Microwave-assisted synthesis of phenanthroimidazole derivatives as stabilizer of c-myc G-quadruplex DNA

Article abstract of DOI:10.1016/j.bmc.2014.09.003

c-myc G-quadruplex DNA, which plays a central role in tumor progression and resistance, has been extensively investigated as potential target of antitumor drugs. In this paper, a series of phenanthroimidazole derives have been synthesized under irradiation of microwave in yields of 51-80%. The antitumor activity of these compounds against various tumor cells has been evaluated, and the results show that these compounds exhibit great inhibition to MDA-MB-231, MCF-7 and Hela cells, especially 5 inhibit the growth of MDA-MB-231 cells with IC₅₀ about 3.6 μM. The further studies show that 5 can bind and stabilize c-myc G4 DNA in π-π stacking mode, which confirmed by the hypochromise in the electronic spectra of 5 with the increasing of c-myc G4 DNA. When dealt with 5, the strength of CD signal attributed to c-myc G4 DNA is decreased and the FRET melting point of c-myc G4 DNA is increased. Moreover, the molecule docking calculation was conducted to show that 5 suitably stack onto the 5′ G-quartet surface, and parallels to the surfaces of the G5 and G-quartet consisting of G7, G11, G16, and G20. As a result, the replication of c-myc oligomers is blocked by 5. In a word, this type of phenanthroimidazole derives can act as potential inhibitor against breast cancer cells by binding and stabilizing c-myc G4 DNA through π-π stacking.

Full text of DOI:10.1016/j.bmc.2014.09.003

Bioorganic & Medicinal Chemistry 22 (2014) 6503–6508
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Microwave-assisted synthesis of phenanthroimidazole derivatives
as stabilizer of c-myc G-quadruplex DNA
b,
Siyan Liao a, Zhao Zhang b, Qiong Wu b, Xicheng Wang c, , Wenjie Mei
⇑
⇑
a Department of Chemistry, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 510182, PR China
b School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
c The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou 510006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2014
Revised 23 August 2014
Accepted 2 September 2014
Available online 28 September 2014
c-myc G-quadruplex DNA, which plays a central role in tumor progression and resistance, has been exten-
sively investigated as potential target of antitumor drugs. In this paper, a series of phenanthroimidazole
derives have been synthesized under irradiation of microwave in yields of 51–80%. The antitumor activity
of these compounds against various tumor cells has been evaluated, and the results show that these com-
pounds exhibit great inhibition to MDA-MB-231, MCF-7 and Hela cells, especially 5 inhibit the growth of
MDA-MB-231 cells with IC50 about 3.6
lM. The further studies show that 5 can bind and stabilize c-myc
Keywords:
Phenanthroimidazole derives
c-myc G-quadruplex DNA
p–p stacking
Molecular docking
G4 DNA in stacking mode, which confirmed by the hypochromise in the electronic spectra of 5 with
p–p
the increasing of c-myc G4 DNA. When dealt with 5, the strength of CD signal attributed to c-myc G4 DNA
is decreased and the FRET melting point of c-myc G4 DNA is increased. Moreover, the molecule docking
calculation was conducted to show that 5 suitably stack onto the 50 G-quartet surface, and parallels to the
surfaces of the G5 and G-quartet consisting of G7, G11, G16, and G20. As a result, the replication of c-myc
oligomers is blocked by 5. In a word, this type of phenanthroimidazole derives can act as potential inhib-
itor against breast cancer cells by binding and stabilizing c-myc G4 DNA through p–p stacking.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
DNA intercalators. There are number of evidence to show that this
type of compound have wide applications as DNA-structural probe,
molecular ‘Light Switch’, DNA-photocleavage reagent and antitu-
mor drugs for their unique electrochemical and photophysical
properties.8 In our previous works, it’s found that imi-
dazo[4,5f][1,10]phenanthroimidazole derivatives can inhibit effec-
tively the growth of human lung cancer A549 cells and induced
tumor cells apoptosis, and down-regulate the expression of
C-MYC protein by this compound have also been observed.11 How-
ever, it’s still not clear whether phenanthroimidazole derives can
inhibit the growth of tumor cells through stabilized the G-quadru-
plex structure of c-myc promoter, and it’s very important to design
novel candidate with high activity and selectivity to tumor cells.
In this paper, a series of phenanthroimidazole derivatives
(Fig. 1) have been synthesized with yield of 87% under microwave
irradiation.12 It’s found that this class of compounds, especially 5
can inhibit the growth of various tumor cells, especially
MDA-MB-231 cells. The binding behavior of 5 with c myc G4
DNA have been investigated by spectroscopy methods, FRET and
PCR-stop assay, and the results show that 5 can stabilize the
G-quadruplex structure and inhibit the replication of c-myc
oligomers. This has been further confirmed by the molecular
docking calculation to suggest that 5 may bind to c-myc G4 DNA
Oncogene c-myc, which is over-expression in a wide variety of
human cancers, plays a critical role as a regulator of gene transcrip-
tion involved in cell proliferation, cell division and metabolism.1–3
The promoter region of c-myc oncogene are G-rich single-strand
DNA and can form G-quadruplex structure via Hoogsten hydrogen
bond in the presence of potassium and sodium ion.4,5 It’s reported
that ligands that stabilize the c-myc overhang into G-quadruplex
can be considered as potential antitumor agents to block the repli-
cation of c-myc. Various of small molecules, such as porphyrins,
quindoline derivatives, carbamide analogs and quarfloxin, have
been described to bind to c-myc G-quadruplex DNA and down-
regulate the expression of C-MYC protein.6–9 Some of these deriv-
atives have been found to induce the apoptosis of tumor cells by
stabilizing c-myc G-quadruplex structure.10 Thus, it’s been devel-
oped to be a promising strategy for the treatment of cancer by sta-
bilizing G-quadruplex structure to suppression activity of c-myc.
Phenanthroimidazole derivatives with enlarged aromatic rings,
which can bind to DNA through p–p stacking, are a type of classic
⇑
Corresponding authors.
E-mail addresses: 13902400598@126.com (X. Wang), wenjiemei@126.com
via p–p stacking in an intercalating mode.
(W. Mei).
http://dx.doi.org/10.1016/j.bmc.2014.09.003
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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S. Liao et al. / Bioorg. Med. Chem. 22 (2014) 6503–6508
R1
R1
H
N
N
O
O
N
N
NH4Ac/HAc
+ OHC
R2
R2
MW 100 oC 20 min
N
N
1 R1=-H R2=-H
4 R1=-Br R2=-H
2 R1=-SO3H R2=-H
5 R1=-H R2=-Br
3 R1=-H R2=-SO3H
Figure 1. Synthesis route of phenanthroimidazole derivatives under the irradiation of microwave.
(d, J = 1.8 Hz, 1H), 8.54–8.50 (m, 1H), 8.50–8.48 (m, 1H), 8.17–
2. Chemicals and methods
2.1. Chemicals
8.15 (m, 1H), 8.15–8.12 (m, 1H), 7.83 (dd, J = 8.1, 4.3 Hz, 1H). 13C
NMR (126 MHz, d6-DMSO, d ppm) 150.08 (s), 145.82 (s), 142.88
(s), 136.77 (s), 131.79 (s), 129.84 (s), 128.69 (s), 125.42 (s), 58.04
(s), 45.54 (s). 4: Yield, 81.7%; 1H NMR (500 MHz, d6-DMSO, d
ppm) 9.04 (dd, J = 4.3, 1.8 Hz, 1H), 8.86 (d, J = 7.3 Hz, 1H), 7.89 (d,
J = 1.1 Hz, 1H), 7.87 (d, J = 1.1 Hz, 1H), 7.85 (t, J = 2.6 Hz, 1H),
7.85–7.80 (m, 1H), 7.61 (td, J = 7.5, 1.2 Hz, 1H), 7.56–7.49 (m,
1H). 13C NMR (126 MHz, d6-DMSO, d ppm) 151.66 (s), 150.00 (s),
145.64 (s), 135.37 (s), 134.40 (d, J = 8.4 Hz), 133.58 (s), 131.61 (s),
129.93 (s), 125.43 (s), 124.09 (s). 5: Yield, 89.0%; 1H NMR
(500 MHz, d6-DMSO, d ppm) 9.02 (dd, J = 4.3, 1.8 Hz, 1H), 8.89 (d,
J = 1.8 Hz, 1H), 8.88 (d, J = 1.8 Hz, 1H), 8.23–8.21 (m, 1H), 8.21–
8.18 (m, 1H), 7.85–7.79 (m, 2H). 13C NMR (126 MHz, d6-DMSO, d
ppm) 151.50 (s), 149.98 (s), 145.69 (s), 134.08 (s), 131.66 (s),
131.22 (s), 130.12 (s), 125.37 (s), 124.96 (s), 64.81 (s), 58.05 (s).
All reagents and solvents were purchased commercially and
used without further purification unless specially noted. Distilled
water was used in all experiments. c-myc oligomers strand
50-TGAGGGTGGGTGGGTAA-30 was purchased from Sangon Biotech
and formed G-quadruplex conformation as literature by renatur-
ation for 24 h at 4 °C, after denaturation for 5 min at 90 °C.13,14
All aqueous solutions were prepared with doubly distilled water.
The Tris-HCl buffer consisting of Tris and KCl, and the pH value
was adjusted to 7.2 by HCl solution, which was applied to UV
titration, Real time PCR (Biored IQ5) and CD spectra.
2.2. Physical measurements
The phenanthroimidazole derivatives were synthesized by
using Anton Paar monowave 300 microwave reactor. ESI-MS spec-
tra were obtained in methanol on Agilent 1100 ESI-MS system
operating at room temperature. UV–vis absorption spectra were
recorded on a Shimadzu UV-2550 spectrophotometer using 1 cm
path length quartz cuvettes (3 mL). Circular dichroism (CD) spectra
were operated on a Jasco J810 spectropolarimeter. Molecular dock-
ing data were calculated by using Addsol, Autogrid and Auto Tors
tool of the software. FRET assay were determined with a Bio-Radi
Q5 realtime PCR detection system. PCR-stop assay were operated
on AlphaSC Thermal Cycler.
2.4. Biological activity in vitro
All complexes were dissolved in DMSO with stock solution at
1 mM. Cell viability was determined by measuring the ability of
cells to transform MTT to a purple formazan dye.4 Cells were seeded
in 96-well tissue culture plates (3 ꢀ 103 cells/well) for 24 h. The
cells were then incubated with the tested compounds at different
concentrations for 72 h. After incubation, 20 ll/well of MTT solu-
tion (5 mg/ml phosphate buffered saline) was added and incubated
for 5 h. The medium was aspirated and replaced with DMSO
(150 ll/well) to dissolve the formazan salt. The absorbance inten-
sity, which reflects the cell growth condition, was measured at
570 nm using a microplate spectrophotometer (Versamax).
2.3. Synthesis of imidazole[4,5f][1,10] phenanthroline
derivatives
2.5. Drug distribution and location
Phenanthroimidazole derivatives were synthesized according to
the literature procedure with some modification. In general, a mix-
ture of 1,10-phenanthroline-5,6-dione (1.50 mmol), benzaldehyde
(2.25 mmol), ammonium acetate (51.9 mmol) and glacial acetic
acid was heated at 100 °C for 20 min under microwave irradiation.
Then, 20 ml of water was added and the pH value was adjusted to
7.0 at room temperature.15–17 The solution was filtered and dried
in vacuum to obtain a yellow precipitate. The product was purified
in a silicagel column by using ethanol as eluent. (1) Yield, 85.6%; 1H
NMR (500 MHz, d6-DMSO, d ppm) 9.03 (dd, J = 4.3, 1.7 Hz, 1H), 8.94
(dd, J = 8.1, 1.7 Hz, 1H), 8.32–8.26 (m, 1H), 7.83 (dd, J = 8.1, 4.3 Hz,
1H), 7.64–7.57 (m, 1H), 7.54–7.49 (m, 1H). 13C NMR (d6-DMSO, d
ppm) 152.65 (s), 149.87 (s), 145.57 (s), 132.06 (s), 131.76 (s),
131.65 (s), 131.05 (s), 128.29 (s), 125.36 (s), 64.81 (s), 58.05 (s).
(2) Yield, 87.0%; 1H NMR (500 MHz, d6-DMSO, d ppm) 9.06 (dd,
J = 4.4, 1.7 Hz, 1H), 8.41 (dd, J = 7.6, 1.2 Hz, 1H), 8.10–8.04 (m,
1H), 7.92 (ddd, J = 8.0, 4.4, 1.7 Hz, 1H), 7.62 (td, J = 7.5, 1.5 Hz,
1H), 7.54 (td, J = 7.6, 1.4 Hz, 1H), 7.22 (s, 1H), 7.12 (s, 1H), 7.02 (s,
1H). 13C NMR (126 MHz, d6-DMSO, d ppm) 152.36 (s), 149.40 (s),
146.17 (s), 144.07 (s), 132.90 (s), 131.81 (s), 131.08 (s), 129.87
(s), 128.89 (s), 125.96 (s). 3: Yield, 51.4%; 1H NMR (500 d6-DMSO,
d ppm) 9.04 (dd, J = 4.3, 1.8 Hz, 1H), 8.92 (d, J = 1.8 Hz, 1H), 8.90
All complexes were dissolved in DMSO with stock solution at
1 mM. Cells were seeded in 6-well tissue culture plates
(5 ꢀ 104 cells/well) and were dealted with drugs at 10
lM for
24 h. It’s observed that different fluorescence of drugs in cells by
using fluorescence microscope (Leica DMI4000B).18
2.6. Electronic absorption measurements
UV–vis absorption spectra were recorded on
a Shimadzu
UV-2550 spectrophotometer by using 1 cm path length quartz cuv-
ettes (3 mL). The absorption titration of the complex 5 in Tris-HCl
buffer was performed by using a fixed drug concentration to which
increments of the DNA stock solution were added. The concentra-
tion of drugs is 20 lM and c-myc G4 DNA was added by degrees.
Drug-DNA solutions were allowed to incubate for 3 min before
the absorption spectra were recorded.
2.7. CD spectra measurements
CD spectra were recorded on a Jasco J810 Circular Dichroism
(CD) Spectrophotometer with a thermoelectrically controlled cell

S. Liao et al. / Bioorg. Med. Chem. 22 (2014) 6503–6508
6505
holder. The cell path length was 1 cm.19 The CD spectra of c-myc G4
DNA (2 M) with the increase of 5 (1 mM) with 2 L for every
3 min. CD spectra were recorded in the range of 230–600 nm in
Table 1
The inhibitory activity (IC50
human breast cancer cells.
/l
M) of the target compounds and cis-platin against
l
l
Compd
Inhibitory activity/IC50 (lM)
0.5 nm increments with an averaging time of 0.5 s.
MDA-MB-231
MCF-7
Hela
5.1
240.3
62.4
7.9
2.8. FRET melting assays
1
2
74.2
152.3
60.0
36.0
3.6
72.6
161.9
184.3
27.3
22.9
3.0
3
4
5
The fluorescent labeled oligonucleotide, c-myc DNA
(50-FAM-TGGGGAGGGTGGGGAGGGTGGGGAAGG-TAMRA-30, FAM:
carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine)
used as the FRET probes was diluted in Tris-HCl buffer and then
annealed by being heated to 92 °C for 5 min, followed by slowly
cooling to room temperature. Fluorescence melting curves were
determined with a Bio-Radi Q5 realtime PCR detection system,
by using a total reaction volume of 25 mL, with labeled oligonu-
8.5
9.4
cis-platin
36.1
3.2. Drug distribution and location
As shown in Figure 2B, a number of MCF-7 cells were shrinking
to globe after dealt with 5 at 10 M for 24 h. It’s observed that
cleotide (1 lM) and different concentrations of 5 in Tris-HCl
l
buffer.9 A constant temperature was maintained for 30 s prior to
each reading to ensure a stable value. Final analysis of the data
was carried out by using Origin7.5 (Origin Lab Corp.).
there are less than 20% cell membrane surface become rough and
uneven with large and more particles, the cell membrane structure
is incomplete. Especially, the cell surface obviously appeared
numerous holes and displayed the obvious characteristics of apop-
tosis of MCF-7 cells. Besides, 5 entered into MCF-7 cells and exhib-
ited green and blue flurensence, which was located and distributed
in cell cytoplasm and nucleus. As shown in Figure 2A, the fluren-
sence observation for 4 was also found that 4 also exhibited green
and blue flurensence, which displayed similar activity with 5.
Compounds 1–3 with little fluorescence was observed, complex 4
and 5 can be uptook by MCF-7 cells and glowing strong green
and blue fluorescence, a possible reason is mainly due to the
absence of hyperchrome Br atom.
2.9. PCR stop assay
The PCR-stop assay was performed by using
protocol of the previously reported method. The reactions
(40
a modified
l
L) were performed in 1 ꢀ PCR buffer, containing each pair
of c-myc G4 DNA (10 pM), deoxy nucleotide triphosphate
(0.16 mM), Taq polymerase (2.5U), and increasing concentrations
of the compound (0–250 l
M).6,9 The reaction mixtures were in
cubation in a thermocycler under the following cycling condi-
tions: 94 °C for 3 min followed by 30 cycles at 94 °C for 30 s,
58 °C for 30 s, 72 °C for 30 s. Amplified products were resolved
on 15% nondenaturing polyacrylamide gels in 1 ꢀ TBE and silver
stained.
3.3. Electronic titration
Considering c-myc oncogene plays a key role in the prolifera-
tion, apoptosis, invasion and metastasis of tumor cells, the interac-
tion of complex 5 with c-myc G-quadruplex DNA has been
investigated as a promising target for anti-tumor agent.
2.10. Molecular docking study
Electronic spectra titration are one of the most common meth-
ods to evaluate the interaction of small molecules with biology
macromolecules, which are conducted to confirm whether 5 can
bind to c-myc G4 DNA. In general, small molecules exhibit charac-
teristic absorption in electronic spectra which will undergo
hypochromism and red shift in the presence of biology molecules
and the degree of change depend on the binding affinity.20
As shown in Figure 3A, the electronic spectra of 5 in Tris-HCl
buffer(pH = 7.2) solution exhibit the characteristic IL (intraligand
charge transfer) absorption in the range of 250–300 nm with the
maximum at 274 nm. When c-myc G4 DNA was added into the
solutions, obvious hypochromism and red shift was observed.
Automated docking studies were per-formed with three differ-
ent docking algorithms, AutoDock 3.0 (‘Lamarckian’ genetic
algorithm), FlexX 1.10 (incremental construction algorithm, as
implemented in Sybyl 6.8), and GOLD 1.2 (‘Darwinian’ genetic
algorithm). As scoring is a very important second aspect of auto-
mated docking method-ologies, it was decided to investigate the
effect of rescoring: the process of reprioritization of docking solu-
tions (primarily ranked by the ‘native’ scoring function imple-
mented in the docking program) with an additional stand-alone
scoring function.
The hypochromism at IL absorption is about 9.4% (Dk = 2.5 nm),
indicating that 5 can bind to c-myc G4 DNA.
3. Results and discussion
3.1. Biological activity
3.4. Circular dichroism spectra
The antitumor activity of synthetic phenanthroimidazole
derivatives against MDA-MB-231 highly metastatic human breast
adenocarcinoma cells and MCF-7 lowly metastatic human breast
cancer cells, and cervical cancer Hela cells was evaluated by
MTT assay, as shown in Table 1. It’s found that all of these com-
pounds, exhibit certain inhibition to various tumor cells after 72 h
treatment, especially 4 and 5 displayed great antitumor activity.
The inhibitory activity (IC50) of 5 against MDA-MB-231 cells is
Circular dichroism (CD) spectra, which is a powerful stool to
investigate the conformation change of biology molecules, was
been further carried out to confirmed the binding of 5 with
c-myc G4 DNA, as shown in Figure 3B. In general, G-quadruplex
DNA will exhibit a positive CD signal in the range of 250–300 nm,
with the maximum at 264 nm.21 Upon the addition of 5, the
decreased signals of c-myc G4 DNA in the CD spectra were
observed. The CD signal for c-myc G4 DNA at 264 nm gradually
receded with the addition of 5 and the CD signal strengths
decreased 23.0%. The change is in consistent with the induction
of the guanine-rich DNA to form the G-quadruplex DNA structure
by compound, and the results has been treated as evident of
about 3.6
lM, which is far more less to that of cis-platin
(36.1 M). These results shown that phenanthroimidazole deriva-
l
tives can effectively inhibit the growth of tumor cells, and the
para-orienting complex exhibits better antitumor activity than
ortho-orienting.

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S. Liao et al. / Bioorg. Med. Chem. 22 (2014) 6503–6508
A
B
Figure 2. The distribution and location of complex 4 (A) and 5 (B) in MDA-MB-231 cells by using fluorescence microscope.
Figure 3. (A) The electronic spectra of 5 in absence and in presence of c-myc G-quadruplex DNA. [5] = 5 ꢀ 10ꢁ5 M, [c-myc G-quadruplex DNA] = (0–40) ꢀ 10ꢁ4 M. Arrow
shows the absorbance changing upon increasing c-myc G-quadruplex DNA concentrations. (B) The CD spectra of c-myc G-quadruplex DNA in the absence and presence of
increasing amounts of 5. [c-myc G-quadruplex DNA] = 2 ꢀ 10ꢁ6 M, [5] = (0–118) ꢀ 10ꢁ6 M. Arrow shows the absorbance changing upon increasing 5.
binding behavior for the ligand and indicated that the G-quadru-
3.6. Molecular docking
plex DNA structure was stabilized by ligand 5.
Molecular docking study was performed to investigate the
possible binding interaction between 5 and c-myc G-quadruplex
DNA. The conformation with the lowest binding energy
(ꢁ8.04 kcal/mol) was selected as the best binding conformation
from the first cluster including 64 docking conformations.7 The
binding energy is made up of two components, intermolecular
energy (ꢁ8.34 kcal/mol) and torsional energy (0.30 kcal/mol),
and intermolecular energy is contributed by two parts as follows:
the sum (ꢁ8.38 kcal/mol) of van der Waals energy, hydrogen
bond energy and desolvation energy, and the electrostatic energy
(0.04 kcal/mol). In addition, 5 is hydrophobic with CLogP = 4.66
calculated by ChemBioDraw software. This property just suits to
the corresponding hydrophobic environment inside the G-quad-
ruplex DNA. Therefore, the van der Waals interactions between
3.5. FRET melting point curves
To investigate the thermodynamic stability of the c myc G4 DNA
with 5, a FRET melting point assay was carried out to detect the
melting temperature.
As shown in Figure 4A, the melting point8 of c-myc G4 DNA is
about 43.8 °C, upon the addition of 1.0 and 3.0
lM of 5, the Tm
increased to 47.2 ( Tm = 3.4) and 50.2 ( Tm = 6.4) °C, respectively.
D
D
Moreover, the melting point of the concentration -dependent
melting curves of 5 are showed in Figure 4B. It is found that all
of the
DTm values of the samples incubated with 5 increased
compared with the control value, indicating that 5 could enhance
the thermodynamic stability of c-myc oligomer.22

S. Liao et al. / Bioorg. Med. Chem. 22 (2014) 6503–6508
6507
Control
1
2
3
Concentration (µM)
A
B
Figure 4. (A) FRET melting profiles of 0.2 lM c-myc G-quadruplex DNA with 5; (B) The melting rising trend with the increasing of 5.
5 and c-myc G4 DNA may play a dominating role in binding
affinity.
Compound 5 is largely in the same plane with A6, and it respec-
tively parallels to the surfaces of the G5 and G-quartet consisting
Herein, a complete overview of docking binding mode of 5 to
c-myc G4 DNA is presented in Figure 5. It can be clearly seen that
the planarity 5 is suitably stacking onto the 50G-quartet surface.
of G7, G11, G16, and G20. Thus, p–p stacking interactions exist
presumably between 5 and the two surfaces to enhance the
binding affinity. These docking results reasonably explain our
A
B
Figure 5. (A) The binding mode between 5 and c-myc G-quadruplex DNA predicted by docking. (B) Docking conformation of 5 and corresponding surface of c-myc
G-quadruplex DNA, in which the red and blue regions represent oxygen and nitrogen atoms respectively, whereas grey and white regions represent carbon and hydrogen atoms.
Figure 6. Effect of 5 on the hybridization of c-myc G-quadruplex DNA in the PCR-stop assay, [5] = 0.0–40 ꢀ 10ꢁ6 M.

6508
S. Liao et al. / Bioorg. Med. Chem. 22 (2014) 6503–6508
experimental fact that 5 could bind to c-myc G4 DNA in intercalat-
ing mode.
Technology Project of Guangzhou (2012J2200034, 2013J4100072),
Science Research Program of Guangzhou Municipal Colleges
(2012C219) and Foundation of Guangzhou Medical University
(2010C15). Project supported by The Joint Natural Sciences Fund
of The Department of Science and Technology and The First
Affiliated Hospital of Guangdong Pharmaceutical University
(GYFYLH201309).
3.7. PCR-stop
PCR-stop assay have also been conduct to determine whether
these complexes were inhibit the activity of Taq polymerase by
stabilizing the G-quadruplex DNA structure of tested oligomer. In
the presence of some G-quadruplex DNA stabilizers, the template
sequence was induced into a G-quadruplex DNA structure that
blocked the hybridization and the detection of the final PCR prod-
uct. And the result was illustrated in Figure 6.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.09.003.
As shown in Figure 6, the single strand c-myc G-quadruplex
DNA will hybrid with a complementary strand in the presence of
Taq polymerase (Control). When 5 was added into the solution, a
G-quadruplex DNA structure was induced to block the hybridiza-
tion with a complementary strand. What’s more, the inhibitory
effect increased with increasing concentration.4,5 As a result, the
50–30 extension with Taq polymerase was inhibited and double-
stranded DNA PCR product was decreased.
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4. Conclusions
In conclusion, a series of phenanthroimidazole derivatives have
been prepared in average yield range of 50–80% under microwave
irradiation. It’s found that these compounds can effectively inhibit
the growth of tumor cells, especially 5 exhibit the best antitumor
activity against MDA-MB-231 cells. Further studies indicated that
5 can interact and stabilize the G-quadruplex structure of c-myc.
In addition, the molecular docking calculation suggested that 5
may bind to the base pair through p–p stacking, as a result to block
the replication of c-myc oligomer. In a word, these studies not only
offer the reliable binding behavior of 5 with c-myc G4 DNA but also
provide insights into the possible anticancer mechanism at
molecular level, and thus they will lay the foundation for the
rational design of new compounds with higher affinity to c-myc
G-quadruplex DNA.
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We are pleased to thank the financial support of the Natural
Science Foundation of Guangdong Province (S2011040000131),
Name of the Science and Technology Foundation of Guangdong
Province (20070327029), Guangdong Province Science and Tech-
nology Plan (20771044), Guangzhou City Science and Technology
Plan (2011J5200017), Medical Science and Technology Research
Foundation of Guangdong Province (WSTJJ20101231), Science and