4-(1H-Imidazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol-based G-quadruplex DNA binding agents: Telomerase inhibition, cytotoxicity and DNA-binding studies

Article abstract of DOI:10.1016/j.bmc.2012.11.059

Four novel 4-(1H-imidazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol derivatives 1-4 have been synthesized, and their G-quadruplex DNA-binding interactions, telomerase inhibition, antiproliferative activity, cell cycle arrest, and apoptotic induction were studied. All compounds show the preferential h-telo, c-myc, and c-kit2 G-quadruplex binding affinity and the G-quadruplex versus duplex selectivity. In the case of the same G-quadruplex target, the compound 1 exhibits better stabilization effect (ΔT_m) than the other three compounds and also gives 80.2% inhibition of telomerase activity at 7.5 μM. All compounds can promote selectively the formation of parallel G-quadruplex structure of both c-myc and c-kit2 without addition of any cations. Four compounds display the cytotoxicity activities against HeLa and HepG2 cells by MTT assay with IC₅₀ values of about 10^-6 and 10 ^-5 M, respectively, and cause a substantial decrease in the G ₂/M-phase cell population and a significant increase in the number of apoptotic cells.

Full text of DOI:10.1016/j.bmc.2012.11.059

Bioorganic & Medicinal Chemistry 21 (2013) 3379–3387
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
4-(1H-Imidazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol-based
G-quadruplex DNA binding agents: Telomerase inhibition,
cytotoxicity and DNA-binding studies
a
a
a
a
Chun-Ying Wei a,b, , Jun-Hong Wang , Ye Wen , Jie Liu , Li-Hua Wang
⇑
a Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, PR China
b State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Four novel 4-(1H-imidazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol derivatives 1–4 have been synthesized,
and their G-quadruplex DNA-binding interactions, telomerase inhibition, antiproliferative activity, cell
cycle arrest, and apoptotic induction were studied. All compounds show the preferential h-telo, c-myc,
and c-kit2 G-quadruplex binding affinity and the G-quadruplex versus duplex selectivity. In the case of
Received 8 September 2012
Revised 26 November 2012
Accepted 27 November 2012
Available online 11 December 2012
the same G-quadruplex target, the compound 1 exhibits better stabilization effect (
DTm) than the other
three compounds and also gives 80.2% inhibition of telomerase activity at 7.5 M. All compounds can
l
Keywords:
G-quadruplex
Telomere
Promoter
Phenanthroline
promote selectively the formation of parallel G-quadruplex structure of both c-myc and c-kit2 without
addition of any cations. Four compounds display the cytotoxicity activities against HeLa and HepG2 cells
by MTT assay with IC50 values of about 10ꢀ6 and 10ꢀ5 M, respectively, and cause a substantial decrease in
the G2/M-phase cell population and a significant increase in the number of apoptotic cells.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
their metal complexes have been reported be able to better stabi-
lize the human telomeric G-quadruplex than the duplex DNA.34–42
G-quadruplex-forming DNA sequences have been identified in
the human telomere1–3 as well as the promoters of several onco-
genes including c-myc,4 kras,5 c-kit,6,7 and bcl-2.8 More recently,
RNA G-quadruplexes were found in the regions of G-rich telomer-
ic repeat-containing RNA (TERRA),9,10 the 50-end of human telo-
merase RNA (hTR)11 and 50-untranslated regions (UTR) of
mRNAs.12–14 The telomeric G-quadruplex DNA-recognizing small
molecules can inhibit cancer cell growth via mechanisms that
may involve disruption of the telomere and/or the prevention of
telomere extension.15–19 Small molecules that bind and stabilize
the c-myc and c-kit G-quadruplexes also suppress transcription
of the proto-oncogenes c-myc20–22 and c-kit.23–25 Consequently,
the G-quadruplexes formed by the telomere and the promoters
of oncogenes are potential molecular targets for anticancer
drugs.26–28 G-quadruplexes in the 50 UTR of mRNAs modulate
translation and may provide yet another class of targets for small
molecule intervention.12,13,26
For example, platinum(II) phenanthroline complex induces a high
degree of quadruplex DNA stabilization and inhibits telomerase
activity,34 another two phenanthroline compounds exhibit better
affinity and selectivity for G-quadruplex DNA than duplex,35 bis-
phenanthroline Ni(II) and Cu(II) complexes promote the G-quadru-
plex folding and inhibit the activity of telomerase,36 and Pt(II)
phenanthroimidazole complexes display a significantly greater
binding affinity and selectivity for quadruplex than duplex.37,38
Also our previous studies have indicated that three phenanthro-
line-based derivatives induce the formation of antiparallel G-quad-
ruplex structure and inhibit the telomerase activity.39
Until now some studies have reported the binding affinity and
selectivity of phenanthroline-based compounds for different bio-
logically relevant G-quadruplex-forming DNA sequences, whereas
studies on effects of G-quadruplex-binding ligands on cell cycle
and apoptosis have been rarely reported.43–47 Herein, we report
the synthesis and characterization of four novel 4-(1H-imi-
dazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol derivatives (Scheme
1). Their binding interactions with human telomeric (h-telo) and
promoter (c-kit2 and c-myc) G-quadruplex DNAs and duplex DNA
were studied by FRET melting assay, competitive dialysis, absorp-
tion titration, and CD spectroscopy. Furthermore, influences of these
compounds on telomerase activity, cell proliferation, cell cycle, and
apoptosis were evaluated.
To date, a large number of small molecules have been designed
to target G-quadruplex DNAs.29–33 1,10-Phenanthroline derivatives
can bind to duplex DNA due to their planar structure. Recently, the
small organic molecules containing phenanthroline scaffold and
⇑
Corresponding author.
E-mail address: weichuny@sxu.edu.cn (C.-Y. Wei).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.059

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C.-Y. Wei et al. / Bioorg. Med. Chem. 21 (2013) 3379–3387
Scheme 1. Synthetic route to compounds 1–4.
targets cannot be directly compared using
quadruplex target, the compound 1 exhibits better stabilization
potential than the other compounds, the Tm values were found
to be 17.8, 13.1, and 10.5 °C, for h-telo, c-myc, and c-kit2 G-quad-
ruplexes, respectively, at 2 M concentration, while the compound
4 exhibits the lowest stabilization potential, and the observed Tm
is 6.8 °C for h-telo, 4.2 °C for c-myc, and 4.0 °C for c-kit2 at the
same concentration. The different stabilization potential of four
compounds may be due to different basicity, polarity, and size of
amino alkyl side chains.39,53 All the compounds show preference
for stabilizing G-quadruplex DNAs over duplex DNA using compet-
itive FRET melting assay in the presence of 10-fold excess of duplex
DTm. For the same G-
2. Results and discussion
2.1. Synthesis
D
l
The general synthetic method for compounds 1–4 is described
in Scheme 1. 1,10-Phenanthroline-5,6-dione48–50 and 4-(1H-imi-
dazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol (p-hpip)50–52 were
prepared following the literature methods. The final products were
prepared from ligand p-hpip and 2-dimethylaminoethyl chloride
hydrochloride (1), N-(2-chloroethyl)piperidine hydrochloride (2),
N-(2-chloroethyl)pyrrolidine hydrochloride (3), or 4-(2-chloro-
ethyl)morpholine hydrochloride (4). Structures of all these final
products were confirmed by 1H and 13C NMR, IR, elementary anal-
ysis, and ESI-MS. The detailed synthetic procedures of p-hpip and
compounds 1–4 are given in the Experimental.
D
DNA (ds26, 2
of ds26 (5 M), the compound 2 also stabilizes the structures of
h-telo, c-myc, and c-kit2 G-quadruplexes, with a slight decrease
in Tm (2.6, 0.9, and 1.6 °C, respectively) compared to G-quadru-
plex alone, whereas the compound 1 stabilizes only structures of
both c-myc and c-kit2 G-quadruplexes (with a decrease in Tm
of 0.3 and 1.5 °C, respectively). In the case of three G-quadruplex
targets, the larger decrease in Tm values (27–76%) was observed
lM). Furthermore, in the presence of 25-fold excess
l
D
2.2. Stabilization potential and binding affinity of the
compounds to G-quadruplex DNAs
D
D
FRET melting analysis was used to determine the G-quadruplex
stabilization and selectivity of the series of ligands to particular G-
quadruplex target. Here the stabilization potential of four com-
pounds (1–4) to h-telo, c-myc, and c-kit2 G-quadruplex DNAs
was first studied by FRET melting assay (Figs. S1–S3, Supplemen-
for compounds 3 and 4 in the presence of 25-fold excess of ds26,
suggesting that both compounds possess moderate binding selec-
tivity for h-telo, c-myc, and c-kit2 G-quadruplexes versus duplex.
The binding interactions of the compounds with G-quadruplex
DNAs were further measured by absorption titration experiment.
Isosbestic points, slight blue-shift, and hypochromic effects were
observed in the absorption spectra for compound 1 upon titration
with G-quadruplexes as depicted in Figure 2. The blue-shift about
1–2 nm may arise from the absorbance of the increasing amount of
DNA. The hypochromic phenomenon is attributed to the strong
interaction between the compound 1 and G-quadruplex DNA.
Using Eq. 1, the binding constants (Kb) of compound 1 with h-telo,
c-myc, and c-kit2 G-quadruplexes were calculated to be
(1.01 0.09), (1.44 0.12), and (0.86 0.03) ꢁ 106 Mꢀ1 at 25.0 °C,
respectively (Table 2).
tary data). The shifts in melting temperature values (
ven in Figure 1 and Table 1.
DTm) are gi-
Four compounds show moderate stabilization of G-quadruplex-
es, with Tm values range 6.8–17.8, 4.2–13.1, and 4.0–10.5 °C for
h-telo, c-myc, and c-kit2, respectively, in the concentration range
1–3 M. Because each DNA target gives a different melting tem-
perature (Tm) value, the selectivity among different quadruplex
D
l
To investigate whether there is a binding selectivity between G-
quadruplex and duplex, a parallel absorption titration experiment
of compound 1 with ctDNA was performed (Fig. S4, Supplementary
data). The Kb value for the ctDNA-compound 1 was determined to
be (6.6 0.3) ꢁ 104 Mꢀ1, which is approximately 15.3-, 21.8-, and
13.0-fold lower than those for complexes of compound 1 with h-
telo, c-myc, and c-kit2 G-quadruplexes, respectively. Similarly,
the binding constants of the other compounds for h-telo, c-myc,
and c-kit2 G-quadruplexes and for ctDNA were also determined,
and the data are summarized in Table 2. These data reveals that
the binding affinities of four compounds to G-quadruplex DNAs
are preferential over duplex DNA. The correlation between stabil-
ization (DTm) and equilibrium binding (Kb) is not straightforward
and thus there is no simple relationship between them.
Figure 1. Increase of melting temperature (
quadruplex DNAs in the presence of compounds 1, 2, 3, and 4 by FRET melting
assay. Concentrations of each compound are 1, 2, and 3
each G-quadruplex target.
DTm) of h-telo, c-myc, and c-kit2 G-
The relative binding affinities of compounds 1, 2, and 3 to differ-
ent DNA structures were further evaluated by competition dialysis
assay, and the results obtained in PBS buffer containing
lM from right to left for

C.-Y. Wei et al. / Bioorg. Med. Chem. 21 (2013) 3379–3387
3381
Table 1
G-quadruplex stabilization potential (DTm) by FRET melting assay
D
Tm (°C) at 2 lM compound concentration
Compounds
h-teloa
h-telo+ds26a
Mc
c-mycb
c-myc+ds26b
Mc
c-kit2a
c-kit2+ds26a
Mc
2
l
5
l
Md
2
l
5
l
Md
2
l
5 l
Md
1
2
3
4
17.8
12.6
13.5
6.8
14.5
10.8
13.1
6.6
12.0
10.0
7.4
13.1
9.1
6.8
12.8
8.3
7.2
12.8
8.2
4.7
10.5
8.8
6.0
10.2
9.3
4.7
9.0
7.2
4.0
2.1
2.4
4.2
4.0
1.0
4.0
3.1
a
b
c
With 100 mM KCl.
With 40 mM KCl.
With 2
With 5
l
l
M duplex DNA (ds26).
M duplex DNA (ds26).
d
Figure 2. Absorption titration spectra of compound 1 (30 lM) with increasing amounts of h-telo (A), c-myc (B), and c-kit2 (C) G-quadruplex DNAs in 10 mM tris–HCl buffer
containing 100 mM KCl (pH 7.4). The arrow indicates the absorbance changes upon increasing DNA concentration. Insert is the curve fit by Eq. 1. Absorbance was monitored
at 315 nm.
Table 2
G-quadruplex and ctDNA binding constants (Kb) measured by Eq. 1
Kb (ꢁ106 Mꢀ1
c-myc
)
a
Compounds
h-telo
c-kit2
ctDNA
1
2
3
4
1.01 0.09
1.17 0.12
0.93 0.06
0.36 0.04
1.44 0.12
1.35 0.11
1.26 0.13
0.64 0.08
0.86 0.03
0.77 0.06
0.61 0.15
0.39 0.03
0.066 0.003
0.081 0.002
0.11 0.02
0.14 0.06
a
Values are means S.E.
Figure 3. Relative affinities of compounds 1–3 to h-telo, c-myc, and c-kit G-
quadruplexes and ctDNA determined by competitive dialysis. The amount of bound
compound was plotted against each DNA structure as a bar. 1 (red), 2 (green), and 3
(blue).
100 mM KCl are depicted in Figure 3. All the compounds bind pref-
erentially to h-telo, c-myc, and c-kit2 G-quadruplex DNAs, demon-
strating that these have the selectivity for quadruplex over duplex.
However, in the case of particular G-quadruplex target, the binding
selectivity of each compound is not remarkable, especially for com-
pounds 2 and 3. In addition, for all the compounds the bound
amount for c-kit2 G-quadruplex is the highest, which fact is incon-
sistent with results obtained by absorption titration. The difference
may result from significant non-specific binding of the compounds
to c-kit2 G-quadruplex. The number of experimental points in
Scatchard plot shown in the inset in Figure 2C is lower than that
of spectra in the titration, because the initial data points from
the absorption titration deviate from the linear plot and were

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C.-Y. Wei et al. / Bioorg. Med. Chem. 21 (2013) 3379–3387
deleted from Scatchard plot-these data may arise from non-specific
binding. However, the similar phenomenon is not observed for
both h-telo and c-myc G-quadruplexes. Because the bound com-
pound concentration obtained in the equilibration dialysis includes
a sum of specific and non-specific binding, this results in the larger
amount of the bound compound for c-kit2 than for h-telo and
c-myc.
characteristic of G-quadruplex DNA in the hybrid form.56 With
the exception of the compound 4, a slight intensity increase of
the positive peak at 290 nm was observed upon addition of the
other compounds. The CD spectra of both c-myc and c-kit2 G-
quadruplexes show a parallel conformation in the presence of
100 mM K+, with a positive peak at 262 nm and a negative peak
at 241 nm. Upon addition of 2 mol equiv of the compounds 1–4
to either c-myc or c-kit2 G-quadruplex, the compound-induced
CD spectral changes were less prominent. In summary, the binding
of four compounds fails to affect significantly the conformation of
h-telo, c-myc, and c-kit2 G-quadruplexes in buffer containing
100 mM KCl.
2.3. Promoting the formation of parallel G-quadruplex DNAs
G-quadruplexesformedfromh-telo,c-kit2,andc-mycsequencesin
the presence of compounds 1–4 were monitored by CD spectroscopy
(Fig.4).TheCDspectrumofc-kit2sequenceintheabsenceofanyadded
saltshowsamajorpositivepeakat262 nm,aminorpeakat294 nm,anda
negativebandat240 nm.Upontitrationofthecompounds1–4,acon-
centration-dependentincreaseofintensityat262 nmisnotaccompa-
niedbyassociatedincreaseofthepeakintensityat294 nm,andthese
featuresareindicativeofselectiveinductionofaparallelG-quadruplex
structure.7,54Inaddition,theCDspectrumofc-mycsequenceintheab-
senceofanyaddedsaltwasfoundtohaveapositivepeakat260 nmanda
negative band at 240 nm. Upon addition of the compound (2 mol e-
quiv),theincreaseofintensityat262 nmwasobservedsuggestingthat
compounds1–4inducetheformationofaparallelG-quadruplexstruc-
ture (Fig. S5A, Supplementary data).55 In contrast, for h-telo a com-
pound-dependent induction of G-quadruplex structure change was
not observed even in the presence of 2 mol equiv of the compound
(Fig. S5B, Supplementary data). These results show that four com-
pounds are able to promote selectively the formation of parallel G-
quadruplexstructuresofbothc-kit2andc-mycandthereforestabilize
theirstructuresundercation-deficientconditions.
2.4. Telomerase inhibition of compounds in cell-free system
Human telomeric G-quadruplex-binding ligands are known to
inhibit telomerase activity. The above results obtained by FRET
melting assay and the moderate stabilization potential of the com-
pounds for h-telo G-quadruplex prompted us to investigate if these
compounds would also show telomerase inhibitory ability using a
modified telomerase repeat amplification protocol assay (TRAP-LIG
assay).39,57 As shown in Figure 5, except the compound 4, the other
compounds were able to inhibit telomerase activity, but the effects
were different. Formation of G-quadruplex structure in TS primer
sequence hinders its elongation by telomerase and thus the inten-
sity of the ladder decreases. Based on this principle, the compound
1 is a strong inhibitor that inhibits 80.2% of telomerase activity at
7.5
l
M, whereas 21.5% and 14.4% inhibitions are obtained for com-
M) and 3 (10 M), respectively.
pounds 2 (15
l
l
Thecurrentresultsindicatethatthepotencyofinvitrotelomerase
inhibitionisinaccordancewiththestabilizationpotential.Incompar-
isonwithcompounds2and3,thecompound1stabilizesthestructureof
theh-teloG-quadruplexmoreefficientlyandthusexhibitsastronger
To understand whether G-quadruplex DNA undergoes confor-
mational changes during compound–DNA interaction, CD spectra
of h-telo, c-kit2, and c-myc G-quadruplexes in the absence and
presence of compounds 1–4 at [compound]/[DNA] ratios of 2 were
determined (Fig. S6, Supplementary data). The CD spectrum of free
h-telo in buffer containing KCl has a negative band at 235 nm and a
positive band at 290 nm with a shoulder near 265 nm, which is
inhibitoryeffectonthetelomeraseactivity(80.2%at7.5
pound4presentsapoorinhibitoryactivity(3.4%at15
l
M).Thecom-
l
M)owingtoits
lowerstabilizationpotentialand/orweakbindingaffinitytoh-teloG-
quadruplexDNA.
Figure 4. CD spectra of 5 lM c-kit2 DNA in the presence of different concentrations of compounds 1 (A), 2 (B), 3 (C), and 4 (D) in 10 mM tris–HCl buffer without any added
salts.

C.-Y. Wei et al. / Bioorg. Med. Chem. 21 (2013) 3379–3387
3383
2.5. In vitro cytotoxicity
respectively. All compounds inhibit the growth of HeLa cells better
than that of HepG2 cells. The compound 4 is also a potent antipro-
liferative agent, especially for HeLa cells, due to other reasons such
as differences in cell uptake, alternative binding position, or cell
modification .
2.5.1. Antiproliferative activity
The in vitro antiproliferative activity of compounds 1–4 was
evaluated against two human cancer cell lines HeLa and HepG2
by treating cells with various concentrations for 72 h. The number
of viable cells, determined by MTT assay, decreases with increasing
concentrations of compounds 1–4 in both tested lines (Fig. 6),
which shows that compounds 1–4 display significant antitumor
2.5.2. Cell cycle analysis and detection of apoptosis
All of the target compounds were further evaluated for their
effects on the cell cycle and for the apoptotic induction in both
HeLa and HepG2 cells by flow cytometric assay that enables quan-
tification of the total cellular population in the different phases of
the cell cycle (G0/G1, S, and G2/M), as well as the sub-G1 apoptotic
population.
activity with the IC50 values of 1.0, 0.60, 0.80, and 0.69 ꢁ 10ꢀ6
M
for HeLa cells and 9.1, 8.7, 11, and 16 ꢁ 10ꢀ6 M for HepG2 cells,
Four compounds delay the cell cycle progression of both HeLa
and HepG2 cells upon treatment for 48 and 30 h, respectively, at
their corresponding IC50 values and arrest cells in G0/G1 and/or S
phases. This causes a substantial decrease in the G2/M phase cell
population that was zero for HepG2 cells and near to zero for HeLa
cells. The treatment also leads to a significant increase in the num-
ber of apoptotic cells, as shown by an increase in the subG0/G1 con-
tent from 12.8% to 53.5% for HeLa cells and from 2.64% to 55.4% for
HepG2 cells (Table 3, Figs. S7 and S8, Supplementary data).
It was reported that apoptosis of AML cells induced by telo-
mestatin might be related to the reduction of telomere length,43
whereas RHPS4 is a potent in vitro telomerase inhibitor and blocks
the cells in the G2/M phase of the cell cycle, but it fails to reduce
telomere length in tumor cells.44,45 2,6-Pyridine-dicarboxamide
derivatives displaying a strong selectivity for G4 structures and
strong inhibition of telomerase inhibit cell proliferation and then
induce apoptosis, but these effects are not associated with telo-
mere shortening.46 HXDV, a G-quadruplex stabilizer, exhibits
Table 3
Flow cytometric analysis of HeLa and HepG2 cell cycles upon the 48 and 30 h
treatment, respectively, with compounds 1–4 at their corresponding IC50
concentrationa
Compounds
G0/G1 (%)
S (%)
G2/M (%)
subG0/G1 (%)
HeLa
Control
56.8 2.1
69.2 1.9
62.7 1.9
50.9 1.0
59.8 1.3
26.9 0.9
22.9 1.1
33.9 1.1
48.4 1.2
40.2 1.2
16.3 0.8
7.9 0.2
3.4 0.2
0.7 0.08
0
12.8 0.2
31.0 0.5
53.5 0.5
25.1 1.2
19.6 0.7
1
2
3
4
HepG2
Control
65.3 2.1
75.2 1.9
49.5 2.3
66.5 1.0
76.4 1.4
24.0 0.9
24.8 1.1
50.5 1.4
33.5 1.2
25.4 1.2
10.7 0.4
2.64 0.2
30.5 0.62
49.9 1.4
52.2 1.2
55.4 0.7
1
2
3
4
0
0
0
0
Figure 5. TRAP assay performed in the presence of increasing concentrations of
compounds 1 (a), 2 (b), 3 (c) and 4 (d). The positive control was run with telomerase
without ligand. The negative control was run without either telomerase or ligand.
Positive and negative control lanes are indicated by + and ꢀ labels, respectively.
a
Values are means S.E. of three independent experiments.
Figure 6. Effect of compounds 1–4 on HeLa (A) and HepG2 (B) cell viability. Both cells were treated with various concentrations of compounds for 72 h. The values are
means S.E. of three independent experiments.

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C.-Y. Wei et al. / Bioorg. Med. Chem. 21 (2013) 3379–3387
antiproliferative activity by a mode of action independent on telo-
merase and also inhibits the cell cycle progression causing a M
phase cell cycle arrest.47 In our present work, the compound 1 is
a potent telomerase inhibitor compared to compounds 2, 3, and
4, but all the compounds arrest the cell cycle and induce the cell
apoptosis. In the case of our current results, the relationship be-
tween telomerase inhibition and the cell cycle and apoptosis is un-
clear. In addition, the moderate stabilization potential and high
binding affinities of compounds for both c-kit and c-myc G-quad-
ruplexes may interfere with the cell function including arrest of
cell cycle and induction of apoptosis due to suppression of gene
expression. Meanwhile, the possible interactions of the compound
with RNA G-quadruplexes also inhibit gene expression and further
affect cell function. Thus the in-depth investigation is deserved to
be performed in future work.
the product was recrystallized from methanol. For p-hpip,50–52
a
mixture of 4-hydroxybenzaldehyde (3.5 mmol, 0.43 g), 1,10-phe-
nanthroline-5,6-dione (2.5 mmol, 0.53 g), ammonium acetate
(50 mmol, 3.9 g) and glacial acetic acid (7 mL) were refluxed for
2 h. The cooled deep red solution was diluted with 25 mL water,
and neutralized with ammonium hydroxide to obtain the precipi-
tates. This mixture was then filtered and the precipitates were
washed with water, dried and recrystallized from ethanol and ace-
tonitrile to get p-hpip as a pale yellow solid (0.65 g, 84%). 1H NMR
(300 MHz, DMSO-d6): d (ppm) 13.52 (s, 1H), 9.98 (s, 1H), 9.03 (d,
2H), 8.94 (d, J = 7.8 Hz, 2H), 8.13 (d, J = 8.4 Hz, 2H), 7.84 (m, 2H),
6.99 (d, J = 8.4 Hz, 2H). 13C NMR (75 MHz, DMSO-d6): d (ppm)
167.1, 159.3, 155.6, 151.4, 137.7, 136.1, 131.3, 129.2, 123.9.
4.2.1. 2-[4-(1H-Imidazo[4,5-f]-1,10-phenanthrolin-2-
yl)phenoxy]-N,N-dimethyl-ethanamine (1)
NaH (60% dispersion in mineral oil) (3.43 mmol, 0.13 g) was
added to the solution of p-hpip (0.7 mmol, 0.21 g) in dry and de-
gassed DMF (20 mL), and the resulting mixture was heated with
stirring for 30 min at 110 °C under N2 atmosphere. A solution of
2-dimethylaminoethyl chloride hydrochloride (0.7 mmol, 0.20 g)
in DMF (40 mL) was added dropwise to the resulting mixture over
a period of 1 h, during which time the reaction solution turned
from deep red to transparent brown-orange. This solution was fur-
ther refluxed for 72 h under nitrogen. After the reaction mixture
was cooled, the solid was filtered off and the filtrate was concen-
trated on a rotary evaporator to yield a brown-yellow solid. This
solid was dissolved in methanol, to which either HCl (aq) or NEt3
was added until the pH value of solution reached 7, the resulting
solution was evaporated under reduced pressure to obtain a solid.
This solid was purified by chromatographic column on silica gel
using MeOH–CHCl3–glacial acetic acid (1:2:0.02, v/v/v) as an elu-
ent to give the compound 1 as a yellow solid (38%, 0.1 g). 1H
3. Conclusions
Inthispaper,fournovelcompoundshavebeensynthesized,which
showamoderatestabilizationpotentialforG-quadruplexes.Forapar-
ticularG-quadruplextarget,thecompound1appearstobeapotentG-
quadruplexDNAstabilizationagentwith
10.5 °C for h-telo, c-myc, and c-kit2, respectively, at 2
tion, which also exhibits 80.2% telomerase inhibitory activity at
7.5 M. All compounds also exhibit selectivity for h-telo, c-kit2, and
D
Tm ofabout17.8,13.1,and
lM concentra-
l
c-myc G-quadruplexes over duplex DNA, moreover the compound 2
can recognize selectively and stabilize h-telo, c-kit2, and c-myc G-
quadruplexeseveninthepresenceof25-foldexcessofds26,although
thecompound1onlystabilizesbothc-kit2andc-mycG-quadruplexes
underthesamecondition.Fourcompoundscanalsoinducetheforma-
tionofaparallelG-quadruplexstructureforbothc-mycandc-kit2un-
der cation-deficient conditions. The antiproliferative effect of all
compoundswasobservedonHeLaandHepG2celllinesinaconcentra-
tion-dependentresponsepattern,withIC50valuesof10ꢀ6and10ꢀ5 M,
respectively. These compounds cause the cell cycle arrest at S and/or
G0/G1 phases and induce cell apoptosis at their corresponding IC50
concentration.
NMR (300 MHz, DMSO-d6):
d (ppm) 9.01 (m, 4H), 8.29 (d,
J = 8.4 Hz, 2H), 7.82 (m, 2H), 7.14 (d, J = 8.4 Hz, 2H), 4.16 (m, 2H),
2.67 (m, 2H), 2.25 (m, 6H). 13C NMR (75 MHz, DMSO-d6): d
(ppm) 160.0, 151.9, 148.0, 143.5, 130.6, 128.5, 123.8, 122.4,
115.2, 66.4, 58.1, 46.0. IR (KBr, cmꢀ1): 3411, 2939, 2769, 1577,
1523, 1483, 1448, 1352, 1249, 1180, 1031, 837, 804, 740, 649.
ESI(+)-MS (m/z): 384.58 (100%), [M+H]+; 789.33 (100%),
[2M+Na]+. Anal. Calcd for C23H21ON5: C, 72.05; H, 5.52; N, 18.26.
Found: C, 72.10; H, 5.38; N, 18.38.
4. Materials and methods
4.1. Materials
All biochemical reagents and chemical solvents were purchased
from commercial sources. DMF was distilled over standard drying
agents and degassed with nitrogen prior to use. IR spectra were re-
corded on a Shimadzu FTIR 8400S spectrometer. 1H and 13C NMR
spectra were taken on a Bruker DRX300 spectrometer at room tem-
perature. ESI-MS spectra were obtained using a LCQ instrument
(Finnigan, USA). Elemental analysis was made on an Elementar
Vario MICRO instrument (Germany). For all experiments, the com-
pound was dissolved in DMSO as 10 mM stock solution and further
dilutions were made in the corresponding buffer.
Calf thymus (ct) DNA was purchased from Sigma, the quadru-
plex-forming oligonucleotides dG3(T2AG3)3 (h-telo), d(CG3CG3CGC-
GAG3AG4) (c-kit2) and d(TGAG3TG4AG3TG4A2) (c-myc) were
purchased from SBS Genetech Co., Ltd (Beijing, China). The HeLa
(human cervix epitheloid carcinoma) cells and the HepG2 (human
hepatocellular carcinoma) cells were bought from Boster Bio-Engi-
neering Limited Company (China).
4.2.2. 2-[4-[2-(1-Piperidyl)ethoxy]phenyl]-1H-imidazo[4,5-f]-
1,10-phenanthroline (2)
Compound 2 was prepared from ligand p-hpip and N-(2-chloro-
ethyl)piperidine hydrochloride as described for compound 1 above.
The product was purified by chromatographic column on silica gel
using MeOH–CHCl3–glacial acetic acid (1:2:0.02, v/v/v) as an elu-
ent. Yellow solid (0.08 g, 29%). 1H NMR (300 MHz, DMSO-d6): d
(ppm) 8.99–8.92 (m, 4H), 8.31 (d, J = 8.1 Hz 2H), 7.75 (m, 2H),
7.07 (d, J = 8.1 Hz, 2H), 4.13 (m, 2H), 2.67 (m, 6H), 1.49 (m, 4H),
1.37 (m, 2H). 13C NMR (75 MHz, DMSO-d6): d (ppm) 159.2, 151.6,
147.3, 142.5, 130.4, 127.9, 123.2, 121.7, 114.4, 65.4, 57.1, 54.2,
25.3, 23.7. IR (KBr, cmꢀ1): 3411, 2933, 2852, 1577, 1526, 1483,
1443, 1413, 1359, 1251, 1180, 1043, 1012, 837, 740, 650. ESI(+)-
MS (m/z): 424.50 (58%), [M+H]+; 446.42 (64%), [M+Na]+; 869.33
(100%), [2M+Na]+. Anal. Calcd for C26H25ON5: C, 73.73; H, 5.95;
N, 16.54. Found: C, 73.57; H, 5.83; N, 16.73.
4.2.3. 2-[4-(2-Pyrrolidin-1-ylethoxy)phenyl]-1H-imidazo[4,5-f]-
1,10-phenanthroline (3)
4.2. Synthesis
Compound 3 was prepared from ligand p-hpip and N-(2-chloro-
ethyl)pyrrolidine hydrochloride as described for compound 1
above. The product was purified by chromatographic column on
silica gel using MeOH–CHCl3–glacial acetic acid (1:4:0.02, v/v/v)
4-(1H-Imidazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol (p-hpip)
the 1,10-phenanthroline-5,6-dione was prepared by oxidation of
1,10-phenanthroline following the literature methods,48–50 and

C.-Y. Wei et al. / Bioorg. Med. Chem. 21 (2013) 3379–3387
3385
as an eluent. Yellow solid (0.07 g, 25%). 1H NMR (300 MHz, DMSO-
d6): d (ppm) 9.05 (m, 2H), 9.00 (m, 2H), 8.36 (d, J = 8.4 Hz, 2H), 7.84
(m, 2H), 7.13 (d, J = 8.4 Hz, 2H), 4.17 (t, J = 5.7 Hz, 2H), 2.83 (t,
J = 5.7 Hz, 2H), 2.55 (m, 4H), 1.70 (m, 4H).13C NMR (75 MHz,
DMSO-d6): d (ppm) 161.9, 153.4, 149.5, 142.5, 130.6, 126.4,
124.6, 121.7, 117.2, 68.9, 56.4, 31.6, 25.5. IR (KBr, cmꢀ1): 3411,
2925, 2873, 1579, 1524, 1481, 1452, 1402, 1359, 1249, 1178,
1074, 1045, 835, 740, 659. ESI(+)-MS (m/z): 410.42 (100%),
[M+H]+. Anal. Calcd for C25H23ON5: C, 73.34; H, 5.66; N, 17.10.
Found: C, 73.18; H, 5.57; N, 17.31.
were carried out by the stepwise addition of either ctDNA or G-
quadruplex DNA solution to a cell containing 30, 30, 20, and
25 lM compounds 1, 2, 3, and 4, respectively, and the spectrum
was collected after equilibration for 10 min at 25 °C. Prior to use,
the oligomers h-telo (d[AG3(T2AG3)3]), c-kit2 ([CG3CG3CGC-
GAG3AG4]), and c-myc (d[TGAG3TG4AG3TG4A2]) dissolved in PBS
buffer containing 100 mM KCl were heated to 95 °C for 5 min, then
gradually cooled to room temperature and incubated at 4 °C over-
night. The formation of G-quadruplex was confirmed by CD spec-
tra. CtDNA was dissolved in buffer and kept at 4 °C for 48 h, a
solution of ctDNA in the buffer gave a ratio of UV absorbances of
about 1.9 at 260 and 280 nm, indicating that the DNA is sufficiently
free of protein. The titration was terminated when the wavelength
and intensity of the absorption band for compounds did not change
any more upon three successive additions of DNA. The binding
4.2.4. 4-[2-[4-(1H-Imidazo[4,5-f]-1,10-phenanthrolin-2-
yl)phenoxy]ethyl]morpholine (4)
Compound 4 was prepared from ligand p-hpip and 4-(2-chloro-
ethyl)morpholine hydrochloride as described for compound 1
above. The product was purified by chromatographic column on
silica gel using C2H5OH–glacial acetic acid (1:0.02, v/v) and
MeOH–CHCl3–glacial acetic acid (1:4:0.02, v/v/v) as an eluent. Yel-
low solid (0.09 g, 31%). 1H NMR (300 MHz, DMSO-d6): d (ppm) 9.00
(m, 4H), 8.32 (m, 2H), 7.78 (m, 2H), 7.09 (m, 2H), 4.18 (m, 2H), 3.61
(m, 4H), 2.70 (m, 2H), 2.28 (m, 4H). 13C NMR (75 MHz, DMSO-d6): d
(ppm) 161.8, 153.1, 148.6, 143.1, 133.6, 129.9, 123.1, 120.0, 116.6,
68.1, 67.4, 58.9, 57.9. IR (KBr, cmꢀ1): 3421, 2931, 2854, 1580, 1524,
1446, 1427, 1344, 1249, 1178, 1116, 1024, 948, 835, 742, 663.
ESI(+)-MS (m/z): 245.42 (100%), [M+2Na+H+H2O]3+; 973.17 (58%),
[2M+5H2O+CH3OH]. Anal. Calcd for C25H23O2N5: C, 70.57; H,
5.45; N, 16.46. Found: C, 70.33; H, 5.32; N, 16.69.
constant, Kb, was determined from a [DNA]/(ea
plot according to
ꢀef) versus 1/(ebꢀef)
½DNAꢂ=ðea
ꢀ
ef Þ ¼ ½DNAꢂ=ðeb
ꢀ
ef Þ þ 1=½ðeb
ꢀ
ef ÞKbꢂ; ð1Þ
where [DNA] is the concentration of DNA in base pairs for ctDNA
and quartets for G-quadruplex DNA, ea is the extinction coefficient
observed for the spectral band at a given DNA concentration, ef is
the extinction coefficient of the compound free in solution, eb is
the extinction coefficient of the compound when fully bound to
DNA.
4.5. Competition dialysis analysis
A PBS buffer containing 100 mM KCl (pH 7.4) was used for all
experiments. For each competition dialysis assay, 400 mL of dialy-
4.3. FRET melting assay
sis solution containing 1
a beaker. A volume of 0.5 mL at 40
l
M compounds 1, 2, or 3 was placed into
FRET melting assay was carried out on a Varian Cary Eclipse
fluorescence spectrometer equipped with a Peltier temperature
control accessory with excitation at 483 nm and detection at
500–700 nm. Both excitation and emission slits were 5 nm. The
G-quadruplex-forming sequences comprise the human telomeric
DNA (h-telo, 50-FAM-d(G3[T2AG3]3)-TAMRA-30) and two promoter
DNAs (c-kit2, 50-FAM-d(CG3CG3CGCGAG3AG4)-TAMRA-30, and c-myc,
50-FAM-d(TGAG3TG4AG3TG4A2)-TAMRA-30. Donor fluorophore
FAM: 6-carboxyfluorescein, and acceptor fluorophore TAMRA:
6-carboxytetramethylrhodamine), which was diluted from stock
to the correct concentration (400 nM) in 10 mM K2HPO4/KH2PO4
buffer (PBS) (pH 7.4) containing either 100 mM KCl (for h-telo
and c-kit2) or 40 mM KCl (for c-myc) and then annealed by heating
to 90 °C for 5 min, followed by cooling to room temperature. Be-
cause the Tm value of c-myc G-quadruplex alone is above 80 °C in
buffer containing 100 mM KCl, it is difficult to measure accurately
their Tm values in the presence of compounds, thus the buffer solu-
tion containing 40 mM KCl was used for c-myc FRET experiment.
Samples were prepared by adding aliquoting 1 mL of the annealed
G-quadruplex (at 2ꢁ concentration, 400 nM) into cells, followed by
addition of buffer or the compound solutions (at 2ꢁ concentration)
1 mL, and further incubated for 1 h, thus the final concentration of
lM monomeric unit of each of
the DNA samples was pipetted into a separate 0.5 mL dialysis bag.
The entire dialysis bag was then placed in the beaker containing
the dialysis solution. The contents were allowed to equilibrate with
continuous stirring for 24 h at 22–23 °C. After equilibration, DNA
samples were carefully removed to microfuge tubes and were trea-
ted with a final concentration of 1% (w/v) triton x-100. The total
concentration of compound within each dialysis bag was then
determined by absorption spectroscopy using extinction coeffi-
cients at 350 nm of 5005, 2490, and 3302 Mꢀ1 cmꢀ1 for compounds
1, 2, and 3, respectively. An appropriate correction was made to ac-
count for volume changes due to the addition of triton x-100 solu-
tion. The amount of bound drug was determined by difference
(Cb = CtꢀCf, where Cf is the concentration of bulk drug solution, Ct
is the total drug concentration that enters the dialysis bag, and
Cb is the concentration of drug bound to the nucleic acid). The data
were plotted as a bar graph using Origin 7.5. Competitive dialysis
for every sample was repeated at least three times.
4.6. CD spectroscopy
CD spectra were recorded on a dual-beam DSM 1000 CD spec-
trophotometer (Olis, Bogart, GA). Each measurement was the aver-
age of three repeated scans recorded from 220 to 320 nm with a
0.1 cm quartz cell at 25 °C. The scanning rate (22 nm minꢀ1) was
automatically selected by the Olis software as a function of the sig-
nal intensity to optimize data collection. G-quadruplex or oligomer
G-quadruplex is 0.2
tored in the presence of various concentrations of compounds (0,
1, 2, and 3 M) without and with 2.0 and 5.0 M double stranded
lM. The melting of G-quadruplex was moni-
l
l
DNA (ds26, 50-CAATCGGATCGAATTCGATCCGATTG-30) competitor.
Fluorescence readings at 533 nm were recorded at intervals of 2–
3 °C over the range 5–95 °C, with a constant temperature being
maintained for 5 min prior to each reading to ensure a stable value.
Final analysis of the data was carried out using Origin 7.5 (Origin-
Lab Corp.).
at a final concentration of 5 lM in the presence of various concen-
trations of the compounds was tested. After each addition of the
compound, the reaction was stirred and allowed to equilibrate
for at least 10 min (until no elliptic changes were observed) and
the CD spectrum was collected. A background CD spectrum of cor-
responding buffer solution was subtracted from the average scan
for each sample. Final analysis of the data was carried out using
Origin 7.5.
4.4. Absorption spectroscopy
Absorption spectra were measured on a Cary 50 spectropho-
tometer with a 1 cm path length quartz cell. Absorption titrations

3386
C.-Y. Wei et al. / Bioorg. Med. Chem. 21 (2013) 3379–3387
4.7. TRAP assay
(2007, 2011-016), and the Selected Research Project Supported
by Shanxi Scholarship Council of China (2009).
Theabilityofcompoundstoinhibittelomeraseinacell-freesystem
wasassessedwiththeTRAP-LIGassayfollowingpreviouslypublished
procedures.39,57 Protein extracts from exponentially growing HeLa
Supplementary data
cellswereused.Briefly,0.1 l
gofTSforwardprimer(50-AATCCGTCGAG-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.059.
CAGAGTT-30)waselongatedbytelomerase(500 ngproteinextract)in
TRAP buffer (20 mM Tris–HCl (pH 8.3), 68 mM KCl, 1.5 mM MgCl2,
1 mM EGTA, and 0.05% Tween 20) containing 125
lM dNTPs and
0.05 gBSA.Themixwasaddedtotubescontainingfreshlypreparedli-
l
References and notes
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outfor20 minat30 °C,followedby94 °Cfor5 minandafinalmainte-
nanceofthemixtureat20 °C.Topurifytheelongatedproductandtore-
move the bound ligands, the QIA quick nucleotide purification kit
(Qiagen) was used according to the manufacturer’s instructions. The
purifiedextendedsampleswerethensubjectedtoPCRamplification.
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