Synthesis and biological evaluation of disubstituted amidoxanthones as potential telomeric G-quadruplex DNA-binding and apoptosis-inducing agents

Article abstract of DOI:10.1016/j.bmc.2015.12.025

A series of disubstituted xanthones was obtained by cationic modification of xanthone's C2 and C7 with amine groups of different pKa values. Modified structures by using moieties with high pKa values had good antitumor activity according to the MTT assay, AO/EB staining and flow cytometry assay, especially bis-dimethylamine derivative (5a). Further study indicated that compound 5a had good binding activity to telomeric G-quadruplex DNA, as detected by using spectroscopy methods, melting profiles, polymerase chain reaction stop assay and molecular modeling study. The results suggested that the antitumor activity of 5a might be associated with its stabilization of G-quadruplex DNA, which could be developed as new G-quadruplex DNA stabilizer and potent antitumor agents.

Full text of DOI:10.1016/j.bmc.2015.12.025

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of disubstituted amidoxanthones
as potential telomeric G-quadruplex DNA-binding
and apoptosis-inducing agents
⇑
Rui Shen , Yujiao Chen, Ziqian Li, Hui Qi, Yitian Wang
College of Pharmaceutical Sciences, Key Laboratory of Pharmaceutical Quality Control of Hebei Province, Hebei University, Baoding 071002, People’s Republic of 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 disubstituted xanthones was obtained by cationic modification of xanthone’s C2 and C7 with
amine groups of different pKa values. Modified structures by using moieties with high pKa values had
good antitumor activity according to the MTT assay, AO/EB staining and flow cytometry assay, especially
bis-dimethylamine derivative (5a). Further study indicated that compound 5a had good binding activity
to telomeric G-quadruplex DNA, as detected by using spectroscopy methods, melting profiles, poly-
merase chain reaction stop assay and molecular modeling study. The results suggested that the antitumor
activity of 5a might be associated with its stabilization of G-quadruplex DNA, which could be developed
as new G-quadruplex DNA stabilizer and potent antitumor agents.
Received 2 November 2015
Revised 12 December 2015
Accepted 15 December 2015
Available online xxxx
Keywords:
Xanthone
Antitumor evaluation
Apoptosis
Ó 2015 Elsevier Ltd. All rights reserved.
G-quadruplex
1
. Introduction
G-quadruplexes are viewed as emerging therapeutic targets in
ity.14–17 The previous results indicated that xanthones based on
symmetrical three-membered ring scaffolds showed antiprolifera-
tive activity in vitro. Furthermore, the different alkyl substitute
groups in xanthones would affect the DNA binding mode and affin-
1
oncology. These special secondary DNA structures are formed
with a minimum of four guanine-rich repeats in DNA or RNA
sequences. They consist of a stack of planar guanine tetramers
called G-quartets that are stabilized by Hoogsteen hydrogen bonds
and monovalent cation coordination.2,3 If telomeres that comprise
large numbers of simple guanine-rich tandem repeats at the ends
of eukaryotic chromosomes could form G-quadruplex structures,
telomerase which is activated in 85–90% of cancer cells and inac-
tive in normal cells, will not catalyse telomeres extension and
maintain cellular immortalization. Therefore, inhibition of oncoge-
nesis and progression through stabilization of G-quadruplex struc-
tures could be a novel anticancer strategy.4 More and more
researchers have paid attentions to develop molecular ligands for
stabilizing G-quadruplex structures to inhibit the activity of telom-
1
8
ity, for example, dimethylamine substitute would bind the DNA
by electrostatic interaction with the negative grooves and showed
1
9
good biological activity. In order to further improve the antitu-
mor activity and reduce the cytotoxicity to normal cells, a series
of disubstituted amidoxanthones with two terminal amino sub-
stituents at C2 and C7 positions on the xanthone ring were synthe-
sized. These compounds comprise xanthone scaffold, which may
form p–p stacking interaction with guanine tetrads, and two side
chains with amine group at the chains termini, which may form
electrostatic interaction with the negative grooves of quadruplex
DNA. Consequently, the xanthone derivatives synthesized may be
as potent G-quadruplexes stabilizers and antitumor agents.
–6
7
–11
erase in vitro.
2
. Chemistry
Xanthone derivatives, which have planar tricyclic chromophore
frameworks (Fig. 1), may form interactions with guanine tetrads of
quadruplex DNA. Group of substituted xanthone derivatives exhi-
bits excellent antitumor activity and has been developed as good
G-quadruplexes stabilizers and antitumor agents, such as
Xanthone (1) was prepared according to the literatures20,21 with
some improvements. Nitration of xanthone and reduction of nitro
to amino was carried out by following known procedures.2
Acetylation of 2,7-diaminoxanthone (3) by chloroacetyl chloride
was stirred at room temperature until TLC indicated completion
of reaction to afford 2,7-bis(2-chloroacetamido)-xanthone (4).24
Then 4 reacted with secondary amine in ethanol to obtain the
2,23
1
2
13
HXO2pip and HELIXA4C. We have been focusing on searching
and developing xanthone derivatives with potent antitumor activ-
2
5
target molecules 5a–5e (Scheme 1).
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmc.2015.12.025
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
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2
R. Shen et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
revealed that compounds 5a–5e could induce the apoptosis of
tumor cells. The dosedependent relationship existed in the mor-
phologic features of the apotoic HeLa cells, which was consistent
with the previous MTT assays.
Figure 1. Xanthone.
3
.1.3. Flow cytometry analysis
In view of the aforementioned results, compound 5a was
selected as the representative to analyze the apoptosis ratios quan-
titatively by using fluorescein isothiocyanate (FITC)-conjugated
Annexin V and propidium iodide (PI) double staining method on
3
3
3
. Result and discussion
.1. Biological activity assay
2
+
a flow cytometer. Annexin V, a Ca dependent phospholipid-bind-
ing protein had a high affinity for the membrane phospholipid
phosphatidylserine (PS), that was translocated from the inside to
the outside of the cell membrane in the early stage of apoptosis.
And the membranes of dead and damaged cells could be perme-
able to PI. Accordingly, cells stained with Annexin V-FITC and PI
could be classified as intact cells (the lower left quadrant;
.1.1. In vitro antitumor potency
The antitumor potency of xanthones against four tumor cell
lines including HeLa (cervical cancer), A549 (lung cancer), SGC-
901 (gastric cancer) and MCF-7 (breast cancer) cells were evalu-
7
ated by MTT assay.
The inhibitory effect of compounds 5a–5e was increased in a
dosedependent manner as illustrated in Figure 2 and the IC50 val-
ues were listed in Table 1. It is evident from the results that xan-
thones showed certain inhibition activity against four human
tumor cell lines. By comparing the IC50 values, compounds 5a–5c
could inhibit growth of four cancer cells efficiently. Especially,
the compound 5a exhibited the highest and most uniform level
of antiproliferative activity, with an IC50 value < 25 lM for all
tested cell lines. The other compounds 5d and 5e had relatively
weak inhibitory activity toward four cancer cells. Probably because
ꢀ
ꢀ)
Annexin /PI , early apoptotic cells (the lower right quadrant;
+
ꢀ
Annexin /PI ), late apoptotic cells (the upper right quadrant;
+
+
ꢀ
Annexin /PI ) or necrotic cells (the upper left quadrant; Annexin /
PI ).
+
29
As shown in Figure 4, the percentages of the late apoptotic cells
increased from 0.36% to 60.78%, while the percentages of the early
apoptotic cells were also increased from 0.11% to 32.07%. It illus-
trated that compound 5a could induce apoptosis of HeLa cells obvi-
ously, and the apoptosis ratios increased in a dosedependent
manner. AO/EB staining and flow cytometry analysis results sug-
gested that compound 5a inhibited HeLa cell growth may be via
induction of cell apoptosis.
the amine substituting groups of compounds 5a–5e had different
pKa values (Table S1, Supplementary material)2
6,27
and configura-
tion, which might affect the relative properties of compounds, thus
they showed the different antiproliferative activity.
3
3
.2. Measurement of G-quardruplex binding ability
3
.1.2. Morphological analysis by AO/EB staining
To determine whether the inhibitory activity of xanthones was
.2.1. CD spectra and CD melting profiles
CD spectra technique is a highly sensitive method for determin-
related to the apoptosis-inducing effect, morphological assay of
cell death was performed with AO/EB staining method. AO could
pass through cell membrane, but EB could not,28 therefore,
AO/EB was usually applied to investigate whether cells underwent
cell death via apoptosis and /or necrosis.
Effect of xanthones on the morphological changes of HeLa cells
was illustrated in Figure 3. HeLa cells exhibited the typical apopto-
sis features, such as obvious chromatin condensation and slight
ing the effect of small molecules binding on DNA conformation. It
has been reported that HTG21 has formed a hybrid-type of quadru-
plex DNA containing parallel and anti-parallel structure in the
+
presence of K , with a large positive band at 290 nm, a shoulder
around 270 nm, a small positive band at 255 nm, and a minor neg-
+
ative band near 234 nm. While in the presence of Na , HTG21 has
formed an anti-parallel structure with a positive band around
3
0,31
2
95 nm and a negative band around 265 nm.
Upon the addition of compounds 5a–5e to the HTG21 solution
nuclear fragmentation (yellow green), at 10
lM 5a or 20 lM 5b–
5
e treatment. With increasing xanthones concentration, apoptotic
+
with K , the CD spectra showed significant changes, with a max-
imum band at 292 nm increased and shifted toward 289 nm, and
the shoulder at 270 nm decreased and merged into the band at
cells with typical nuclear fragmentation (orange) were increased,
whereas the control cells did not appear visible changes in cell
nucleus and cell membrane integrity. The results of AO/EB staining
Scheme 1. Reagents and conditions: (i) CuO/K
reflux.
2
CO
3
, reflux; (ii) POCl
3
, reflux; (iii) HNO
3
, H
2
SO
4
; (iv) Na
2
Sꢁ9H
2 2 2
O, H O, 70 °C; (v) chloroacetyl chloride; (vi) R NH, EtOH, NaI,
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R. Shen et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
Figure 2. Inhibitory effects of compounds 5a–5e (0, 2.5, 5, 10, 20, 40 and 80
lM) on human tumor cells proliferation in vitro. Error bars represent the standard deviation.
2
89 nm. Meanwhile, the small positive band at 255 nm decreased
agreement with the above antitumor potency evaluation results.
We presumed that dimethylamine moiety at the chains termini
of the compound 5a, which had high pKa value (Table S1, Supple-
mentary material) and a simple structure was more likely to be
protonated and developed a cationic charge, forming electrostatic
interaction with the negative grooves of quadruplex DNA easily.
and a minor negative band near 240 nm increased (Fig. 5a). These
changes indicated that HTG21 was affected upon interaction with
the compounds, then transformed into other conformations and
stabilized by compounds. Especially, compound 5a showed the
most visible effect on HTG21 conformation. The results were in
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4
R. Shen et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
base pairs of DNA would lead to bathochromic shifts and
The IC50 values of compounds 5a–5e against various human tumor cells
15–18
hypochromicity on the electronic absorption spectra.
To eval-
Compounds
IC50
(l
M)
uate the G-quadruplexes binding capacity of disubstituted amidox-
anthones, the representative spectra of 5a in the absence and
presence of G-quadruplex DNA were investigated. As shown in Fig-
ure 7, in the absence of G-quadruplex, compound 5a showed a
HeLa
A549
SGC-7901
MCF-7
5
5
5
5
5
a
b
c
d
e
13.22
20.43
48.22
>100
>100
18.74
19.9
21.5
80.53
>100
17.29
32.24
35.86
84.85
86.65
22.98
26.61
34.91
>100
strong
p
–
p
⁄ transitions band around 210 nm and a medium n–
p⁄ transitions band around 265 nm. Upon addition of G-quadru-
83.94
plex, the absorption band around 210 nm exhibited visible
hypochromicities. It suggested that the xanthone scaffold of com-
pound 5a probably formed
tetrads of the G-quadruplex DNA.
From the absorption data, the binding constant Kb was deter-
mined by using the following Eq. 1.
p–p stacking interaction with guanine
As further proof, titration of HTG21 with compound 5a in the
+
+
presence of K or Na was performed. The results were illustrated
+
in Figure 5b and c. Presence of K , the CD spectra with a maximum
positive band increased and shifted, and the shoulder at 270 nm
gradually decreased and merged into the band at 290 nm (Fig. 5b).
While presence of Na , the CD spectra changed as well, with the
½DNAꢂ=ð a
e
ꢀ
ef Þ ¼ ½DNAꢂ=ð b
e
ꢀ
ef Þ þ 1=Kbð b
e
ꢀ
ef
Þ
ð1Þ
+
positive and negative band both increased, suggesting that an
anti-parallel structure may be stabilized by compound 5a (Fig. 5c).
where [DNA] was the concentration of DNA in base pairs, the appar-
ent absorption coefficient ( ) was obtained by calculating Aobsd
compound]. The terms and correspond to the extinction
coefficient of free and the fully bound compounds, respectively.
In plots of [DNA]/( ) versus [DNA], K was given by the ratio
of slope to the intercept. (Fig. 7 inset). The binding constant K for
e
a
/
2
95 nm was chosen to study the influence of the compound on
[
e
f
e
b
the stability of G-quadruplex DNA. It was consistent with previous
33
3
2
reports. The normalized CD intensity of HTG21 with 5 lM com-
e
a
ꢀ
e
f
b
pound 5a in buffer at 295 nm versus the temperature was showed
b
in Figure 6. The transition temperature of the G-quadruplex
4
ꢀ1
compound 5a was (2.89 ± 0.12) ꢃ 10 M . While the absorption
of 5a exhibited weak hypochromicities around 210 nm with
increasing duplex DNA concentration (Fig. S1, Supplementary
+
increased from 55.1 to 60.8 °C in Na buffer and increased from
+
6
4.6 to 70.3 °C in K buffer, at a concentration ratio [compound]/
[
DNA] = 1:1. The melting temperature increases indicated a stabi-
material). And the binding constant K
(
b
to duplex DNA was
0.46 ± 0.09) ꢃ 10 M , The results indicated that 5a had better
G-quadruplex DNA binding ability than duplex DNA.
lizing effect. These data indicated compound 5a could form stable
interactions with G-quadruplexes resulting in the formation of
bound compound whose thermal stabilities were markedly
enhanced from native G-quadruplexes.
4
ꢀ1
3
.2.3. Fluorescence emission spectra
Concomitant with the hypochromism in the absorption spectra
3
.2.2. Electronic absorption spectra
Generally, when xanthone derivatives intercalated into DNA,
of compound 5a upon addition of HTG21, an obvious fluorescent
emission decreases at 450 nm occured (Fig. 8). This phenomenon
might be attributed to the electrostatic quenching. Because the
phosphate groups of G-quadruplex DNA with a negatively charge
may form a polyanion. And the amine moiety at the chains termini
of 5a was more likely to be protonated and develop a cationic
charge, which could be quenched by the high negative charge of
G-quadruplex DNA easily. While upon addition duplex DNA, the
fluorescent emission of compound 5a exhibited a small decreases
at 450 nm, showing a weak surface binding (Fig. S2, Supplemen-
tary material).
the stacking interaction between aromatic chromophore and the
3
.2.4. NMR spectroscopy
In order to further investigate the binding property between
xanthone derivatives and G-quadruplex at a molecular level, 1
H
NMR experiment was carried out with an intermolecular four-
stranded G-quadruplex DNA formed from [d(TTAGGG)]
(HT-6)34
a single repeat sequence of the human telomere. Figure 9 showed
4
,
1
4
the H NMR spectra of [d(TTAGGG)] in the downfield-shifted
region at 25 °C. The imino protons signals assigned to G4–G6 were
resolved in the 10–12 ppm, which belonged to Hoogsteen-bound
guanine imino proton of the G quartets. These imino protons sig-
nals exhibited line broadening and upfield shifts with an increasing
compound 5a concentration, suggesting that
formed between the aromatic surface of 5a and the terminal G-tet-
rad(s) of the [d(TTAGGG)] . The changes were similar to those of
p–p interaction
4
the known G-quadruplex ligands with a planar aromatic ring for
which these signals shift upfield upon addition of ligands.35
3
.2.5. PCR-stop assay
The amplification of HTG21 telomeric G-quadruplex sequence
in vitro by using PCR-stop assay provide the further support for
quadruplex DNA may be stabilized by compound 5a. With the
Figure 3. Fluorescent microscopic observation of HeLa cells treated with
compounds 5a–5e (0, 5, 10, 20 and 40 lM) for 24 h with AO/EB staining.
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R. Shen et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 4. Apoptosis rate of HeLa cells. Cells were treated with 0, 10, 20, 40
lM of compound 5a for 24 h and stained with Annexin V-FITC and PI, followed by flow cytometry
analysis.
Figure 5. CD spectra of HTG21 + compounds 5a–5e (2 Mol equiv) in K buffer (a); HTG21 + compound 5a (0–1 Mol equiv) in K+ buffer (b); HTG21 + compound 5a
+
(
0–1 Mol equiv) in Na+ buffer (c).
Figure 6. Normalized CD melting curves for G-quadruplex DNA (N), G-quadruplex
Figure 7. Electronic absorption spectra of compound 5a upon addition of HTG21.
compound 5a] = 5 M, [HTG21] = 0, 1, 2, 3, 4, 5 M. The arrow indicates the
absorbance decreases upon increasing DNA concentration. The inset is plot of
DNA]/( ) versus [DNA] for the titration of DNA to compound 5a.
+
DNA + 5a (j), in Na buffer (pH 7.4); G-quadruplex DNA (d), G-quadruplex DNA
[
l
l
+
+
5a (.), in K buffer (pH 7.4). [HTG21] = 5
l
M, [compound 5a] = 5
lM. The stability
of G-quadruplex DNA was assessed by CD at 295 nm.
[
b f
e ꢀe
(Fig. 10). The results further demonstrated that compound 5a
increasing concentration of compound 5a, the final double-
stranded DNA PCR product decreased, which indicated that 5a sta-
bilized the structure of HTG21 G-quadruplex and inhibited PCR
amplification (Fig. 10). A control experiment with an oligomer
including two mutations in one of the guanine repeats (HTG21mu)
was performed to confirm whether the compound would affect Taq
DNA polymerase. In that case, no inhibition was observed at the
highest concentration under the same reaction conditions
was able to stabilize the structures of HTG21 G-quadruplexes.
3.3. Molecular modeling
Compound 5a was selected for molecular modeling study to
understand the binding mode between the G-quadruplex DNA
and the xanthone derivatives better. The human telomeric G-
quadruplex structure (PDB ID: 1KF1) was used as an initial model.
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6
R. Shen et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
For simplification, the ratio of 5a and G-quadruplex was assigned
to 1:1. As shown in Figure 11, 5a interacted with G-quadruplex
with a similar binding mode: the aromatic xanthone scaffold of
5
a was stacked onto the quartet, stabilized by p–p stacking inter-
action, and one side-chain interacted with one of the G-quadruplex
grooves, while the other did not reach another groove. The calcula-
tion showed that the binding free energy
ꢀ
D
G
of 5a was
4.40 kcal mol . The docking study was agreement with the
above results of G-quardruplex binding experiments.
ꢀ1
4
. Conclusions
We have prepared a series of disubstituted xanthones deriva-
tives with terminal ammonium substituents at C2 and C7 positions
on the xanthone ring as G-quardruplex stabilizing anticancer
agents. Most compounds showed good cytotoxicity against cancer
cells. Especially, compound 5a displayed the highest cytotoxicity,
which might be associated with its stabilization of G-quadruplex
DNA. The structure optimization and further mechanism study is
in progress.
Figure 8. Fluorescence emission spectra of 5a upon addition of HTG21
(
5
k
ex = 320 nm, kem = 350–600 nm). [compound 5a] = 5
, 6, 7, 8, 9, 10 M. The arrow indicates the fluorescence decrease upon increasing
DNA concentration. The inset is plot of F/F versus [HTG21]/[compound].
lM, [HTG21] = 0, 1, 2, 3, 4,
l
5
5
. Experimental section
0
.1. Materials and measurements
Microculture tetrazolium (MTT), acridine orange (AO) and
ethidium bromide (EB) were purchased from Sigma–Aldrich Co.
LLC. The Taq DNA polymerase was purchased from New England
Biolabs (Beijing) Ltd. All oligomers/primers and deoxyribonucle-
oside triphosphate (dNTP) were purchased from Sangon Biotech
(
Shanghai) Co. Ltd. Other chemicals were reagent grade without
further purification.
1H and 13C NMR spectra were recorded using a Bruker AVANCE
III 600 spectrometer; Mass spectra (MS) were recorded on an Agli-
ent 1200/6310 instrument with an ESI mass detector. Elemental
analyses were conducted on a Vario EL analyzer. The electronic
absorption spectra were recorded by using a Varian Cary 100 spec-
trophotometer, the fluorescence emission spectra were recorded
by using a Hitachi F-4600 spectrofluorimeter and the circular
Figure 9. 1H NMR spectra of [d(TTAGGG)]
TTAGGG)] ]} = 0, 0.5 and 1 in 0.2 mM [d(TTAGGG)]
buffer (pH 7.0), and 150 mM KCl at 25 °C. Only the imino proton signals are shown.
for which R5a {[compound 5a]/[[d
, 25 mM potassium phosphate
4
(
4
4
dichroic (CD) spectra were recorded by using
a Bio-Logic
MOS-450 spectropolarimeter, respectively. The polymerase chain
reaction (PCR) was performed on a L96G/Y thermal cycler. The
electrophoresis bands of DNA were imaged by using a Tanon-
1600 gel imager under UV light. The absorbance of MTT assay was
recorded on a Bio-Tek Microplate Reader. Morphological analysis
was observed under an Olympus fluorescence microscope. Flow
Cytometry (FCM) was recorded on FACS Aria II Flow Cytometer.
The measurements of G-quadruplex DNA binding properties
were carried out in Tris–HCl buffer (10 mM Tris and adjusted to
pH 7.4 with HCl) including KCl or NaCl. PCR stop assay was carried
out in Thermopol buffer (20 mM Tris–HCl, 10 mM KCl, 10 mM
Figure 10. Effect of compound 5a (0, 2.5, 5, 7.5, 10, 12.5 and 15 lM) on the
PCR-stop assay with HTG21 or HTG21mu.
(
4 2 4 4
NH ) SO , 2 mM MgSO , 0.1% Triton X-100, pH 8.8). Xanthones
were first dissolved in a minimum amount of DMSO (0.5% of the
final volume) and then diluted with the corresponding buffer to
the required concentrations.
5
.2. General aminolysis procedure of compound (5a–5e)
To a stirred refluxing suspension of 2, 7-bis(2-chloroacetami-
do)-xanthone 4 (113.0 mg, 0.5 mmol) and NaI (100 mg) in EtOH
(
(
10 mL) was added dropwise appropriate secondary amine
1.0 mL) in EtOH (10 mL). The mixture was stirred under reflux
for 6 h, cooled down to room temperature, and diluted with dis-
tilled water. The precipitate was filtered, washed with ether, dried
and evaporated to afford 5a–5e.
Figure 11. Molecular modeling for the compound 5a and G-quadruplex. Pictures
were generated by using PyMOL.
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R. Shen et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
5
.2.1. 2,7-Bis(2-(dimethylamino)acetamido)-xanthone (5a)
and treated with 0, 5, 10, 20 and 40
l
M of compounds 5a–5e,
1
Yelid 89%, white yellow solid. H NMR (600 MHz, CDCl
s, 2H), 8.43–8.41 (dd, 2H), 8.07 (d, 2H), 7.49–7.48 (d, 2H), 3.11 (s,
3
): d 9.40
respectively. After 24 h incubation, cells were stained with AO/EB
dyemix (100 g/mL AO and 100 g/mL EB) for 10 min and then
(
l
l
4
1
4
6
H), 2.40 (s, 12H) ppm; 13C NMR (151 MHz, CDCl
69.08, 152.63, 134.25, 127.32, 121.34, 118.84, 115.40, 63.50,
3
): d 176.61,
washed with PBS to remove background staining. After that, the
apoptotic cells were viewed under a fluorescent microscope.
+
24 4 4
6.04; ESI-MS: m/z 419.9 [M+Na] ; Anal. Calcd [C21H N O ]: C,
3.62; H, 6.10; N, 14.13 Found: C, 63.91; H, 5.93; N, 14.58.
5.3.3. Flow cytometry analysis
5
HeLa cells were plated in 6-well plates at a density of 3 ꢃ 10
5
.2.2. 2,7-Bis(2-(diethylamino)acetamido)-xanthone (5b)
cells per well and grown in DMEM culture medium at 37 °C in a
1
Yelid 93%, white solid. H NMR (600 MHz, CDCl
3
): d 9.70 (s, 2H),
water-atmosphere (5% CO
with 0, 10, 20 and 40 M of compound 5a, respectively. After
24 h incubation, the cells were trypsinized, collected, washed with
cold PBS buffer, resuspended gently in 200 L binding buffer, and
stained with 10 L Annexin V-FITC and 10 L PI in the dark for
20 min. Finally, 300 L binding buffer was added into the samples
2
) for 24 h. Then the cells were treated
8
(
.48–8.47 (d, 2H), 8.07 (s, 2H), 7.52–7.51 (d, 2H), 3.21 (s, 4H), 2.70
l
s, 8H), 1.15–1.13 (t, 12H) ppm; 13C NMR (151 MHz, CDCl3): d
1
5
76.73, 170.47, 152.63, 134.30, 127.29, 121.36, 118.89, 115.16,
7.96, 49.01, 12.41; ESI-MS: m/z 475.7 [M+Na] ; Anal. Calcd
l
l
+
l
[
C
H
25 32
N
4
O
4
]: C, 66.35; H, 7.13; N, 12.38 Found: C, 65.95; H,
l
7
.23; N, 12.55.
to analyze by flow cytometry.
5
.2.3. 2,7-Bis(2-pyrrolidinoacetamido)-xanthone (5c)
5.4. Measurement of G-quardruplex binding ability
Yelid 86%, orange solid. 1H NMR (600 MHz, CDCl
3
): d 9.41 (s,
2
3
H), 8.45–8.43 (dd, 2H), 8.04–8.03 (d, 2H), 7.49–7.48 (d, 2H),
.30 (s, 4H), 2.71 (s, 8H), 1.88 (s, 8H) ppm; 13C NMR (151 MHz,
5.4.1. CD spectra and CD melting profiles
0
0
The oligomer HTG21 (5 -GGGTTAGGGTTAGGGTTAGG G-3 ) at a
CDCl3):d176.79, 169.58, 152.65, 134.30, 127.50, 121.28, 118.84,
final concentration of 5 lM was dissolved in buffer containing xan-
+
1
15.42, 59.66, 54.69, 24.11; ESI-MS: m/z 471.7 [M+Na] ; Anal.
Calcd [C25 ]: C, 66.95; H, 6.29; N, 12.49 Found: C, 66.52;
H, 6.46; N, 12.98.
thones to be tested. The oligomer HTG21 was annealed by heating
at 95 °C for 5 min, and then slowly cooled to room temperature
and incubated at 4 °C overnight. The CD spectra were scanned in
the range of 230–320 nm at 25.0 °C. The optical chamber of the
CD spectrometer was deoxygenated with dry nitrogen before use
and kept in a nitrogen atmosphere during experiments. Scans were
accumulated and automatically averaged. The spectra represented
the average of three scans without the buffer background.
28 4 4
H N O
5
.2.4. 2,7-Bis(2-morpholinoacetamido)-xanthone (5d)
Yelid 87%, white yellow solid. H NMR (600 MHz, CDCl
1
3
): d 9.32
(
3
s, 2H), 8.46–8.44 (dd, 2H), 8.08–8.07 (d, 2H), 7.54–7.52 (d, 2H),
1
3
.84–3.83 (t, 8H), 3.20 (s, 4H), 2.67 (s, 8H) ppm;
C NMR
(
1
151 MHz, CDCl3): d 176.57, 168.27, 152.72, 134.05, 127.52,
21.32, 118.93, 115.52, 66.99, 62.45, 53.91; ESI-MS: m/z 504.0
The CD melting profiles were collected at 295 nm. The temper-
ature ranged from 40 to 90 °C, and the heating rate was 1 °C per
minute. The melting temperature (T ) was defined as the temper-
m
+
[
M+Na] ; Anal. Calcd [C25
H
28
N
4
O
6
]: C, 62.49; H, 5.87; N, 11.66
Found: C, 63.15; H, 5.33; N, 11.98.
ature of the midtransition point.
5
.2.5. 2,7-Bis(2-piperidinoacetamido)-xanthone (5e)
Yelid 88%, yellow solid. 1H NMR (600 MHz, CDCl
): d 9.51 (s,
5.4.2. Electronic absorption spectra
Electronic absorption spectra experiments were performed by
fixing the concentration of compound 5a at 5 M while varying
the concentration of HTG21. The oligomer HTG21 was thermally
treated as described above. Fitting was completed by using an
3
2
3
H), 8.45–8.43 (dd, 2H), 8.05–8.04 (d, 2H), 7.50–7.49 (d, 2H),
.11 (s, 4H), 2.57 (s, 8H), 1.70–1.66 (m, 8H), 1.50 (s, 4H) ppm;
l
1
3
3
C NMR (151 MHz, CDCl ) d:176.71, 169.31, 152.65, 134.32,
1
27.45, 121.37, 118.87, 115.36, 62.76, 55.06, 26.27, 23.62; ESI-
Origin 7.5 spreadsheet, where value of the binding constant K
b
was calculated.
+
MS: m/z 501.6 [M+Na] ; Anal. Calcd [C27
H
32
N
4
4
O ]: C, 68.05; H,
6
5
5
.77; N, 11.76 Found: C, 68.36; H, 6.23; N, 11.38.
5
.4.3. Fluorescence emission spectra
Fluorescence emission spectra experiments were performed by
fixing the concentration of compound 5a at 5 M while varying the
.3. Biological activity assay
l
.3.1. In vitro antitumor potency
concentration of HTG21. The oligomer HTG21 was thermally trea-
ted as described above. Fitting was completed by using an Origin
7.5 spreadsheet.
Standard MTT assay procedures36 were used for assessing the
antiproliferative activity of compounds 5a–5e. DMSO at 0.5% con-
centration in the culture medium was used as the control. Briefly,
HeLa, A549, SGC-7901 and MCF-7 cells were seeded into 96-well
5.4.4. NMR spectroscopy
4
1
plates at density of 5 ꢃ 10 cells per well and grown in DMEM cul-
The H NMR spectra of oligonucleotides were recorded at 25 °C
ture medium at 37 °C in a water-atmosphere (5% CO
2
), respectively.
2 2
in a 90% H O/10% D O solution containing 150 mM KCl and 25 mM
After overnight incubation, the cells were treated with different con-
centrations of xanthones (0, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 M) for
4 h. 10 L MTT (5 mg/mL) was added to each well and incubated
continually at 37 °C for 4 h. The medium was removed and DMSO
was added to dissolve the formazan crystals. Subsequently, absor-
bance was recorded at 570 nm. All experiments were performed in
triplicate and each experiment was repeated at least three times.
Cell growth inhibition rate (%) was calculated using the following
equation: Inhibitory rate (%) = (1ꢀAtreatment/Acontrol) ꢃ 100%.
potassium phosphate buffer (pH = 7.0). Water suppression
was achieved by the Watergate method. The oligonucleotide
d(TTAGGG) was purified by using HPLC, and the concentration
was 0.2 mM for the NMR measurements.
l
4
l
5.4.5. PCR-stop assay
The PCR-stop assay was performed according to the previously
3
7
0
reported method. The forward primer HTG21or HTG21mu (5 -
0
GGGTTAGGGTTAAAGTTAGGG-3 ), and its reverse primer
RevHTG21 (5 -ATCGCTTCTCGTCCCTAACC-3 ) were used in PCR
0
0
5
.3.2. Morphological analysis by AO/EB staining
Morphological analysis was performed by AO/EB dual staining
amplifications. The reaction were performed in 1 ꢃ Thermopol
buffer, containing 0.4 lM of each primer, 0.2 mM of dNTP and
assay. A monolayer of HeLa cells was cultured in 24-well plates
1.5 U DNA Taq polymerase. Reaction mixtures were incubated in
Please cite this article in press as: Shen, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.12.025

8
R. Shen et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
a thermocycler, with the following cycling conditions: 94 °C for
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3
7
min, followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and
2 °C for 30 s. Amplified products were resolved on 15% non-
6
7
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SYBR Green staining.
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1
1
1
The crystal structure of 22-mer telomeric G-quadruplex (PDB
ID: 1KF1, RCSB Protein Data Bank) was used as an initial model
to study the interaction between the xanthone and telomeric
DNA. Docking studies were performed with the AUTODOCK 4.2
program. Water molecules were removed from the PDB file, non-
polar hydrogen atom of the telomeric G-quadruplex were added to
their corresponding carbon atoms, and partial atomic charges were
3
8
1
1
1
3
9
assigned, by using ADT. The dimensions of the active site box,
which was placed at the center of the G-quadruplex, were set to
1
2
6
0 Å ꢃ 60 Å ꢃ 60 Å with the grid points 0.375 Å apart. The Lamar-
ckian genetic algorithm (LGA) was used to perform docking calcu-
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population size: 150), with a maximum number of 25,000,000
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7,000, and a mutation rate of 0.02. 100 independent docking runs
2
2
a
2
2
2
2
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Acknowledgements
We are grateful for the financial support of the Natural Science
Foundation of Hebei Province of China (B2012201132) and the
Scientific Research Foundation of Hebei University (2010-204).
We are also grateful for the kind help of Professor Yali Song and
Guan Zhou on Molecular modeling study.
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