanted S180 or HepG2 carcinoma were presented in Table 2.
The results revealed that compound 8d significantly decreased the
tumor weights of S180 and HepG2 tumor-bearing mice. The potent
activity was showed with inhibitory rates 40.6% and 48.5% for S180
and HepG2 at the dosage of 60 mg/kg/day, respectively.
3. Conclusions
In this study, two series of phenstatin-stavudine derivatives
were designed, synthesized and biologically evaluated as potential
telomerase inhibitors. Among the 13 compounds, compounds 8d
exhibited potent activities against Hela, SMMC-7721, HepG2 and
SGC-7901 cells. In vivo studies showed that compound 8d displayed
potent anticancer activity with inhibition tumor growth of S180
and HepG2 tumor-bearing mice. Moreover, compound 8d showed
high inhibitory activity against telomerase. Annexin V/propidium
iodide assay results revealed that compound 8d could induce
apoptosis in SGC-7901 cells. Further mitochondrial membrane
potential assay indicated that the dissipation of MMP might
participate in apoptosis induced by compound 8d.
2.7. Telomerase activity
To confirm if the synthesized compounds performed anticancer
activity via telomerase inhibition as designed, some title com-
pounds were evaluated by a modified TRAP assay using an extrac-
tion from SGC-7901 cells. Modified TRAP is a powerful technique to
determine small molecules inhibiting telomere elongation qualita-
tively and quantitatively [22]. The results were summarized in
Table 3. Among them, compounds 8a and 8d showed potent
inhibitory activities against telomerase with IC50 values at 6.43 and
4. Experimental section
4.1. Chemistry
7.01
mM, better than positive control staurosporine.
The reactions were monitored by thin layer chromatography

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J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739
Fig. 4. Collapse of mitochondrial membrane potential of SGC-7901 cells treated by compound 8d of 1 mM, 2 mM and 4 mM for 24 h, respectively, and stained by JC-1. Selected fields
illustrated the corresponding live cells (orange-red), apoptotic cells (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
(TLC) on pre-coated silica GF254 plates. Melting points were
determined on a XT4MP apparatus (Take Corp., Beijing, China), and
are uncorrected. 1H and 13C NMR spectra were recorded on a
Brucker AM-300 (300 MHz) spectrometer with CDCl3 or DMSO-d6
as the solvent and TMS as the internal standard. Chemical shifts are
To a solution of compounds 4a~4e (8 mmol) in MeOH (25 mL) was
added NaOAc (36 mmol). The reaction mixture was refluxed for
30e60 min, then the solvent was evaporated. The obtained residue
was added H2O (10 mL) with stirring for 2e5 h, filtered, and dried to
afford compounds 5a~5e as white or off-white powders.
reported in
d (parts per million) values. Coupling constants J were
reported in Hz. Mass spectra were performed on an Agilent 1260-
6221 TOF mass spectrometer. Stavudine was purchased from WuXi
AppTec, the stereo is known: 1-((2R,5S)-5-(hydroxymethyl)-2,5-
dihydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione. All re-
agents were purchased from standard commercial suppliers and
treated with standard methods before use. Solvents were dried in a
routine way and redistilled.
4.3. General procedure for preparation of compounds 8
To a dry DCM (10 mL) solution of stavudine (4.46 mmol) and
succinic anhydride (6.69 mmol) in an ice bath was added TEA
(10.8 mmol). The mixture was stirred for 8e12 h at room temper-
ature, then distilled to remove DCM and TEA. The residue was
purified by crystallization from ethanol to give stavudine succinic
acid monoester 7.
To a DCM (10 mL) solution of 7 (0.5 mmol) was added DCC
(0.7 mmol), DMAP (0.5 mmol), the mixture was stirred 10 min at
0
added. The reaction solution was stirred overnight at room tem-
perature. The mixture was filtered and filtrate was washed twice
with water, dried over anhydrous Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by chromatography on a
silica gel column (petroleum ether: ethyl acetate, 10:1 / 1:1) to
give title crude compounds, the product was recrystallized from
suitable solvent to give title compounds 8a~8e (Scheme 2) as solids.
4.2. Synthesis of benzophenone analogues intermediates 5
ꢀC, then corresponding benzophenone analogues 5 (1.0 eq.) was
Guaiacol (10.2 mmol) and chloroacetyl chloride (14.6 mmol)
were placed into a flask under N2 atmosphere. The mixture was
heated to 120e130 ꢀC for 5e8 h with stirring. The mixture was
cooled to r.t. and carefully added to cold water, then was extracted
with CH2Cl2 (3 ꢁ 30 mL). The combined organic phase was washed
sequentially with water, saturated NaHCO3, brine, dried with
anhydrous Na2SO4. The solvent was evaporated in vacuo and yiel-
ded yellow oil, which was recrystallized with ethanol to afford
white needle-like crystals compound 2.
To an Eaton's reagent (50 mL) [23] ice bath solution was added
benzoic acid derivatives 3 (67.4 mmol) and compound 2 (45 mmol).
The reaction mixture was allowed to stand at 25e50 ꢀC for 8e10 h,
then cooled and poured into ice water (150 mL). The mixture was
extracted with CH2Cl2 (3 ꢁ 30 mL). The combined organic phase was
washed sequentially with water, saturated NaHCO3, brine, dried with
anhydrous Na2SO4. The solvent was evaporated in vacuo and the
obtained residue was refluxed in ethanol, then cooled to r.t. and
filtered through a Büchner funnel to afford off-white powders 4a~4e.
4.3.1. 8a: 2-methoxy-5-(2,4,5-trimethoxybenzoyl)phenyl((2S,5R)-
5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5-
dihydrofuran-2-yl)methyl succinate
Light yellow powder solid, yield, 85%; mp 177e178 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
(ppm): 8.30 (s, 1H, NH), 7.71 (dd, J ¼ 8.6, 2.1 Hz,
1H, ArH), 7.52 (d, J ¼ 2.1 Hz, 1H, ArH), 7.24 (s, 1H, pyrimidine C6eH),
6.97 (m, 3H, ArH and 10-H), 6.56 (s, 1H, ArH), 6.27 (d, J ¼ 6.0 Hz, 1H,
30-H), 5.88 (d, J ¼ 6.0 Hz, 1H, 20-H), 5.04 (m, 1H, 40-H), 4.47 (dd,
J ¼ 12.3, 4.0 Hz, 1H, 50-H), 4.25 (dd, J ¼ 12.3, 3.1 Hz, 1H, 500-H), 3.96

J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739
735
Fig. 5. Inhibition of cell cycle progress in SGC-7901 cells treated with compound 8d for 48 h. Cells were fixed with ethanol and stained with PI. Cell cycle distribution was analyzed
by flow cytometry. Analyses were performed at least 3 times, and a representative experiment was presented.
Table 2
Antitumor effects of compound 8d against tumor growth on the S180 and HepG2 xenograft mice.a
Models
Groups
Animal number (End, n)
Body weight (g)
Beginning
Tumor weight (g)
Inhibition rate (%)
End
S180
Control
8d
Control
8d
10
10
10
10
19.38 1.07
19.99 1.26
19.15 1.01
19.87 0.51
24.00 1.78
24.25 1.55
25.17 1.60
26.34 1.67
2.02 0.19
1.20 0.17*
2.01 0.09
1.04 0.28**
40.6
48.5
HepG2
a
Mice were inoculated with S180 or HepG2 subcutaneously into the right front armpit and randomly divided into four test groups. The mice were daily treated by
compound 8d (60 mg/kg/day), or normal saline (NS, 10 mL/kg) by oral gavage for ten consecutive days. Data were analyzed using SPSS11.0. Significant difference between each
treatment and the control are shown as P < 0.05 (*) and P < 0.01 (**).
(s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.70 (s, 3H,
OCH3), 3.04e2.89 (m, 2H, CH2), 2.74 (m, 2H, CH2), 1.92 (s, 3H, py-
rimidine C5eCH3). TOF-HRMS: calcd for C31H32N2NaO12 [MþNa]þ,
647.1849; found 647.1849.
(300 MHz, CDCl3)
d
(ppm): 8.36 (s, 1H, NH), 7.80 (dd, J ¼ 8.6, 5.5 Hz,
2H, ArH), 7.71 (dd, J ¼ 8.5, 2.1 Hz, 1H, ArH), 7.57 (d, J ¼ 2.1 Hz, 1H,
ArH), 7.24 (s, 1H, pyrimidine C6eH), 7.16 (t, J ¼ 8.6 Hz, 2H), 7.03 (d,
J ¼ 8.5 Hz, 1H, ArH), 6.98 (m, 1H, 10-H), 6.28 (d, J ¼ 5.9 Hz, 1H, 30-H),
5.89 (d, J ¼ 5.9 Hz, 1H, 20-H), 5.04 (m, 1H, 40-H), 4.47 (dd, J ¼ 12.3,
4.0 Hz, 1H, 50-H), 4.26 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500-H), 3.91 (s, 3H,
OCH3), 3.04e2.87 (m, 2H, CH2), 2.75 (m, 2H, CH2), 1.92 (s, 3H, py-
rimidine C5eCH3). TOF-HRMS: calcd for C28H25FN2NaO9 [MþNa]þ,
575.1427; found 575.1427.
4.3.2. 8b: 5-(4-fluorobenzoyl)-2-methoxyphenyl((2S,5R)-5-(5-
methyl-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-2,5-
dihydrofuran-2-yl)methyl succinate
Off-white powder solid, yield, 90%; mp 192e193 ꢀC; 1H NMR

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Table 3
4.4. General procedure for the synthesis of title compounds
11a~11h
Inhibitory activities of some compounds against
telomerase.a
Compound
IC50 (mM)
To an acetone (15 mL) solution of corresponding benzophenone
analogues (0.314 mmol) in an ice bath was added anhydrous
K2CO3 (0.377 mmol) and ethyl bromoacetate (0.377 mmol). The
mixture was heated to 60e65 ꢀC with stirring. The reaction was
monitored by TLC. When the starting material had completely
disappeared, the mixture was distilled to remove acetone. The
residue was added H2O (20 mL) and ethyl acetate (20 mL), then
stirred over 10e15 min. The product was extracted into ethyl ac-
etate (2 ꢁ 20 mL), the organic extract washed with water, and the
resulting organic liquor evaporated to dryness on a rotavaporator
and obtained crude compounds 9. The crude compounds 9
(1 mmol) were dissolved in methanol (10 mL), 2 N NaOH (2 mL)
solution and stirred at r.t. for 2e3 h. After completion of the re-
action, the mixture was acidified with HCl (1 N), then the solvent
was concentrated to a low volume. The precipitated product was
granulated at 10 ꢀC for a further hour. The title compounds 10 was
collected by filtration, washed with water, and dried to give as
solid.
To a DCM (15 mL) solution of compounds 10 (0.266 mmol) was
added EDC$HCl (0.319 mmol), HOBt (0.319 mmol), and Stavudine
(0.266 mmol), the mixture was stirred 30 min at 0 ꢀC. After TEA
(0.319 mmol) was added, the reaction solution was stirred over-
night at r.t. The mixture was washed twice with saturated NH4Cl,
NaHCO3 and water, dried with Na2SO4, filtered, and concentrated in
vacuo. The residue was recrystallized from suitable solvent to give
title compounds 11a~11h (Scheme 3) as solids.
8a
8c
8d
11f
6.43 1.19
28.91 1.95
7.01 1.14
noc
Staurosporineb
8.50 0.37
a
Telomerase supercoiling activity.
Staurosporine is reported as a control.
b
c
The standard deviation (SD) of three time independent
tests. No, not observed in the tested concentration range
0e60 mm.
4.3.3. 8c: 2-methoxy-5-(4-nitrobenzoyl)phenyl((2S,5R)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5-
dihydrofuran-2-yl)methyl succinate
Off-white powder solid, yield, 92%; mp 180e181 ꢀC;1H NMR
(300 MHz, CDCl3)
d
(ppm): 8.39 (s, 1H, NH), 8.34 (d, J ¼ 8.7 Hz, 2H,
ArH), 7.89 (d, J ¼ 8.7 Hz, 2H, ArH), 7.72 (dd, J ¼ 8.6, 2.1 Hz, 1H, ArH),
7.59 (d, J ¼ 2.1 Hz, 1H, ArH), 7.23 (s, 1H, pyrimidine C6eH), 7.05 (d,
J ¼ 8.6 Hz, 1H, ArH), 6.98 (m, 1H, 10-H), 6.28 (d, J ¼ 6.0 Hz, 1H, 30-H),
5.89 (d, J ¼ 5.9 Hz, 1H, 20-H), 5.05 (s, 1H, 40-H), 4.44 (dd, J ¼ 12.3,
4.1 Hz, 1H, 50-H), 4.28 (dd, J ¼ 12.3, 3.1 Hz, 1H, 500-H), 3.93 (s, 3H, O
CH3), 3.04e2.91 (m, 2H, CH2), 2.75 (m, 2H, CH2), 1.92 (s, 3H, py-
rimidine C5eCH3). TOF-HRMS: calcd for C28H25N3NaO11 [MþNa]þ,
602.1383; found 602.1383.
4.3.4. 8d: 2-methoxy-5-(2-methoxybenzoyl)phenyl((2S,5R)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5-
dihydrofuran-2-yl)methyl succinate
4.4.1. 11a: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(2-methoxy-5-(3,4,5-
trimethoxybenzoyl)phenoxy)acetate
Off-white powder solid, yield, 90%; mp 187e188 ꢀC; 1H NMR
Off-white powder solid, yield, 82%; mp 170e171 ꢀC; 1HNMR
(300 MHz, CDCl3)
d
(ppm): 8.38 (s, 1H, NH), 7.70 (dd, J ¼ 8.6, 2.1 Hz,
(300 MHz, CDCl3)
d
(ppm):
d
8.57 (s,1H, NH), 7.49 (dd, J ¼ 8.6,1.9 Hz,
1H, ArH), 7.56 (d, J ¼ 2.1 Hz, 1H, ArH), 7.45 (dd, J ¼ 11.2, 4.5 Hz, 1H,
ArH), 7.33 (dd, J ¼ 7.5, 1.6 Hz, 1H, ArH), 7.25 (s, 1H, pyrimidine
C6eH), 7.08e6.93 (m, 4H, ArH and 10-H), 6.27 (d, J ¼ 6.0 Hz, 1H, 30-
H), 5.88 (d, J ¼ 5.7 Hz, 1H, 20-H), 5.04 (s, 1H, 40-H), 4.48 (dd, J ¼ 12.3,
4.0 Hz, 1H, 50-H), 4.25 (dd, J ¼ 12.3, 3.2 Hz, 1H, 500-H), 3.88 (s, 3H, O
CH3), 3.74 (s, 3H, O CH3), 3.05e2.85 (m, 2H, CH2), 2.73 (dd, J ¼ 9.1,
4.9 Hz, 2H, CH2), 1.92 (s, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz,
1H, ArH), 7.35 (d, J ¼ 1.9 Hz, 1H, ArH), 7.22 (d, J ¼ 1.2 Hz, 1H, py-
rimidine C6eH), 7.00 (s, 2H, ArH), 7.00e6.97 (m, 1H, 10-H), 6.95 (d,
J ¼ 8.6 Hz, 1H, ArH), 6.24 (d, J ¼ 6.0 Hz, 1H, 30-H), 5.91 (d, J ¼ 5.4 Hz,
1H, 20-H), 5.05 (m, 1H, 40-H), 4.78 (s, 2H, CH2), 4.51 (dd, J ¼ 12.2,
4.3 Hz, 1H, 50-H), 4.35 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500-H), 3.96 (s, 3H, O
CH3), 3.94 (s, 3H, O CH3), 3.89 (s, 6H, 2OCH3), 1.91 (d, J ¼ 1.0 Hz, 3H,
pyrimidine C5eCH3). TOF-HRMS: calcd for C29H30N2NaO11
[MþNa]þ, 605.1744; found 605.1744.
CDCl3)
d (ppm): 194.21 (C]O, diphenyl ketone), 171.65 (OeC]O),
170.00 (OeC]O), 163.64 (NHeC]O,dihydropyrimidine), 157.10,
155.06 (NeC]O,dihydropyrimidine), 150.74, 139.21, 135.56 (CH2-
dihydropyrimidine), 133.34, 131.74, 130.86, 130.07, 129.36, 128.63,
127.20, 124.36, 120.51, 111.45, 111.36, 111.09 (CH-dihydropyr-
imidine), 89.81 (CH, 2-furan), 84.16 (CH, 5-furan), 64.97 (OCH2),
56.12 (OCH3), 55.62 (OCH3), 28.92 (CH2), 28.67 (CH2), 12.53 (Me-
4.4.2. 11b:((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(2-methoxy-5-(2,4,5-
trimethoxybenzoyl)phenoxy)acetate
Off-white powder solid, yield, 82%; mp 172e174 ꢀC; 1H NMR
dihydropyrimidine). TOF-HRMS: calcd for
C29H28N2NaO10
(300 MHz, CDCl3)
d (ppm): 8.30 (s, 1H, NH), 7.39 (m, 2H, ArH), 7.25
[MþNa]þ, 587.1636; found 587.1632.
(s, 1H, pyrimidine C6eH), 6.99 (m, 1H, 10-H), 6.93 (s, 1H, Ar), 6.88 (d,
J ¼ 9.0 Hz, 1H, ArH), 6.57 (s, 1H, ArH), 6.25 (d, J ¼ 6.0 Hz, 1H, 30-H),
5.91 (d, J ¼ 6.0 Hz, 1H, 20-H), 5.05 (s, 1H, 40-H), 4.76 (s, 2H, CH2), 4.53
(dd, J ¼ 12.2, 4.1 Hz, 1H, 50-H), 4.35 (dd, J ¼ 12.3, 3.0 Hz, 1H, 500-H),
3.97 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.70 (s, 3H,
OCH3), 1.90 (s, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3)
4.3.5. 8e: 5-(2-chlorobenzoyl)-2-methoxyphenyl((2S,5R)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5-
dihydrofuran-2-yl)methyl succinate
Off-white powder solid, yield, 85%; mp 175e176 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
(ppm): 8.34 (s, 1H, NH), 7.66 (dd, J ¼ 8.6, 2.1 Hz,
d (ppm): 193.87 (C]O, diphenyl ketone), 168.35 (OeC]O), 163.64
1H, ArH), 7.58 (d, J ¼ 2.0 Hz, 1H, ArH), 7.47e7.40 (m, 2H, ArH),
7.37e7.28 (m, 2H, ArH), 7.25 (s, 1H, pyrimidine C6eH), 6.97e6.99
(m, 2H, ArH and 10-H), 6.27 (d, J ¼ 6.0 Hz, 1H, 30-H), 5.89 (d,
J ¼ 5.9 Hz, 1H, 20-H), 5.04 (s, 1H, 40-H), 4.46 (dd, J ¼ 12.3, 4.0 Hz, 1H,
50-H), 4.25 (dd, J ¼ 12.3, 3.2 Hz, 1H, 500-H), 3.89 (s, 3H, O CH3),
2.99e2.88 (m, 2H, CH2), 2.79e2.67 (m, 2H, CH2), 1.91 (s, 3H, py-
rimidine C5eCH3). TOF-HRMS: calcd for C28H25ClN2NaO9 [MþNa]þ,
591.1141; found 561.1143.
(NHeC]O,dihydropyrimidine), 153.50, 152.72, 152.13, 150.74
(NeC]O,dihydropyrimidine), 146.73, 143.02, 135.47 (CH2-dihy-
dropyrimidine), 132.98, 131.32, 127.50, 126.86, 119.88, 114.14, 113.19,
111.30, 110.53 (CH-dihydropyrimidine), 97.58, 89.84 (CH, 2-furan),
83.86 (CH, 5-furan), 66.04 (OCH2), 65.44 (OCH2), 56.74 (OCH3),
56.51 (OCH3), 56.17 (OCH3), 56.01 (OCH3), 12.42 (Me-dihydropyr-
imidine). TOF-HRMS: calcd for C29H30N2NaO11 [MþNa]þ, 605.1744;
found 605.1744.

J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739
737
4.4.3. 11c:((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(5-(4-fluorobenzoyl)-2-
methoxyphenoxy)acetate
(300 MHz, CDCl3)
d
(ppm): 8.36 (s, 1H, NH), 7.55 (dd, J ¼ 8.5, 2.2 Hz,
1H, ArH), 7.47 (d, J ¼ 2.3 Hz, 1H, ArH), 7.43 (d, J ¼ 1.7 Hz, 1H, ArH),
7.27e7.35 (m, 2H, ArH), 7.24 (s, 1H, pyrimidine C6eH), 6.99 (brs, 1H,
10-H), 6.90 (d, J ¼ 8.5 Hz, 1H, ArH), 6.25 (d, J ¼ 6.0 Hz, 1H, 30-H), 5.93
(d, J ¼ 5.9 Hz, 1H, 20-H), 5.07 (s, 1H, 40-H), 4.78 (s, 2H, CH2), 4.54 (dd,
J ¼ 12.2, 4.2 Hz, 1H, 50-H), 4.36 (dd, J ¼ 12.2, 3.0 Hz, 1H, 500-H), 3.94
(s, 3H, OCH3), 1.91 (s, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz,
Off-white powder solid, yield, 80%; mp 180e181 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
(ppm): 8.44 (s, 1H, NH), 7.78 (dd, J ¼ 8.7, 5.4 Hz,
2H, ArH), 7.43 (dd, J ¼ 8.4, 1.9 Hz, 1H, ArH), 7.37 (d, J ¼ 1.9 Hz, 1H,
ArH), 7.22 (d, J ¼ 1.2 Hz,1H, pyrimidine C6eH), 7.16 (t, J ¼ 8.6 Hz, 2H,
ArH), 7.00e6.96 (m, 1H, 10-H), 6.94 (d, J ¼ 8.4 Hz, 1H, ArH), 6.24 (d,
J ¼ 6.0 Hz, 1H, 30-H), 5.91 (d, J ¼ 6.0 Hz, 1H, 20-H), 5.06 (s, 1H, 40-H),
4.78 (s, 2H, CH2), 4.52 (dd, J ¼ 12.2, 4.4 Hz, 1H, 50-H), 4.36 (dd,
J ¼ 12.2, 3.1 Hz, 1H, 500-H), 3.95 (s, 3H, OCH3), 1.90 (d, J ¼ 0.9 Hz, 3H,
CDCl3)
d (ppm): 191.77 (C]O, diphenyl ketone), 168.11 (OeC]O),
163.66 (NHeC]O,dihydropyrimidine), 154.76 (NeC]O,dihy-
dropyrimidine), 150.75, 147.35, 140.18, 135.40 (CH2-dihydropyr-
imidine), 133.92, 132.89, 131.53, 131.50, 130.07, 128.74, 127.73,
127.59,120.54,113.42,111.32 (CH-dihydropyrimidine),110.94, 89.89
(CH, 2-furan), 83.84 (CH, 5-furan), 65.93 (CH2), 65.54 (CH2), 56.18
(OCH3), 12.47 (Me-dihydropyrimidine). TOF-HRMS: calcd for
pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3)
d (ppm): 193.59 (C]
O, diphenyl ketone), 168.27 (OeC]O), 163.68 (NHeC]O,dihy-
dropyrimidine), 153.59 (NeC]O,dihydropyrimidine), 150.75,
147.02, 135.38 (CH2-dihydropyrimidine), 134.09, 134.05-132.83-
132.36, 132.24, 129.90, 127.60, 126.78, 115.57, 115.28, 115.02, 111.29
(CH-dihydropyrimidine), 110.65, 89.92 (CH, 2-furan), 83.85 (CH, 5-
furan), 66.05 (CH2), 65.50 (CH2), 56.11 (OCH3), 12.45 (Me-dihy-
dropyrimidine). TOF-HRMS: calcd for C26H23FN2NaO8 [MþNa]þ,
533.1331; found 533.1333.
C
26H22BrClN2NaO8 [MþNa]þ, 627.0145; found 627.0145.
4.4.7. 11g: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(2-methoxy-5-(2-
methoxybenzoyl)phenoxy)acetate
Off-white solid, purified by flash chromatography (PE/EtOAc,
1:1); yield, 77%; mp 170e171 ꢀC; 1H NMR (300 MHz, CDCl3)
4.4.4. 11d:((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(5-(4-bromobenzoyl)-2-
methoxyphenoxy)acetate
d
(ppm): 8.54 (s, 1H, NH), 7.54e7.41 (m, 2H, ArH), 7.35 (dd, J ¼ 8.4,
1.9 Hz, 1H, ArH), 7.31e7.27 (m, 1H, ArH), 7.26 (d, J ¼ 1.2 Hz, 1H,
pyrimidine C6eH), 7.09e6.98 (m, 3H, ArH and 10-H),6.86 (d,
J ¼ 8.5 Hz, 1H, ArH), 6.24 (d, J ¼ 6.0 Hz, 1H, 30-H), 5.91 (d, J ¼ 5.8 Hz,
1H, 20-H), 5.06 (s, 1H, 40-H), 4.77 (s, 2H, CH2), 4.53 (dt, J ¼ 13.4,
4.9 Hz, 1H, 50-H), 4.34 (dd, J ¼ 12.3, 3.0 Hz, 1H, 500-H), 3.91 (s, 3H,
OCH3), 3.75 (s, 3H, OCH3), 1.90 (d, J ¼ 0.8 Hz, 3H, pyrimidine
C5eCH3). TOF-HRMS: calcd for C27H26N2NaO9 [MþNa]þ, 545.1533;
found 545.1533.
Off-white powder solid, yield, 80%; mp 177e179 ꢀC; 1H NMR
(300 MHz, CDCl3)
d (ppm): 8.45 (s, 1H, NH), 7.62 (s, 4H, ArH), 7.42
(dd, J ¼ 8.4, 1.9 Hz, 1H, ArH), 7.37 (d, J ¼ 1.9 Hz, 1H, ArH), 7.22 (d,
J ¼ 1.1 Hz, 1H, pyrimidine C6eH), 7.01e6.96 (m, 1H, 10-H), 6.94 (d,
J ¼ 8.4 Hz, 1H, ArH), 6.24 (d, J ¼ 6.0 Hz, 1H, 30-H), 5.91 (d, J ¼ 5.6 Hz,
1H, 20-H), 5.06 (s, 1H, 40-H), 4.78 (s, 2H, CH2), 4.52 (dd, J ¼ 12.2,
4.4 Hz, 1H, 50-H), 4.36 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500-H), 3.95 (s, 3H,
OCH3), 1.90 (d, J ¼ 0.8 Hz, 3H, pyrimidine C5eCH3). 13C NMR
4.4.8. 11h: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-2,5-dihydrofuran-2-yl)-methyl 2-(5-(2-chlorobenzoyl)-2-
methoxyphenoxy)acetate
(75 MHz, CDCl3)
d (ppm): 192.87 (C]O, diphenyl ketone), 167.22
(OeC]O), 162.71 (NHeC]O,dihydropyrimidine), 152.73 (NeC]
O,dihydropyrimidine), 149.76, 146.03, 135.62 (CH2-dihydropyr-
imidine), 134.35, 131.79, 130.54, 130.25, 128.57, 126.61, 126.02,
125.86, 113.89, 110.26, 109.66 (CH-dihydropyrimidine), 88.89 (CH,
2-furan), 82.82 (CH, 5-furan), 65.00 (OCH2), 64.49 (OCH2), 55.11
(OCH3), 11.45 (Me-dihydropyrimidine). TOF-HRMS: calcd for
Off-white powder solid, yield, 78%; mp 180e181 ꢀC; 1H NMR
(300 MHz, CDCl3) d (ppm): 8.53 (s, 1H, NH), 7.48e7.38 (m, 3H, ArH),
7.29e7.37 (m, 3H, ArH), 7.25 (d, J ¼ 1.0 Hz, 1H, pyrimidine C6eH),
7.01e6.96 (m, 1H, 10-H), 6.89 (d, J ¼ 8.5 Hz, 1H, ArH), 6.24 (d,
J ¼ 6.0 Hz, 1H, 30-H), 5.92 (d, J ¼ 5.7 Hz, 1H, 20-H), 5.06 (s, 1H, 40-H),
4.78 (s, 2H, CH2), 4.54 (dd, J ¼ 12.2, 4.2 Hz, 1H, 50-H), 4.35 (dd,
J ¼ 12.2, 3.1 Hz, 1H, 500-H), 3.93 (s, 3H, OCH3), 1.91 (d, J ¼ 0.8 Hz, 3H,
C
26H23BrN2NaO8 [MþNa]þ, 593.0537; found 593.0537.
4.4.5. 11e: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(2-methoxy-5-(4-
nitrobenzoyl)phenoxy)acetate
pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3)
d (ppm): 193.53 (C]
O, diphenyl ketone), 168.18 (OeC]O), 163.63 (NHeC]O,dihy-
dropyrimidine), 154.44 (NeC]O,dihydropyrimidine), 150.72,
147.22, 138.55, 135.44 (CH2-dihydropyrimidine), 132.93, 131.05,
131.00, 130.02, 129.36, 128.85, 127.62, 127.54, 126.71, 113.52, 111.31
(CH-dihydropyrimidine), 110.85, 89.86 (CH, 2-furan), 83.84 (CH, 5-
furan), 65.95 (CH2), 65.50 (CH2), 56.12 (OCH3), 12.44 (Me-dihy-
dropyrimidine). TOF-HRMS: calcd for C26H23ClN2NaO8 [MþNa]þ,
549.1035; found 549.1035.
Yellow powder solid, yield, 78%; mp 170e171 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
(ppm): 8.40 (s, 1H, NH), 8.31 (d, J ¼ 8.6 Hz, 2H,
ArH), 7.87 (d, J ¼ 8.6 Hz, 2H, ArH), 7.40 (dd, J ¼ 8.4, 1.9 Hz, 1H, ArH),
7.35(d, J ¼ 1.9 Hz,1H, ArH), 7.20 (s, 1H, pyrimidine C6eH), 7.02e6.93
(m, 2H, 10-H and ArH), 6.22 (d, J ¼ 6.0 Hz, 1H, 30-H), 5.89 (d,
J ¼ 5.6 Hz, 1H, 20-H), 5.05 (s, 1H, 40-H), 4.75 (s, 2H, CH2), 4.51 (dd,
J ¼ 12.2, 4.3 Hz, 1H, 50-H), 4.34 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500-H), 3.93
(s, 3H, OeCH3), 1.88 (s, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz,
4.5. Cell proliferation assays
CDCl3)
d (ppm): 193.12 (C]O, diphenyl ketone), 168.26 (OeC]O),
163.79 (NHeC]O,dihydropyrimidine), 154.49 (NeC]O,dihy-
dropyrimidine), 150.84, 149.69, 147.43, 143.50, 135.46 (CH2-dihy-
dropyrimidine), 132.85, 130.46, 128.91, 127.79, 127.43, 123.60,
114.80, 111.32 (CH-dihydropyrimidine), 110.88, 90.09 (CH, 2-furan),
83.96 (CH, 5-furan), 66.11 (CH2), 65.65 (CH2), 56.32 (OCH3), 12.59
The antiproliferative activity evaluation was conducted by using
a modified procedure as described in the literature [24]. 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 mLꢂ1 with the complete medium, 100
mL of the ob-
(Me-dihydropyrimidine). TOF-HRMS: calcd for
C26H23N3NaO10
tained 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 antiproliferation assess-
ment. Tested samples at pre-set concentrations were added to 6
wells with Doxorubicin (AMD) co-assayed as a positive reference.
[MþNa]þ, 560.1276; found 560.1276.
4.4.6. 11f: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-2,5-dihydrofuran-2-yl)-methyl 2-(5-(5-bromo-2-
chlorobenzoyl)-2-methoxyphenoxy)acetate
After 48 h exposure period, 25
MTT was added to each well. After 4 h, the medium was replaced by
m
L of PBS containing 2.5 mg mLꢂ1 of
Off-white powder solid, yield, 80%; mp 168e169 ꢀC; 1H NMR

738
J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739
150
m
L DMSO to dissolve the purple formazan crystals produced.
Sciences, and the cells were cultured in RPMI-1640 medium, which
was supplemented with 10% heat-inactivated fetal bovine serum,
100 U/mL penicillin and 100 U/mL streptomycin and cultured in an
atmosphere of 5% CO2 at 37 ꢀC. Cells were collected for the exper-
iments in the logarithmic growth phase. To establish the tumor-
bearing mouse model, the cell lines were harvested and inocu-
lated subcutaneously into the right armpit region of the mice. On
the 7th day, the tumor ascrites were obtained and washed with
sterile PBS. Under sterile condition, the tumor ascrites were diluted
with sterile normal saline to 1 ꢁ 1010/L cell suspension. Tumor
ascites were maintained in vivo in mice by transplantation of 0.2 mL
of ascites (2 ꢁ 106 cells) from the infected mice to the non-infected
mice.
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's test.
The IC50 value was defined as the concentration at which 50% of the
cells could survive.
4.6. Apoptosis analysis
Apoptosis was detected by flow cytometric analysis of annexin V
staining. Annexin V-FITC/PI assay was performed as previously
described. Briefly, adherent SGC-7901 cells were harvested and
suspended in the annexin-binding buffer (1 ꢁ 106 cells/mL). Then,
cells were incubated with V-FITC buffer and incubated with
annexin V-FITC for 15 min and then stained by PI for 5 min at 4 ꢀC in
the dark and immediately analyzed by flow cytometry. The data are
presented as biparametric dot plots showing PI red fluorescence vs
annexin V-FITC green fluorescence.
4.9.2. Tumor xenograft model in vivo
Each Kunming mouse (male or female, weight 20 2 g) were
inoculated with seven-day-old ascrite (0.2 mL, 2 ꢁ 106 cells) sub-
cutaneously into the right front armpit. 24 h after implantation of
tumor cells, the mice were randomly divided into four test groups
with 10 mice per group. Each mouse was weighed immediately
after inoculation. The mice were treated by oral gavage with test
samples (60 mg/kg/day) or normal saline (NS, 10 mL/kg) for ten
days once daily. On day 11, the mice were sacrificed via cervical
dislocation, and the mouse and tumor were excised and weighed
for evaluating the tumor growth inhibition. The tumor inhibitory
rate was calculated by the following formula:
4.7. MMP assays
Exponentially growing gastric cancer cells were cultured in six-
well plates (2 ꢁ 105/well) and treated with 1, 2, 4
mM compound 8d
for 48 h. After incubation, adherent cells were detached with 0.5%
trypsin. The detached and suspended cells were harvested in the
DMEM medium. Then, cells were stained with JC-1 in a humidified
incubator with 5% CO2, 37 ꢀC for 0.5 h in the dark. At last cells were
washed in JC-1 dyeing buffer twice and analyzed by FACSVERSE
(BD) Flow Cytometer.
ꢀ
ꢁ
Wcontrol ꢂ Wtreated
Tumor inhibitory rate rate ð%Þ ¼
ꢁ 100%
Wcontrol
(1)
4.8. Cell cycle analysis
Wcontrol and Wtreated were the average tumor weights of the control
and treated mice, respectively.
The distribution of cells at specific cell cycle stages was evalu-
ated by Flow Cytometry. 2 ꢁ 105 cells/well were cultured in six-well
plates and treated with 1, 2, 4 mM compound 8d in a humidified
incubator with 5% CO2, 37 ꢀC for 48 h, and cells incubated in me-
dium without compound 8d served as controls. After incubation,
adherent cells were detached with 0.5% trypsin. The detached and
suspended cells were harvested in complete medium and centri-
fuged at 300 g for 5 min. Pellets were washed with PBS twice and
fixed with ice cold 70% ethanol for 1 h at ꢂ20 ꢀC, treated with
RNAse and subsequently stained with propidium iodide. The cells
were analyzed by FC 500 (BECKMAN) Flow Cytometer analyzer.
4.10. Telomerase activity assays
Compounds 8a, 8c, 8d and 11f were tested in a search for small
molecule inhibitors of telomerase activity by using the TRAP-PCR-
ELISA assay. In detail, the SGC-7901 cells were firstly maintained
in RPMI 1640 buffer (Hyclone, Miami, FL, USA), supplemented with
10% fetal bovineserum (GIBCO, New York, 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 0.082 l g/mL, respectively. After 24 h
treatment, the cells were harvested by cell scraper orderly
4.9. Evaluation of the antitumor activities in vivo
4.9.1. Animals and cell lines
Kunming mice (SPF, male or female, 20 2 g) were purchased
from the experimental animal center of China Pharmaceutical
University. Animals were housed in a temperature (22 2 ꢀC) and
relatively humidity (50%)-controlled room on a 12 h light/dark
cycle, given free access to food and water, and acclimatized for at
least one week prior to use. All the animal experiments were per-
formed in accordance with the Regulations of the Experimental
Animal Administration issued by the State Committee of Science
and Technology of China. Efforts were made to minimize the
number of animals used and their suffering. Animals were main-
tained in accordance with the Guides of Center for Developmental
Biology, Anhui Medical University for the Care and Use of Labora-
tory Animals and all experiments used protocols approved by the
institutions' subcommittees on animal care. Cell lines used for
evaluation of the in vivo antitumor activity in this study included
two tumor cell lines, namely S180 (sarcoma tumor cell line), HepG2
(liver carcinoma cell line). All of cell lines were purchased by the
Shanghai Institutes for Biological Sciences, Chinese Academy of
following by washed once with PBS. The cells were lysed in 150 mL
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 ꢂ80 ꢀC.
The TRAP-PCR-ELISA assay was performed using a telomerase
detection kit (Roche, Basel, Switzerland) according to the manu-
facturer's protocol. In brief, 2
48
mL of cell extracts were mixed with
m
L TRAP reaction mixtures. TRAP primers and Taq polymerase
and incubated at 25 ꢀC for 30 min. PCR was then initiated at 94 ꢀC,
120 s for predenaturation and performed using 35 cycles each
consisting of 94 ꢀC for 30 s, 50 ꢀC for 30 s, 72 ꢀC for 90 s. Then 20
mL
of PCR products were hybridized to 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

J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739
739
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fragments 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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The authors wish to thank the National Natural Science Foun-
dation of China (No. 21272008, 21572003), Science and Techno-
logical Fund of Anhui Province for Outstanding Youth
(1408085J04).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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