12-N-Methylated 5,6-dihydrobenzo[c]acridine derivatives: A new class of highly selective ligands for c-myc G-quadruplex DNA

Article abstract of DOI:10.1016/j.ejmech.2012.03.034

12-N-Methylated and non-methylated 5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized as new series of c-myc G-quadruplex binding ligands. Their interactions with c-myc G-quadruplex were evaluated using fluorescence resonance energy tra

Full text of DOI:10.1016/j.ejmech.2012.03.034

European Journal of Medicinal Chemistry 53 (2012) 52e63
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
12-N-Methylated 5,6-dihydrobenzo[c]acridine derivatives: A new class of highly
selective ligands for c-myc G-quadruplex DNA
Sheng-Rong Liao, Chen-Xi Zhou, Wei-Bin Wu, Tian-Miao Ou, Jia-Heng Tan, Ding Li, Lian-Quan Gu,
*
Zhi-Shu Huang
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou University City, Waihuan East Road 132, Guangzhou 510006, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
12-N-Methylated and non-methylated 5,6-dihydrobenzo[c]acridine derivatives were designed and
synthesized as new series of c-myc G-quadruplex binding ligands. Their interactions with c-myc G-
quadruplex were evaluated using fluorescence resonance energy transfer (FRET) melting assay, circular
dichroism (CD) spectroscopy, surface plasmon resonance (SPR), polymerase chain reaction (PCR) stop
assay, and molecular modeling. Compared with the non-methylated derivatives, 12-N-methylated
derivatives had stronger binding affinity and stabilizing ability to c-myc G-quadruplex structure, and
could more effectively stack on the G-quartet surface. All these derivatives had high selectivity for c-myc
G-quadruplex DNA over duplex DNA. The reverse transcription (RT) PCR assay showed that compound
21c could down-regulate transcription of c-myc gene in Ramos cell line containing NHE III1 element, but
had no effect in CA46 cell line with NHE III1 element removed.
Received 15 January 2012
Received in revised form
16 March 2012
Accepted 17 March 2012
Available online 30 March 2012
Keywords:
c-myc G-Quadruplex
5,6-Dihydrobenzo[c]acridine derivatives
Selectivity
Ó 2012 Elsevier Masson SAS. All rights reserved.
12-N-Methylation
Binding affinity
1. Introduction
which some are alkaloid derivatives or their methylated products,
and the latter showed better potency.
Guanine-rich sequences can self-associate into planar guanine
quartets (G-quartets) that stack on each other to form unusual
structures called G-quadruplexes [1,2]. G-Quadruplexes can be
stabilized by cations (preferring Kþ) and widely distribute in telo-
meric regions [3,4] and gene promoters such as c-myc [5,6], c-kit
[7], bcl-2 [8], and VEGF [9]. The formation or stabilization of G-
quadruplexes in these regions may result in a series of biological
processes such as inhibition of telomerase [10], prevention of
telomere elongation [11], avoidance of chromosomal alignment
and recombination [3], and down-regulation of gene expression
[12]. Ligands that can selectively bind to and stabilize G-quadruplex
can interfere with telomere maintenance [13] or regulate gene
expression [14,15], therefore are promising lead compounds for
cancer treatment [16,17]. Several series of c-myc G-quadruplex
ligands have been developed, including macrocyclic [18e20], large
coplanar [14,18,20e22], and flexible molecules [20,23e25], in
Acridine is a fused polycyclic aromatic molecule, which is a main
scaffold in some natural acridine alkaloids [26]. Its derivatives such
as amasacrine and quinacrine have been originally used as effective
DNA-intercalating agents [27]. Recent years, acridine derivatives
have been modified as telomeric [28e34] or c-myc [18,20] G-
quadruplex ligands, in which some excellent ligands have been
discovered, such as BRACO-19 [20], BOQ1 [32], and RHPS4 [35].
Although most of these ligands have shown high telomerase
inhibitory activity (IC50 < 1.0
mM), they have exhibited moderate
stabilizing ability (
D
Tm w 10 ꢀC) and low selectivity to G-quad-
ruplex structures. It should be noted that an effective G-quadruplex
binding ligand should have not only good bioactivity, but also high
selectivity, in order to avoid acute toxicity and intolerable side
effects in normal tissues.
With this aim in mind and based on the previous studies, we
designed and synthesized a new class of crescent-shaped 5,6-
dihydrobenzo[c]acridine derivatives (Fig. 1) as c-myc G-quad-
ruplex ligands. Unlike the previous completely coplanar acridine
molecules, our new acridine derivatives have a saturated CeC bond
at the 5,6-position forming a slightly-twisted planar ring scaffold
with non-methylated (Fig. 1A) and methylated (Fig. 1B) structures.
The rationale for designing these derivatives are as follows: At first,
it is interesting to know whether these partially-saturated acridine
derivatives can act as G-quadruplex binding ligands, with good
Abbreviations: FRET, fluorescence resonance energy transfer; CD, circular
dichroism; SPR, surface plasmon resonance; NMR, nuclear magnetic resonance;
PCR, polymerase chain reaction; RT, reverse transcription; MTT, methyl thiazolyl
tetrazolium.
* Corresponding author. Tel./fax: þ86 20 39943056.
E-mail address: ceshzs@mail.sysu.edu.cn (Z.-S. Huang).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.034

S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
53
5
4
1
R1
R2
R1
R2
3
2
6
R3
N
R3
N
7
N
O
O
N
O
O
8
9
12
R4
R4
n
n
l
C
11
10
B
A
Fig. 1. Scaffold of 5,6-dihydrobenzo[c]acridine derivatives.
selectivity toward G-quadruplex DNA over duplex DNA. Secondly,
some previous studies [35,36] have shown that the methylation of
the nitrogen atom in the center of the ligands could strongly
enhance their binding and stabilizing activity toward G-quadruplex,
therefore, it is interesting to know whether the methylation of our
new acridine derivatives can also increase their interactions with G-
quadruplex. Thirdly, some studies have shown [32,37] that the fused
product 10 or 11 had dechlorination, hydrolysis of sulfonic group,
and alkylation with dibromoalkanes to afford compound 12e14.
Then the bromine atom was replaced with amine to yield final
products 15a, 15b, 16a, 17a, and 17c. Compound 12e14 could also
be methylated with methyl triflate on the nitrogen atom [42], anꢁd
then the bromine atom was replaced with amine, and lastly OTf3
was exchanged into Clꢁ using anion exchange resin [43] to yield
final products 20aee, 21b, and 21c (Scheme 2).
crescent-shaped planar structure could offer effective
p-orbital
stacking on the G-quadruplex, therefore it is interesting to know
whether our nearly planar crescent-shaped ligands can also effec-
tively interact with G-quadruplex through p-orbital stacking. Lastly,
3. Result and discussion
it is interesting to know whether the 1,3-dioxole ring is better for the
ligands to interact with G-quadruplex than the two methoxy
groups. Therefore, we synthesized a series of 5,6-dihydrobenzo[c]
acridine derivatives (Fig. 1), and studied their interactions with c-
myc G-quadruplex DNA through FRET-melting, CD spectroscopy,
SPR assay, PCR-stop assay, RT-PCR assay, and molecular modeling.
3.1. Stabilizing ability and selectivity studies with FRET
The stabilizing ability of 5,6-dihydrobenzo[c]acridine deriva-
tives on c-myc G-quadruplex DNA was evaluated with FRET-melting
experiments [44]. The c-myc gene FPu18T (50-FAM-AG3TG4AG3TG4-
TAMRA-30) was used as the G-quadruplex DNA model. The FRET-
melting data (Table 1) showed that the
compounds were in a wide range from 5.3 ꢀC to 25.4 ꢀC. The FRET
results indicated that the methylated derivatives ( Tm values of
11.6e25.4 ꢀC) had higher stabilizing ability than the non-
methylated ones (
Tm values of 5.3e11.2 ꢀC), which suggested
DTm values for all the
2. Chemistry
D
The 5,6-dihydrobenzo[c]acridine derivatives were synthesized
as follows: 1,2-dimethoxybenzene 1 was acylated with dihy-
drofuran-2,5-dione 2 using AlCl3 as catalyst in nitrobenzene at
0e60 ꢀC to afford compound 3, which was then deoxidized [38] and
intra-cyclized to afford intermediate 5. The intermediate 5 was
demethylated with 48% HBr to give compound 6, which was
cyclized with dibromomethane under KF to yield intermediate 7
(Scheme 1) [39].
Compound 8 was synthesized by following a previously repor-
ted procedure [40], which then had the oxidation of its formyl
group and the reduction of its nitro group to yield intermediate 9
(Scheme 2). Next the intermediate 5 or 7 was condensed with 9 in
phosphorus oxychloride under reflux condition [41], and the
D
that the central positive charge made an important contribution for
the methylated derivatives to interact with c-myc G-quadruplex,
and implied the nitrogen atom at the 12-N-position had weak
tendency of protonation. Among all the methylated derivatives, 21b
and 21c with the 2,3-dimethoxy moiety had the highest potency to
stabilize the c-myc G-quadruplex DNA with
D
Tm values of 25.4 ꢀC
and 23.2 ꢀC respectively, while their counterparts 20b and 20c with
the 1,3-dioxole ring had lower affinity with the
DTm values of
22.2 ꢀC and 21.4 ꢀC, respectively, indicating the 1,3-dioxole ring was
unfavorable for the interaction of 5,6-dihydrobenzo[c]acridine
derivatives with G-quadruplex. Similar results were obtained for
O
O
O
O
O
O
O
a
b
+
O
O
H
CO2
H
CO2
O
1
4
2
3
H
H
O
O
O
O
O
c
e
d
O
O
O
O
6
7
5
Scheme 1. Synthesis of intermediates 5 and 7. Reagents and conditions: (a) AlCl3/nitrobenzene, 0e60 ꢀC, 3 h; (b) Et3SiH/CF3CO2H, reflux, 2 h; (c) PPA/DCM, reflux, 2 h; (d) 48% HBr,
125 ꢀC, 4 h; (e) CH2Br2, KF/DMF, 140 ꢀC, 6 h.

54
S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
Scheme 2. Synthesis of compounds 15a, 15b, 16a, 17a, 17c, 20aee, 21b, and 21c. Reagents and conditions: (a) (i) KMnO4/acetone, rt, 2 h; (ii) Fe, acetic acid/ethanol/water, rt, 4 h; (b)
5 or 7, POCl3, reflux, 8 h; (c) (i) H2,10% Pd/C, DMF/ethanol, 60 ꢀC, 3 h; (ii) NaOH, dioxane/water, 100 ꢀC, 2 h; (iii) dibromoalkanes, K2CO3/acetonitrile, 60 ꢀC, 3 h; (d) amine, K2CO3/
acetonitrile, 60 ꢀC, 3e6 h; (e) methyl triflate/toluene, rt, 1 h; (f) (i) amine/DCM, cat. KI, 2 day; (ii) dowex (Cl).
those non-methylated compounds, and the compounds 17a and
17c with two methoxy groups had the highest Tm values. For
Furthermore, the selectivity of the derivatives for G-quadruplex
DNA over duplex DNA was also evaluated by using FRET-based
competition assay, and the binding affinity of the ligands to G-
quadruplex was challenged by non-fluorescent complementary
DNA (self-complementary ds26 DNA: 50-GTTAGCCTAGCTTAAGC-
TAGGCTAAC-30) [24,45]. As shown in Fig. 3, in the presence of 15 or
D
methylated 5,6-dihydrobenzo[c]acridine derivatives (20aee, 21b,
and 21c) with amino side chains at their 8-position, their stabilizing
ability appeared to be mostly similar. The only exception is ligand
20e with a cyclohexylamino group at the end of its side chain,
which might be due to its weak protonation ability because of the
steric hindrance of cyclohexylamino group.
50 folds competitor of ds26, the DTm values of FPu18T raised by the
ligands were only slightly affected, showing that the complemen-
tary DNA had negligible effect on the binding between the ligands
and G-quadruplex DNA.
The melting temperature of FPu18T treated with various
concentrations (0.1e6.0 mM) of 21c was also monitored with FRET-
melting assay as shown in Fig. 2, which showed that the melting
temperature of c-myc G-quadruplex was concentration-dependent.
The interaction of all the derivatives with duplex DNA was also
examined with the FRET-melting experiments. The oligomer F10T
(50-FAM-TATAGCTA-TA-HEG-TATAGCTATA-TAMRA-30) that could
self-associate into a hairpin structure was used as a duplex DNA
3.2. Binding affinity and selectivity studies with SPR
Being surprised to the FRET results of the 5,6-dihydrobenzo[c]
acridine derivatives interacting with the c-myc G-quadruplex DNA,
we further studied the binding affinity of these acridine derivatives
on c-myc G-quadruplex DNA using surface plasmon resonance (SPR)
methods with biotinylated c-myc DNA and duplex DNA attached to
a streptavidin-coated sensor chip [46,47]. The compounds with
a range of concentrations were injected simultaneously to the
model [24]. As shown in Table 1, the
DTm values for F10T treated
with all the derivatives were very low, indicating that all
compounds exhibited poor ability to stabilize the duplex DNA and
thus had good selectivity to G-quadruplex DNA over duplex DNA.

S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
55
Table 1
G-Quadruplex DNA and duplex DNA stabilization temperatures (
DTm) obtained from FRET-melting and equilibrium binding constants (KD) measured with SPR.
Compound
n
R1, R2
NR3R4
FRET (
D
Tm/ꢀC)
SPR (KD/mM)
FPu18Ta
F10Tb
G4c
Duplexd
15a
15b
16a
17a
17c
20a
20b
20c
20d
20e
21b
21c
2
2
3
3
3
3
3
3
3
3
3
3
eOCH2Oe
eOCH2Oe
eOCH2Oe
CH3Oe, CH3Oe
CH3Oe, CH3Oe
eOCH2Oe
eOCH2Oe
eOCH2Oe
eOCH2Oe
eOCH2Oe
CH3Oe, CH3Oe
CH3Oe, CH3Oe
Diethylamino
Pyrrolidino
Diethylamino
Diethylamino
Piperidino
Diethylamino
Pyrrolidino
Piperidino
6.3 ꢂ 0.5
5.3 ꢂ 0.4
1.6 ꢂ 0.3
1.0 ꢂ 1.0
1.0 ꢂ 0.7
0.4 ꢂ 0.0
1.0 ꢂ 1.0
1.2 ꢂ 0.8
1.1 ꢂ 0.8
0.5 ꢂ 0.3
0.5 ꢂ 0.2
0.9 ꢂ 0.8
1.4 ꢂ 0.0
0.8 ꢂ 0.2
2.6 ꢂ 0.3
1.4 ꢂ 0.2
3.0 ꢂ 0.6
4.4 ꢂ 0.7
2.8 ꢂ 0.5
0.6 ꢂ 0.1
0.4 ꢂ 0.1
0.6 ꢂ 0.4
0.8 ꢂ 0.2
1.2 ꢂ 0.2
0.6 ꢂ 0.1
0.6 ꢂ 0.1
e
e
e
e
e
e
e
e
e
e
e
e
6.2 ꢂ 0.4
11.2 ꢂ 1.7
11.0 ꢂ 1.3
21.8 ꢂ 1.1
22.2 ꢂ 0.6
21.4 ꢂ 1.4
19.0 ꢂ 1.6
11.6 ꢂ 1.2
25.4 ꢂ 0.7
23.2 ꢂ 1.6
N-methylpiperazino
Cyclohexylamino
Pyrrolidino
Piperidino
a
D
Tm values for incubation of 0.2
m
M FPu18T with and without 2.0
m
M compound and 0.2 mM KCl,
D
Tm ¼ Tm (DNA þ ligand) ꢁ Tm (DNA). The values were obtained as the
Tm ¼ Tm (DNA þ ligand) ꢁ Tm (DNA). The values were obtained as the
The values were obtained with SPR as the mean ꢂ SD from at least two independent measurements.
mean ꢂ SD from at least three independent measurements.
b
DTm values for incubation of 0.2
mM F10T with and without 2.0 mM compound and 0.2 mM KCl, D
mean ꢂ SD from at least three independent measurements.
c
d
No obvious binding even with 10 mM of compounds.
immobilized c-myc DNA and duplex DNA with blank reference
(Fig. S1). The binding constants were determined through equilib-
rium analysis as shown in Table 1. The KD values of the derivatives
interacting with c-myc G-quadruplex DNA fell into a range of
3.3. Binding property studies with CD
The circular dichroism (CD) experiment was performed with
oligonucleotide Pu27 50-TGGGGAGGGTGGGGAGGGTGGGGAAGG-30
as the c-myc sequence according to a previous study [21]. The
conformational property of c-myc G-quadruplex DNA treated with
increasing concentrations (0.5e10 eq.) of the derivatives (15a, 15b,
16a, 17a, 17c, 20aee, 21b, and 21c) in the presence or absence of Kþ
(100 mM) were studied by using CD spectroscopy. The CD result
showed that the conformation of c-myc G-quadruplex had parallel
structure in the presence or absence of Kþ (100 mM) with a positive
signal at 262 nm and a negative signal at 242 nm (Fig. S2). This c-
myc G-quadruplex structure had little change when treated with
various concentrations of the derivatives either in the presence or
in the absence of Kþ (100 mM) (Fig. S2).
0.4e4.4
binding affinity to c-myc G-quadruplex. Our SPR results showed that
the methylated compounds had higher KD values (0.4e1.2 M) than
non-methylated ones (1.4e4.4 M), which was consistent with the
mM, which demonstrated that all the derivatives had a high
m
m
FRET results, indicating that the positive charge played a significant
role in binding with c-myc G-quadruplex. Both FRET and SPR results
showed that the pyrrolidino group of 20b or 21b was optimal for
their interaction with c-myc G-quadruplex DNA, while the intro-
duction of the cyclohexylamino group (20e) was unfavorable for the
binding interaction. Besides, the SPR data indicated that all the
derivatives had no obvious binding to duplex DNA even at
a concentration of 10 mM (Table 1, Fig. S1), which was consistent
with the FRET results. The results from above FRET and SPR exper-
iments all supported that these acridine derivatives could selec-
tively recognize c-myc G-quadruplex DNA over duplex DNA. The
methylated derivatives had good activity in binding on and stabi-
lizing G-quadruplex DNA, which could act as a new class of highly
selective G-quadruplex binding ligands.
3.4. Inhibition of amplification in the promoter region of c-myc by
5,6-dihydrobenzo[c]acridine derivatives
The inhibition of amplification of c-myc gene by the derivatives
in vitro was investigated with Pu27 (50-TGGGGAGGGTGGGGAGG
GTGGGGAAGG-30) as the c-myc DNA sequence by using PCR-stop
Fig. 2. Concentration-dependent melting curve for compound 21c upon binding to
FPu18T. The concentration of the FPu18T remained at 0.2
with 0.2 mM KCl, pH 7.2. The concentrations of compound 21c were 0.1, 0.5, 1.0, 2.0,
4.0, and 6.0 M.
m
M in 10 mM TriseHCl buffer
Fig. 3. Competitive FRET results for 5,6-dihydrobenzo[c]acridine derivatives without
and with 3
mM or 10 mM of duplex DNA competitor (ds26). The concentration of FPu18T
m
was 0.2 M.
m

56
S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
Fig. 4. Effect of 5,6-dihydrobenzo[c]acridine derivatives on the PCR-stop assay with Pu27. The white ladders were the PCR products. (A) The concentrations of compounds 15a, 15b,
16a, 17a, and 17c were at 50 M. (B) The increasing concentrations of compounds 20aee, 21b, and 21c were at 1, 5, 10, and 25 M, respectively.
m
m
assay [21]. The compounds of various concentrations (1.0, 5.0, 10.0,
and 25.0 M) were used, and the PCR products were separated with
PAGE and stained with GelRed (Fig. 4). The concentrations for 50%
inhibition of amplification (IC50) were calculated as shown in
Table 2. The methylated compounds (20aee and 21bec) showed
good inhibitory effect on the hybridization of Pu27 with the IC50
on the loops of the G-quadruplexes through electrostatic interac-
tions with the phosphate backbone. The binding free energy
m
showed that compounds 21c (
D
G ¼ ꢁ7.05 kcal$molꢁ1) and 20c
(D
G ¼ ꢁ6.75 kcal$molꢁ1) had better interactions with G-quadruplex
than compound17c (
D
G ¼ ꢁ6.38 kcal$molꢁ1). Thepositivelycharged
groups of compounds 21c and 20c were located above the negative
oxygen channel of G-quadruplex, while the weakly protonated
nitrogen of compound 17c had a certain degree of deviation from
this negative channel. These results showed that the methylation of
the nitrogen atom provided more effective ligand than its weak
protonation in the design of new G-quadruplex binding ligands.
The above modeling study results showed that the two methoxy
groups of compound 21c could adjust their locations and orienta-
tions based on their surroundings, while the 1,3-dioxole ring of
compound 20c could only orient itself to the groove of G-quad-
values ranged from 4.7 to 14.0
mM (Table 2), while the non-
methylated ones had weak inhibitory activity (IC50 > 50
mM),
which were consistent with the FRET and SPR results and
confirmed the importance of the nitrogen methylation in the
binding of the ligands to the c-myc G-quadruplex DNA.
Compared with compounds 20b and 20c, compounds 21b and 21c
had higher inhibition activity, indicating the 1,3-dioxole ring indeed
was not a better structural building block than the opened o-dime-
thoxyl group for the ligands to interact with G-quadruplex. It might be
the result of the two methoxy groups could adjust themselves better to
accommodate the G-quartet surface, while the rigid 1,3-dioxole ring in
compounds 20b and 20c could not. The PCR result also showed that
compound 20e had lower inhibition activity, which was consistent
with the FRET and SPR results, indicating the cyclohexylamino was not
a suitable group in designing G-quadruplex ligands.
ruplex, which might result in the lowered
DG value of 21c
(ꢁ7.05 kcal$molꢁ1) compared with that of 20c (ꢁ6.75 kcal$molꢁ1).
This result indicated that the two free methoxy groups were more
effective for 5,6-dihydrobenzo[c]acridine derivatives to interact
with G-quadruplex than the rigid 1,3-dioxole ring.
3.6. Cell proliferation assay and inhibition of c-myc DNA
transcription by 5,6-dihydrobenzo[c]acridine derivatives in cancer
cell line
3.5. Molecular modeling studies
In order to better understand how the nearly planar crescent-
shaped ligands interact with c-myc G-quadruplex DNA through
To evaluate the inhibitory activity of the derivatives for cancer
cell lines, the short-term proliferation assay of two cell lines, Ramos
and CA46, treated with compound 21c was studied. The NHE III1
element of c-myc gene was retained in the Ramos cell line, which
was removed together with the P1 and P2 promoters in the CA46
cell line [48,49]. The cells were incubated with compound 21c of
p-orbital stacking, we carried out the modeling study for the binding
of compound 21c, 20c and 17c with the c-myc G-quadruplex (Fig. 5).
The c-myc G-quadruplex model and the ligands were modified
according to the previously reported procedure [45]. The modeling
results showed that these three ligands could stack on both external
G-quartet surfaces, with preference to stack on the 50 G-quartet
surface. As shown in Fig. 5, these three compounds had similar
binding mode with the G-quadruplex, and their nearly planar scaf-
varying concentrations (2.5, 5, and 10 mM), and the experiments
were carried out for four days with cell counts and viability
determined every day. As shown in Fig. 6, the concentration-
dependent inhibition for the proliferation of Ramos by compound
21c was observed, while for cell line CA46 whose NHE III1 element
folds were stacked on the G-quartet surface through
p-orbital
overlapping, while their protonated side chains were oriented
Table 2
The IC50 values of compounds in PCR-stop assay.
Compounds
IC50 M)
15a
15b
16a
17a
17c
20a
20b
20c
20d
20e
21b
21c
(
m
>50
>50
>50
>50
>50
7.5
5.3
5.4
9.5
14.0
4.7
5.0

S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
57
Fig. 5. Modeling studies for the complexes of the ligands (17c, 20c, and 21c) and G-quadruplex. Pictures were generated by using PyMOL.
Fig. 6. Effect of compound 21c on the proliferation of (A) Ramos cells and (B) CA46 cells. The cells were exposed to the indicated concentrations of compound 21c or 0.1% DMSO
(control), respectively. Every day, the cells in control and drug-exposed well were counted. Each experiment was performed three times for each point.
Fig. 7. RT-PCR assay for the effect of compound 21c. Ramos cells and CA46 cells were treated with varying concentrations of compound 21c (0.5, 1, 2.5, and 5
transcriptions of c-myc gene were determined. c-myc mRNA level was normalized to -actin mRNA level for each sample.
mM), and the
b

58
S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
was removed, the cell proliferation was not obviously affected by
compound 21c.
Following the cell proliferation assay experiments, the tran-
scriptions of c-myc gene in Ramos cell line and CA46 cell line were
also studied. Ramos cells and CA46 cells were incubated with
After finishing addition, the mixture was continuously stirred for
10 min at this temperature, and then stirred at 60 ꢀC for about 3 h.
The resulting mixture was slowly added into the crashed ice, and
the precipitate was filtered and dried. The crude product was
recrystallized from the ethyl acetate, and compound 3 was
obtained as a white solid (60 g, 24%), Mp: 161e163 ꢀC (lit:
compound 21c at the concentrations of 0.5, 1, 2.5, and 5 mM for 4
days. The total RNA was extracted and reversely transcripted for
158e160 ꢀC [50]). 1H NMR (400 MHz, CDCl3):
d 10.83 (s, 1H), 7.61
cDNA, which was used as a template for specific PCR amplification
of the c-myc gene with b-actin as a control. As shown in Fig. 7, the
(dd, J ¼ 2 Hz, J ¼ 2 Hz, 1H), 7.53 (d, J ¼ 1.6 Hz, 1H), 6.89 (d, J ¼ 8.4 Hz,
1H), 3.94 (s, 3H), 3.92(s, 3H), 3.28 (t, J ¼ 6.8 Hz, 2H), 2.79 (t,
J ¼ 6.8 Hz, 2H); MS (ESI þ APCI) m/z: 237.1 [M ꢁ H]ꢁ.
ligand 21c showed good concentration-dependent inhibition on
the transcription of c-myc gene in Ramos cells, which was not
observed in CA46 cell line. The above results suggested that the
derivatives might target the G-quadruplex structure in the
promoter region of c-myc gene and hence inhibited its expression.
5.1.2. Synthesis of 4-(3,4-dimethoxyphenyl)butanoic acid (4)
Triethylsilane (0.78 mol, 125 mL) was added into the mixture of
the compound 3 (0.19 mol, 46 g) in CF3CO2H (140 mL), and the
resulting solution was heated under reflux for 2 h. Then the solvent
was removed under reduced pressure, and the residue was poured
into the crashed ice. The precipitate was filtered and dried, and
compound 4 was obtained as a pale solid (42 g, 97.7%), Mp:
4. Conclusion
The interaction between crescent-shaped 5,6-dihydrobenzo[c]
acridine derivatives (15a,15b,16a,17a,17c, 20aee, 21b, and21c)andG-
quadruplex DNA in promoter region of c-myc gene was studied in
detail. According to the FRET, SPR, and PCR-stop results, it could be
concluded that the nitrogen atom in the 5,6-dihydrobenzo[c]acridine
scaffold had weak tendency of protonation under physiological
59e61 ꢀC (lit: 60e61 ꢀC [50]). 1H NMR (400 MHz, CDCl3):
d 11. 80 (s,
1H), 6.79 (d, J ¼ 7.6 Hz, 1H), 6.73 (d, J ¼ 1.6 Hz, 1H), 6.71 (s, 1H), 3.87
(s, 3H), 3.85 (3, 3H), 2.62 (t, J ¼ 7.6 Hz, 2H), 2.38 (t, J ¼ 7.6 Hz, 2H),
1.99e1.91 (m, 2H); MS (ESI þ APCI) m/z: 223.1 [M ꢁ H]ꢁ.
conditions. Besides the
p
-orbital stacking of the scaffold and the elec-
5.1.3. Synthesis of 6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-
one (5)
trostatic interactions of the side chain, the electrostatic interactions
between the positive charge at 12-N-position and the negative oxygen
channel of G-quadruplex made an important contribution for the
derivatives to strongly bind on and stabilize the c-myc G-quadruplex.
Our results also indicated that the two methoxy groups in the 5,6-
dihydrobenzo[c]acridine scaffold could be more suitable for the
derivatives to interact with G-quadruplex than the 1,3-dioxole ring.
Their significant activities of inhibiting the c-myc transcription in
Ramos cell line but not in CA46 cell line might be due to stabilization of
G-quadruplex structure by the 12-N-methylated 5,6-dihydrobenzo[c]
acridine derivatives. Thus we might conclude that methylation on
nitrogen atom in certain molecular scaffold which generates a positive
charge, is important in designing a new G-quadruplex ligand, and the
methylated derivatives with high selectivity toward G-quadruplex
DNA over duplex DNA might become promising lead compounds for
developing anti-cancer agents.
Polyphosphoric acid (210 g) was added into the compound 4
(0.19 mol, 42 g) dissolved in dichloromethane (50 mL), and the
mixture was heated under reflux for 2 h. After the reaction was
finished, the solvent was removed and the residue was slowlyadded
into the crashed ice. The pH value of the solution was adjusted to
7e8, and the precipitate was filtered and dried, and compound 5
was obtained as a white solid (36 g, 93.3%), Mp: 94e96 ꢀC (lit:
96e98 ꢀC [50]). 1H NMR (400 MHz, CDCl3):
3.93 (s, 3H), 3.91 (s, 3H), 2.89 (t, J ¼ 6 Hz, 2H), 2.60 (t, J ¼ 6.4 Hz, 2H),
2.15e2.09 (m, 2H); 13C NMR (101 MHz, CDCl3):
197.10, 153.51,
d 7.51 (s,1H), 6.67 (s,1H),
d
147.96, 139.27, 125.79, 110.22, 108.57, 55.98, 38.50, 29.44, 23.61.
5.1.4. Synthesis of 6,7-dihydroxy-3,4-dihydronaphthalen-1(2H)-
one (6)
The compound 5 (0.19 mol, 34 g) was added into 48% HBr
(500 mL), and the mixture was heated under reflux for 4 h, and then
the mixturewas cooled to room temperature. The brown crystal was
filtered and dried, and compound 6 was obtained (24.6 g, 84.8%),
5. Experimental section
5.1. Synthesis and characterization
Mp: 198e200 ꢀC. 1H NMR (400 MHz, CDCl3):
d 7.67 (s, 1H), 7.16 (s,
1H), 6.75 (s,1H), 6.23 (s,1H), 2.85 (t, J ¼ 6.4 Hz, 2H), 2.59 (t, J ¼ 6.4 Hz,
1H and 13C NMR spectra were recorded using TMS as the internal
standard in DMSO-d6, D2O or CDCl3 with a Bruker BioSpin GmbH
spectrometer at 400 MHz; Mass spectra (MS) were recorded on a Shi-
madzu LCMS-2010A instrument with an ESI-ACPI mass selective
detector, and high resolution mass spectra (HRMS) were recorded on
Shimadzu LCMS-IT-TOF. All the compounds were purified by using
flash column chromatography with silica gel (200e300 mesh) or Al2O3
(200e300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd.
Their purities were proved to be higher than 95% by using analytical
HPLC equipped with Shimadzu LC-20AB system and an Ultimate XB-
2H), 2.12e2.06 (m, 2H); MS (ESI þ APCI) m/z: 177.1 [M ꢁ H]ꢁ.
5.1.5. Synthesis of 7,8-dihydronaphtho[2,3-d][1,3]dioxol-
5(6H)-one (7)
To a reaction flask, compound 6 (0.14 mol, 24.6 g), KF (1.24 mol,
72 g), dibromomethane (0.17 mol, 11.6 mL) and DMF (300 mL) were
added, and the mixture was heated at 140 ꢀC for 6 h. After the
reaction was cooled to room temperature, the solvent was removed
and the residue was extracted with ethyl acetate. The solution was
washed with water, dried, and purified with gel chromatography to
give compound 7 as a white solid (14.2 g, 54%), Mp: 75e78 ꢀC (lit:
C18 column (4.6 ꢃ 250 mm, 5
mm), which was eluted with
methanolewater (35:65e50:50) containing 0.1% TFA at a flow rate of
0.5 mL$minꢁ1. Melting points (Mp) were determined using a SRS-
OptiMelt automated melting point instrument without correction.
75 ꢀC [51]). 1H NMR (400 MHz, CDCl3):
5.99 (s, 2H), 2.86 (t, J ¼ 6 Hz, 2H), 2.58 (t, J ¼ 6.4 Hz, 2H), 2.11e2.05
(m, 2H), 13C NMR (101 MHz, CDCl3):
196.53, 151.99, 146.91, 141.35,
d 7.45 (s, 1H), 6.65 (s, 1H),
d
127.47, 107.91, 106.23, 101.52, 38.61, 30.02, 23.46.
5.1.1. Synthesis of 4-(3,4-dimethoxyphenyl)-4-oxobutanoic acid (3)
AlCl3 (2.65 mol, 353.0 g) was slowly added to the mixture of 1,2-
dimethoxybenzene (1.06 mol, 146.0 g) in nitrobenzene (1.5 L) at
5.1.6. Synthesis of the intermediate 6-amino-3-methoxy-2-
(phenylsulfonyloxy)benzoic acid (9)
KMnO4 (0.33 mol, 53 g) dissolved in water (100 mL) was slowly
added into a solution containing compound 8 (0.22 mol, 76 g) [40]
0
ꢀC over about 0.5 h, and then the dihydrofuran-2,5-dione
(1.27 mol, 127 g) was slowly added to the above mixture over 1 h.

S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
59
and acetone (300 mL) at room temperature, and the resulting
mixture was stirred for about 2 h. After the reaction was completed,
isopropyl alcohol (20 mL) was added, and the mixture was
continuously stirred for another 1 h. The mixture was filtered on
celite, and the solvent was removed under reduced pressure. The
resulting white solid was directly used for the next step.
The obtained white solid was added into acetic acid/ethanol/
water (2:2:1, 500 mL), and Fe powder (3.4 mol, 189 g) was added in
portion. The mixture was stirred at room temperature for about 4 h,
and white solid appeared and was filtered. The solid was dissolved
in 5 M NaOH (100 mL), and the resulting solution was filtered on
celite. After the pH value of the filtrate was adjusted to 5e6, the
precipitate appeared. The crude product was filtered and dried to
give compound 9 as a pale solid (30 g, 41.7%), Mp: 150e153 ꢀC. 1H
dibromoalkanes (5 eq.) and K2CO3 (3 eq.) were added. The mixture
was kept at 60 ꢀC for 3 h, and the solvent was removed, and the
residue was purified by using flash column chromatography to
afford compound 12, 13 or 14 as a light yellow solid.
5.1.8.1. 8-(2-Bromoethoxy)-9-methoxy-5,6-dihydro-[1,3]dioxolo
[40,50:4,5]benzo[1,2-c]-acridine (12). Following the general proce-
dure, compound 10 (2 mmol, 1.0 g) and 1,2-dibromoethane
(10.6 mmol, 2.0 g) were used, and the desired product was
obtained as a light yellow solid (0.26 g, 30%), Mp: 158e160 ꢀC. 1H
NMR (400 MHz, CDCl3):
d
8.31 (s,1H), 8.02 (s,1H), 7.87 (d, J ¼ 9.2 Hz,
1H), 7.41 (d, J ¼ 9.2 Hz, 1H), 6.73 (s, 1H), 6.00 (s, 2H), 4.50 (t,
J ¼ 6.0 Hz, 2H), 4.00 (s, 3H), 3.73 (t, J ¼ 6.0 Hz, 2H), 3.11 (t, J ¼ 7.6 Hz,
2H), 2.91 (t, J ¼ 7.6 Hz, 2H); MS (ESI þ APCI) m/z: 428.0 (93.5%),
430.1 (100%) [M þ H]þ.
NMR (400 MHz, CDCl3):
d
7.89 (d, J ¼ 7.6 Hz, 2H), 7.65 (t, J ¼ 7.6 Hz,
1H), 7.52 (t, J ¼ 7.6 Hz, 2H), 6.92 (d, J ¼ 8.8 Hz, 1H), 6.59 (d,
J ¼ 9.2 Hz, 1H), 3.38 (s, 3H), 13C NMR (101 MHz, DMSO-d6):
d
167.31,
5.1.8.2. 8-(3-Bromopropoxy)-9-methoxy-5,6-dihydro-[1,3]dioxolo
[40,50:4,5]benzo[1,2-c]-acridine (13). Following the general proce-
dure, compound 10 (4 mmol, 2.0 g) and 1,3-dibromopropane
(20 mmol, 3.8 g) were used, and the desired product was
obtained as a light yellow solid (0.62 g, 35%), Mp: 132e135 ꢀC. 1H
143.96, 142.16, 136.34, 136.26, 134.35, 129.21, 128.10, 118.54, 115.50,
110.79, 56.07; MS (ESI þ APCI) m/z: 324 [M þ H]þ.
5.1.7. General procedure for preparation of compounds 10 and 11
The mixture of compound 7 or 5 (1 eq.) and compound 9
(1.5 eq.) was heated under reflux in phosphorus oxychloride for 8 h.
The mixture was cooled down to room temperature, and slowly
poured into the crashed ice. The precipitate was filtered, dissolved
in dichloromethane, washed with saturated NaHCO3, dried, and
purified by using flash column chromatography to give compound
10 or 11 as a light yellow solid.
NMR (400 MHz, CDCl3):
d
8.74 (s, 1H), 8.55 (d, J ¼ 9.6 Hz, 1H), 8.09
(s, 1H), 7.71 (d, J ¼ 9.2 Hz, 1H), 6.83 (s, 1H), 6.14 (s, 2H), 4.36 (t,
J ¼ 5.6, 2H), 4.05 (s, 3H), 3.78 (t, J ¼ 5.6 Hz, 2H), 3.19 (t, J ¼ 7.6 Hz,
2H), 3.00 (t, J ¼ 7.6 Hz, 2H), 2.45e2.39 (m, 2H); MS (ESI þ APCI) m/z:
442.1 (75.5%), 444.1 (100%) [M þ H]þ.
5.1.8.3. 8-(3-Bromopropoxy)-2,3,9-trimethoxy-5,6-dihydrobenzo[c]
acridine (14). Following the general procedure, compound 11
(4.7 mmol, 2.4 g) and 1,3-dibromopropane (25 mmol, 4.7 g) were used,
and the desired product was obtained as a light yellow solid (1.24 g,
5.1.7.1. 7-Chloro-9-methoxy-5,6-dihydro-[1,3]dioxolo[40,50:4,5]benzo
[1,2-c]acridin-8-yl benzenesulfonate (10). Following the general
procedure, compound 7 (0.075 mol, 14.2 g) and compound 9
(0.11 mol, 36 g) were used, and the desired product was obtained as
a light yellow solid (23.7 g, 64%), Mp: 201e204 ꢀC. 1H NMR
57.6%), Mp: 126e127 ꢀC. 1H NMR (400 MHz, CDCl3):
d 8.17 (s, 1H), 8.08
(s,1H), 7.89 (d, J ¼ 8.8 Hz,1H), 7.41 (d, J ¼ 9.2 Hz,1H), 6.76 (s,1H), 4.28 (t,
J ¼ 6.0 Hz, 2H), 4.06 (s, 3H), 4.00 (s, 3H), 3.95 (s, 3H), 3.78 (t, J ¼ 6.4 Hz,
2H), 3.13 (t, J¼ 7.6 Hz, 2H), 2.95 (t, J¼ 7.6 Hz, 2H), 2.45e2.39 (m, 2H); MS
(ESI þ APCI) m/z: 458.1 (100%), 460.1 (94%) [M þ H]þ.
(400 MHz, CDCl3):
d
8.08 (d, J ¼ 8.0 Hz, 1H), 8.00 (s, 1H), 7.87 (d,
J ¼ 7.6 Hz, 2H), 7.66 (t, J ¼ 7.6 Hz, 1H), 7.53 (t, J ¼ 8.0 Hz, 2H), 7.32 (d,
J ¼ 9.2 Hz, 1H), 6.73 (s, 1H), 6.01 (s, 2H), 3.43 (s, 3H), 3.24 (t,
J ¼ 7.6 Hz, 2H), 2.91 (t, J ¼ 7.6 Hz, 2H); 13C NMR (101 MHz, CDCl3):
5.1.9. General procedure for preparing of compound 15a, 15b, 16a,
17a, 17c
d
151.39, 150.35, 149.35, 147.31, 143.15, 137.83, 135.78, 134.19, 133.57,
130.87, 130.76, 130.12, 128.61, 128.49, 127.63, 121.50, 115.86, 107.78,
106.15, 101.25, 55.91, 27.48, 26.02; MS (ESI þ APCI) m/z: 496.0
[M þ H]þ.
The compound 12, 13 or 14 (1 eq.) was dissolved in acetonitrile
(10e100 mL), and the alkyl amine (1.5e10 eq.) and K2CO3 (3 eq.)
were added, and the mixture was stirred at 60 ꢀC for about 3e6 h.
The solvent was removed under reduced pressure, and the residue
was washed with water, extracted with dichloromethane, dried
and purified by using flash column chromatography to give the
desired compound 15a, 15b, 16a or 17a, 17c as a pale solid.
5.1.7.2. 7-Chloro-2,3,9-trimethoxy-5,6-dihydrobenzo[c]acridin-8-yl
benzenesulfonate (11). Following the general procedure, compound
5 (9.7 mmol, 2 g) and compound 9 (15.5 mmol, 5 g) were used, and
the desired product was obtained as a light yellow solid (2.4 g,
48.4%), Mp: 210e211 ꢀC. 1H NMR (400 MHz, CDCl3):
d
8.20 (s, 1H),
5.1.9.1. N,N-Diethyl-2-((9-methoxy-5,6-dihydro-[1,3]dioxolo
8.09 (s, 1H), 7.88 (d, J ¼ 0.8 Hz, 1H), 7.86 (d, J ¼ 1.6 Hz, 1H), 7.66 (t,
J ¼ 7.6 Hz, 1H), 7.53 (t, J ¼ 8 Hz, 2H), 7.33 (d, J ¼ 9.2 Hz, 1H), 6.76 (s,
1H), 4.06 (s, 3H), 3.96 (s, 3H), 3.43 (s, 3H), 3.27 (t, J ¼ 7.6 Hz, 2H),
[40,50:4,5]benzo[1,2-c]acridin-8-yl)oxy)ethanamine (15a). Following
the general procedure, compound 12 (0.14 mmol, 60 mg) and dieth-
ylamine (1.4 mmol, 0.144 mL) were used, and the desired product was
obtained as a pale solid (17 mg, 29%). Purity: 98.4% (by HPLC, MeOH/
2.95 (t, J ¼ 7.6 Hz, 2H); 13C NMR (101 MHz, CDCl3):
d 151.51, 151.03,
150.38, 148.51, 143.17, 137.86, 135.89, 133.55, 132.57, 130.99, 130.80,
129.99, 128.60, 128.49, 126.06, 121.49, 115.89, 110.42, 108.73, 56.18,
55.98, 55.96, 27.01, 26.08; MS (ESI þ APCI) m/z: 512.1 [M þ H]þ.
H2O). Mp: 90e91 ꢀC; 1H NMR (400 MHz, CDCl3):
d 8.26 (s, 1H), 8.01(s,
1H), 7.83 (d, J ¼ 9.2 Hz, 1H), 7.40 (d, J ¼ 9.2 Hz, 1H), 6.73 (s, 1H), 6.00 (s,
2H), 4.25 (t, J ¼ 6.0 Hz, 2H), 3.99 (s, 3H), 3.10e3.07 (m, 2H), 3.00 (s, 2H),
2.92e2.89 (m, 2H), 2.73 (s, 4H), 1.11 (t, J ¼ 6.8 Hz, 6H); 13C NMR
5.1.8. General procedure for preparation of compounds 12e14
Compound 10 or 11 was dechlorinated under H2 and 10% Pd/C
(1 eq. wt %) at atmospheric pressure in DMF/ethanol at 60 ꢀC for 3 h.
The mixture was filtered through celite, and the solvent was
removed. The residue was dissolved in dioxane, and 5 M NaOH
(5 eq.) was added, and the mixture was kept at 100 ꢀC for 2 h. Then
dioxane was removed, and the pH value of the solution was
adjusted to 6e7. A light red solid appeared, which was filtered and
dried. Then the solid was dissolved in acetonitrile, and
(101 MHz, CDCl3): d 151.61,148.66,148.02,147.19,143.37,140.82,134.21,
129.94, 128.97, 127.67, 125.21, 123.41, 116.84, 107.97, 105.95, 101.07,
71.49, 56.74, 52.84, 47.44, 29.26, 28.55,11.72; HRMS (ESI) m/z: calcd for
C25H28N2O4 [M þ H]þ 421.2127, found 421.2122.
5.1.9.2. 9-Methoxy-8-(2-(pyrrolidin-1-yl)ethoxy)-5,6-dihydro-[1,3]
dioxolo[40,50:4,5]benzo[1,2-c]acridine (15b). Following the general
procedure, compound 12 (0.14 mmol, 60 mg) and pyrrolidine
(0.2 mmol, 0.017 mL) were used, and the desired product was

60
S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
obtained as a pale solid (28 mg, 46.7%). Purity: 100% (by HPLC,
MeOH/H2O). Mp: 118e120 ꢀC; 1H NMR (400 MHz, CDCl3):
8.20 (s,
1H), 7.95 (s, 1H), 7.78 (d, J ¼ 9.2 Hz, 1H), 7.34 (d, J ¼ 9.2 Hz, 1H), 6.67
(s, 1H), 5.94 (s, 2H), 4.21 (t, J ¼ 5.6 Hz, 2H), 3.93 (s, 3H), 3.04e3.00
(m, 2H), 2.92 (s, 2H), 2.86e2.82 (m, 2H), 2.62 (s, 4H),1.80 (s, 4H); 13C
compound 13 (1.13 mmol, 500 mg) and methyl triflate (1.3 mmol,
0.15 mL) were used, and the desired product was obtained as
a yellow solid (437 mg, 64%), Mp: 194e197 ꢀC. 1H NMR (400 MHz,
d
CDCl3):
d
8.80 (s, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 7.86 (d, J ¼ 9.2 Hz, 1H),
7.43 (s, 1H), 6.94 (s, 1H), 6.15 (s, 2H), 4.73 (s, 3H), 4.38 (t, J ¼ 5.6 Hz,
2H), 4.08 (s, 3H), 3.76 (t, J ¼ 6.4 Hz, 2H), 3.10 (t, J ¼ 7.6 Hz, 2H), 2.96
(t, J ¼ 7.6 Hz, 2H), 2.46e2.40 (m, 2H); MS (ESI þ APCI) m/z: 456.1
(100%), 458.1 (99.5%) [M ꢁ OTf3]þ.
NMR (101 MHz, CDCl3):
d 151.62, 148.70, 148.18, 147.24, 143.48,
140.96, 134.21, 129.92, 129.06, 127.72, 125.30, 123.58, 117.04, 107.97,
106.01, 101.07, 72.09, 56.85, 55.95, 54.52, 29.30, 28.61, 23.61; HRMS
(ESI) m/z: calcd for C25H26N2O4 [M þ H]þ 419.1971, found 419.1969.
5.1.10.2. 8-(3-Bromopropoxy)-2,3,9-trimethoxy-12-methyl-5,6-dihyd
robenzo[c]acridin-12-ium trifluoromethanesulfonate (19). Following
the general procedure, compound 14 (0.92 mmol, 420 mg) and methyl
triflate (1.9 mmol, 0.21 mL) were used, and the desired product was
obtained as a yellow solid (283 mg, 50%), Mp: 186e187 ꢀC. 1H NMR
5.1.9.3. N,N-Diethyl-3-((9-methoxy-5,6-dihydro-[1,3]dioxolo
[40,50:4,5]benzo[1,2-c]acridin-8-yl)oxy)propan-1-amine
(16a). Following the general procedure, compound 13 (0.59 mmol,
260 mg) and diethylamine (5.9 mmol, 0.61 mL) were used, and the
desired product was obtained as a pale solid (230 mg, 90.2%). Purity:
100% (by HPLC, MeOH/H2O). Mp: 91e93 ꢀC; 1H NMR (400 MHz,
(400 MHz, CDCl3):
d
8.77 (s, 1H), 8.27 (d, J ¼ 9.6 Hz, 1H), 7.82 (d,
J ¼ 10.0 Hz, 1H), 7.57 (s, 1H), 6.93 (s, 1H), 4.79 (s, 3H), 4.37 (t, J ¼ 6.0 Hz,
1H), 4.07 (s, 3H), 4.03 (s, 6H), 3.76 (t, J ¼ 6.4 Hz, 2H), 3.10 (t, J ¼ 7.2 Hz,
2H), 2.96 (t, J ¼ 7.2 Hz, 2H), 2.45e2.40 (m, 2H); MS (ESI þ APCI) m/z:
472.1 (90%), 474.1 (100%) [M ꢁ OTf3]þ.
CDCl3):
d
8.16 (s, 1H), 8.01 (s, 1H), 7.83 (d, J ¼ 9.2 Hz, 1H), 7.40 (d,
J ¼ 9.2 Hz,1H), 6.72 (s,1H), 5.99 (s, 2H), 4.19 (t, J ¼ 6.4 Hz, 2H), 3.98 (s,
3H), 3.10e3.07 (m, 2H), 2.92e2.88 (m, 2H), 2.85e2.81 (m, 2H), 2.67
(q, J ¼ 7.2 Hz, 4H), 2.12e2.05 (m, 2H),1.11 (t, J ¼ 7.2 Hz, 6H); 13C NMR
(101 MHz, CDCl3):
d
151.59, 148.69, 148.04, 147.22, 143.51, 140.98,
5.1.11. General procedure for preparation of compounds 20aee,
21b, 21c
134.18, 129.90, 128.99, 127.42, 125.16, 123.41, 117.13, 107.95, 105.98,
101.06, 72.18, 56.84, 49.79, 46.92, 29.28, 28.56, 27.71, 11.41; HRMS
(ESI) m/z: calcd for C26H30N2O4 [M þ H]þ 435.2284, found 435.2279.
Compound 18 or 19 (1 eq.) was dissolved in dichloromethane,
and amine (1.2e10 eq.) and K2CO3 (3 eq.) with cat. KI were added.
The reaction mixture was stirred for 2 days at room temperature,
and then washed with water and saturated NaCl, dried and purified
by using flash column chromatography. The anion ðOTf3ꢁÞ was
exchanged into chloride form (Clꢁ) with ion-exchange resin, and
the obtained residue was purified again by using Al2O3 chroma-
tography to give the desired compound as a light yellow solid.
5.1.9.4. N,N-Diethyl-3-((2,3,9-trimethoxy-5,6-dihydrobenzo[c]acri-
din-8-yl)oxy)propan-1-amine (17a). Following the general proce-
dure, compound 14 (0.13 mmol, 60 mg) and diethylamine
(0.2 mmol, 0.02 mL) were used, and the desired product was
obtained as a pale solid (20 mg, 34.2%). Purity: 99.7% (by HPLC,
MeOH/H2O). Mp: 95e98 ꢀC; 1H NMR (400 MHz, CDCl3):
d 8.14 (s,
1H), 8.06 (s, 1H), 7.86 (d, J ¼ 9.2 Hz, 1H), 7.40 (d, J ¼ 9.2 Hz, 1H), 6.75
(s, 1H), 4.20 (t, J ¼ 6.0 Hz, 2H), 4.05 (s, 3H), 3.98 (s, 3H), 3.94 (s, 3H),
3.13e3.10 (m, 2H), 2.99e2.92 (m, 4H), 2.82e2.77 (m, 4H), 2.16 (t,
J ¼ 7.6 Hz, 2H), 1.19 (t, J ¼ 7.2 Hz, 6H); 13C NMR (101 MHz, CDCl3):
5.1.11.1. 8-(3-(Diethylamino)propoxy)-9-methoxy-12-methyl-5,6-
dihydro-[1,3]dioxolo[40,50:4,5]benzo[1,2-c]acridin-12-ium
chloride
(20a). Following the general procedure, compound 18 (0.5 mmol,
300 mg) and diethylamine (1.3 mmol, 0.13 mL) were used, and the
desired product was obtained as a yellow solid (60 mg, 25%). Purity:
99.5% (by HPLC, MeOH/H2O). Mp: 149e150 ꢀC; 1H NMR (400 MHz,
d
151.73, 150.30, 148.39, 147.93, 143.41, 140.67, 132.64, 130.14, 127.37,
127.35, 125.22, 123.26, 116.79, 110.63, 108.34, 71.70, 56.73, 56.11,
55.93, 49.66, 46.82, 29.37, 28.03, 26.95, 10.67; HRMS (ESI) m/z:
calcd for C27H34N2O4 [M þ H]þ 451.2591, found 451.2596.
CDCl3):
d
8.79 (s, 1H), 8.71 (d, J ¼ 9.6 Hz, 1H), 7.92 (d, J ¼ 9.6 Hz, 1H),
7.66 (s, 1H), 6.92 (s, 1H), 6.14 (s, 2H), 4.96 (s, 3H), 4.27 (t, J ¼ 6.4 Hz,
2H), 4.04 (s, 3H), 3.07e3.04 (m, 2H), 2.94e2.91 (m, 2H), 2.71e2.68
(m, 2H), 2.59 (q, J ¼ 7.2 Hz, 4H), 2.05e2.01 (m, 2H),1.05 (t, J ¼ 7.2 Hz,
5.1.9.5. 2,3,9-Trimethoxy-8-(3-(piperidin-1-yl)propoxy)-5,6-
dihydrobenzo[c]acridine (17c). Following the general procedure,
compound 14 (0.13 mmol, 60 mg) and piperidine (0.2 mmol,
0.02 mL) were used, and the desired product was obtained as a pale
solid (24 mg, 40%). Purity: 99.9% (by HPLC, MeOH/H2O). Mp:
6H); 13C NMR (101 MHz, CDCl3):
d 153.12, 152.54, 150.46, 147.66,
141.43, 141.01, 135.31, 135.14, 133.59, 123.64, 121.94, 119.64, 116.92,
111.13, 109.00, 102.71, 73.00, 56.74, 49.36, 46.81, 46.75, 29.11, 28.83,
27.88, 11.55; HRMS (ESI) m/z: calcd for C27H33N2O4 [M ꢁ Cl]þ
449.2440, found 449.2432.
128e129 ꢀC; 1H NMR (400 MHz, CDCl3):
d 8.15 (s, 1H), 8.08 (s, 1H),
7.86 (d, J ¼ 9.2 Hz, 1H), 7.40 (d, J ¼ 9.2 Hz, 1H), 6.75 (s, 1H), 4.20 (t,
J ¼ 6.4 Hz, 2H), 4.06 (s, 3H), 3.99 (s, 2H), 3.95 (s, 3H), 3.12 (t,
J ¼ 7.2 Hz, 2H), 2.96e2.92 (m, 2H), 2.67 (s, 2H), 2.49 (s, 4H), 2.13 (s,
5.1.11.2. 9-Methoxy-12-methyl-8-(3-(pyrrolidin-1-yl)propoxy)-5,6-
dihydro-[1,3]dioxolo[40,50:4,5]benzo[1,2-c]acridin-12-ium
chloride
2H), 1.66 (s, 4H), 1.48 (s, 2H); 13C NMR (101 MHz, CDCl3):
d
151.71,
(20b). Following the general procedure, compound 18 (0.165 mmol,
100 mg) and pyrrolidine (0.2 mmol, 0.017 mL) were used, and the
desired product was obtained as a yellow solid (30 mg, 37.5%). Purity:
95.4% (by HPLC, MeOH/H2O). Mp: 125e126 ꢀC; 1H NMR (400 MHz,
150.33,148.45, 148.05, 143.54, 140.96, 132.58, 130.03, 127.52, 127.49,
125.16, 123.42, 117.05, 110.70, 108.46, 72.06, 56.86, 56.15, 56.04,
55.97, 54.21, 29.41, 28.09, 27.49, 25.58, 24.18; HRMS (ESI) m/z: calcd
for C28H34N2O4 [M þ H]þ 463.2597, found 463.2588.
CDCl3):
d
8.83 (s,1H), 8.30 (d, J ¼ 9.6 Hz,1H), 7.85 (d, J ¼ 9.6 Hz,1H), 7.45
(s,1H), 6.94 (s,1H), 6.15 (s, 2H), 4.75 (s, 3H), 4.33 (t, J ¼ 6.4 Hz, 2H), 4.06
(s, 3H), 3.07 (t, J ¼ 7.2 Hz, 2H), 2.96e2.93 (m, 2H), 2.78 (t, J ¼ 7.2 Hz, 2H),
2.62 (s, 4H), 2.17e2.09 (m, 2H),1.83 (s, 4H); 13C NMR (101 MHz, CDCl3):
5.1.10. General procedure for preparation of compounds 18, 19
To a stirred solution of compound 13 or 14 (1 eq.) in toluene,
methyl triflate (1.2e2.0 eq.) was slowly added at room temperature,
and a light yellow solid appeared in 5 min. After the reaction was
continuously stirred for about 1 h, the solid was filtered and dried
to afford the desired compound 18 or 19 as a yellow solid.
d
153.17, 152.56, 150.48, 147.67, 141.43, 141.09, 135.49, 135.13, 133.71,
123.69, 121.87, 119.66, 116.80, 111.11, 109.02, 102.73, 72.60, 56.76, 54.17,
52.75, 46.64, 29.45, 29.08, 28.84, 23.46; HRMS (ESI) m/z: calcd for
C27H31N2O4 [M ꢁ Cl]þ 447.2284, found 447.2275.
5.1.10.1. 8-(3-Bromopropoxy)-9-methoxy-12-methyl-5,6-dihydro-
tri-
fluoromethanesulfonate (18). Following the general procedure,
5.1.11.3. 9-Methoxy-12-methyl-8-(3-(piperidin-1-yl)propoxy)-5,6-
chloride
(20c). Following the general procedure, compound 18 (0.5 mmol,
[1,3]dioxolo[40,50:4,5]benzo[1,2-c]acridin-12-ium
dihydro-[1,3]dioxolo[40,50:4,5]benzo[1,2-c]acridin-12-ium

S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
61
300 mg) and piperidine (0.7 mmol, 0.07 mL) were used, and the
desired product was obtained as a yellow solid (45 mg, 18.4%).
Purity: 98.8% (by HPLC, MeOH/H2O). Mp: 140e142 ꢀC; 1H NMR
piperidine (1.1 mmol, 0.11 mL) were used, and the desired product
was obtained as a yellow solid (65 mg, 44.3%). Purity: 99.9% (by
HPLC, MeOH/H2O). Mp: 170e172 ꢀC; 1H NMR (400 MHz, CDCl3):
(400 MHz, D2O):
d
8.41 (s, 1H), 7.85 (d, J ¼ 9.6 Hz, 1H), 7.70 (d,
d
8.71 (s, 1H), 8.54 (d, J ¼ 9.6 Hz, 1H), 8.01 (s, 1H), 7.83 (d, J ¼ 9.6 Hz,
J ¼ 10 Hz, 1H), 7.21 (s, 1H), 6.97 (s, 1H), 6.04 (s, 2H), 4.39 (s, 3H), 3.97
(t, J ¼ 6.4 Hz, 2H), 3.91 (s, 3H), 2.89 (t, J ¼ 7.6 Hz, 2H), 2.78 (t,
J ¼ 7.6 Hz, 2H), 2.41e2.33 (m, 6H), 1.86e1.79 (m, 2H), 1.48e1.45 (m,
1H), 6.90 (s,1H), 5.12 (s, 3H), 4.27 (t, J ¼ 6.4 Hz, 2H), 4.14 (s, 3H), 4.04
(s, 3H), 4.02 (s, 3H), 3.08e3.05 (m, 2H), 2.96e2.93 (m, 2H), 2.57 (t,
J ¼ 7.6 Hz, 2H), 2.44 (s, 4H), 2.08e2.04 (m, 2H), 1.62e1.58 (m, 4H),
4H), 1.35 (s, 2H); 13C NMR (101 MHz, D2O):
d
153.81, 151.99, 150.02,
1.48e1.43 (m, 2H); 13C NMR (101 MHz, CDCl3):
d 153.98, 153.64,
146.75, 142.44, 140.56, 134.87, 134.56, 134.47, 123.20, 120.85, 119.49,
115.14, 110.11, 109.09, 102.73, 73.45, 56.41, 55.12, 53.61, 44.71, 28.45,
27.94, 26.17, 24.67, 23.30; HRMS (ESI) m/z: calcd for C28H33N2O4
[M ꢁ Cl]þ 461.2440, found 461.2432.
150.33, 148.75, 141.55, 138.39, 135.42, 134.83, 133.78, 123.71, 121.63,
118.89, 116.72, 114.99, 110.81, 72.90, 57.93, 56.74, 56.32, 55.63,
54.66, 46.73, 29.20, 28.37, 27.71, 25.93, 24.34; HRMS (ESI) m/z: calcd
for C29H37N2O4 [M ꢁ Cl]þ 477.2753, found 477.2743.
5.1.11.4. 9-Methoxy-12-methyl-8-(3-(4-methylpiperazin-1-yl)pro-
poxy)-5,6-dihydro-[1,3]dioxolo[40,50:4,5]benzo[1,2-c]acridin-12-ium
chloride (20d). Following the general procedure, compound 18
(0.5 mmol, 300 mg) and N-methyl piperazine (0.63 mmol, 0.07 mL)
were used, and the desired product was obtained as a yellow solid
(40 mg, 16%). Purity: 98.9% (by HPLC, MeOH/H2O). Mp: 137e138 ꢀC;
5.2. Materials
All oligomers/primers used in this study were purchased from
Invitrogen (China). Stock solutions of all the derivatives (10 mM)
were made using DMSO (10%) or double-distilled deionized water.
Further dilutions to working concentrations were made with
double-distilled deionized water.
1H NMR (400 MHz, CDCl3):
d
8.75 (s, 1H), 8.64 (d, J ¼ 9.6 Hz, 1H),
7.91 (d, J ¼ 9.6 Hz, 1H), 7.65 (s, 1H), 6.93 (s, 1H), 6.14 (s, 2H), 4.93 (s,
3H), 4.28 (t, J ¼ 6.4 Hz, 2H), 4.05 (s, 3H), 3.08e3.05 (m, 2H),
2.95e2.92 (m, 2H), 2.65e2.63 (m, 2H), 2.55 (s, 4H), 2.32 (s, 3H),
5.3. FRET-melting assay
2.09e2.04 (m, 6H); 13C NMR (101 MHz, CDCl3):
d
153.23, 152.58,
FRET assay was carried out following previously reported
procedures [44]. The fluorescently labeled oligomer FPu18T (50-
FAM-AGGGTGGGGA-GGGTGGGG-TAMRA-30) and F10T [50-FAM-
150.44, 147.69, 141.34, 141.07, 135.18, 135.15, 133.67, 123.64, 121.88,
119.66, 116.80, 111.14, 109.02, 102.73, 72.64, 56.75, 54.85, 54.82,
52.95, 46.60, 45.82, 29.18, 28.83, 27.55; HRMS (ESI) m/z: calcd for
C28H34N3O4 [M ꢁ Cl]þ 476.2549, found 476.2541.
dTATAGCTATA-HEG-TATA-GCTATA-TAMRA-30]
(HEG
linker:
[(eCH2eCH2eOe)6]) were used as G-quadruplex and duplex DNA
model respectively. The experiments were performed in real-time
PCR apparatus (Roche LigntCycler), allowing the simultaneous
recording of 32 samples. The melting of each sample, which con-
5.1.11.5. 8-(3-(Cyclohexylamino)propoxy)-9-methoxy-12-methyl-
5,6-dihydro-[1,3]dioxolo[40,50:4,5]benzo[1,2-c]acridin-12-ium chlo-
ride (20e). Following the general procedure, compound 18
(0.5 mmol, 300 mg) and cyclohexylamine (5 mmol, 0.57 mL) were
used, and the desired product was obtained as a yellow solid
(130 mg, 51.6%). Purity: 96.1% (by HPLC, MeOH/H2O). Mp:
taining 0.2
buffer, pH 7.2, 0.2 mM KCl, was monitored in the presence or
absence of 2 M compounds by measuring their fluorescence
mM oligonucleotide FPu18T or F10T in 10 mM TriseHCl
m
intensity with excitation at 470 nm and detection at 530 nm, and
the experiment was repeated for three times. Fluorescence read-
ings were taken at an interval of 1 ꢀC over the range 37e99 ꢀC, with
a constant temperature being maintained for 30 s prior to each
reading to ensure a stable value. Final analysis of the data was
carried out using Origin 8.0 (OriginLab Corp.).
To test the binding selectivity of the compounds for their
interactions with the G-quadruplex DNA over duplex DNA, we
added various concentrations of double-stranded DNA (self-
complementary ds26 DNA: 50-GTTAGCCTAGCTTAAGCTAGGCTAAC-
30) as competitor. The T1/2 was defined as the temperature value
when the normalized fluorescence emission is 0.5.
127e128 ꢀC; 1H NMR (400 MHz, CDCl3):
d 8.79 (s, 1H), 8.69 (d,
J ¼ 9.6 Hz, 1H), 7.92 (d, J ¼ 10 Hz, 1H), 7.65 (s, 1H), 6.92 (s, 1H), 6.14
(s, 2H), 4.94 (s, 3H), 4.30 (t, J ¼ 6.4 Hz, 2H), 4.05 (s, 3H), 3.08e3.05
(m, 2H), 2.94e2.89 (m, 4H), 2.50e2.45 (m, 1H), 2.08e2.05 (m, 2H),
1.94e1.91 (m, 2H), 1.75e1.71 (m, 2H), 1.27e1.20 (m, 4H), 1.13e1.06
(m, 2H); 13C NMR (101 MHz, CDCl3):
d
153.17, 152.51, 150.40,
147.60, 141.37, 141.18, 135.48, 135.05, 133.81, 123.63, 121.81, 119.59,
116.75, 111.03, 109.03, 102.70, 72.76, 56.98, 56.75, 46.61, 43.36,
32.92, 30.44, 29.03, 28.80, 25.92, 24.98; HRMS (ESI) m/z: calcd for
C29H35N2O4 [M ꢁ Cl]þ 475.2597, found 475.2589.
5.1.11.6. 2,3,9-Trimethoxy-12-methyl-8-(3-(pyrrolidin-1-yl)pro-
poxy)-5,6-dihydrobenzo[c]acridin-12-ium chloride (21b). Following
the general procedure, compound 19 (0.16 mmol, 100 mg) and
pyrrolidine (0.32 mmol, 0.03 mL) were used, and the desired
product was obtained as a yellow solid (21 mg, 26.3%). Purity: 98.1%
(by HPLC, MeOH/H2O). Mp: 156e159 ꢀC; 1H NMR (400 MHz,
5.4. Surface plasmon resonance
SPR measurements were performed on a ProteOn XPR36 Protein
Interaction Array system (Bio-Rad Laboratories, Hercules, CA) using
a Neutravidin-coated GLH sensor chip [46,47]. In a typical experi-
ment, biotinylated Pu27 was dissolved in filtered and degassed
running buffer (50 mM TriseHCl, pH 7.2, 100 mM KCl). The DNA
samples were then captured (w1000 RU) in flow cells, leaving the
last flow cell as a blank. Ligand solutions (at 10, 5, 2.5, 1.25, 0.625,
CDCl3):
d
8.79 (s, 1H), 8.22 (d, J ¼ 9.6 Hz, 1H), 7.80 (d, J ¼ 9.6 Hz, 1H),
7.52 (s, 1H), 6.93 (s, 1H), 4.76 (s, 3H), 4.31 (t, J ¼ 6.4 Hz, 2H), 4.04 (s,
3H), 4.01 (s, 6H), 3.09e3.06 (m, 2H), 2.97e2.93 (m, 2H), 2.76 (t,
J ¼ 7.6 Hz, 2H), 2.61 (s, 4H), 2.11 (d, J ¼ 6.8 Hz, 2H), 1.82 (s, 4H); 13C
NMR (101 MHz, CDCl3):
d
154.01, 153.70, 150.37, 148.53, 141.69,
0.3125
dilutions from stock solution. Six concentrations were injected
simultaneously at a flow rate of 100
L minꢁ1 for 400 s of associ-
mM) were prepared with running buffer through serial
139.10, 135.41, 135.12, 133.92, 123.72, 121.37, 118.59, 115.68, 113.83,
111.05, 72.77, 57.02, 56.71, 56.37, 54.21, 52.79, 45.57, 29.53, 29.12,
28.25, 23.46; HRMS (ESI) m/z: calcd for C28H35N2O4 [M ꢁ Cl]þ
463.2591, found 463.2590.
m
ation phase, followed with 400 s of dissociation phase at 25 ꢀC. The
GLH sensor chip was regenerated with short injection of 50 mM
NaOH between consecutive measurements. The final graphs were
obtained by subtracting blank sensorgrams from G-quadruplex
sensorgrams. Data were analyzed with ProteOn manager software,
using the Equilibrium method for fitting kinetic data.
5.1.11.7. 2,3,9-Trimethoxy-12-methyl-8-(3-(piperidin-1-yl)propoxy)-
5,6-dihydrobenzo[c]acridin-12-ium chloride (21c). Following the
general procedure, compound 19 (0.27 mmol, 170 mg) and

62
S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
5.5. CD measurements
plates at 1.0 ꢃ 105 per well and exposed to the indicated concen-
trations of the ligand or an equivalent volume of 0.1% DMSO. The
cells in control and drug-exposed wells were counted every day
and the experiment was carried out for 4 days.
CD experiments were performed on
dichroism spectrophotometer (Applied Photophysics) [21].
a chirascan circular
A
quartz cuvette with 1 cm path length was used for the spectra
recorded over a wavelength range of 230e430 at 1 nm bandwidth,
5.9. RNA extraction
1 nm step size, and 0.5 s per point. 1 m
M of the oligomer Pu27 (50-
TGGGGAGGGTGGGGAGGGTGGGGAAGG-30) in 10 mM TriseHCl
buffer, pH 7.2, 100 mM KCl was annealed by heating at 95 ꢀC for
5 min, and then gradually cooled to room temperature and incu-
bated at 4 ꢀC overnight. The CD titration experiment was performed
Cell pellets harvested from each well of the culture plates were
lysed in TRIpure solution. RNA was extracted with M-MLV accord-
ing to manufacturer’s protocol and eluted in distilled, deionized
water with 0.1% diethyl pyrocarbonate (DEPC) to a final volume of
10e50 mL. RNA was quantitated spectra-photometrically and
stored at ꢁ80 ꢀC.
at a fixed Pu27 concentration (1
mM) by adding various concen-
trations (0e10 mol) of the ligands in TriseHCl buffer, 100 mM KCl
m
at 25 ꢀC. After each addition of ligand, the reaction was stirred and
allowed to equilibrate for at least 10 min (until no elliptic changes
were observed), and then a CD spectrum was collected by averaging
two scans. A buffer baseline was collected in the same cuvette, and
subtracted from the sample spectra. Final analysis of the data was
carried out using Origin 8.0 (OriginLab Corp.).
5.10. RT-PCR
Total RNA was used as a template for reverse transcription using
the following protocol: each 20
buffer, 2.5 mM dNTP, 100 pM oligo dT18 primer, 1
transcriptase, DEPC in water (DEPC H2O), and 2
m
L reaction contained 5 ꢃ M-MLV
L M-MLV reverse
g of total RNA.
m
The CD experiment of oligomer Pu27 DNA was also carried out
m
Briefly, RNA and oligo dT18 primer were incubated at 70 ꢀC for
10 min, and then immediately placed on ice. Next, the other
components were added, and incubated at 42 ꢀC for 1 h, and then at
70 ꢀC for 15 min. Finally, the reacted solution was stored at ꢁ20 ꢀC.
at a final concentration of 1
m
M in 10 mM TriseHCl buffer, pH 7.2 at
25 ꢀC containing the tested ligands (0, 3, 5, and 10
mM) in the
absence of KCl. The samples containing Pu27 and various concen-
trations of compounds in 10 mM TriseHCl buffer, pH 7.2, were
annealed by heating at 95 ꢀC for 5 min, and then gradually cooled to
room temperature and incubated at 4 ꢀC overnight. The recording
of CD spectra was carried out following the titration experiment.
Both c-myc and b-actin were amplified by using a real-time PCR
apparatus (Roche LightCycler 2), and the PCR products were
analyzed with electrophoresis on 1% agarose gel at 120 V for
20 min.
5.6. PCR-stop assay
Conflict of interest
The PCR-stop assay was performed with a modified protocol of
a previously reported procedure [24]. The oligomer of Pu27 (50-
TGGGGAGGGTGGGGAGGGTGGGGAAGG-30) and its corresponding
complementary sequence (50-ATCGATCGCTTCTCGTCCTTCCCCA-30)
were used in the experiment. The reactions were performed in
We declare that we have no conflict of interest.
Acknowledgments
1 ꢃ PCR-stop buffer, containing 10
mM of each pair of oligomers,
0.16 M of dNTP, 2.5 U of Taq polymerase, and the indicated amount
We thank the Natural Science Foundation of China (21172272),
the International S&T Cooperation Program of China
(2010DFA34630), and the Science Foundation of Guangzhou
(2009A1-E011-6) for financial support of this study.
m
of the compounds. Reaction mixtures were incubated in a thermo-
cycler, with the following cycling conditions: 94 ꢀC for 2 min, fol-
lowed with 30 cycles of 94 ꢀC for 30 s, 58 ꢀC for 30 s, and 72 ꢀC for
30 s. Amplified products were loaded on 16% non-denaturing poly-
acrylamide gel in 1 ꢃ TBE and GelRed stained.
Appendix A. Supplementary data
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.03.034.
5.7. Molecular modeling
Pu-18B G-quadruplex DNA structure constructed as reported
previously [45] was used as an initial model to study the interaction
between 5,6-dihydrobenzo[c]acridine derivatives and human c-
myc G-quadruplex DNA. The ligands were firstly constructed and
minimized in SYBYL 7.3.5 (Tripos Inc., St. Louis, MO, USA). Docking
studies were carried out using the AUTODOCK 4.2 program. The G-
quadruplex structure was used as an input for the AUTOGRID
program. The grid box was placed at the center of the G-quad-
ruplex. The dimensions of the active site box were set at
60 ꢃ 60 ꢃ 60 Å. Docking calculations were carried out using the
Lamarckian genetic algorithm (LGA). Other parameters were used
according to the default. Hundred of independent docking runs
were carried out. The ligand conformation was chosen based on
both the docked energy and the RMSD (root-mean-square-
deviation).
References
[1] H.J. Lipps, D. Rhodes, G-quadruplex structures: in vivo evidence and function,
Trends Cell Biol. 19 (2009) 414e422.
[2] J.L. Huppert, Four-stranded nucleic acids: structure, function and targeting of
G-quadruplexes, Chem. Soc. Rev. 37 (2008) 1375e1384.
[3] L. Oganesian, T.M. Bryan, Physiological relevance of telomeric G-quadruplex
formation: a potential drug target, Bioessays 29 (2007) 155e165.
[4] Y. Xu, Chemistry in human telomere biology: structure, function and targeting
of telomere DNA/RNA, Chem. Soc. Rev. 40 (2011) 2719e2740.
[5] D. Yang, L.H. Hurley, Structure of the biologically relevant G-quadruplex in the
c-MYC promoter, Nucleosides Nucleotides Nucleic Acids 25 (2006) 951e968.
[6] A.T. Phan, Y.S. Modi, D.J. Patel, Propeller-type parallel-stranded G-quad-
ruplexes in the human c-myc promoter, J. Am. Chem. Soc. 126 (2004)
8710e8716.
[7] S. Rankin, A.P. Reszka, J. Huppert, M. Zloh, G.N. Parkinson, A.K. Todd,
S. Ladame, S. Balasubramanian, S. Neidle, Putative DNA quadruplex formation
within the human c-kit oncogene, J. Am. Chem. Soc. 127 (2005) 10584e10589.
[8] T.S. Dexheimer, D. Sun, L.H. Hurley, Deconvoluting the structural and drug-
recognition complexity of the G-quadruplex-forming region upstream of the
bcl-2 P1 promoter, J. Am. Chem. Soc. 128 (2006) 5404e5415.
[9] D. Sun, K. Guo, J.J. Rusche, L.H. Hurley, Facilitation of a structural transition in
the polypurine/polypyrimidine tract within the proximal promoter region of
the human VEGF gene by the presence of potassium and G-quadruplex-
interactive agents, Nucleic Acids Res. 33 (2005) 6070e6080.
5.8. Proliferation assay
The Ramos cell line and CA46 cell line were used in the short-
term proliferation experiments. Cells were seeded in 24-well

S.-R. Liao et al. / European Journal of Medicinal Chemistry 53 (2012) 52e63
63
[10] P. Boukamp, N. Mirancea, Telomeres rather than telomerase a key target for
anti-cancer therapy? Exp. Dermatol. 16 (2007) 71e79.
telomeric DNA by a bis(quinacridine) macrocycle, J. Am. Chem. Soc. 125
(2003) 4732e4740.
[11] J.F. Riou, L. Guittat, P. Mailliet, A. Laoui, E. Renou, O. Petitgenet, F. Megnin-
Chanet, C. Helene, J.L. Mergny, Cell senescence and telomere shortening
induced by a new series of specific G-quadruplex DNA ligands, Proc. Natl.
Acad. Sci. U.S.A. 99 (2002) 2672e2677.
[33] M. Read, R.J. Harrison, B. Romagnoli, F.A. Tanious, S.H. Gowan, A.P. Reszka,
W.D. Wilson, L.R. Kelland, S. Neidle, Structure-based design of selective and
potent G quadruplex-mediated telomerase inhibitors, Proc. Natl. Acad. Sci.
U.S.A. 98 (2001) 4844e4849.
[12] D. Sun, L.H. Hurley, The Importance of negative superhelicity in inducing the
formation of G-quadruplex and i-motif structures in the c-myc promoter:
implications for drug targeting and control of gene expression, J. Med. Chem.
52 (2009) 2863e2874.
[34] J.L. Mergny, L. Lacroix, M.P. Teulade-Fichou, C. Hounsou, L. Guittat, M. Hoarau,
P.B. Arimondo, J.P. Vigneron, J.M. Lehn, J.F. Riou, T. Garestier, C. Helene,
Telomerase inhibitors based on quadruplex ligands selected by a fluorescence
assay, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 3062e3067.
[13] S. Neidle, Human telomeric G-quadruplex: the current status of telomeric G-
quadruplexes as therapeutic targets in human cancer, FEBS J. 277 (2010)
1118e1125.
[35] M.K. Cheng, C. Modi, J.C. Cookson, I. Hutchinson, R.A. Heald, A.J. McCarroll,
S. Missailidis, F. Tanious, W.D. Wilson, J.L. Mergny, C.A. Laughton, M.F. Stevens,
Antitumor polycyclic acridines. 20. Search for DNA quadruplex binding
[14] T.M. Ou, J. Lin, Y.J. Lu, J.Q. Hou, J.H. Tan, S.H. Chen, Z. Li, Y.P. Li, D. Li, L.Q. Gu,
Z.S. Huang, Inhibition of cell proliferation by quindoline derivative (SYUIQ-05)
through its preferential interaction with c-myc promoter G-quadruplex,
J. Med. Chem. 54 (2011) 5671e5679.
[15] H.J. Kang, H.J. Park, Novel molecular mechanism for actinomycin D activity as
an oncogenic promoter G-quadruplex binder, Biochemistry 48 (2009)
7392e7398.
selectivity in a series of 8,13-dimethylquino[4,3,2-kl]acridinium salts:
telomere-targeted agents, J. Med. Chem. 51 (2008) 963e975.
[36] Y.J. Lu, T.M. Ou, J.H. Tan, J.Q. Hou, W.Y. Shao, D. Peng, N. Sun, X.D. Wang,
W.B. Wu, X.Z. Bu, Z.S. Huang, D.L. Ma, K.Y. Wong, L.Q. Gu, 5-N-Methylated
quindoline derivatives as telomeric G-quadruplex stabilizing ligands: effects
of 5-N positive charge on quadruplex binding affinity and cell proliferation,
J. Med. Chem. 51 (2008) 6381e6392.
[16] S. Balasubramanian, L.H. Hurley, S. Neidle, Targeting G-quadruplexes in gene
promoters: a novel anticancer strategy? Nat. Rev. Drug Discov. 10 (2011)
261e275.
[17] S. Neidle, The structures of quadruplex nucleic acids and their drug
complexes, Curr. Opin. Struc. Biol. 19 (2009) 239e250.
[18] V. Gabelica, E.S. Baker, M.P. Teulade-Fichou, E. De Pauw, M.T. Bowers, Stabi-
lization and structure of telomeric and c-myc region intramolecular G-
quadruplexes: the role of central cations and small planar ligands, J. Am.
Chem. Soc. 129 (2007) 895e904.
[19] M.W. Freyer, R. Buscaglia, K. Kaplan, D. Cashman, L.H. Hurley, E.A. Lewis,
Biophysical studies of the c-MYC NHE III1 promoter: model quadruplex
interactions with a cationic porphyrin, Biophys. J. 92 (2007) 2007e2015.
[20] T. Lemarteleur, D. Gomez, R. Paterski, E. Mandine, P. Mailliet, J.F. Riou, Stabili-
zation of the c-myc gene promoter quadruplex by specific ligands’ inhibitors of
telomerase, Biochem. Biophys. Res. Commun. 323 (2004) 802e808.
[21] T.M. Ou, Y.J. Lu, C. Zhang, Z.S. Huang, X.D. Wang, J.H. Tan, Y. Chen, D.L. Ma,
K.Y. Wong, J.C. Tang, A.S. Chan, L.Q. Gu, Stabilization of G-quadruplex DNA and
down-regulation of oncogene c-myc by quindoline derivatives, J. Med. Chem.
50 (2007) 1465e1474.
[22] A. Rangan, O.Y. Fedoroff, L.H. Hurley, Induction of duplex to G-quadruplex
transition in the c-myc promoter region by a small molecule, J. Biol. Chem.
276 (2001) 4640e4646.
[23] J. Dash, Z.A. Waller, G.D. Pantos, S. Balasubramanian, Synthesis and binding
studies of novel diethynyl-pyridine amides with genomic promoter DNA G-
quadruplexes, Chem.–Eur. J. 17 (2011) 4571e4581.
[37] O. Baudoin, F. Gonnet, M.P. Teulade-Fichou, J.P. Vigneron, J.C. Tabet, J.M. Lehn,
Molecular recognition of nucleotide pairs by a cyclo-bis-intercaland-type
receptor molecule: a spectrophotometric and electrospray mass spectrom-
etry study, Chem.dEur. J. 5 (1999) 2762e2771.
[38] J.E. Nordlander, M.J. Payne, F.G. Njoroge, V.M. Vishwanath, G.R. Han,
G.D. Laikos, M.A. Balk, A short enantiospecific synthesis of 2-amino-6,7-
dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN), J. Org. Chem. 50 (1985)
3619e3622.
[39] A.J. Walz, R.J. Sundberg, Synthesis of 8-methoxy-1-methyl-1H-benzo[de][1,6]
naphthyridin-9-ol (isoaaptamine) and analogues, J. Org. Chem. 65 (2000)
8001e8010.
[40] J.B. Press, V.T. Bandurco, E.M. Wong, Z.G. Hajos, R.M. Kanojia, R.A. Mallory,
E.G. Deegan, J.J. Mcnally, J.R. Roberts, M.L. Cotter, D.W. Graden, J.R. Lloyd,
Synthesis of 5,6-dimethoxyquinazolin-2(1H)-ones, J. Heterocycl. Chem. 23
(1986) 1821e1828.
[41] C. Ronco, G. Sorin, F. Nachon, R. Foucault, L. Jean, A. Romieu, P.Y. Renard,
Synthesis and structure-activity relationship of Huprine derivatives as human
acetylcholinesterase inhibitors, Bioorg. Med. Chem. 17 (2009) 4523e4536.
[42] J. Styskala, P. Cankar, M. Soural, J. Hlavac, P. Hradil, J. Vicar, V. Simanek,
Synthesis of isodecarine, Heterocycles 73 (2007) 769e775.
[43] Y. Ma, T.M. Ou, J.H. Tan, J.Q. Hou, S.L. Huang, L.Q. Gu, Z.S. Huang, Synthesis and
evaluation of 9-O-substituted berberine derivatives containing aza-aromatic
terminal group as highly selective telomeric G-quadruplex stabilizing
ligands, Bioorg. Med. Chem. Lett. 19 (2009) 3414e3417.
[44] A. De Cian, L. Guittat, M. Kaiser, B. Sacca, S. Amrane, A. Bourdoncle, P. Alberti,
M.P. Teulade-Fichou, L. Lacroix, J.L. Mergny, Fluorescence-based melting
assays for studying quadruplex ligands, Methods 42 (2007) 183e195.
[45] Y. Ma, T.M. Ou, J.H. Tan, J.Q. Hou, S.L. Huang, L.Q. Gu, Z.S. Huang, Quinolino-
benzo-[5, 6]-dihydroisoquindolium compounds derived from berberine:
a new class of highly selective ligands for G-quadruplex DNA in c-myc
oncogene, Eur. J. Med. Chem. 46 (2011) 1906e1913.
[24] D. Peng, J.H. Tan, S.B. Chen, T.M. Ou, L.Q. Gu, Z.S. Huang, Bisaryldiketene
derivatives: a new class of selective ligands for c-myc G-quadruplex DNA,
Bioorg. Med. Chem. 18 (2010) 8235e8242.
[25] J. Dash, P.S. Shirude, S.T.D. Hsu, S. Balasubramanian, Diarylethynyl amides that
recognize the parallel conformation of genomic promoter DNA G-quad-
ruplexes, J. Am. Chem. Soc. 130 (2008) 15950e15956.
[26] M. Wainwright, Acridine-a neglected antibacterial chromophore, J. Anti-
microb. Chemother. 47 (2001) 1e13.
[27] G. Cholewinski, K. Dzierzbicka, A.M. Kolodziejczyk, Natural and synthetic
acridines/acridones as antitumor agents: their biological activities and
methods of synthesis, Pharmacol. Rep. 63 (2011) 305e336.
[28] M. Laronze-Cochard, Y.M. Kim, B. Brassart, J.F. Riou, J.Y. Laronze, J. Sapi,
Synthesis and biological evaluation of novel 4,5-bis(dialkylaminoalkyl)-
substituted acridines as potent telomeric G-quadruplex ligands, Eur. J. Med.
Chem. 44 (2009) 3880e3888.
[29] Y.T. Fu, B.R. Keppler, J. Soares, M.B. Jarstfer, BRACO19 analog dimers with
improved inhibition of telomerase and hPot 1, Bioorg. Med. Chem. 17 (2009)
2030e2037.
[30] N.H. Campbell, M. Patel, A.B. Tofa, R. Ghosh, G.N. Parkinson, S. Neidle, Selec-
tivity in ligand recognition of G-quadruplex loops, Biochemistry 48 (2009)
1675e1680.
[31] N.H. Campbell, G.N. Parkinson, A.P. Reszka, S. Neidle, Structural basis of DNA
quadruplex recognition by an acridine drug, J. Am. Chem. Soc. 130 (2008)
6722e6724.
[46] W.B. Wu, S.H. Chen, J.Q. Hou, J.H. Tan, T.M. Ou, S.L. Huang, D. Li, L.Q. Gu,
Z.S. Huang, Disubstituted 2-phenyl-benzopyranopyrimidine derivatives as
a new type of highly selective ligands for telomeric G-quadruplex DNA, Org.
Biomol. Chem. 9 (2011) 2975e2986.
[47] E.M. Rezler, J. Seenisamy, S. Bashyam, M.Y. Kim, E. White, W.D. Wilson,
L.H. Hurley, Telomestatin and diseleno sapphyrin bind selectively to two
different forms of the human telomeric G-quadruplex structure, J. Am. Chem.
Soc. 127 (2005) 9439e9447.
[48] T. Simonsson, M. Henriksson, c-myc Suppression in Burkitt’s lymphoma cells,
Biochem. Biophys. Res. Commun. 290 (2002) 11e15.
[49] L.M. Facchini, L.Z. Penn, The molecular role of myc in growth and trans-
formation: recent discoveries lead to new insights, FASEB J. 12 (1998)
633e651.
[50] A. Hamada, Y.A. Chang, N. Uretsky, D.D. Miller, Dopaminergic agonists:
comparative actions of amine and sulfonium analogues of dopamine, J. Med.
Chem. 27 (1984) 675e680.
[51] R. Beugelmans, J. Chastanet, H. Ginsburg, L. Quintero-Cortes, G. Roussi, Studies
in SRN1 series. Part 14. Direct synthesis of benzo[c]phenanthridines and
benzo[c]phenanthridones via SRN1 reactions, J. Org. Chem. 50 (1985)
4933e4938.
[32] M.P. Teulade-Fichou, C. Carrasco, L. Guittat, C. Bailly, P. Alberti, J.L. Mergny,
A. David, J.M. Lehn, W.D. Wilson, Selective recognition of G-quadruplex