Pyridostatins selectively recognize two different forms of the human telomeric G-quadruplex structures and their anti-tumor activities in vitro

Article abstract of DOI:10.1016/j.tet.2015.05.081

G-quadruplex as a therapeutic target to develop novel anti-cancer agents has attracted a growing interest. Among all the ligands of G-quadruplexes, pyridostatin derivatives play a very important role. Here, we first reported the recognition of the fundamental skeleton pyridostatin I, which was simply synthesized. Compared to pyridostatin II comprising terminal amino groups, pyridostatin I selectively stabilized intramolecular anti-parallel telomeric G-quadruplex by raising the melting temperature about 20 °C at 295 nm of H22, while pyridostatin II preferred to stabilize intermolecular parallel telomeric G-quadruplex by raising the melting temperature about 25 °C at 265 nm of H7, maybe due to the suited size measurements between G-quadruplex hosts and pyridostatin guests. MTT assays indicated that pyridostatin II had better cytotoxic effects against HCT-8 and A549 cell lines obviously, indicating positively charged side chains may be required for improving the water solubility and cellular uptake of the apolar central skeleton.

Full text of DOI:10.1016/j.tet.2015.05.081

Accepted Manuscript
Pyridostatins Selectively Recognize Two Different Forms of the Human Telomeric G-
Quadruplex Structures and Their Anti-tumor Activities in Vitro
Li-Xia Wang, Qian Shang, Qian Li, Jun-Feng Xiang, Yan Liu, Ai-Jiao Guan, Hong-Xia
Sun, Li-Jia Yu, Ya-Lin Tang
PII:
S0040-4020(15)00788-7
10.1016/j.tet.2015.05.081
TET 26799
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 23 March 2015
Revised Date: 19 May 2015
Accepted Date: 22 May 2015
Please cite this article as: Wang L-X, Shang Q, Li Q, Xiang J-F, Liu Y, Guan A-J, Sun H-X, Yu L-J, Tang
Y-L, Pyridostatins Selectively Recognize Two Different Forms of the Human Telomeric G-Quadruplex
Structures and Their Anti-tumor Activities in Vitro, Tetrahedron (2015), doi: 10.1016/j.tet.2015.05.081.
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Pyridostatins Selectively Recognize Two
Different Forms of the Human Telomeric G-
Quadruplex Structures and Their Anti-
tumor Activities in Vitro
Leave this area blank for abstract info.
Li-Xia Wang, Qian Shang, Qian Li, Jun-Feng Xiang, Yan Liu, Ai-Jiao Guan, Hong-Xia Sun, Li-Jia
Yu, Ya-Lin Tang*
State Key laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry, Chinese Academy of
Sciences (ICCAS), Beijing 100190, China.
NH2
O
O
O
N
O
N
O
N
NH
N
HN
N
N
O
NH
HN
O
N
N
Pyridostatin II
Pyridostatin I

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Pyridostatins Selectively Recognize Two Different Forms of the Human
Telomeric G-Quadruplex Structures and Their Anti-tumor Activities in Vitro
Li-Xia Wang a, Qian Shang a, Qian Li a, Jun-Feng Xiang a, Yan Liu a, Ai-Jiao Guan a, Hong-Xia Sun a,
Li-Jia Yu a, Ya-Lin Tang a,*
a Beijing National Laboratory for Molecular Sciences (BNLMS), Center for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and
Stable Species, Institute of Chemistry Chinese Academy of Sciences, Beijing 100190, P.R. China. E-mail: tangyl@iccas.ac.cn; Fax: 86-10-6252-2090; Tel: 86-
10-6252-2090.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
G-quadruplex as a therapeutic target to develop novel anti-cancer agents has attracted a growing
interest. Among all the ligands of G-quadruplexes, pyridostatin derivatives play a very important
role. Here, we first reported the recognition of the fundamental skeleton pyridostatin I, which
was simply synthesized. Compared to pyridostatin II comprising terminal amino groups,
pyridostatin I selectively stabilized intramolecular anti-parallel telomeric G-quadruplex by
raising the melting temperature about 20 oC at 295 nm of H22, while pyridostatin II preferred to
stabilize intermolecular parallel telomeric G-quadruplex by raising the melting temperature
about 25 oC at 265 nm of H7, maybe due to the suited size measurements between G-quadruplex
hosts and pyridostatin guests. MTT assays indicated that pyridostatin II had better cytotoxic
effects against HCT-8 and A549 cell lines obviously, indicating positively charged side chains
may be required for improving the water solubility and cellular uptake of the apolar central
skeleton.
Available online
Keywords:
G-quadruplex
recognition
stabilization
pyridostatin derivatives
anti-cancer drug
2009 Elsevier Ltd. All rights reserved
.
1. Introduction
Unlike
dsDNA,
G-quadruplex
exhibits
structural
polymorphism depending on the number of strands involved in
the structure, strand orientation and variations in loop size and
sequence.16 G-quadruplex structures could be summarized by
three conformation: anti-parallel, parallel, and hybrid-type with
both parallel and anti-parallel features. Human telomeric
nucleotide sequence can provide all the three different
conformations. For example, four T7 d(TTGGGTT) stands could
bind together to form an intermolecular parallel G-quadruplex
structure.17 A single DNA H24 d(TTAGGG)4 give intramolecular
G-quadruplexes: an anti-parallel conformation in the presence of
Na+, a parallel conformation in the presence of PEG200, and a
hybrid-type conformation in the presence of K+.18-21 The
conformer of G-quadruplex plays an important role in providing
special sites for recognition of small-molecule ligands, to be
Telomeres are essential chromosomal components to protect
the end of eukaryotic chromosomes and maintain proper
replication.1 The G-quadruplex structure formed by human
telomeric DNA repeats has been witnessed to inhibit telomerase
activity in vitro.2-4 G-tetrad of G-quadruplex is a square planar
alignment of four guanines connected by cyclic Hoogsteen
hydrogen bonding of guanine bases and plays a very important
role in recognizing planar aromatic ligands through π-π stacking
mainly, that brings the ligands into, or very close to, the G-
quadruplex host.5-9 Molecules that induce and stabilize G-
quadruplex structures have the potential to interfere with
telomere replication and serve as anti-cancer agents.10-15
Therefore, there are growing interests on G-quadruplex as a
therapeutic target to develop novel anti-cancer agents.
applied
in
assisting
the
catalytic
transformation22
Corresponding author. Tel.: +010-62622090; fax: +010-82617304; e-mail: tangyl@iccas.ac.cn.

2
Tetrahedron
o
and designing new anti-cancer agents.10-15 Pyridostatin
temperature about 5 C. In the contrast, pyridostatin II shows a
strong ability to stabilize G-quadruplex H7 by raising the
melting temperature about 25 oC, but a weak ability to stabilize
G-quadruplex H22.
ACCEPTED MANUSCRIPT
derivatives comprising terminal amino groups have been
designed to promote the folding of telomeric G-quadruplexes.23-
24
So far as we know, what role does the fundamental skeleton
pyridostatin I (Scheme 1) play in recognizing G-quadruplex has
never been described. For comparison, we also successfully
prepared another new molecule pyridostatin II, which contained
a (2-dimethylamino)ethoxy group linked to each quinoline
fragment without no need of amino-protection, but the structure
was similar to the reported probe by Balasubramanian et. al.23-24
In our research, we found that pyridostain I selectively stabilized
intramolecular anti-parallel telomeric G-quadruplex, whereas
pyridostatin II with terminal amino groups stabilized
intermolecular parallel telomeric G-quadruplex.
(a)
10
8
H22
H22: I
H22: II
6
4
2
0
2. Results and Discussion
20
30
40
50
60
70
80
90
Telomeric
sequences
d[TTAGGGT]
(H7)
and
Temperature (oC)
d[AGGG(TTAGGG)3] (H22) have been chosen to prepare the
various conformations of telomeric G-quadruplexes. CD spectra
has been first used to confirm the effect of pyridostatin
derivatives on the conformational transition of telomeric G-
quadruplexes. The CD spectra of parallel G-quadruplexes
showed a negative band at 240 nm and a positive band at 265
nm; anti-parallel G-quadruplexes showed a negative band at 260
nm and a positive band at 295 nm; and hybrid-type G-
quadruplexes showed a negative band at 240 nm and two
positive bands at 265 and 290 nm. The addition of pyridostatin I-
II did not cause a transition of the CD spectra of all the telomeric
G-quadruplexes investigated, indicating that pyridostatin I-II did
not alter the conformations, which facilitates the study on
telomeric G-quadruplexes recognition of pyridostatin I-II. The
synthetic pathways for new pyridostatin I-II are shown in
Experimental Section, and some key intermediates of
pyridostatin II are shown in Supporting Information.
(b)
6
5
4
3
2
1
0
H7
H7: I
H7: II
20
30
40
50
60
70
80
90
Temperature (oC)
Fig. 1. CD melting profiles of: (a) G-quadruplex of H22 (5 µM) with
pyridostatin I or II; (b) G-quadruplex of H7 (5 µM) with pyridostatin I and
II in Tris-HCl buffer (20 mM, pH 7.4, 100 mM NaCl).
NH2
Fluorescence Spectroscopy
O
O
O
The fluorescence spectra of pyridostatin I and II binding with
intramolecular and intermolecular telomeric G-quadruplexes are
respectively given in Fig. 2. The interaction of pyridostatin I-II
with these G-quadruplexes resulted in concomitant discrepancy
on their spectra. The increasing amout of DNA led to the gradual
quenching of fluorescence emissions in Tris-HCl buffer. For
intermolecular parallel G-quadruplex H7, the presence of which
with more than 4 equiv. relative to pyridostatin I led to the
obivious discrepancy. While, intramolecular anti-parallel G-
quadruplex H22 with the ratio of 1:1 gave nearly no peak at λem
= 444 nm of pyridostatin I. For pyridostatin II, both of the
chosen telomeric G-quadruplexes H7 and H22 led to its decrease
of the fluorescence intensity at 361 nm, but the H7 was the better
one. These results clewed that pyridostatin I selectively
recognized intramolecular anti-parallel G-quadruplex over
intermolecular parallel G-quadruplex, while pyridostatin II
prefered to recognize intermolecular parallel G-quadruplex.
N
O
N
O
N
NH
N
HN
N
N
O
NH
HN
O
N
N
II
I
Scheme 1. Structures of pyridostatin I and II.
Selectively Recognition for Different Telomeric G-
Quadruplexes of Pyridostatin I and II.
Circular Dichroism Spectroscopy
Pyridostatin I as a neutral molecule was prepared from one-
step reaction between 2,6-pyridinedicarbonyl dichloride and 2-
aminoquinoline. To evaluate the effect of pyridostatin I on the
telomeric G-quadruplexes, we applied circular dichroism to
monitor the melting temperature (Tm) of Hum G-quadruplexes.
The change of Tm in the folded and unfolded quadruplex
structure upon interacting with ligand provides evidence of
thermal stabilization of DNA structure. Telomeric G-quadruplex
was incubated with pyridostatin I at 2 equal molar ratio for more
than 12 h, the Tm values for varied G-quadruplexes were
calculated as shown in Fig.1a. The pyridostatin I shows the
preference for the intramolecular anti-parallel G-quadruplex, and
selectively stabilizes G-quadruplex H22, raising the melting
temperature about 20 oC monitored at 295 nm. Whereas,
(a)
pyridostatin
I shows weak ability to stabilize parallel
intramolecular G-quadruplex H7 by raising the melting

3
(a)
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1800
1600
1400
1200
1000
800
H7
H22
600
400
(b)
200
0
0:1
1:1
2:1
3:1
4:1
Ratio of [DNA] : [I]
(b)
650
600
550
500
450
400
H7
H22
0:1
1:1
2:1
3:1
4:1
Ratio of [DNA] : [II]
Fig. 3. Optimized models of: (a) complex between pyridostatin I and H22
anti-parallel G-quadruplex; (b) complex between pyridostatin II and H7
parallel G-quadruplex. The G is colored in green, the T is colored in light
blue, the A is colored in red, and the ligands are colored in yellow.
Fig. 2. The fluorescence emission spectra of pyridostatin I (3 µM) and its
complexes with (a) intermolecular parallel G-quadruplex H7 and (b)
intermolecular anti-parallel G-quadruplex H22 in Tris-HCl buffer (20 mM,
pH 7.4, 100 mM NaCl).
The anti-tumor activities of pyridostatin I-II.
Binding modes of pyridostatin derivatives targeting to
telomeric G-quadruplexes.
Considerable circumstantial evidence suggests that these
structures can exist in vivo in specific regions of the genome
including the telomeric ends of chromosomes and oncogene
regulatory regions. Recent studies have demonstrated that small
molecules can facilitate the formation of, and stabilize, G-
quadruplexes. In our research, pyridostatin I and II showed the
discrepant recognition of telomeric G-quadruplex, thus possibly
led to the different inhibition of anticancer cells. IC50 values i.e.,
the effective concentration at which 50% growth of the cancer
cells was inhibited, were calculated to evaluate the anticancer
activities. Human colonic carcinoma cell line (HCT-8), human
non-small cell lung cancer cell line (A549), and human
hepatocellular carcinoma cell line (BEL-7402) were used in the
cytotoxicity test. All the experiments were carried out in
triplicates, and the results were shown in Table 1.
Pyridostatins showing different preference for human
telomeric G-quadruplex structures, prompted us to investigate
the binding modes between pyridostatins and G-quadruplex
structures. To investigate the binding modes between
pyridostatin I-II and intermolecular or intramolecular G-
quadruplex structure, qualitative molecular modeling using
molecular mechanics energy minimizations was studied.
Optimized models of the complexes between pyridostatin I-II
and G-quadruplexes H22 or H7 were illustrated in Fig. 3.
Molecular modeling in the ratio of 1:1 shows that the end-
stacking mode is favored for pyridostatin I binding to the
intramolecular G-quadruplex H22 while the groove binding
mode is favored for pyridostatin II binding to the intermolecular
G-quadruplex H7, the binding energy was -59.93 kcal/mol and -
66.62 kcal/mol, respectively. In comparision, the binding energy
for pyridostatin I binding to the G-quadruplex H7 was -45.32
kcal/mol (Fig. S1a); while the binding energy for pyridostatin II
binding to the G-quadruplex H22 was -56.27 kcal/mol (Fig.
S1b). Although the pyridostatin I and II showed similar
interaction mode and non-bonded interaction energy, the
torsional energy of the pyridostatin II was much higher than
pyridostatin I. The relatively high torsional energy made the
pyridostatin II unstable when binding to the intramolecular G-
quadruplex H22. Therefore, the pyridostatin I was more favored
binding to intramolecular G-quadruplex H22 than the
pyridostatin II. Whereas, the pyridostatin II had a preference for
intermolecular G-quadruplex H7, the multi-amino-appendages of
which endowed with positive charges made good matches with
the shape and volume of the G-quadruplex H7 active sites.
Table 1. IC50 values of pyridostatin I and II on human cancer
cell lines.a
IC50 values in µM
Compound
HCT-8
> 100
BEL-7402
> 100
A549
> 100
I
II
5-Fu
9.19 ± 0.18
87.33 ± 0.93
> 100
53.97 ± 0.30
4.66 ± 0.64
69.96 ± 1.87
a IC50 is the drug concentration effective in inhibiting 50% of the cell
growth measured by the MTT assay after 72 h drug exposure.
As shown in Table 1, pyridostatin II shows significant
cytotoxic activities on HCT-8 and A549 cell lines, better than I
and 5-Fu (positive control), but little effect on BEL-7402, maybe
related to the content and the types of telomeric G-quadruplexes

4
Tetrahedron
ACCEPTED MANUSCRIPT
in the chosen samples.29 Pyridostatin I doesn’t give good
2H), 5.32 (s, br, 1H), 4.46 (t, J = 5.2 Hz, 4H), 4.19 (t, J = 4.8 Hz,
2H), 3.62 (t, J = 4.8 Hz, 2H), 3.01 (t, J = 5.2 Hz, 2H), 2.49 (s,
12H); 13C NMR (100 MHz, CDCl3) δ 167.9, 162.8, 161.9, 158.3,
156.7, 148.8, 148.4, 130.2, 125.5, 122.0, 121.9, 117.9, 112.5,
90.4, 80.1, 68.8, 67.3, 57.8, 46.4, 41.3, 28.6; HRMS (ES)
calculated for C40H49N8O7 ([M + H]+) m/z: 753.37197, found
753.37187.
results as a cancer inhibitor, maybe due to the limited solubility
in water leading to the very high IC50 values, and meanwhile
indicates that multi-amino appendages on pyridostatin II
providing positively charged side chains may be required for
improving the water solubility and cellular uptake of the apolar
central skeleton.
Step ii: The resulting product of step i (300 mg, 0.4 mmol)
was dissolved in 4 ml DCM and 2 ml TFA and stirred at rt. for
2h. The solvents was removed in vacuo and the crude product
was crystallized from MeOH, and yielded a white solid
II·3CF3COOH (400 mg, 100%): 1H NMR (400 MHz, DMSO)
δ 8.36 (d, J = 7.6 Hz, 2H), 8.16 (s, 2H), 7.97 (d, J = 7.6 Hz, 4H),
7.82 (t, J = 6.8 Hz, 2H), 7.57 (t, J= 6.8 Hz, 2H), 4.71 (s, 4H),
4.55 (s, 2H), 3.77 (s, 4H), 3.56 (s, 2H), 2.99 (s, 12H); 13C NMR
(75 MHz, DMSO) δ 167.1, 163.6, 162.0, 152.6, 151.5, 147.4,
131.4, 127.3, 125.3, 122.9, 119.4, 115.3, 112.8, 95.6, 63.4, 55.6,
43.2, 31.1; HRMS (ES) calculated for C35H41N8O5 ([M + H]+)
m/z: 653.31946, found 653.31944.
3. Conclusion
In summary we have identified two new pyridostatin
compounds I and II as potent G-quadruplex stabilizing small
molecules. Pyridostain I selectively stabilized intramolecular
anti-parallel G-quadruplex H22, whereas pyridostatin II with
terminal amino groups stabilized intermolecular parallel G-
quadruplex H7, maybe due to the suited size measurements
between G-quadruplex hosts and pyridostatin guests. MTT
assays indicated that pyridostatin II had good cytotoxic effects
against HCT-8 and A549 cell lines, and had a potential
application in the exploration of anticancer drugs.
NHBoc
O
NH2
4. Experimental Section
O
O
O
N
4.1. Compounds and Materials
i
OH
OH
II-a
O
N
O
N
ii
N
Preparation of pyridostatin I.
+
O
NH
HN
O
N
N
N
O
Pyridostatin I was prepared from the reaction between 2-
aminoquinoline (1.40 g, 9.50 mmol) and 2,6-pyridine dicarbonyl
dichloride (1.00 g, 4.75 mmol) in 40 mL of dry toluene under
reflux for 4 hours (Scheme 2). The white precipitate was
collected by filtration and washed with acetone and 5% NaHCO3
to give pyridostatin I (1.80 g) in 87% yield. 1H NMR (600 MHz,
DMSO) δ H 12.21 (s, 2H), 8.53 (s, 4H), 8.49 (d, J = 7.8 Hz, 2H),
8.35 (t, J = 7.8 Hz, 1H), 8.03 (dd, J = 7.8, 3.6 Hz, 4H), 7.82 (t, J
= 7.8 Hz, 2H), 7.61 (t, J = 7.8 Hz, 2H). 13C NMR (150 MHz,
DMSO) δ 163.4, 151.2, 148.9, 146.0, 139.9, 138.8, 130.4, 128.0,
126.8, 126.3, 126.0, 125.6, 115.4. HRMS (EI) calculated for
C25H17N5O2 [M]+ m/z: 419.1382, found 419.1387.
II
N
NH2
II-b
Scheme 3. Synthetic route of pyridostatin II. Note: i, 1-chloro-N, N′,
2-trimethyl-1-propenylamine, DCM, 2 h at 0 ºC then TEA, 1 h at 0 ºC then
II-b, 12 h, rt. 41%. ii, DCM/ TFA( 2: 1), 100%.
The oligonucleotides H22 and H7 were purchased from
Invitrogen (Beijing, China), purified by PAGE. All organic
reagents were purchased from Beijing J&K Chemical Scientific
Company or Beijing InnoChem Science&Technology Co.,Ltd.
4.2. Spectra
O
O
O
N
O
N
NMR Spectra
N
+
N
Cl
Cl
NH
HN
Chemical shifts were given on the δ scale (ppm) and were
referenced to the residual solvent signals; Coupling constants J
were reported in hertz (Hz). The abbreviations s, d, and m were
used for singlet, doublet, and multiplet, respectively.
Toluene, reflux
87%
I
N
NH2
CD Spectra
Scheme 2. Synthetic route of pyridostatin I.
CD spectra were collected from 200 to 350 nm on a Jasco-815
automatic recording spectropolarimeter with a 1-cm pathlength
quartz cell. Spectra were collected with scan speed of 1000 nm
min-1. Each spectrum was the average of three scans.
Preparation of pyridostatin II.
Pyridostatin II was prepared according to the reported similar
procedure,24 shown as in Scheme 3. The key intermediates II-a
and II-b were prepared starting from chelidamic acid
monohydrate and 2-aminoquinolinone, respectively (see
Supporting Information).
Fluorescence Spectra
Fluorescence spectra were recorded on a Hitachi F4500
spectrofluorometer (Japan) in a 1-cm pathlength quartz cell at
room temperature. Xenon arc lamp was used as the excitation
light source. The excitation and emission slits were both 10 nm.
Excitation was set at 320 nm, and emission was collected from
335 to 600 nm.
Step i: Compound II-a (326 mg, 1 mmol) and 1-chloro-
N,N′,2-trimethyl-1-propenyl-amine (280 ꢀl, 2.1 mmol) were
dissolved in 10 ml DCM at 0 ºC and stirred for 2 h.
Triethylamine (347 ꢀl, 2.5 mmol) was added dropwise and the
solution stirred for 1 h at 0 ºC. A solution of II-b (462 mg, 2
mmol) in 5 ml DCM was added slowly and the resulting mixture
stirred at rt overnight. The solvent was removed in vacuo and the
crude product was crystallized from MeCN yielded a white
powder (310 mg, 41.2%) : 1H NMR (400 MHz, CDCl3) δ 11.46
(s, 2H), 8.19 (s, 2H), 8.14 (d, J = 8.4 Hz, 2H), 7.87 (s, 2H), 7.85
(d, J = 8.4 Hz, 2H), 7.67 (t, J= 7.6 Hz, 2H), 7.42 (t, J = 7.6 Hz,
4.3. Binding Mode Models
The 3D coordinates of Hum7 and H22 DNA G-quadruplex
structures were retrieved from the RCSB Protein Data Bank
(PDB CODE: 1NZM26 and 3T5E27). The G-quadruplex
structures were prepared for docking as described.28 The

5
pyridostatin I-II molecule structures were optimized with
C., Munshi, N. C. Mol. Cancer. Ther. 2003, 2, 825–833.
ACCEPTED MANUSCRIPT
MMFF force field using the Discovery Studio 3.5 (Accelrys
Software Inc., San Diego). The molecular docking studies were
carried out by using the LigandFit module of Discovery Studio
3.5 following the protocols developed for DNA G-quadruplex
and ligand docking.28
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4.4. MTT assays
16. Neidle, S., Balasubramanian, S. Quadruplex Nucleic Acids, ed. RSC
publishing, Cambridge, 2006.
Three human solid tumor cell lines including human non-
small cell lung cancer cell line (A549), human colonic carcinoma
cell line (HCT-8), and human hepatocellular carcinoma cell line
(BEL-7402) were used in the cytotoxicity test. The cells were
cultured in RPMI-1640 medium supplemented with 10% fetal
bovine serum (FBS), 100 ꢀg/mL of streptomycin, and
100 ꢀg/mL of penicillin in an atmosphere of 5% CO2 at 37 °C.
Cytotoxicity of all the compounds were determined by MTT
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assay
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; MTT, Sigma). Briefly, the suspension of 5000 cells per
well was plated in 96-well culture plates with culture medium
which was incubated for 24 h at 37 °C in a water-atmosphere
(5% CO2). The compounds with desired concentrations were
obtained by dissolving in DMSO and diluting with culture
medium. And then the diluted solution of complexes was added
to the wells, and the cells were incubated for 48 h at 37 °C. After
that, 10 ꢀL of a freshly diluted MTT solution (5 mg/mL in PBS)
were added to each well and the plates were incubated for 4.5 h.
The media with MTT solution were removed with DMSO
solution (100 ꢀL). The absorbance was measured at 540 nm with
an automatic microplate ELISA reader. The IC50 value was
determined from the chart of cell viability (%) against dose of
complex added (ꢀM).
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2003. 334, 25-36.
27. Collie, G. W., Promontorio, R., Hampel, S. M., Micco, M., Neidle, S.,
Parkinson, G. N. J. Am. Chem. Soc, 2012, 134, 2723-2731.
28. Haider, S., Neidle, S. Methods Mol Biol, 2010, 608, 17-37.
29. The states of the related samples are so complex that we just could
provide a possible explanation.
Acknowledgement
The authors wish to thank the National Natural Science
Foudation of China (Grant No. 21302188, 81072576, 91027033)
and Chinese Academy of Sciences (Grant No. KJCX2-EW-N06-
01 and XDA09030307 ) for financial support.
Supplementary data
Supplementary datas including synthetic procedures of II-a
and II-b, and optimized models for the complexes: 1) between
pyridostatin I and G-quadruplex H7; and 2) between pyridostatin
II and G-quadruplex H22, and characterization datas of all the
new compounds.
References
1. de Lange, T. Genes Dev. 2005, 19, 2100-2110.
2. Organisian, L., and Bryan, T. M. BioEssays. 2007, 29, 155–165.
3. Kelland, L. R. Eur. J. Cancer. 2005, 41, 971–979.
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627-636.
7. Murat, P., Singh, Y., Defrancq, E. Chem. Soc. Rev. 2011, 40, 5293-5307.
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18, 1973-1983.
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6836.
10. Chen, Y. W., Yang, D. Z. Current Protocols in Nucleic Acid Chemistry.
2012, 17.5.1-17.5.17.
11. Chu, B., Yuan, G., Zhou, J. Qu Y., Zhu, P. Drug Dev. Res. 2008, 69,
235–241.
12. Shammas, M. A., Shmookler Reis, R. J., Akiyama, M., Koley, H.,
Chauhan, D., Hideshima, T., Goyal, R. K., Hurley, L. H., Anderson, K.

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Supporting Information
Pyridostatins Selectively Recognize Two Different Forms of the
Human Telomeric G-Quadruplex Structures and Their Antitumor
Activities in Vitro
Lixia Wang, Qian Shang, Qian Li, Junfeng Xiang, Yan Liu, Aijiao Guan,
Hongxia Sun, Lijia Yu, Yalin Tang*
National Laboratory for Molecular Sciences, Center for Molecular Sciences, State
Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of
Chemistry Chinese Academy of Sciences, Beijing 100190, P.R. China. Fax: 86 10
6252 2090; Tel: 86 10 8261 7304; E-mail: tangyl@iccas.ac.cn

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Preparation of the key intermediates of pyridostatin II:
NHBoc
O
O
O
N
a-c
O
O
O
N
H
OH
OH
OH
OH
II-a
Steps: (a) H2SO4, MeOH, reflux. (b) N-Boc-ethanolamine, PPh3, DIAD, THF, rt. (c) aq. NaOH,
MeOH, 0 ºC.
Dimethyl-4-[(Tetrahydro-2-pyrany)oxy]-2-6-pyridinedicarboxylate
Chelidamic acid monohydrate (97%, 52.0 g, 0.275 mol) and a catalytic amount of
sulfuric acid (10 mL) were dissolved in 700 mL of methanol and the mixture was
refluxed for 20 h. After it was cooled to room temperature, the reaction mixture was
neutralized with aqueous sodium carbonate solution and then acidified by
concentrated hydrochloric acid. Water was added and the resulting mixture was
extracted with dichloromethane to give a yellow solid. Crude dimethyl chelidamate
was recrystallized from methanol and gave a white solid1 (39.2 g, 67.6%).1H NMR
(400MHz, CDCl3) δ 7.33(s, 2H), 4.03 (s, 6H).
Dimethyl-4-(2-tert-butoxycarbonylamino-ethoxy)-2,6-pyridinedicarboxylate
Chelidamic acid dimethyl ester (1.06 g, 5 mmol), N-Boc-ethanolamine (1.21 g, 7.5
mmol) and triphenylphosphine (2.62 g, 10 mmol) were dissolved in 30 ml freshly
distilled THF and cooled to 0 °C. DIAD (1.52 g, 7.5 mmol) was added dropwise
under argon. The mixture was allowed to warm to rt 12 h. The solvent was removed in
vacuo and the product purified by column chromatography to obtain the title
compound (1.07 g, 60.5%) as a white powder2. 1H NMR (400 MHz, CDCl3) δ 7.79 (s,
2H), 4.93 (s, br, 1H), 4.19 (t, J= 5.0 Hz, 2H), 3.99 (s, 6H), 3.58 (m, 2H), 1.43 (s, 9H).
4-(2-tert-butoxycarbonylamino-ethoxy)-2,6-pyridinedicarboxylic acid II-a:
Compound 2 (354 mg, 1mmol) was dissolved in MeOH (5ml), and then 2 M aq. soln.
of NaOH (80mg, 2mmol) was added dropwise at 0 ºC.After finishing the dropping ,
the reaction mixture was neutralized with concentrated hydrochloric acid to PH ≈ 1.

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Water was added and the resulting mixture was extracted with dichloromethane to
give a white solid2 (300 mg, 92.0%). 1H NMR (400 MHz, D2O) δ H 7.82 (s, 2H),
4.61(t, J = 5.6 Hz, 2H), 4.46 (t, J = 5.6 Hz, 2H), 1.23 (s, 9H).
O
N
O
d
N
H
NH2
N
NH2
II-b
Step d: N, N-Dimethylethanolamine, PPh3, DIAD, THF, rt.
4-(Dimethylamino-ethoxy)-quinolin-2-ylamine II-b
2-amino-quinolinone (800 mg, 5 mmol), N,N′-Dimethylethanolamine (667.5 mg, 7.5
mmol) and triphenylphosphine (2.62 g, 10 mmol) were dissolved in 30 ml freshly
distilled THF and cooled to 0 °C. DIAD (1.52 g, 7.5 mmol) was added dropwise
under argon. The mixture was allowed to warm to rt and stirred for 20h. The solvent
was removed in vacuo and the product purified by column chromatography to obtain
1
a white powder (539 mg, 46.7%). H NMR (400 MHz, CDCl3) δ H 7.98 (d, J = 8.0
Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H),
6.02 (s, 1H), 4.82 (s, br, 2H), 4.19 (t, J = 5.6 Hz, 2H), 2.87 (t, J = 5.6 Hz, 2H), 2.38 (s,
13
6H); C NMR (100 MHz, CDCl3) δ 162.7, 158.2, 148.4, 130.2, 125.5, 122.0, 121.9,
117.8, 90.0, 67.0, 57.9, 46.2; HRMS (ES) calculated for C13H18N3O ([M + H]+) m/z:
232.14459, found 232.14444.
Reference:
(1). Bradshaw J. S., Colter, M. L., Nakatsuji, Y., Spencer, N. O., Brown, M. F., Izatt,
R. M., Arena, G., Tse, P. K., Wilson, B. E., Lamb, J. D., Dalley, N. K. Proton-ionizable
crown compounds. 2. Synthesis, complexation properties, and structural studies of
macrocyclic polyether-diester ligands containing a 4-hydroxypyridine subcyclic unit.
J. Org. Chem, 1985, 50, 4865-4872.
(2). Rodriguez, R., Müller, S., Yeoman, J. A., Trentesaux, C., Riou, J.-F. and
Balasubramanian, S. A novel small molecule that alters shelterin integrity and triggers

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a DNA-damage response at telomeres. J. Am. Chem. Soc. 2008, 130, 15758-15759.
(a)
(b)
Fig. S1. Optimized models of: (a) complex between pyridostatin I and G-quadruplex H7; (b)
complex between pyridostatin II and G-quadruplex H22. The G is colored in green, the T is
colored in light blue, the A is colored in red, and the ligands are colored in yellow.

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