Disubstituted 1,8-dipyrazolcarbazole derivatives as a new type of c-myc G-quadruplex binding ligands

Article abstract of DOI:10.1016/j.bmc.2012.03.031

A series of 1,8-dipyrazolcarbazole (DPC) derivatives (6a-6d, 7a-7d) designed as G-quadruplex ligands have been synthesized and characterized. The FRET-melting and SPR results showed that the DPC derivatives could well recognize G-quadruplex with strong discrimination against the duplex DNA. In addition, the DPC derivatives showed much stronger stabilization activities and binding affinities for c-myc G-quadruplex rather than telomeric G-quadruplex. Therefore, their interactions with c-myc G-quadruplex were further explored by means of CD spectroscopy, PCR-stop assay, and molecular modeling. In cellular studies, all compounds showed strong cytotoxicity against cancer cells, while weak cytotoxicity towards normal cells. RT-PCR assay showed that compound 7b could down-regulate c-myc gene expression in Ramos cell line, while had no effect on c-myc expression in CA46 cell line with NHE III₁ element removed, indicating its effective binding with G-quadruplex on c-myc oncogene in vivo.

Full text of DOI:10.1016/j.bmc.2012.03.031

Bioorganic & Medicinal Chemistry 20 (2012) 2829–2836
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Disubstituted 1,8-dipyrazolcarbazole derivatives as a new type of c-myc
G-quadruplex binding ligands
Wei-Jia Chen, Chen-Xi Zhou, Pei-Fen Yao, Xiao-Xiao Wang, Jia-Heng Tan, Ding Li, Tian-Miao Ou,
⇑
Lian-Quan Gu, Zhi-Shu Huang
School of Pharmaceutical Sciences, Sun Yat-Sen University, 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:
A series of 1,8-dipyrazolcarbazole (DPC) derivatives (6a–6d, 7a–7d) designed as G-quadruplex ligands
have been synthesized and characterized. The FRET-melting and SPR results showed that the DPC deriv-
atives could well recognize G-quadruplex with strong discrimination against the duplex DNA. In addition,
the DPC derivatives showed much stronger stabilization activities and binding affinities for c-myc
G-quadruplex rather than telomeric G-quadruplex. Therefore, their interactions with c-myc G-quadruplex
were further explored by means of CD spectroscopy, PCR-stop assay, and molecular modeling. In cellular
studies, all compounds showed strong cytotoxicity against cancer cells, while weak cytotoxicity towards
normal cells. RT-PCR assay showed that compound 7b could down-regulate c-myc gene expression in
Received 29 January 2012
Revised 13 March 2012
Accepted 14 March 2012
Available online 21 March 2012
Keywords:
G-Quadruplex
c-myc Oncogene
Ramos cell line, while had no effect on c-myc expression in CA46 cell line with NHE III
indicating its effective binding with G-quadruplex on c-myc oncogene in vivo.
1
element removed,
1
,8-Dipyrazolcarbazole derivatives
Binding ligand
Selectivity
Ó 2012 Elsevier Ltd. All rights reserved.
1
. Introduction
onto the terminal tetraplex of the quadruplex providing extra sta-
1
3,14
bilization.
Their side chains usually have positive charge, or
G-Quadruplexes are DNA secondary structures formed from
can become protonated at physiological pH, which is another com-
mon feature of providing electrostatic interactions with the nega-
tively charged phosphate residues in the grooves.
planar arrangements of four guanines stabilized by Hoogsteen
1
15
hydrogen bonding and monovalent cations. G-Quadruplexes have
received much attention recently due to their putative existence in
telomeres and in the promoter regions of oncogenes such as c-myc.
Human telomeric DNA, located at the end of chromosomes, con-
Some carbazole alkaloids have been isolated from Clausena
harmandiana and Clausena excavate, which have shown significant
2
1
6,17
cytotoxicity against cancer cell lines.
Recently, a carbazole
sists of tandem repeats of sequence d[(TTAGGG)
as the single strand. Ligands capable of inducing and stabilizing
G-quadruplex formation of these telomeric DNA could inhibit telo-
merase activity and interference with telomere biology. On the
other hand, the c-Myc oncoprotein is an important transcription
factor that plays a pivotal role in cell proliferation and survival.
n
], which ends
derivative, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide
(BMVC), has been found to be a good G-quadruplex stabilizer,
which has shown an increase in G-quadruplex melting tempera-
ture by 13 °C and has had a potent inhibitory effect on telomerase
3
4
,5
1
8
activity. So we think that the molecular architecture of carbazole
offers an attractive template for the design of G-quadruplex bind-
ing ligand. Besides, previous studies have shown that expanding
the size of aromatic planar surface of molecules might enhance
their binding potency and selectivity on G-quadruplex DNA over
duplex DNA, because square aromatic surface of G-quartet is much
6
The nuclear hypersensitivity element III
1 1
(NHE III ), upstream of
the P1 promoter of c-myc, controls 80–90% of the c-myc transcrip-
7
,8
1
tion level. The NHE III , a G (guanine)-rich strand of the DNA
containing a 27 base pair sequence, can form intramolecular G-
quadruplex structures and functions as a transcriptional repressor
1
9,20
larger than that of the Watson–Crick base pairs.
We hypothe-
9
element. The transcription of c-myc can be inhibited by the stabil-
sized that introduction of other aromatic groups to carbazole core
could further improve its quadruplex stabilizing property. It should
be mentioned that pyrazole has been found as a pharmacophore in
some active biological molecules, and pyrazole derivatives have
ization of G-quadruplexes by using specific G-quadruplex binders.
Several small molecular ligands have been reported to stabilize
G-quadruplex, including cationic porphyrins, quindoline derivatives,
and platinum complexes.1
0–12
These aromatic moieties
p
-stack
been extensively studied as anticancer.
and antimicrobial
21,22
anti-inflammatory,
23
2
4,25
molecules. The pyrazole ring represents a
good pharmacophore for the synthesis of biologically active com-
pounds with varying activity and good safety profiles. Therefore,
we designed a series of 1,8-dipyrazolcarbazole (DPC) derivatives,
⇑
Corresponding author. Tel./fax: +86 20 39943056.
E-mail address: ceshzs@mail.sysu.edu.cn (Z.-S. Huang).
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.031

2
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W.-J. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2829–2836
these compounds showed stronger effect on the thermal stability
of c-myc G-quadruplex ( : 4.4–18.1 °C) than telomeric G-quad-
ruplex ( : 1.2–11.9 °C).
N
N
DT
m
DT
m
N
N
FRET-melting experiments were also used to examine the bind-
N
H
N
N
ing selectivity of the compounds. F10T (5 -FAM-dTATAGCTATA-
0
N
N
0
HEG-TATAGCTATA-TAMRA-3 ) is a self-complementary hairpin
N
H
3
3
duplex DNA. As shown in Table 1, the derivatives showed weak
effect on the thermal-stability of the duplex DNA F10T ( <1 °C),
BMVC
1,8-dipyrazolcarbazole
DT
m
suggesting their poor binding with the duplex DNA. To further
characterize the selectivity of these ligands for G-quadruplex, com-
petitive FRET experiment was carried out, and a duplex oligomer
Figure 1. Structures of BMVC and 1,8-dipyrazolcarbazole.
0
0
ds26 (5 -CAATCGGATCGAATTCGATCCG-ATTG-3 ) was used in this
3
4
which had two pyrazole groups for enhancing their anticancer
activity through their stacking interactions with G-quadru-
study. Excess duplex DNA ds26 was mixed with c-myc G-quadru-
plex FPu18T before FRET-melting assay with ligands for val-
ues determination. The result of competitive FRET-melting assay
was shown in Figure 2, and the values for compounds were
p–p
DT
m
plex. As shown in Figure 1, intramolecular hydrogen bond is
formed in these designed molecules, which improves the planarity
of the molecular structure through enhanced p-delocalization. Two
cationic side chains of the 1,8-dipyrazolcarbazole core could inter-
act with the grooves and loops of the G-quadruplex, which may be
necessary to enhance its G-quadruplex binding potency and selec-
tivity, as well as its aqueous solubility. The interactions between
the DPC derivatives and c-myc G-quadruplex were examined by a
series of biophysical and biochemical methods.
DT
m
not significantly decrease, which indicated that the DPC derivatives
could specifically stabilize c-myc G-quadruplex without significant
effect for duplex DNA.
2
.3. Studies for the binding and selectivity of the DPC
derivatives on G-quadruplex with Surface Plasmon Resonance
Surface Plasmon Resonance (SPR) experiments were carried out
to study the interactions between DPC derivatives and G-quadru-
2
2
. Results and discussion
3
5
plex quantitatively (Fig. S2).
As shown in Table 1, compounds
.1. Chemistry
6a–6c, 7a–7c exhibited high affinities for c-myc G-quadruplex
and telomeric G-quadruplexes. No obvious binding was found for
The synthetic pathway for the DPC derivatives was shown in
compounds 6d and 7d even at concentration of 20
lM, which
Scheme 1. Nitration of carbazole with cupric nitrate, acetic acid
and acetic anhydride gave a mixture of 1-nitro-, 1,6-dinitro-, and
was consistent with very low values obtained from FRET melt-
DT
m
ing experiment. SPR experiments also indicated that compounds
with longer side chains showed higher G-quadruplex binding affin-
ities except compounds with morpholine terminal group. In addi-
tion, the carbazole derivatives showed much stronger binding
affinities for c-myc G-quadruplex than telomeric G-quadruplex.
For example, compounds 7a showed more than 20 times binding
affinity for c-myc G-quadruplex over telomeric G-quadruplex. For
the study of their binding affinities for duplex DNA, the incubation
of these compounds with duplex DNA immobilized on the chips
showed no significant binding interactions. These results were in
parallel with FRET-melting data showing the DPC derivatives can
be considered as a new class of selective G-quadruplex binding
ligands.
3
2
3
3
,6-dinitrocarbazoles from which the desired 3,6-dinitrocarbazole
could be isolated by using chromatography.2 The 1,8-dibromo-
,6-dinitrocarbazole 3 was obtained through the reaction of
,6-dinitrocarbazole 2 with bromine in sulfuric acid.27 The 1,8-
6
dibromo-3,6-dinitrocarbazole 3 was then coupled with pyrazole
to give 1,8-dipyrazol-3,6-dinitrocarbazole 4. A large excess of pyr-
2
8
azole had to be used, but it was possible to recycle this material.
The reduction of 1,8-dipyrazol-3,6-dinitrocarbazole 4 with tin
powder and hydrochloric acid produced intermediate 1,8-dip-
yrazol-3,6-diaminocarbazole, which was reacted with acyl chloride
to give compounds 5a–5b.26 Finally, the desired products 6a–6d
and 7a–7d were obtained through aminolysis of the compounds
5
a and 5b with appropriate secondary amines under reflux.
From both FRET-melting and SPR results, the DPC derivatives
showed much stronger stabilization activities and binding affini-
ties for c-myc G-quadruplex rather than telomeric G-quadruplex.
Therefore, the interaction of the DPC derivatives and c-myc G-
quadruplex was further explored by means of CD spectroscopy,
PCR-stop assay, and molecular modeling.
2
.2. Studies for the stabilization and selectivity of the DPC
derivatives on G-quadruplex with FRET assays
The effect of the DPC derivatives on the stabilization of G-quad-
ruplex was evaluated by using a FRET-melting assay.29 The human
telomeric G-quadruplex DNA (F21T) and the oncogene promoter
G-quadruplex c-myc (FPu18T) were used in this assay. The results
of the FRET-melting experiments were shown in Table 1, which
indicated that these compounds had a wide range of G-quadruplex
stabilization activity towards telomeric G-quadruplex and c-myc
G-quadruplex. The compounds with longer side chains showed
better G-quadruplex stabilizing properties except those with mor-
2.4. Studies of the binding property of the DPC derivatives on c-
myc G-quadruplex with Circular Dichroism
To further confirm the above results, and determine the confor-
mational change of G-quadruplex induced by the DPC derivatives,
3
6
Circular Dichroism (CD) experiment was carried out. CD spec-
troscopy was used to examine the effect of 7a, 7b and 7c on
c-myc G-quadruplex in the presence and absence of stabilizing salt.
pholine terminal group. The difference of the
caused by several reasons, for example, compound 6d and 7d had
m
DT values could be
0
0
Oligonucleotide Pu18 (5 -AGGGTGGGGAGGGTGGGG-3 ) was used
two morpholine rings on the side chains, the oxygen atom on the
as the c-myc sequence. As shown in Figure 3A, in the presence of
100 mM K , Pu18 showed a positive signal at about 262 nm and
+
morpholine ring made compound 6d and 7d more polar than the
others, the basicity of side chain amines was different.3
0,31
Similar
a negative signal at 240 nm, which suggested a typical parallel G-
quadruplex structure. Upon addition of compounds to Pu18, no
significant change was observed, which indicated that the DPC
derivatives could still maintain the parallel c-myc G-quadruplex
structure in the presence of potassium ion.
results had been obtained for tri-substituted isoalloxazine and tri-
3
2
arylpyridine derivatives, and the derivatives with a morpholine
ring on the side chain had lower values than those with amino
alkyl side chains. On the other hand, it should be mentioned that
DT
m

W.-J. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2829–2836
2831
O N
NO2
2
O N
NO2
O N
NO
2
2
2
i
ii
iii
N
H
N
N
O
N
H
N
H
N
H
N
N
Br
Br
1
2
3
4
H
N
H
N
H
H
N
Cl
n
Cl
R
n
n R
n
N
O
O
O
iv
v
vi
N
H
N
H
N
N
N
N
N
N
N
N
5
5
a n = 1
b n = 2
6a-d n = 1
7a-d n = 2
Compounds
R
a
b
c
d
O
N
N
N
N
Scheme 1. Synthesis of the DPC derivatives. Reagents and conditions: (i) Cu(NO
Cu
3
)
2
, acetic anhydride, acetic acid (90 °C); (ii) Br
2
, concentrated H
2
SO
4
(90 °C); (iii) pyrazole,
2
O, N-methyl-2-pyrrolidone, Ar (200 °C); (iv) Sn, concentrated HCl, acetic acid (reflux); (v) chloroacetyl chloride or 3-chloropropionyl chloride (70 °C); (vi) substituted
alkylamine, EtOH (reflux).
Table 1
A
10
8
pu18 + KCl
m
G-Quadruplex stabilization (DT ) potential obtained from FRET-melting and equi-
librium binding constants (K
D
) measured with SPR
pu18 + KCl + 7a
pu18 + KCl + 7b
pu18 + KCl + 7c
a
Compounds
FRET (D
T
m
/°C)
D
SPR (K /lM)
6
FPu18T
F21T
F10T
Pu18
HTG21
Duplex
—b
4
6
6
6
6
7
7
7
7
a
b
c
d
a
b
c
9.3
5.5
7.4
6.3
4.0
3.6
1.2
10.7
9.7
0.3
0.1
0.6
0.2
0.5
0.3
0.3
0.3
0.694
1.46
2.03
2.97
8.07
6.86
b
—
—
—
—
—
—
2
b
b
b
b
4.4
—
—
0
b
15.1
12.1
18.1
7.3
0.131
0.135
0.233
2.64
1.88
2.59
b
-2
-4
-6
b
11.9
4.2
b
b
b
d
—
—
—
a
D
T
m
= T
m
(DNA + ligand) ꢀ T
m
(DNA). The concentrations of FPu18T, F21T, and
M, and the concentrations of compounds 1.0 lM.
240
260
280
300
320
340
F10T were 0.2
l
b
No significant binding was found for addition of up to 20 lM ligand, which
Wavelength (nm)
might indicate no specific interactions between the ligand and the DNA.
8
6
4
2
0
2
B
pu18
pu18 + 7b
no competitor
1
1
1
1
1
8
6
4
2
0
3
μM ds26
10 μM ds26
8
6
4
2
0
Δ
-
240
260
280
300
320
340
Wavelength (nm)
6
d
6b
6c
6d
7a
7b
7c
7d
Compounds
Figure 3. (A) Change of the CD spectra of Pu18 in the presence of KCl. The
concentrations of 7a, 7b and 7c were 25 M. The concentration of the Pu18
remained at 5 M in 10 mM Tris–HCl buffer, 100 mM KCl, pH 7.2. (B) Change of the
CD spectra of Pu18 in the absence of KCl. The concentration of 7b was 25 M. The
concentration of the Pu18 remained at 5 M in 10 mM Tris–HCl buffer, pH 7.2.
l
Figure 2. Competitive FRET results for the DPC derivatives (1
with 15-fold (3 M) or 50-fold (10 M) excess of duplex DNA competitor (ds26).
The concentration of FPu18T was 0.2 M.
l
M), without and
l
l
l
l
l
l

2
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W.-J. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2829–2836
The CD spectroscopy of Pu18 in the absence of metal ion was
also studied. As shown in Figure 3B, The CD spectra of Pu18 with-
out any metal cation exhibited a small negative peak at 250 nm
and a large positive peak at 275 nm. After the treatment with 7b
(
25 lM), the positive peak shifted to 262 nm while the negative
peak shifted to 240 nm. These changes in the CD spectra indicated
that 7b could effectively induce conformational change of G-
quadruplex.
2
.5. Molecular modeling studies
To further investigate the mode of binding, molecular modeling
for the binding of 7a on the model of the NHE III intramolecular G-
1
quadruplex loop isomer was performed. Nucleotides G2–G5 in the
Pu27-mer were not involved in the G-quadruplex structure, so the
truncated sequence Pu18-mer (A6-G23 in Pu27-mer) was used for
subsequent studies. The previously built Pu-18B G-quadruplex
structure was used as the initial model to study the interaction be-
tween compounds and human c-myc G-quadruplex DNA.37 Our re-
sults showed that 7a could stack on both external G-quartet
Figure 5. Effects of the DPC derivatives 6a–d, 7a–d, on the hybridization of the
Pu27 in the PCR-stop assay.
Table 2
The IC50 values of the DPC derivatives in PCR-stop assay
0
planes. Preferable binding site of 7a was found to be 5 -terminal
Compounds
IC50 M)
6a
6b
6c
6d
7a
7b
7c
7d
G-quartet plane, with a calculated binding energy of ꢀ8.3 kcal/
mol. As shown in Figure 4, the ligand molecule effectively filled
much of the space in the plane of the G-quartet between the phos-
phate groups. The two positively-charged side chains were prop-
erly oriented and directed into the DNA groove towards the
negatively charged phosphate diester backbone, and formed elec-
trostatic interactions and hydrogen bonds.
(
l
5.2
>20
>20
>20
3.6
4.9
6.3
>20
amplification. The pyrrolidine group of 6a or 7a was optimal for
their interactions with G-quadruplex, while the morpholine group
of 6d or 7d was unfavorable for the binding interaction.
2
.6. Inhibition of amplification in the promoter region of c-myc
2.7. MTT assay
by the DPC derivatives
By using an MTT assay, the cytotoxicity of compounds against
HL60, HepG2, and NCI-H460 cancer cell lines was determined,
while their toxicity to normal cells was evaluated by using the nor-
mal cell line ECV-304 (Table 3). All compounds displayed strong
cytotoxicity against cancer cells, but showed a weak cytotoxicity
against normal epithelial cell ECV-304, indicating that all the deriv-
atives had relatively good selectivity on tumor cells, although some
of them had little effect on c-myc G-quadruplex binding and stabil-
ization, which may be due to other factors.
To get quantitative estimates of G-quadruplex stabilization by
the DPC derivatives, PCR stop assay was performed.
37,39
0
Pu27 (5 -
0
TGGGGAGGGTGGGGAGGGTGGGGAAGG-3 ) was chosen as the
testing oligonucleotide. The formation of the G-quadruplex can
block its hybridization with a complementary strand and leads to
the double-stranded DNA PCR product being undetectable. The re-
sults were shown in Figure 5 and Table 2. Compounds 6a, 7a–c
showed high inhibitory effect on the hybridization of Pu27, and
their IC50 values ranged from 3.6 to 6.3
showed weak inhibitory effect on the hybridization of Pu27 (IC50
20 M). These results were correlated with the values and
binding affinities obtained from SPR in Table 1, indicating that
the stability and binding interactions on c-myc G-quadruplex in-
duced by these compounds were important for inhibiting PCR
lM. Other compounds
2.8. Inhibition of c-myc expression in cancer cell line
>
l
DT
m
A reverse transcriptase-polymerase chain reaction (RT-PCR)
was performed to determine the impact of complexes on the
3
7
mRNA level of c-myc oncogene. Two Burkitt’s lymphoma cell
0
Figure 4. Top view (A) and side view (B) of compound 7a forming
p–p
stacking interactions with the 5 G-quartet. The ligand side chains are directing in the grooves. Pictures
were generated by PYMOL.38

W.-J. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2829–2836
2833
Table 3
cells may not be limited to their effect on c-myc G-quadruplex.
Other possible G-quadruplex genomic targets could be involved
in drug action. Our previous research reported that the quindoline
derivative SYUIQ-05 had relatively stronger binding affinity to c-
myc G-quadruplex DNA in vitro, and it exhibited its antitumor
activity on tumor cells mainly through its interaction with c-myc
IC50 values (lM) of the DPC derivatives against tumor cells
Compounds
HL60
NCI-H460
HepG2
ECV-304
6
a
1.9
2.4
2.0
3.9
2.2
1.2
1.9
1.7
2.7
3.3
2.5
3.9
3.5
2.7
2.6
2.8
2.7
5.6
3.0
6.1
4.2
5.1
2.7
6.9
22.2
27.3
22.8
29.3
25.2
25.0
23.3
26.2
6
b
6
c
6
d
42
in vivo. The DPC derivatives were identified as G-quadruplex li-
7
a
gand for both telomere and c-myc, but these compounds showed
much stronger stabilization activities and binding affinities for c-
myc G-quadruplex rather than telomeric G-quadruplex. To further
clarify molecular mechanism for antiproliferative properties of the
DPC derivatives, more detailed in vivo investigations are now
underway.
7
b
7
c
7
d
A
B
4
4
. Experimental section
.1. Synthesis and characterization
1H and 13C NMR spectra were recorded using TMS as the inter-
6 3
nal standard in DMSO-d or CDCl with a Bruker BioSpin GmbH
spectrometer at 400 MHz and 100 MHz, respectively. Mass spectra
(MS) were recorded on a Shimadzu LCMS-2010A instrument with
an ESI or ACPI mass selective detector, and high resolution mass
spectra (HRMS) were recorded on a Shimadzu LCMS-IT-TOF. Melt-
ing points (mp) were determined using a SRS-OptiMelt automated
melting point instrument without correction.
Ramos
CA46
1
00
8
6
4
2
0
0
0
0
0
4
.1.1. 3,6-Dinitro-9H-carbazole (2)
A homogeneous mixture of Cu(NO
3
)
2
ꢁ2.5H
2
O (30 mmol), acetic
acid (20 mL), and acetic anhydride (30 mL) was prepared at room
temperature. To this solution were added carbazole (25 mmol)
in small portions over 10 min. Temperature was maintained at
1
5–20 °C during addition of carbazole. The temperature was al-
-myc
lowed to rise to room temperature over a period of 30 min and
then to 90 °C. Reaction was continued with stirring for a period
of 30 min at this temperature. The mixture was poured into
0
1
2.5
5
10
Concentration of 7b (μM)
2
50 mL of distilled water with constant stirring. The precipitate
was collected by filtration, and washed five times each with about
00 mL of distilled water. The filtrate was dried in vacuumat. Chro-
matography on silica gel, eluting with petroleum/EtOAC (3:1) gave
Figure 6. (A)
transcription by compound 7b in Ramos and CA46 cells. Amplified products were
91 bp for c-myc and 541 bp for b-actin. (B) Relative c-myc expression level was
A RT-PCR assay revealed dose-dependent inhibition of c-myc
1
1
measured by using a densitometer.
2
7
1
2
(5.23 g, 81%) as a yellow solid. mp >300 °C (lit. mp >300 °C). H
lines with different translocation break points within the c-myc
NMR (400 MHz, DMSO-d ) d 12.69 (s, 1H), 9.50 (d, J = 2.1 Hz, 2H),
6
were tested. The Ramos cell line retains the NHE III
1
during trans-
8.40 (dd, J = 9.0, 2.1 Hz, 2H), 7.77 (d, J = 9.0 Hz, 2H). ESI-MS m/z:
258[M+H] .
+
location, while the CA46 cell line has lost this element together
with the P1 and P2 promoters.4
the NHE III element deleted, 7b showed no effect on c-myc expres-
sion, while for the Ramos cell line with this element, 7b could low-
er the c-myc expression. As shown in Figure 6 and 2.5 M of 7b
0,41
For the CA46 cell line with
1
4.1.2. 1,8-Dibromo-3,6-dinitro-9H-carbazole (3)
A mixture of 3,6-dinitrocarbazole (9 mmol), bromine (3 mL),
l
and H SO (30 mL) was warmed for 15 min at 90 °C. Then addi-
2
4
started to inhibit the expression of c-myc in Ramos cell line, and
2 4
tional H SO (6 mL) were added. The resulting mixture was heated
could significantly suppress the c-myc mRNA level at a concentra-
at 90 °C for 6 h. After cooling to room temperature, the brown reac-
tion mixture was poured onto ice and filtered to provide 3 (2.1 g,
tion of 10
. Conclusion
A series of the DPC derivatives were designed, synthesized and
lM.
2
7
1
5
6%) as a yellow-green solid. Mp >300 °C. (lit. mp >300 °C). H
3
6
NMR (400 MHz, DMSO-d ) d 12.72 (s, 1H), 9.49 (d, J = 2.1 Hz, 2H),
+
8
.54 (d, J = 2.1 Hz, 2H). ESI-MS m/z: 416 [M+H] .
found to be selective G-quadruplex binding ligands. The terminal
pyrrolidino group was proved to be optimal for G-quadruplex
binding, while the terminal morpholine group was proved to be
poor for G-quadruplex binding. Further cellular studies showed
that all compounds had strong cytotoxicity against cancer cells
with low toxicity towards the normal cells. Compound 7b showed
significant inhibition of c-myc gene expression in cultured cells,
presumably through the stabilization of the c-myc G-quadruplex
structure. Because of the complicated network of various genes
in vivo, the cytotoxicity of the carbazole derivatives against cancer
4.1.3. 3,6-Dinitro-1,8-di(1H-pyrazol-1-yl)-9H-carbazole (4)
A mixture of compound 3 (4.8 mmol), Cu O (0.48 mmol) and
pyrazole (48 mmol) were heated together with NMP (15 ml) at
2
200 °C under argon for 24 h. After cooling to room temperature,
the mixture was poured into H
2
O and filtered to provide a yellow
solid. Chromatography on Al
2
O
3
basic, eluting with CH Cl gave 4
2
2
1
as a yellow-green solid (0.81 g, 43%). Mp >300 °C. H NMR
(400 MHz, DMSO-d ) d 12.83 (s, 1H), 9.35 (d, J = 1.8 Hz, 2H), 9.02
6
(d, J = 2.6 Hz, 2H), 8.76 (d, J = 1.9 Hz, 2H), 8.01 (d, J = 1.7 Hz, 2H),
+
6.74–6.71 (m, 2H). ESI-MS m/z: 390 [M+H] .

2
834
W.-J. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2829–2836
37 9 2
ESI-HRMS m/z: calcd for C32H N O
[M+H]+ 580.3143, found
580.3169.
4
.1.4. General procedure: preparation of 5a and 5b
Compound 4 (2.0 mmol) were placed into a round-bottom flask
together with Sn (metal, 10 mmol), acetic acid (10 mL), and con-
centrated hydrochloric acid (2.5 mL). The mixture was refluxed
for 30 h under a blanket of nitrogen. The resulting solution was
cooled, and poured into an aqueous solution of 1.2 g of NaOH in
4.1.5.3.
N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)-
Compound 5a was
bis(2-(diethylamino)acetamide) (6c).
treated with diethylamine following the general procedure to give
5
0 mL of water. The precipitate was filtered, washed free of alkali
the desired product 6c as a white solid with a yield of 23%. Mp
1
with water, and dried in vacuum for 24 h. The product was purified
by extraction with THF, evaporation of THF under vacuum to give a
solid residue (0.48 g, 73%). Then solid residue in chloroacetyl chlo-
ride (10 mL) or 3-chloropropionyl chloride (10 mL) was heated at
6
151–153 °C. H NMR (400 MHz, DMSO-d ) d 11.61 (s, 1H), 9.80
(s, 2H), 8.63 (d, J = 2.5 Hz, 2H), 8.54 (d, J = 1.3 Hz, 2H), 8.10 (d,
J = 1.6 Hz, 2H), 8.00 (d, J = 1.7 Hz, 2H), 6.72–6.68 (m, 2H), 3.23 (s,
4H), 2.68 (q, J = 7.1 Hz, 8H), 1.09 (q, J = 7.1 Hz, 12H). 13C NMR
7
0 °C for 8 h. After cooling to 0 °C, the mixture was diluted with
3
(101 MHz, CDCl ) d 169.11, 140.00, 129.24, 127.90, 126.37,
distilled ether. The resulting solution was filtered, washed with
ether and water, and then evaporated under vacuum. The crude so-
lide was chromatographed on silica gel eluting with petroleum/
EtOAC (2:1) to give 5a and 5b.
124.22, 123.72, 108.10, 106.92, 106.13, 57.14, 47.98, 11.51. ESI-
+
HRMS m/z: calcd for
C
30
H
37
N
9
O
2
[M+H] 556.3143, found
556.3168.
4
.1.5.4.
N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)-
Compound 5a was trea-
bis(2-morpholinoacetamide) (6d).
4
.1.4.1.
bis(2-chloroacetamide) (5a).
a white solid 5a (0.48 g, 68%) was obtained. Mp: 286–287 °C.
NMR (400 MHz, DMSO-d ) d 11.64 (s, 1H), 10.54 (s, 2H), 8.59 (d,
J = 2.5 Hz, 2H), 8.35 (d, J = 1.3 Hz, 2H), 8.02 (d, J = 1.7 Hz, 2H),
N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)-
ted with morpholine following the general procedure to give the
Following General procedure,
1
desired product 6d as a white solid with a yield of 39%. Mp 250–
H
1
2
8
51 °C. H NMR (400 MHz, DMSO-d
6
) d 11.60 (s, 1H), 9.85 (s, 2H),
6
.60 (d, J = 2.4 Hz, 2H), 8.45 (d, J = 1.3 Hz, 2H), 8.05 (d, J = 1.5 Hz,
+
2H), 8.00 (d, J = 1.5 Hz, 2H)), 6.73–6.70 (m, 2H), 3.71 (t, J = 4.0 Hz,
6
.73–6.71 (m, 2H), 4.36 (s, 4H). ESI-MS m/z: 482 [M+H] .
1
3
8
H), 3.21 (s, 4H), 2.58 (t, J = 4.0 Hz,8H); C NMR (101 MHz, CDCl
3
)
d 166.89, 140.12, 128.97, 128.05, 126.36, 124.20 123.80, 108.36,
4
.1.4.2. N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)bis
1
C
07.09, 106.27, 66.08, 61.54, 52.92. ESI-HRMS m/z: calcd for
(
3-chloropropanamide) (5b).
white solid 5b (0.45 g, 61%) was obtained. mp: 268–269 °C.
NMR (400 MHz, DMSO-d ) d 11.55 (s, 1H), 10.29 (s, 2H), 8.53 (d,
J = 2.4 Hz, 2H), 8.34 (d, J = 1.5 Hz, 2H), 8.01 (d, J = 1.8 Hz, 2H),
Following General procedure, a
+
30 33 9 4
H N O [M+H] 584.2728, found 584.2738.
1
H
6
4
.1.5.5.
N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)-
Compound 5b
bis(3-(pyrrolidin-1-yl)propanamide) (7a).
8
2
.01 (d, J = 1.6 Hz, 2H), 6.72–6.68 (m, 2H), 3.96 (t, J = 6.3 Hz, 4H),
.91 (t, J = 6.3 Hz, 4H). ESI-MS m/z: 510 [M+H] .
was treated with pyrrolidine following the general procedure to
+
give the desired product 7a as a white solid with a yield of 32%.
1
Mp 263–265 °C; H NMR (400 MHz, DMSO-d
6
) d 11.49 (s, 1H),
4
.1.5. General procedure: preparation of 6a–d, and 7a–d
To a stirred suspension of the chloride compounds 5a or 5b
1
0.23 (s, 1H), 8.51 (d, J = 2.1 Hz, 2H), 8.31 (d, J = 1.3 Hz, 2H), 7.99
(
(
1
1
5
d, J = 1.6 Hz, 2H), 7.98 (d, J = 1.5 Hz, 2H), 6.71–6.67 (m, 2H), 2.79
t, J = 7.0 Hz, 4H), 2.56 (t, J = 7.0 Hz, 4H), 2.54–2.51 (m, 8H), 1.73–
(
0.3 mmol) in EtOH (25 mL) was added dropwise appropriate
1
3
amine (2.0 mL) in EtOH (5 mL). The mixture was stirred and heated
under reflux for 8–12 h, and the reaction was monitored periodi-
cally by using TLC, cooled to 0 °C, and diluted with distilled water.
The resulting solution was filtered, washed with ether and water,
then evaporated under vacuum. The crude solid was purified by
.70 (m, 8H);
30.08, 127.79, 126.31, 124.16, 123.60, 108.53, 107.63, 106.07,
37 9 2
2.28, 50.58, 33.65, 22.81. ESI-HRMS m/z: calcd for C32H N O
3
C NMR (101 MHz, CDCl ) d 169.88, 139.95,
+
[
M+H] 580.3143, found 580.3144.
using Al
and 7a–d.
2 3 2
O with CHCl /MeOH (50:1–20:1) elution to afford 6a–d
4
.1.5.6.
N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)-
Compound 5b
bis(3-(piperidin-1-yl)propanamide) (7b).
was treated with piperidine following the general procedure to
4
.1.5.1.
N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)-
Compound 5a was
give the desired product 7b as a white solid with a yield of 42%.
1
bis(2-(pyrrolidin-1-yl)acetamide) (6a).
Mp 166–167 °C. H NMR (400 MHz, DMSO-d ) d 11.49 (s, 1H),
6
treated with pyrrolidine following the general procedure to give
10.29 (s, 2H), 8.50 (d, J = 2.5 Hz, 2H), 8.28 (d, J = 1.5 Hz, 2H), 7.99
(d, J = 1.7 Hz, 2H), 7.97 (d, J = 1.6 Hz, 2H), 6.71–6.68 (m, 2H), 2.67
(t, J = 6.9 Hz, 4H), 2.53 (t, J = 6.9 Hz, 4H), 2.45–2.41 (m, 8H), 1.55–
the desired product 6a as a white solid with a yield of 22%. Mp
1
2
45–247 °C. H NMR (400 MHz, DMSO-d
6
) d 11.58 (s, 1H), 9.80
1
3
(
s, 2H), 8.59 (d, J = 2.4 Hz, 2H), 8.49 (d, J = 1.2 Hz, 2H), 8.08 (d,
J = 1.5 Hz, 2H), 8.00 (d, J = 1.5 Hz, 2H), 6.71–6.68 (m, 2H), 3.33 (s,
H), 2.69–2.66 (m, 8H), 1.83–1.79 (m, 8H). 13C NMR (101 MHz,
DMSO-d6) d 168.98, 141.15, 131.38, 128.23, 126.87, 124.45,
23.87, 109.35, 108.51, 107.66, 59.64, 53.85, 23.56. ESI-HRMS m/
1.52 (m, 8H), 1.44–1.40 (m, 4H). C NMR (101 MHz, CDCl ) d
3
169.79, 139.92, 130.24, 127.71, 126.29, 124.11, 123.59, 107.95,
4
107.40, 106.07, 53.45, 52.70, 31.53, 25.31, 23.24. ESI-HRMS m/z:
+
calcd for C34H41N O [M+H] 608.3456, found 608.3479.
9
2
1
+
z: calcd for C30
H
33
N
9
O
2
[M+H] 552.2830, found 552.2848.
4.1.5.7.
N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)-
Compound 5b was
bis(3-(diethylamino)propanamide) (7c).
4
.1.5.2.
N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)-
Compound 5a was
treated with diethylamine following the general procedure to give
bis(2-(piperidin-1-yl)acetamide) (6b).
treated with piperidine following the general procedure to give
the desired product 7c as a white solid with a yield of 53%. Mp
1
271–272 °C. H NMR (400 MHz, CDCl ) d 11.44 (s, 1H), 11.24 (s,
3
the desired product 6b as a white solid with a yield of 55%. Mp
2H), 8.04 (d, J = 2.0 Hz, 2H), 7.92–7.89 (m, 4H), 7.82 (d, J = 1.7 Hz,
2H), 6.48–6.44 (m, 2H), 2.81 (t, J = 5.8 Hz, 4H), 2.70 (q, J = 7.1 Hz,
8H), 2.54 (t, J = 5.8 Hz, 4H), 1.13 (t, J = 7.1 Hz, 12H). 13C NMR
(101 MHz, CDCl3) d 169.36, 139.86, 129.84, 127.63, 126.29,
124.10, 123.41, 108.64, 107.57, 106.02, 47.82, 45.19, 31.90, 28.67
1
1
60–162 °C. H NMR (400 MHz, DMSO-d
6
) d 11.59 (s, 1H), 9.76
(
s, 2H), 8.61 (d, J = 2.4 Hz, 2H), 8.48 (d, J = 1.3 Hz, 2H), 8.06 (d,
J = 1.5 Hz, 2H), 8.00 (d, J = 1.6 Hz, 2H), 6.71–6.67 (m, 2H), 3.14 (s,
1
3
4
H), 2.56–2.52 (m, 8H), 1.67–1.61 (m, 8H), 1.47–1.41 (m, 4H).
C
+
3
NMR (101 MHz, CDCl ) d 167.86, 140.02, 129.2, 127.96, 126.38,
9.77. ESI-HRMS m/z: calcd for C32H41N O [M+H] 584.3456, found
9
2
1
24.25, 123.74, 108.26, 107.03, 106.15, 61.84, 54.04, 25.34, 22.65.
584.3468.

W.-J. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2829–2836
2835
4
.1.5.8.
N,N’-(1,8-Di(1H-pyrazol-1-yl)-9H-carbazole-3,6-diyl)-
Compound 5b was
4.2.4. Molecular modeling
The previously built Pu-18B G-quadruplex structure was used
as the initial model to study the interactions between compounds
bis(3-morpholinopropanamide) (7d).
treated with morpholine following the general procedure to give
the desired product 7d as a white solid with a yield of 25%. Mp
3
7
and human c-myc G-quadruplex. Ligand structures were con-
structed by adopting the empirical Gasteiger–Huckel (GH) partial
atomic charges, and then were optimized (Tripos force field) with
a nonbond cutoff of 12 Å and a convergence of 0.01 kcal mol /Å
over 10,000 steps using the Powell conjugate-gradient algorithm.
Docking studies were carried out using the AUTODOCK 4.0 program.
The G-quadruplex structure was used as an input for the AUTO-
GRID program. The grid box was placed at the center of the G-
quadruplex. The dimensions of the active site box were set at
1
2
64–266 °C; H NMR (400 MHz, DMSO-d
6
): d 11.51 (s, 1H), 10.23
(
s, 2H), 8.52 (d, J = 2.2 Hz, 2H), 8.32 (d, J = 1.5 Hz, 2H), 8.00 (d,
ꢀ1
J = 1.5 Hz, 2H), 7.97 (d, J = 1.5 Hz, 2H), 6.69–6.72 (m, 2H), 3.59 (t,
J = 4.2 Hz, 8H), 2.70 (t, J = 6.8 Hz, 4H), 2.56 (t, J = 6.8 Hz, 4H), 2.45
1
3
(
t, J = 4.2 Hz, 8H). C NMR (101 MHz, CDCl
3
) d 169.31, 140.04,
29.78, 127.97, 126.28, 124.44, 123.45, 108.36, 107.55, 106.21,
6.07, 53.31, 51.92, 31.31. ESI-HRMS m/z: calcd for C32
1
6
37 9 4
H N O
+
[
M+H] 612.3041, found 612.3052.
6
0 ꢂ 60 ꢂ 60 Å. Docking calculations were carried out using the
4
4
.2. Biological assays
Lamarckian genetic algorithm (LGA). Initially, we used a popula-
tion of random individuals (population size: 150), a maximum
number of 25,000,000 energy evaluations, a maximum number of
generations of 27,000, and a mutation rate of 0.02. Hundred inde-
pendent docking runs were carried out for each ligand. The result-
ing positions were clustered according to a root-mean-square
criterion of 0.5 Å.
.2.1. FRET-melting assay
FRET assay was carried out on a real-time PCR apparatus follow-
ing previously published procedures. The fluorescently labeled oli-
gonucleotides FPu18T (5 -FAM-AGGGTGGGGA-GGGTGGGG-TAM
0
0
0
0
RA-3 ), F21T (5 -FAM-d(GGG[TTAGGG]
3
)-TAMRA-3 ) and F10T:
0
0
5
-FAM-dTAT-AGCTATA-HEG-TATAGCTATA-TAMRA-3 (donor fluo-
rophore FAM is 6-carboxy-fluorescein; acceptor fluorophore TAMRA is
-carboxytetramethyl-rhodamine; HEG linker is [(–CH –CH –O–) ])
were used as the FRET probes. Fluorescence melting curves were
determined with a Roche LightCycler 2 real-time PCR machine,
4.2.5. PCR-stop assay
6
2
2
6
The polymerase stop assay was performed as described previ-
3
7
0
ously. Sequences of the tested oligomers were Pu27 (5 -TG
0
GGGAGGGTGGGGAGGGTGGGG-AAGG-3 ) and the corresponding
using a total reaction volume of 20
gonucleotide in Tris–HCl buffer (10 mM, pH 7.2) containing
l
L, with 0.2
l
M of labeled oli-
complementary sequence. The reactions were performed in 1 ꢂ
PCR buffer, containing 10 lM of each pair of oligomers, 0.16 mM
0
4
.2 mM or 60 mM KCl. Fluorescence readings with excitation at
70 nm and detection at 530 nm were taken at intervals of 1 °C
dNTP, 2.5 U Taq polymerase, and the indicated amount of the com-
pounds. Reaction mixtures were incubated in a thermocycler, with
the following cycling conditions: 94 °C for 3 min, followed with 30
cycles of 94 °C for 30 s, 5 °C for 30 s, and 72 °C for 30 s. Amplified
products were resolved on 15% nondenaturing polyacrylamide gels
in 1 ꢂ TBE and silver stained.
over the range 37–99 °C, with a constant temperature being main-
tained for 30 s prior to each reading to ensure a stable value. The
melting of the G-quadruplex was monitored alone or in the
presence of various concentrations of compounds and/or of
double-stranded competitor ds26 (ds26 DNA: 5 -GTTAGCCTAGCTT
AAGCTAGGCTAAC-3 ). Final analysis of the data was carried out
0
0
4.2.6. Cell culture
HL60 leukemia cell line, ECV-304 epithelial cell, HepG2 hepa-
toma cell line, NCI-H460 lung cancer, Ramos, and CA46, were ob-
tained from the Experimental Animal Center of Sun Yat-sen
University. The cell cultures were maintained in RPMI-1640 med-
ium supplemented with fetal bovine serum (10%), glutamine
using Origin8.0 (OriginLab Corp.).
4
.2.2. 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. In a typical experi-
ment, biotinylated duplex DNA, biotinylated HTG21 and biotinyla-
ted Pu18 were folded in filtered and degassed running buffer
1%), Penicillin (100 U/mL) and Streptomycin (1%) in 25 cm2 cul-
(
ture flasks at 37 °C in a humidified atmosphere with 5% CO
2
.
(
Tris–HCl, 50 mM, pH 7.2, 100 mM KCl). The DNA samples were
4.2.7. MTT assay
then captured (ꢀ1000 RU) in flow cells 1 and 2, leaving the third
HL60 leukemia cell line, ECV-304 epithelial cell, HepG2 hepa-
toma cell line and NCI-H460 lung cancer cell line were seeded on
flow cell as a blank. Ligand solutions (at 0.3125, 0.625, 1.25, 2.5,
3
5
, 10
l
M) were prepared with running buffer by serial dilutions
96-well plates (1.0 ꢂ 10 /well), and exposed to various concentra-
from stock solutions. Six concentrations were injected simulta-
tions of derivatives. After 48 h of treatment at 37 °C in a humidified
ꢀ
1
neously at a flow rate of 100 mL min for 150 s of association
phase, followed with 300 s of dissociation phase at 25 °C. The
GLH sensor chip was regenerated with short injection of 1 M NaCl
between consecutive measurements. The final graphs were ob-
tained by subtracting blank sensorgrams from the duplex or quad-
ruplex sensorgrams. Data were analyzed with ProteOn manager
software, using the Langmuir model for fitting kinetic data.
2
atmosphere of 5% CO , 10 lL of 5 mg/mL methyl thiazolyl tetrazo-
lium (MTT) solution was added to each well and further incubated
for 4 h. The cells in each well were then treated with dimethyl sulf-
oxide (DMSO) (200 lL for each well), and the optical density (OD)
was recorded at 570 nm. All drug doses were parallel tested in trip-
licate, and the IC50 values were derived from the mean OD values of
the triplicate tests versus drug concentration curves.
4
.2.3. CD measurements
4.2.8. RNA extraction
Ramos and CA46 cells were seeded in six-well plates at 100,000
cells/well, and compounds were added at final concentration of 1,
0
0
The oligomer Pu18 (5 -AGGGTGGGGAGGGTGGGG-3 ) at a final
concentration of was resuspended in Tris–HCl buffer
10 mM, pH 7.2) containing the derivatives to be tested. The sam-
5 lM
(
2.5, 5, 10 lM or DMSO control. After incubation for 4 days, cells
ples were heated to 95 °C, then gradually cooled to room temper-
ature, and incubated at 4 °C for at least 6 h. The CD spectra were
recorded on a Chirascan (AppliedPhotophysics) spectrophotome-
ter, using 0.5 s-per-points from 220 to 450 nm and 1 nm band-
width. The CD spectra were obtained by averaging two scans.
Final analysis of the data was carried out using Origin 8.0 (Origin-
lab Corp.).
were harvested, and the RNA was extracted according to the man-
ufacturer’s instructions. Cell pellets harvested from each well of
the culture plates were lysed in Redzol solution. RNA was extracted
with M-MLV according to manufacturer’s protocol, and eluted in
distilled, deionized water with 0.1% diethyl pyrocarbonate (DEPC)
to a final volume of 10–50 mL. RNA was quantitated spectrophoto-
metrically and stored at ꢀ80 °C.

2
836
W.-J. Chen et al. / Bioorg. Med. Chem. 20 (2012) 2829–2836
10. Seenisamy, J.; Bashyam, S.; Gokhale, V.; Vankayalapati, H.; Sun, D.; Siddiqui-
4
.2.9. RT-PCR
Jain, A.; Streiner, N.; Shin-Ya, K.; White, E.; Wilson, W. D.; Hurley, L. H. J. Am.
Chem. Soc. 2005, 127, 2944.
1. Lu, Y. J.; Ou, T. M.; Tan, J. H.; Hou, J. Q.; Shao, W. Y.; Peng, D.; Sun, N.; Wang, X.
D.; Wu, W. B.; Bu, X. Z.; Huang, Z. S.; Ma, D. L.; Wong, K. Y.; Gu, L. Q. J. Med.
Chem. 2008, 51, 6381.
The quantity of the RNA was measured by using UV spectrom-
etry. Synthesis of cDNA was performed using 2
tal RNA was used as a template for reverse transcription using the
following protocol: each 20
L reaction contained 5 ꢂ M-MLV buf-
fer, 2.5 mM dNTP, 50 M oligo dT18 primer, 1 L M-MLV reverse
transcriptase, DEPC in water (DEPC H O), and 2 g of total RNA.
Briefly, RNA and oligo dT18 primer was incubated at 70 °C for
0 min and then immediately placed on ice, after which the other
components were added and incubated at 42 °C for 1 h and then at
0 °C for 15 min. Finally, the reacted solution was stored at ꢀ20 °C.
For each cDNA sample, one 20 L reaction was set up on ice, con-
lg of total RNA. To-
1
l
12. Wu, P.; Ma, D. L.; Leung, C. H.; Yan, S. C.; Zhu, N.; Abagyan, R.; Che, C. M.
Chemistry 2009, 15, 13008.
l
l
2
l
13. Patel, D. J.; Phan, A. T.; Kuryavyi, V. Nucleic Acids Res. 2007, 35, 7429.
1
4. Monchaud, D.; Teulade-Fichou, M. P. Org. Biomol. Chem. 2008, 6, 627.
5. Moorhouse, A. D.; Haider, S.; Gunaratnam, M.; Munnur, D.; Neidle, S.; Moses, J.
E. Mol. Biosyst. 2008, 4, 629.
1
1
16. Taufiq-Yap, Y. H.; Peh, T. H.; Ee, G. C.; Rahmani, M.; Sukari, M. A.; Ali, A. M.;
Muse, R. Nat. Prod. Res. 2007, 21, 810.
7
17. Songsiang, U.; Thongthoom, T.; Boonyarat, C.; Yenjai, C. J. Nat. Prod. 2011, 74,
l
208.
taining 10 ꢂ PCR buffer, 2.5 mM dNTP, primer c-mycA, primer c-
mycS, and rTaq. Both c-myc and b-actin were amplified by using
PCR, and the PCR products were analyzed by using electrophoresis
on 1% agarose gel at 120 V for 20 min. Photograghs were taken
with multilmage light cabinet. The quantity of the c-myc expres-
sion was normalized based on the control gene b-actin, and the
expression of c-myc in Ramos cells was compared with the expres-
sion of c-myc in CA46 cells.
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C.; Sansonna, P.; Villa, M.; Pierce, B. S.; Pulici, M.; Giordano, P.; Martina, K.;
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We declare that we have no conflict of interest.
Acknowledgements
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This work was supported by National Natural Science Foundation
of China (Nos. U0832005, 90813011, 21172272, 81001400),
the International S&T Cooperation Program of China (No.
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