9-N-Substituted berberine derivatives: Stabilization of G-quadruplex DNA and down-regulation of oncogene c-myc

Article abstract of DOI:10.1016/j.bmc.2008.07.029

A series of 9-N-substituted berberine derivatives (2a-j) were synthesized and evaluated as a new class of G-quadruplex binding ligands. G-quadruplex of DNA had been proven to be the transcription controller of human c-myc gene. The interaction of 9-N-substituted berberine derivatives with G-quadruplex DNA in c-myc was examined via EMSA, CD spectroscopy, FRET-melting method, PCR-stop assay, competitive dialysis, cell proliferation assay, and RT-PCR assay. The experiment results indicated that these derivatives could selectively induce and stabilize the formation of intramolecular parallel G-quadruplex in c-myc, which led to down-regulation of transcription of the c-myc in the HL60 lymphomas cell line. The related structure-activity relationships were also discussed.

Full text of DOI:10.1016/j.bmc.2008.07.029

Bioorganic & Medicinal Chemistry 16 (2008) 7582–7591
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
9-N-Substituted berberine derivatives: Stabilization of G-quadruplex DNA
and down-regulation of oncogene c-myc
*
Yan Ma, Tian-Miao Ou, Jin-Qiang Hou, Yu-Jing Lu, Jia-Heng Tan, 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 9-N-substituted berberine derivatives (2a–j) were synthesized and evaluated as a new class of
G-quadruplex binding ligands. G-quadruplex of DNA had been proven to be the transcription controller of
human c-myc gene. The interaction of 9-N-substituted berberine derivatives with G-quadruplex DNA in
c-myc was examined via EMSA, CD spectroscopy, FRET-melting method, PCR-stop assay, competitive dial-
ysis, cell proliferation assay and RT-PCR assay. The experiment results indicated that these derivatives
could selectively induce and stabilize the formation of intramolecular parallel G-quadruplex in c-myc,
which led to down-regulation of transcription of the c-myc in the HL60 lymphomas cell line. The related
structure–activity relationships were also discussed.
Received 31 May 2008
Revised 11 July 2008
Accepted 12 July 2008
Available online 18 July 2008
Keywords:
9-N-Substituted berberine derivatives
Synthesis
c-myc G-quadruplex
Down-regulation of transcription
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
point for G-quadruplex stabilizing agent, quarfloxin (CX-3543),
which targets ribosomal RNA biogenesis in cancer cells and is cur-
The human oncogene c-myc plays important role in many cellu-
lar events, and the overexpression of the oncogene gene is related
to the increasing of cellular proliferation in a variety of different
malignant tumors, including breast, colon, cervix, small-cell lung,
osteosarcomas, glioblastomas and myeloid leukemias.1,2 Up to
90% of the transcription of c-myc is controlled by the P1 and P2
promoter, and a nuclease hypersensitivity element III1 (NHE III1)
with guanine-rich (G-rich) sequence (Fig. 1) is located at P1.3,4
The previous study demonstrated that the G-quadruplex presented
in the promoter region of the c-myc functioned as a transcriptional
repressor element,1,5–7 consequently controlling the transcription
of c-myc via the G-quadruplex structure had emerged as an attrac-
tive target for anti-cancer therapeutic strategies (Fig. 1).
rently in clinical trials as an antitumor agent.12
Berberine, an alkaloid isolated from Chinese herbs, was initially
used as anti-microbial agent. Berberine and its derivatives were
subsequently evaluated as inhibitors of the topoisomerase I and
II which were linked to anti-cancer activity.13–15 Recently, 13-
substituted berberine derivatives were reported by Neidle’s group
that had the abilities to selectively bind to G-quadruplex over dou-
ble-stranded DNA,16 and inhibited telomerase activity by binding
The strategy to find new chemical entities that were able to selec-
tivelyinterferewith c-myc expression by the formation/stabilization
of the specific G-quadruplexes had potential applications.1,8 A num-
ber of G-quadruplex ligands, such as porphyrins,2,8,9 perylenes,4 had
been developed and shown to induce and/or stabilize the G-quadru-
plex structure. The quindoline derivatives had been identified by our
group to have the abilities in inducing/stabilizing the G-quadruplex
structure in the NHE III1 sequence and down-regulation of transcrip-
tion of the c-myc in cancer cell line.10,11 A potential of G-quadruplex
stabilizing compounds as anti-cancer agents is to refer a good case in
Figure 1. Location of the NHE III1 in the c-myc gene and proposed biological
function of G-quadruplex in this region. The numbering of the purine-rich
Pu27-mer sequence and the truncated Pu18-mer are shown. When the G-
quadruplex structure formed in the promoter region of c-myc, the transcription
will be blocked.
* Corresponding authors. Tel./fax: +86 20 39332678 (L.-Q.G.); +86 20 39332679
(Z.-S.H.).
E-mail addresses: cesglq@mail.sysu.edu.cn (L.-Q. Gu), ceshzs@mail.sysu.edu.cn
(Z.-S. Huang).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.029

Y. Ma et al. / Bioorg. Med. Chem. 16 (2008) 7582–7591
7583
to G-quadruplex DNA.17 Later, our group demonstrated that 9-O-
substituted berberine derivatives could induce and stabilize the
anti-parallel G-quadruplex structure in the telomere DNA in the
presence or absence of metal cations.18
2.2. Inducing G-quadruplex by 9-N-substituted berberine
derivatives
EMSA was performed to identify whether the 9-N-substituted
berberine derivatives induced the formation of c-myc G-quadru-
plex structures (Pu27, 50-TGGGGAGGGTGGGGAGGGTGGGG-AAG
G-30, see Fig. 1). The oligomer Pu27 was incubated with the deriv-
atives 2a–j, respectively, for at least 4 h in a Tris–HCl buffer
(50 mM, pH 7.2).
According to the previous gel-shift data obtained under similar
experimental conditions and the shift mobility of the Pu27
G-quadruplex induced by potassium ions, the major bands were
identified as higher order structures (higher order) and intramolec-
ular G-quadruplex structures (intra-G). As shown in Figure 3, all
the derivatives showed interaction with the oligomer Pu27 with-
out potassium cation. The major band was similar to the band in
the presence of KCl reported previously,1 indicated that the 9-N-
substituted berberine derivatives might induce the formation of
intramolecular G-quadruplex.
In this work, a series of 9-N-substituted berberine deriva-
tives were designed and synthesized, and the interactions be-
tween the derivatives and NHE III1 DNA were investigated
using electrophoretic mobility shift assay (EMSA), circular
dichroism spectroscopy (CD), fluorescence resonance energy
transfer-melting (FRET-melting) method, polymerase chain
reaction-stop assay (PCR-stop assay), competition dialysis
method, cell proliferation assay and reverse transcription-poly-
merase chain reaction (RT-PCR). All the results showed that
the 9-N-substituted berberine derivatives could selectively sta-
bilize the formation of intramolecular parallel G-quadruplex
in c-myc DNA, thus led to down-regulation of the transcription
of c-myc in cancer cell line.
2. Results and discussion
To identify the interaction of the 9-N-substituted berberine
derivatives with c-myc G-quadruplex, CD spectroscopy of Pu18
was performed after treatment with berberine and its 9-N-substi-
tuted derivatives. The CD spectra of Pu18 without any metal cat-
ions at room temperature exhibited a small negative peak at
240 nm and a large positive peak at 275 nm. After the treatment
with the 9-N-substituted berberine derivatives, the positive peak
shifted to 262 nm and the negative peak at 240 nm was increased,
which was similar with the CD spectra of Pu18 in the presence of
K+ (Fig. 5), but no significant difference of CD spectra could be ob-
served while equivalent berberine was added (Fig. 4A). The results
indicated that the interaction abilities of the 9-N-substituted ber-
berine derivatives with G-quadruplex structure were much stron-
ger than the berberine.
The CD spectra of Pu18 titrated with 2j in a Tris–HCl buffer was
shown in Figure 4B. It could be found that the positive peak was
gradually shifted from 275 nm to about 262 nm with an increase
of the concentration of 2j from 1 to 5 mol equivalences, and other
derivatives exhibited the similar results with the derivative 2j. The
above results combined with the EMSA results indicated that the
9-N-substituted berberine derivatives could have the ability to pro-
mote the forming of G-quadruplex in the promoter region of hu-
man oncogene c-myc, and the G-quadruplex structures could be
similar with that in the presence of K+.
The conformational property of the Pu18 G-quadruplex DNA in-
duced by the 9-N-substituted berberine derivatives in the presence
of 100 mM K+ and 100 mM Na+ was also monitored by the CD spec-
troscopy. As shown in Figure 5, in the presence of K+, Pu18 only
showed a signal at about 262 nm. Upon addition berberine or 2j
to the Pu18, no significant change was appeared but the peak min-
or decreased. When added 2j to Na+ system, the major signal
shifted from 270 nm to about 262 nm, indicated 2j could induce
Pu18 to form the G-quadruplex structure like in the K+ system.7
Comparing the results in Figures 4 and 5, we could give a same
2.1. Design and synthesis of the 9-N-substituted berberine
derivatives
Berberine with N+-containing aromatic moiety (Scheme 1) ap-
peared suitable for p–p stacking interactions with a G-quartet. In
our previous studies, introduction of a side chain with terminal
amino group in the 9-position of the berberine led to significant in-
crease stabilization effect on telomeric G-quadruplex DNA and
inhibitory activity on telomerase.18 It could be boiled down to
the terminal amino group in the side chain strengthen the electro-
static interactions with the phosphate backbone of G-quadruplex
DNA.19 Molecular modeling studies of interactions between ber-
berine or berberine derivative 2j and G-quadruplex structure in
the NHE III1 (A truncated sequence Pu18-mer was used for this
work since nucleotides G2–G5 in the Pu27-mer not involved in
the G-quadruplex structure,1,11 Fig. 1.) indicated that the deriva-
tives containing 9-N-substituted side chain could have higher
binding affinity with G-quadruplex compared to berberine, be-
cause of hydrophobic forces, electrostatic interactions, and hydro-
gen-bond interactions of side chain (Fig. 2). For these reasons,
several 9-N-substituted berberine derivatives were designed and
synthesized.
Compounds 2a–j were prepared by nucleophilicoromatic dis-
placement reaction of berberine.20,21 Treatment of berbeine with
primary amines produced amino-isoquinolinium derivatives. The
synthetic route of 9-N-substituted berberine derivatives 2a–j was
shown in Scheme 1.
The berberine was added in a solution of anhydrous ethanol
with constituent amine, and the mixture was stirred and refluxed
for 2–4 h. Subsequently the mixture was concentrated and purified
on Al2O3 column with yield of 20–51%. The yield decreased with
the time prolonging. All of these compounds were characterized
by NMR, ESI-MS, and element analysis.
compound
n
2
2
R
compound
n
3
3
R
4
5
N(CH3)2
N(CH3)2
2a
2b
2f
O
O
O
O
Cl
7
Cl
N
N
O
N
N
O
2g
i
1
R
NH
O
9
O
O
13
2c
2d
2e
2
2
2
2h
2i
3
3
6
N
N
CH2
n
N
OH
11
NH2
C
2j
1
2a-j
Scheme 1. Reagents and conditions: (i) NH2(CH2)nR, ethanol, reflux, 2–4 h.

7584
Y. Ma et al. / Bioorg. Med. Chem. 16 (2008) 7582–7591
Figure 2. Models for the G-quadruplex–ligand complexes. View onto the plane of the 30 surface of Pu18B G-quadruplex.11 (A) Berberine making
with the guanine tetrad. (B) 9-Substituted berberine derivative 2j making stacking interactions with G-quartet, its long side chain making hydrophobic interactions with
p–p stacking interactions
p–p
G-quartet, and the terminal amino group in side chain making electrostatic interactions and hydrogen-bond interactions with the negative electrostatic potential
phosphodiester backbone. Pictures were created with PyMOL.22
14
A
Pu18
12
Pu18+25 μM berberine
10
8
Pu18+25 μM 2j
6
4
2
0
Figure 3. Polyacylamide gel electrophoresis of the oligomer Pu27 (10
lM) without
(lane no drug) and with (left lanes) berberine derivatives 2a–j (10 M), respectively.
l
-2
-4
The major bands were identified as higher order structures (higher order) and
intramolecular G-quadruplex structures (intra-G). KCl band: the Pu27 was incu-
bated in the Tris–HCl buffer (pH 7.2) containing 100 mM KCl in the absence of any
drugs.
220
240
260
280
300
320
340
Wavelength (nm)
14
12
10
8
B
conclusion that the conformational property of the Pu18 G-quad-
ruplex in the case of 2j was similar to that in the presence of K+.
The results of other 9-N-substituted derivatives showed the similar
properties.
titration with 2j
Pu18
Pu18 + 5 μM 2j
Pu18 + 10 μM 2j
Pu18 + 15 μM 2j
Pu18 + 20 μM 2j
Pu18 + 25 μM 2j
6
2.3. Thermodynamic stability of the c-myc G-quadruplex in the
presence of 9-N-substituted berberine derivatives
4
2
The stability of c-myc G-quadruplex DNA treated with the ber-
berine and its 9-N-substituted derivatives were studied by FRET-
melting assay. The oligomer Pu18 containing a fluorophore at the
0
-2
50-end and
a
fluorescence quencher at the 30-end (FPu18T,
50-FAM-AGGGTGGGGA-GGGTGGGG-TAMRA-30) was used in this
assay. Fluorescence quenching curves were determined under
different conditions with variant ion concentrations. It could be
seen that the Tm values were much higher in K+-containing buffers
than the same concentration of Na+-containing buffers, indicated
that the G-quadruplex structure was more stable in the presence
of potassium.
220
240
260
280
300
320
340
Wavelength (nm)
Figure 4. CD spectra of Pu18 in Tris–HCl buffer (10 mM, pH 7.2). (A) CD spectra of
Pu18 in the absence of cations, the blank sample without drug was shown by solid
line, the sample with 25
the sample with 25 M 2j was shown by open circle symbols (s). (B) Change of the
CD spectra of Pu18 in the presence of 2j. The concentrations of 2j were 0 (line), 5
(h), 10 (j), 15 (s), 20 (d) and 25 (4) M. The concentration of the Pu18 remained
at 5 M in a Tris–HCl buffer, 10 mM, pH 7.2, in the absence of metal ions.
lM berberine was shown by solid circle symbols (d), and
l
l
The melting temperature of Pu18 was monitored by FRET-
melting assay in the presence of various concentrations (0.2–
l
3.0 lM) of 9-N-substituted berberine derivatives in Tris–HCl buf-
fer containing of 0.2 mM K+. The results of FRET-melting assay
for 2j at different concentrations was shown in Figure 6A. It
was clear that 2j could raise the melting temperature of G-quad-
ruplex by about 29 °C, indicated an obvious stabilization effect of
2j on G-quadruplex in Pu18. As shown in Figure 6B, the stabil-
ization of G-quadruplex DNA was depended on the concentration
of 2j.
The Tm values of the Pu18 G-quadruplex treated with the ber-
berine and its 9-N-substituted derivatives were calculated from
the melting curves and listed in Table 1. The Tm values of the
derivatives 2a–j were 12–29 °C, while a Tm of 1.4 °C for Pu18
G-quadruplex induced by berberine indicating that the derivatives
D
D

Y. Ma et al. / Bioorg. Med. Chem. 16 (2008) 7582–7591
7585
F10T induced by the derivatives was slightly affected, indicatingthat
the derivatives were high selective G-quadruplex binding ligands.
From the data of Table 1, 2j had more distinct binding affinity
with G-quadruplex structure than other derivatives. These results
demonstrated that the structural difference in the side chains of
the derivatives showed significantly different effects on the
binding affinity with G-quadruplex DNA. The derivative 2j with a
1,6-diaminohexyl side chain at the 9-position possessed higher
binding affinity with G-quadruplex DNA. The derivatives with a
1,2-diaminoethyl side chain (2a, 2b, and 2c) or with a 1,3-diami-
no-propyl side chain (2f, 2g, 2h, and 2i) had good binding affinity
with G-quadruplex, while the derivative 2d with a side chain
containing a terminal hydroxyl group, showed a comparatively
8
6
4
2
0
Pu18
Pu18 +KCl
Pu18+KCl+25 μM 2j
Pu18+ NaCl
PU18+NaCl+25 μM 2j
240
260
280
300
320
340
lower
containing a terminal phenyl group also showed a good binding
affinity with G-quadruplex DNA, the Tm value achieved is 20 °C.
DTm value. Interestingly, the derivative 2e with a side chain
Wavelength (nm)
D
Figure 5. CD spectra of Pu18 in the presence of cations. The blank sample without
drug or cations was shown by solid line, the control samples were shown by solid
circle symbols (d, in the presence of K+) and solid square symbols (j, in the
2.4. Inhibition of amplification in the promoter region of c-myc
by berberine derivatives
presence of Na+), and the samples with 25
l
M 2j was shown by open circle symbols
(s, in the presence of K+) and open square symbols (h, in the presence of Na+). The
concentration of the Pu18 remained at 5
the presence of 100 mM metal ions.
lM in a Tris–HCl buffer, 10 mM, pH 7.2, in
The induction of biologically relevant G-quadruplex formation
in the Pu27 by the berberine derivatives was investigated using
PCR-stop assay. In the presence of the derivatives, the single-strand
Pu27 was folded into a G-quadruplex structure and blocked the
hybridization with its complementary strand (Pu27rev). In that
case, the 50 to 30 extension by the Taq polymerase was inhibited
and the final double-stranded DNA product could not be detected
by electrophoresis separation.
1.0
A
no drug
0.2 μM 2j
0.4 μM 2j
0.8
0.8 μM 2j
1.0 μM 2j
2.0 μM 2j
3.0 μM 2j
The berberine derivatives at various concentrations of 1.0, 2.5,
4.0, and 5.0 lM were used in this assay. The PCR products were
0.6
separated by agarose electrophoresis and silver stained (Fig. 8).
The concentrations of derivatives for inhibition of amplification
by 50% (IC50) were listed in Table 2. These results were correlated
0.4
0.2
0.0
to the
DTm values in Table 1, which indicated that the stability of c-
myc G-quadruplex induced by berberine derivatives was an impor-
tant factor for inhibiting the gene amplification.
40 45 50 55 60 65 70 75 80 85 90 95
Temperature (oC)
To further confirm the inhibitory effects of berberine deriva-
tives against the stabilization of Pu27 G-quadruplex, a parallel
experiment using an oligomer Pu27-13, 14 (5-TGGGGAGGGTGG
AAAGGGTGGGGAAGG-3) which could not form the G-quadruplex
structure was performed, while no inhibition was observed under
B
35
30
25
20
15
10
5
2i
2j
such circumstances even at the highest concentration of 5.0
(Fig. 8B).
lM
2.5. Selectivity for G-quadruplex DNA and other DNA structures
by berberine derivatives
T
To evaluate the selectivity of the berberine derivatives for G-
quadruplex and other DNA structures, a competition dialysis
experiment was performed using different types of DNA. Among
the DNA used in the present study, the Pu18 and HTG21 could form
the intramolecular G-quadruplex structures, and the HT-7 could
form the intermolecular G-quadruplex structures. The HTC21
could form the i-motif structure, the 2d(T)21/d(A)21 was associated
to a triplex structure, the d(T)21/d(A)21 and HTG21/HTC21 were
associated to a duplex structure, the d(A)21 and d(T)21 were sin-
gle-strand purine and pyridine structures, respectively, and the
HTG21mu was mutant HTG21 structure which could not form
the G-quadruplex structure. Higher binding affinity was reflected
by the higher concentration of bound ligands accumulated in the
dialysis cassette containing the specific form of DNA.
C20oC
0.8
C20oC
0
0.2
0.4
1
2
3
Compound (μM)
Figure 6. (A) Stabilization effects of the Pu18 in the presence of 2j at various
concentrations. (B) Concentration dependency of Pu18 stabilization ( Tm). Data
was presented of 2i and 2j onto Pu18 in potassium buffer. The concentration of the
Pu18 remained at 0.2 M in a Tris–HCl buffer, 10 mM, pH 7.2, in the presence of
0.2 mM K+, the concentrations of 2i and 2j were 0.2, 0.4, 0.8, 1.0, 2.0, and 3.0
M.
D
l
l
possessed a much stronger stabilizing ability to the Pu18 G-quad-
ruplex than berberine (Fig. 7).
Furthermore, competitive FRET experiments were performed to
gain further insights into their selectivity between G-quadruplex
(Pu18) and duplex DNA (F10T, 50-FAM-TATAGCTATA-HEG-TATAGC-
TATA-TAMRA-30). As shown in Table 1, the thermal stabilization of
The data in Figure 9 were shown as bar charts in which the
amount of bound ligand with 10 structurally different nucleic acids
was plotted. In this assay, the various nucleic acids were dialyzed
simultaneously against a free ligand solution. The amount of the
bound ligand was directly proportional to the binding constant

7586
Y. Ma et al. / Bioorg. Med. Chem. 16 (2008) 7582–7591
Table 1
The melting temperatures of the Pu18 or the duplex F10T treated with the berberine derivatives
a
b
c
d
a
b
c
d
Compound
Tm (°C)
D
Tm (°C)
Tm (°C)
D
Tm (°C)
Compound
Tm (°C)
D
Tm (°C)
Tm (°C)
D
Tm (°C)
None
2a
2b
2c
2d
54
73
72
74
66
74
0
58
58
59
58
58
59
—
0
1
0
0
1
2f
2g
2h
2i
74
71
78
78
83
55
20
17
24
25
29
1
59
58
59
60
60
60
1
0
1
2
2
2
19
18
20
12
20
2j
2e
Berberine
a,b
Tm and
Tm and
D
D
Tm values of 0.2
Tm values of 0.2
l
l
M Pu18 incubated in the presence of KCl 0.2 mM, compound 2.0
M F10 T incubated in the presence of KCl 0.2 mM, compound 2.0
l
l
M.
M.
c,d
results, it was clearly that these derivatives could selectively bind
to the quadruplex over duplex.
30
25
20
15
10
5
2.6. Inhibition of the transcription of c-myc by the berberine
derivatives in the cancer cell line
To evaluate the inhibitory abilities of the derivatives on the
transcription of c-myc in the cancer cell line, proliferation assays
were carried firstly. Figure 10 showed the cell viability of HL60
lymphomas cells treated with derivative 2j of increasing concen-
trations for 8 days. An inhibition of cell proliferation was found
on day 2 after treatment, and a dose-dependent decrease of cell
proliferation appeared after treatment with the derivative (Fig.
T
0
2a 2b 2c 2d 2e 2f 2g 2h 2i 2j ber
10). The derivative 2j at the concentrations of 5 and 10 lM showed
Compound
a totally inhibition effect on the cell proliferation.
According to the data in above proliferation assays, the treated
concentrations in the transcription assay were chosen as 0.3, 0.7,
Figure 7. Stabilization of the Pu18 (0.2 lM) treated with the derivatives at 2.0 lM
in the Tris–HCl buffer (pH 7.2) containing 0.2 mM KCl, the result was plotted as a
bar graph.
1.7, 3.3 and 6.7 lM to minimize the cell toxicity effect by the deriv-
for each conformer of DNA. As shown in Figure 9, the derivative 2j
binding preferentially with G-quadruplex DNA, whereas much
weaker affinity for other types of DNA structures.
A high binding selectivity is an essential criterion for the use of
G-quadruplex ligands in complex environments. Hence the selec-
tivities of the derivatives for G-quadruplex DNA over double strand
atives. For the transcription assay of c-myc in cancer cell lines,
about 5 ꢀ 105 HL60 cells were seeded into 6-well plates and the
derivative 2j at variant concentrations were also added, and after
4-day incubation the total RNA was extracted and reverse tran-
scripted to cDNA. The cDNA was then used as a template for spe-
cific PCR amplification of the c-myc sequence and controlled by
b-actin.
were evaluated again by FRET-melting assay. The Tm and
DTm val-
ues of the duplex F10T treated with the 9-N-substitued berberine
derivatives were shown in Table 1. The treatment with the deriva-
tives to duplex had a weak effect on the thermal stability of the du-
As shown in Figure 11, the decreasing/disappearing of c-myc
PCR products was significant when treated with derivative 2j.
The derivative 2j at the concentrations of 0.7–1.7 lM started to in-
plex DNA (DTm < 2 °C) implied the derivatives were not the typical
hibit the transcription of c-myc, which were consistent with the
duplex DNA ligands. Compared with Pu18 FRET-melting assay
PCR-stop assay results.
Figure 8. Effects of the berberine derivatives 2a–j on the hybridization of the Pu27 in the PCR-stop assay. (A) 9-N-Substituted berberine derivatives with different
concentration at 1.0 M, 2.5 M, 4.0 M and 5.0 M were added to Pu27 oligomer, as indicated according to Section 4. The band (arrow symbols) presented the 43 bp double-
stranded PCR product. (B) The parallel experiments were performed using all these derivatives in oligomer Pu27-13, 14 at a concentration of 5.0 M.
l
l
l
l
l

Y. Ma et al. / Bioorg. Med. Chem. 16 (2008) 7582–7591
7587
Table 2
The IC50 values of berberine and its derivatives (2a–j) from the PCR-stop assay
4. Experimental
Compound
IC50 M)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
1 H NMR spectra were recorded at 300 MHz on a Mercury-Plus
spectrometer using TMS as an internal standard in DMSO-d6 or
CD3OD/CDCl3; MS spectra were recorded on a Shimadzu LCMS-
2010A instrument with an ESI mass selective detector. Elemental
analysis was carried out on an Elementar Vario EL CHNS Elemental
Analyzer. Flash column chromatography was performed with silica
gel (200–300 mesh) purchased from Qingdao Haiyang Chemical
Co. Ltd or alumina from Sinopharm Chemical Reagent Co. Ltd.
Thin-layer chromatography was carried out with Merck silica gel
60F254 glass plates.
(l
3.9
7.1
4.0
5.5
3.6
3.3
4.8
3.2
2.3
2.0
2.7. Cytotoxicity against tumor cells of the berberine
derivatives
Table 3 showed the IC50 values (cytotoxicity potency indexes) of
derivatives 2a–j against two types of tumor cells using MTT cyto-
toxicity assay. The berberine exhibited low or insignificant cyto-
toxicity to human tumor cells with the IC50 values of more than
The CD spectra were recorded on a Chirascan (Applied Photo-
physics) spectrophotometer, PCR-stop assay was carried out on
the Eppendorf thermocycler for DNA amplification (Mastercycler
personal, 5332). FRET-melting assay was recorded on a real-time
PCR apparatus (Roche Light Cycler). All oligomers/primers used
in this study were purchased from Invitrogen (China), and their se-
quences were listed in Table 4. Acrylamide/bisacrylamide solution
and N,N,N0,N0-tetramethyl-ethylenediamine were purchased from
Sigma. Taq DNA polymerase was purchased from Sangon, China.
The total RNA isolation kit and the two-step RT-PCR kit were pur-
chased from SBS Genetech, China. Stock solutions of all the deriv-
atives (10 mM) were made using DMSO (10%) or double-distilled
water. Further dilutions to working concentrations were made
with double-distilled water.
100 lM, and all the derivatives had much more potent cytotoxicity
with significantly lower IC50 values on tested tumor cells. Among
all the derivatives, 2e and 2j were most potent in inhibiting the tu-
mor cell proliferation with the lowest IC50 values of 2
lM and
4
l
M, respectively. As for the structure–activity relationship, the
derivative 2j bearing the longer side chains had a higher cytotoxic
activity than the other derivatives. The terminal phenyl group of
the derivative 2e could participate in
p–p stacking interactions
with the base group and showed a higher cytotoxic activity. The
cytotoxicity assay results of these derivatives were consistent with
the stabilization ability of derivatives on G-quadruplex.
3. Conclusions
Berberine chloride was isolated from Chinese herbal medicine
(‘Huang-Lian’) and recrystallized from hot water. Compound 2a–j
were synthesized followed the procedures below.
A new class of 9-N-substituted berberine derivatives (2a–j)
were designed and semisynthesized in a convenient method. The
interaction of berberine and berberine derivatives with the G-
quadruplex DNA in the promoter region of the c-myc had been
investigated in detail. Our results indicated that 9-N-substituted
berberine derivatives could significantly induce and stabilize the
parallel G-quadruplex structure formation in the presence or ab-
sence of metal cations. The derivatives with different structures
had different abilities to stabilize the c-myc G-quadruplex. Intro-
ducing of 9-N-substitutes, such as a 1,6-diaminohexyl side chain,
into the 9-position of berberine improves the selectivity binding
with G-quadruplex and increases the inhibitory effect on the
hybridization, resulting in blocking the gene expression. 9-N-Sub-
stisuted berberine derivatives also showed obvious inhibitory ef-
fect on the transcription of c-myc in the cancer cell line, and
higher cytotoxicity against tumor cells comparing with berberine.
This study suggested that the berberine derivatives might be po-
tential lead compounds for the development of new anti-cancer
agent.
4.1. General procedures for the preparation of 2a–j
To a solution of berberine (0.37 g, 1 mmol) in anhydrous etha-
nol, the substituent amine (4–10 mmol) were added and the mix-
ture was stirred for 2–4 h at 78 °C and the reaction was monitored
by TLC. The reaction mixture was concentrated in vacuo. The resid-
ual oil was purified on Al2O3 column with CHCl3/CH3OH (100:1–
30:1) as eluent to afford the proposed compound.
4.1.1. 9-N-20-(N0,N0-Dimethylamino)ethylberberine (2a)
Berberine was treated with 2-(N0,N0-dimethylamino)ethylamine
according to general procedure to give the desired product 2a as a
red solid, yield 35%. 1H NMR (300 MHz, CDCl3) d: 11.18 (s, 1H), 8.47
(s, 1H), 7.54 (d, 1H, J = 8.6), 7.91 (s, 1H), 7.04 (d, 1H, J = 8.4), 6.76 (s,
1H), 6.06 (s, 2H), 5.00 (t, 2H, J = 6.1), 3.96 (s, 3H), 4.15 (t, 2H,
J = 6.8), 3.31 (t, 2H, J = 6.1), 3.13 (t, 2H, J = 6.8), 2.76 (s, 6H);
2j
berberine
i-motif
HT-7
i-motif
HT-7
Pu18
Pu18
HTG21
HTG21
2(dT21):dA21
HTG21:HTC21
dT21:dA21
HTG21mu
dT21
2(dT21):dA21
HTG21:HTC21
dT21:dA21
HTG21mu
dT21
dA21
dA21
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Bound ligand (μM)
Bound ligand (μM)
Figure 9. Results of competition dialysis experiment. One micromolar solution of 2j or berberine was dialyzed against 10 different nucleic acid structures (45
The amount of 2j (left) or berberine (right) bound to each structure was plotted as a bar graph.
lM) for 24 h.

7588
Y. Ma et al. / Bioorg. Med. Chem. 16 (2008) 7582–7591
solid, yield 47%. 1H NMR (300 MHz, DMSO-d6) d: 10.02 (s, 1H),
blank
2000000
1500000
1000000
500000
0
8.67 (s, 1H), 7.87, 7.90 (d, 1H, J = 8.10), 7.74 (s, 1H), 7.48, 7.51 (d,
1H, J = 8.10), 7.05 (s, 1H), 6.14 (s, 2H), 4.78 (t, 2H, J = 6.0), 3.97 (s,
3H), 3.63 (m, 4H), 3.19 (t, 2H, J = 6.0). ESI-MS m/z: 365.40 [MꢁCl]+.
Anal. Calcd for C21H21N2O4+: C, 62.92; H, 5.28; N, 6.99. Found: C,
62.87; H, 5.16; N, 6.85.
0.2 μM 2j
0.5 μM 2j
1.0 μM 2j
3.0 μM 2j
5.0 μM 2j
10.0 μM 2j
4.1.5. 9-N-20-(Phenyl)ethylberberine (2e)
Berberine was treated with 2-phenylethylamine according to
general procedure to give the desired product 2e as a red solid,
yield 51%. 1H NMR (300 MHz, CDCl3) d: 11.57 (s, 1H), 7.88 (s,
1H), 7.53, 7.50 (d, 1H, J = 8.2), 7.30 (m, 5H), 7.16 (m, 1H), 7.14,
7.13 (d, 1H, J = 8.2), 6.80 (s, 1H), 6.07 (s, 2H), 5.06 (t, 2H, J = 6.3),
4.06 (t, 2H, J = 7.3), 3.92 (s, 3H), 3.40 (t, 2H, J = 6.3), 3.10–3.18 (t,
2H, J = 7.3); 13CNMR (300 Hz) 150.35, 148.50, 148.12, 146.57,
140.03, 139.77, 135.61, 133.43, 129.90, 129.38, 128.38, 126.09,
125.51, 120.73, 118.57, 117.60, 114.02, 108.86, 104.93, 102.29,
57.328, 54.410, 48.375, 38.187, 28.328; ESI-MS m/z: 425.45
[MꢁCl]+. Anal. Calcd for C27H25N2O3+: C, 70.35; H, 5.47; N, 6.08.
Found: C, 70.05; H, 5.37; N, 6.00.
1
2
3
4
5
Day
6
7
8
Figure 10. Effect of derivative 2j on cell proliferation. In vitro growth curves of the
HL60 lymphomas cells untreated and treated with 0 (j), 0.2 (h), 0.5 (4), 1.0 (N), 3.0
(s), 5.0 (d) and 10.0 (ꢀ)
lM of the derivative 2j.
ESI-MS m/z: 392.55 [MꢁCl]+. Anal. Calcd for C23H26N3O3+: C, 64.55;
H, 6.12; N, 9.82. Found: C, 64.45; H, 6.07; N, 9.74.
4.1.6. 9-N-30-(N0,N0-Dimethylamino)propylberberine (2f)
4.1.2. 9-N-20-(4-Morphiline)ethylberberine (2b)
Berberine was treated with 3-(N0,N0-dimethylamino)propyl-
amine according to general procedure to give the desired product
2f as a red solid, yield 40%. 1H NMR (300 MHz, CDCl3) d: 11.39 (s,
1H), 7.88 (s, 1H), 7.52 (d, 1H, J = 8.5), 7.28 (s, 1H), 7.14 (d, 1H,
J = 8.5), 6.79 (s, 1H), 6.07 (s, 2H), 5.09 (t, 2H, J = 6.1), 3.94 (s, 3H),
3.89 (t, 2H, J = 6.5), 3.16 (t, 2H, J = 6.1), 2.63 (t, 2H, J = 6.5), 2.3 (s,
6H), 2.0 (m, 2H). ESI-MS m/z: 406.55 [MꢁCl]+. Anal. Calcd for
Berberine was treated with 2-(4-morphiline)-ethylamine
according to general procedure to give the desired product 2b as
a red solid, yield 45%. 1H NMR (300 MHz, CDCl3) d: 11.33 (s, 1H),
7.89 (s, 1H), 7.52, 7.55 (d, 1H, J = 8.4), 7.28 (s, 1H), 7.13, 7.16 (d,
1H, J = 8.4), 6.79 (s, 1H), 6.07 (s, 2H), 5.12 (t, 2H, J = 6.0), 3.96 (s,
3H), 4.02 (t, 2H, J = 6.7), 3.70, 3.73 (t, 4H), 3.17 (t, 2H, J = 6.0),
2.83–2.87 (t, 2H, J = 6.7), 2.63 (t, 4H). ESI-MS m/z: 434.55 [MꢁCl]+.
Anal. Calcd for C25H28N3O4+: C, 63.89; H, 6.01; N, 8.94. Found: C,
63.80; H, 6.15; N, 8.87.
C
24H28N3O3+: C, 65.22; H, 6.39; N, 9.51. Found: C, 65.02; H, 6.34;
N, 9.42.
4.1.7. 9-N-30-(4-Morphiline)propylberberine (2g)
4.1.3. 9-N-20-(1-Piperidine)ethylberberine (2c)
Berberine was treated with 3-(4-morphiline)propylamine
according to general procedure to give the desired product 2g as
a red solid, yield 37%. 1H NMR (300 MHz, CDCl3) d: 11.46 (s, 1H),
7.89 (s, 1H), 7.52, 7.55 (d, 1H, J = 8.5), 7.28 (s, 1H), 7.14, 7.17 (d,
1H, J = 8.5), 6.79 (s, 1H), 6.07 (s, 2H), 5.08 (t, 2H, J = 6.6), 3.95 (s,
3H), 3.89 (t, 2H, J = 6.8), 3.81 (t, 4H), 3.16 (t, 2H, J = 6.6), 2.81 (t,
2H, J = 6.8), 2.72 (t, 4H), 2.15 (t, 2H); ESI-MS m/z: 448.60 [MꢁCl]+.
Anal. Calcd for C26H30N3O4+: C, 64.52; H, 6.25; N, 8.68. Found: C,
64.34; H, 6.11; N, 8.61.
Berberine was treated with 2-(1-piperidine)-ethylamine
according to general procedure to give the desired product 2c as
a red solid, yield 45%. 1H NMR (300 MHz, CD3OD/CDCl3) d: 10.96
(s, 1H), 7.90 (s, 1H), 7.47, 7.44 (d, 1H, J = 8.20), 7.09 (s, 1H), 7.17,
7.20 (d, 1H, J = 8.20), 6.71 (s, 1H), 6.00 (s, 2H), 5.05 (t, 2H, J = 6.2),
3.89 (s, 3H), 3.12 (t, 2H, J = 6.2), 2.69 (t, 2H), 2.44 (m, 6H), 1.51
(m, 4H), 1.39 (m, 2H); ESI-MS m/z: 432.60 [MꢁCl]+. Anal. Calcd
for C26H30N3O3+: C, 66.73; H, 6.46; N, 8.98. Found: C, 66.55; H,
6.35; N, 8.19.
4.1.4. 9-N-20-(2-Hydroxyl)ethylberberine (2d)
4.1.8. 9-N-30-(1-Piperidine)propylberberine (2h)
Berberine was treated with 2-(2-hydroxyl)-ethylamine accord-
ing to general procedure to give the desired product 2d as a red
Berberine was treated with 3-(1-piperidine)propylamine
according to general procedure to give the desired product 2h as
1.0
0.8
0.6
0.4
0.2
0.0
A
B
2j
μm
0
0.3 0.7 1.7 3.3 6.7
-actin
β
c-myc
0
1
2
3
4
5
6
7
Concentration of 2j (μM)
Figure 11. RT-PCR to determine the transcription of c-myc in the HL60 cell line treated with derivatives 2j. (A) HL60 cells were treated with medium (no drug), 0.3, 0.7, 1.7,
3.3 and 6.7 M of the derivative for 4 days, and the total RNA was extracted and subjected to reverse transcription, followed by PCR for c-myc and b-actin (control). Amplified
l
products were 191 bp for c-myc and 541 bp for b-actin. (B) The optical density of each band by using Quantity One software (Biorad). The graph showed the relative
expression of c-myc as compared with the ‘no drug’ sample for each time point versus concentration of the derivative.

Y. Ma et al. / Bioorg. Med. Chem. 16 (2008) 7582–7591
7589
Table 3
Docking studies were carried out using the AUTODOCK 4.0 pro-
gram.23 Using ADT,24 non-polar hydrogens of c-myc G-quadruplex
were merged to their corresponding carbons and partial atomic
charges were assigned. The non-polar hydrogens of the ligands
were merged, and rotatable bonds were assigned. The resulting
G-quadruplex structure was used as an input for the AUTOGRID
program. The grid box was placed at the center of the G-quadru-
plex. The dimensions of the active site box were set at
50 ꢀ 50 ꢀ 50 Å. Docking calculations were carried out using the
Lamarckian genetic algorithm (LGA). Other parameters were used
default. Hundred independent docking runs were carried out for
berberine and compound 2j. Considering both the docked energy
and the rmsd (root-mean-square-deviation), ligand conformation
was chosen. The resulting structures were then imported into SYB-
YL package and minimized.
IC50 cytotoxicity values (lM) of the berberine derivatives against tumor cells (data
derived from the mean of three independent assays)
Compound
IC50
(NCI,
IC50
(GLC,
Compound
IC50
(NCI,
IC50
(GLC, lM)
lM)
lM)
lM)
2a
2b
2c
2d
2e
36
50
22
35
2
56
50
38
51
18
2f
2g
2h
2i
29
85
38
22
4
64
102
69
32
9
2j
a red solid, yield 41%. 1H NMR (300 MHz, CD3OD/CDCl3) d: 11.34(s,
1H), 7.88 (s, 1H), 7.53, 7.51 (d, 1H, J = 8.5), 7.28 (s, 1H), 7.16, 7.13
(d, 1H, J = 8.5), 6.79 (s, 1H), 6.1 (s, 2H), 5.1 (t, 2H, J = 6.3), 3.94 (s,
3H), 3.91 (t, 2H, J = 7.2), 3.17 (t, 2H, J = 6.3), 2.87 (t, 2H), 2.76 (t,
2H, J = 7.2), 2.12 (t, 2H), 1.60 (m, 2H), 1.26 (m, 6H). ESI-MS m/z:
446.60 [MꢁCl]+. Anal. Calcd for C27H32N3O3+: C, 67.28; H, 6.69; N,
8.72. Found: C, 67.15; H, 6.54; N, 8.62.
4.3. Polyacrylamide gel electrophoresis
The oligomer Pu27 at a final concentration of 10
to 95 °C for 10 min in Tris–HCl buffer (10 mM, pH 7.2). After the
solution was gradually cooled to room temperature, a 1 L stock
solution of the derivatives was added to each sample to produce
the specified concentrations at a total volume of 10 L. The reac-
tion mixture was incubated overnight at 4 °C. After incubation,
L of loading buffer (50% glycerol, 0.25% bromophenol blue,
lM was heated
4.1.9. 9-N-30-(1-Pyrrole)propylberberine (2i)
l
Berberine was treated with 3-(1-pyrrole) propylamine accord-
ing to general procedure to give the desired product 2i as a red so-
lid, yield 43%. 1H NMR (300 MHz, CD3OD/CDCl3) d: 11.33 (s, 1H),
7.88 (s, 1H), 7.52, 7.49 (d, 1H, J = 8.5), 7.28 (s, 1H), 7.15, 7.11 (d,
1H, J = 8.5), 6.78 (s, 1H), 6.06 (s, 2H), 5.08 (t, 2H, J = 6.4), 3.93 (s,
3H), 3.90 (t, 2H, J = 6.9), 3.16 (t, 2H, J = 6.4), 2.84 (t, 2H, J = 6.9),
2.73 (t, 4H), 2.10 (t, 2H), 1.84 (m, 4H). ESI-MS m/z: 432.60 [MꢁCl]+.
Anal. Calcd for C26H30N3O3+: C, 66.73; H, 6.46; N, 8.98. Found: C,
66.65; H, 6.39; N, 8.85.
l
2
l
and 0.25% xylene cyanol) was added to each mixture. Ten microli-
ter aliquots of each sample were subsequently analyzed by native
15% PAGE. The gels were silver-stained and photos were taken.
The control assays were repeated as above in a Tris–HCl buffer
(10 mM, pH 7.2) containing neither derivatives nor metal ion or
100 mM KCl.
4.1.10. 9-N-60-Amino hexylberberine (2j)
4.4. CD measurements
Berberine was treated with 1,6-hexyldiamine according to gen-
eral procedure to give the desired product 2j as a red solid, yield
43%. 1H NMR (CD3OD, 300 Hz) d: 11.03 (s, 1H), 8.10 (s, 1H), 7.91
(s, 1H), 7.53 (d, 1H, J = 8.2), 7.16 (d, 1H, J = 8.2), 6.78 (s, 1H), 6.05
(s, 2H), 5.05 (t, 2H, J = 6.5), 3.92 (s, 3H), 3.78 (t, 2H, J = 6.8), 3.19
(t, 2H, J = 6.5), 2.93 (t, 2H, J = 6.8), 1.94 (s, 2H), 1.75 (m, 4H), 1.26
(m, 4H). ESI-MS m/z: 420.55 [MꢁCl]+. Anal. Calcd for
C25H30N3O3+: C, 65.85; H, 6.63; N, 9.22. Found: C, 65.35; H, 6.56;
N, 9.12.
The oligomer Pu18 at a final concentration of 5 lM was resus-
pended in Tris–HCl buffer (10 mM, pH 7.2) containing the deriva-
tives to be tested. The samples were heated to 95 °C, then
gradually cooled to room temperature, and incubated at 4 °C for
at least 6 h. The CD spectra were recorded on Chirascan (Applied
Photophysics) spectrophotometer, using 0.5 s-per-points from
220 to 450 nm and 1 nm bandwidth. The CD spectra were obtained
by taking the average of two scans made from 220 to 450 nm.
4.2. Molecular modeling
4.5. FRET-melting assay
The Pu-18B G-quadruplex structure built by Tian-Miao Ou et
al.11 was used as the initial model to study the interaction between
berberine or the berberine derivatives and human c-myc DNA. Li-
gands were constructed in SYBYL 7.3.5 (Tripos Inc., St. Louis, MO,
USA). The compounds were charged using the Gasteiger-Huckel
computational method. Stepwise minimizations using the Tripos
force field were subsequently carried out to a convergence of
0.01 kcal molꢁ1/Å over 5000 steps.
Denaturation of the oligomer FPu18T (50-FAM-AGGGTGGG-
GAGGGTGGGG-TAMRA-30). In the experiments presented here, a
real-time PCR apparatus (Roche LigntCycler) was used, allowing
the simultaneous recording of 32 samples. Fluorescence measure-
ments with the Pu18 oligonucleotide (0.2 lM) were studied in the
buffer (pH 7.2) containing metal ion. The melting of the G-quadru-
plex DNA was monitored alone or in the presence of the derivatives
Table 4
Sequences of oligomers (primers) used in the present study
Oligomer
Sequence
Description
Pu27
50-TGGGGAGGGTGGGGAGGGTGGGGAAGG-30
50-ATCGATCGC TTCTCGTCCTTCCCCA-30
50-TGGGGAGGGTGGAAAGGGTGGGGAAGG-30
50-AGGGTGGGGAGGGTGGGG-30
Partial sequence in the promoter of oncogene c-myc that may form G-quadruplex
Complementary sequence of Pu27 in PCR-stop assay
Pu27rev
Pu27-13,14
Pu18
Mutant oligomer of Pu27 that may not form G-quadruplex, whose mutant sites were 13 and 14
Truncated sequence of Pu27 that involved in the G-quadruplex forming
Partial sequence in human telomere that may form the G-quadruplex structure
Complementary sequence of TSG4 that may form an i-motif structure
Upstream primer for c-myc in RT-PCR
Downstream primer for c-myc in RT-PCR
Upstream primer for b-actin as control in RT-PCR
Downstream primer for b-actin as control in RT-PCR
TSG4
50-GGGTTAGGGTTAGGGTTAGGG-30
50-CCTAACCCTAACCCTAACCC-30
HTC21
c-mycA
c-mycS
b-actinA
b-actinS
50-TGGTGCTCCATGAGGAGACA-30
50-GTGGCACCTCTTGAGGACCT-30
50-GTTGCTATCCAGGCTGTGC-30
50-GCATCCTGTCGGCAATGC-30

7590
Y. Ma et al. / Bioorg. Med. Chem. 16 (2008) 7582–7591
Table 5
Nucleic acid conformation and samples used in competition dialysis experiments
Conformation
DNA/oligonucleotide
e )
(Mꢁ1 cmꢁ1
Single-strand purine
Single-strand pyrimidine
Single strand
Duplex DNA
Duplex DNA
d(A)21
d(T)21
255,400
170,700
HTG21mu (50-GGGTTAGAGTTAGGGTTAGGG-30)
d(T)21/d(A)21
12,000
14,800
HTG21/HTC21
Triplex DNA
(dT21)2/d(A)21
17,200
Quadruplex DNA
Quadruplex DNA
Quadruplex DNA
i-motif
Pu18 (50-AGGGTGGGGAGGGTGGGG-30)
TSG4 (50-GGGTTAGGGTTAGGGTTAGGG-30)
HT-7 (50-TTAGGGT-30)
149,520
172,000
69,800
HTC21 (50-CCCTAACCCTAACCCTAACCC-30)
148,720
at 2
lM, by measuring the fluorescence of fluorescein. To test the
increasing concentrations ranging from 0 lM to 10 lM were added
binding selectivity of the compound to the quadruplex structure
over double-stranded DNA, double-stranded DNA F10T: (50-FAM
TATAGCTATA-HEG-TATAGCTATA-TAMRA-30) was designed and
measured, the melting temperature Tm is the mid-point of a melt-
ing curve at which the complex is 50% dissociated.
to the culture medium, which were then left for 4 days. Then, at
day 5, the compounds were re-added to medium and left for an
additional 4 days. Cell counts and viability (Trypan blue dye exclu-
sion) were determined daily, from day 1 to day 8 of culture. The
compounds dose inhibition cell proliferations by 50% (IC50) were
calculated at 8 day of treatment.
4.6. PCR-stop assay
4.10. RNA extraction
The PCR-stop assay was performed with a modified protocol of
the previous study.8 Sequences of the test oligomers, including
Pu27, Pu27-13,14, and the corresponding complementary se-
quence used in the current study were presented in Table 4. The
For RNA extraction, about 5 ꢀ 105 HL60 cells were seeded in 6-
well plates, and the freshly dissolved derivative 2j at increasing
concentrations were added into the plates After 4-day cultured,
the cell pellets were lysed in TRIzol solution and the total RNA
was extracted according to the protocol and eluted in distilled,
deionized water with 0.1% diethyl pyrocarbonate (DEPC) to a final
reactions were performed in 1ꢀ PCR buffer, containing 10
lmol
of each pair of oligomers, 0.16 mM dNTP, 2.5 U Taq polymerase,
and the indicated amount of the derivatives in Figure 8. Reaction
mixtures were incubated in a thermocycler, with the following cy-
cling conditions: 94 °C for 3 min, followed by 30 cycles of 94 °C for
30 s, 58 °C for 30 s, and 72 °C for 30 s. Amplified products were re-
solved on 15% nondenaturing polyacrylamide gels in 1ꢀ TBE and
silver stained.
volume of 50 lL. RNA was quantitated spectrophotometrically and
stored at ꢁ80 °C.
4.11. RT-PCR
Total RNA was used as a template for reverse transcription
4.7. Competition dialysis
using the following protocol: each 20
M-MLV buffer, 500 M dNTP, 100 pmol oligo dT primer, 100 U of
M-MLV reverse transcriptase, DEPC in water (DEPC-H2O), and
g of total RNA. Mixtures were incubated at 42 °C for 60 min
l
L reaction contained 1ꢀ
l
A Tris–HCl buffer (10 mM, pH 7.2) containing 100 mM NaCl was
used for all experiments. For each competition dialysis assay,
300 mL of dialysate solution containing the derivative 2j or berber-
1
l
for reverse transcription and then at 92 °C for 10 min to inactivate
ine or 2i at 1
l
M was placed into a beaker. A volume of 500
l
L at
the enzyme. PCR was performed according to the following proto-
45 M monomeric unit (single strands, double strands, i-motif,
l
col: each 20
0.15 M b-actin primers, 1.5
merase, 0.1% DEPC–H2O, and 3
tions were incubated in a Thermal Cycler as follows: 95 °C for
5 min, followed by 36 cycles of 95 °C for 1 min, 50 °C for 1 min,
and 72 °C for 1 min. The amplified products were separated on a
1.5% agarose gel, and photos were taken on a Gel Doc 2000 Imager
System.
l
L reaction contained 1ꢀ PCR buffer, 500
M c-myc primers, 1 U of Taq poly-
L of the cDNA template. The reac-
lM dNTPs,
triplet or quartet) of each of the nucleic acids samples was pipetted
into a separate 0.5 mL DispoDialyzerÒ (Spectrum Laboratories,
Inc.). A panel of 10 different nucleic acid structures used was listed
in Table 5. All the 10 dialysis units were then placed in the beaker
l
l
l
containing dialysate solution (Tris–HCl containing 1 lM deriva-
tive). The contents were allowed to equilibrate with continuous
stirring for 24 h at room temperature. At the end of the equilibra-
tion period, DNA samples were carefully removed to microfuge
tubes, and treated with 1% SDS. The ligand concentration in each
sample was determined by UV absorbance.
Acknowledgements
We thank the Natural Science Foundation of China (20772159),
the NSFC/RGC Joint Research Scheme (Grant 30731160006), the
Science Foundation of Guangzhou (2006Z2-E402), the Science
Foundation of Zhuhai (Grant PC20041131), and the NCET for finan-
cial support of this study.
4.8. Cell culture
The human lymphomas cell line HL60 was purchased from the
Center of Experiment Animal of Sun Yat-sen University. The cells
cultures were maintained in RPMI-1640 medium supplemented
with 10% fetal bovine serum, 100 U/mL penicillin, and 100 lg/mL
streptomycin in 25 cm2 culture flasks at 37 °C humidified atmo-
References and Notes
sphere with 5% CO2.
1. Siddiqui-Jain, A.; Grand, C. L.; Bearss, D. J.; Hurley, L. H. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 11593.
2. Seenisamy, J.; Bashyam, S.; Gokhale, V.; Vankayalapati, H.; Sun, D.; Siddiqui-
Jain, A.; Streiner, N.; Shinya, K.; White, E.; Wilson, W. D.; Hurley, L. H. J. Am.
Chem. Soc. 2005, 127, 2944.
3. Simonsson, T.; Pecinka, P.; Kubista, M. Nucleic Acids Res. 1998, 26, 1167.
4. Rangan, A.; Fedoroff, O. Y.; Hurley, L. H. J. Biol. Chem. 2001, 276, 4640.
4.9. Proliferation assay
For the proliferation assay, about 5000 HL60 cells were seeded
in a 24-well microplate, and the freshly dissolved derivative 2j at

Y. Ma et al. / Bioorg. Med. Chem. 16 (2008) 7582–7591
7591
5. Freyer Matthew, W.; Buscaglia, R.; Kaplan, K.; Cashman, D.; Hurley Laurence,
H.; Lewis Edwin, A. Biophys. J. 2007, 92, 2007.
14. Kim, S. A.; Kwon, Y.; Kim, J. H.; Muller, M. T.; Chung, I. K. Biochemistry 1998, 37,
16316.
6. Maiti, S.; Chaudhury, N. K.; Chowdhury, S. Biochem. Biophys. Res. Commun. 2003,
310, 505.
7. Seenisamy, J.; Rezler, E. M.; Powell, T. J.; Tye, D.; Gokhale, V.; Joshi, C. S.;
Siddiqui-Jain, A.; Hurley, L. H. J. Am. Chem. Soc. 2004, 126, 8702.
8. Lemarteleur, T.; Gomez, D.; Paterski, R.; Mandine, E.; Mailliet, P.; Riou, J.-F.
Biochem. Biophys. Res. Commun. 2004, 323, 802.
9. Grand, C. L.; Han, H.; Munoz, R. M.; Weitman, S.; Von Hoff, D. D.; Hurley, L. H.;
Bearss, D. J. Mol. Cancer Ther. 2002, 1, 565.
10. Zhou, J.-L.; Lu, Y.-J.; Ou, T.-M.; Zhou, J.-M.; Huang, Z.-S.; Zhu, X.-F.; Du, C.-J.;
Bu, X.-Z.; Ma, L.; Gu, L.-Q.; Li, Y.-M.; Chan, A. S.-C. J. Med. Chem. 2005, 48,
7315.
15. Kang, M. R.; Chung, I. K. Mol. Pharmacol. 2002, 61, 879.
16. Gornall, K. C.; Samosorn, S.; Talib, J.; Bremner, J. B.; Beck, J. L. Rapid Commun.
Mass Spectrom. 2007, 21, 1759.
17. Franceschin, M.; Rossetti, L.; D’Ambrosio, A.; Schirripa, S.; Bianco, A.; Ortaggi,
G.; Savino, M.; Schultes, C.; Neidle, S. Bioorg. Med. Chem. Lett. 2006, 16, 1707.
18. Zhang, W.-J.; Ou, T.-M.; Lu, Y.-J.; Huang, Y.-Y.; Wu, W.-B.; Huang, Z.-S.;
Zhou, J.-L.; Wong, K.-Y.; Gu, L.-Q. Bioorg. Med. Chem. 2007, 15, 5493.
19. Harrison, R. J.; Cuesta, J.; Chessari, G.; Read, M. A.; Basra, S. K.; Reszka, A. P.;
Morrell, J.; Gowan, S. M.; Incles, C. M.; Tanious, F. A.; Wilson, W. D.; Kelland, L.
R.; Neidle, S. J. Med. Chem. 2003, 46, 4463.
20. Naruto, S.; Mizuta, H.; Nishimura, H. Tetrahedron Lett. 1976, 17, 1597.
21. Hassner, A.; Munger, P.; Belinka, B. A. Tetrahedron Lett. 1982, 23, 699.
22. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San
Carlos, CA, 2002.
11. Ou, T.-M.; Lu, Y.-J.; Zhang, C.; Huang, Z.-S.; Wang, X.-D.; Tan, J.-H.; Chen, Y.; Ma,
D.-L.; Wong, K.-Y.; Tang, J. C.-O.; Chan, A. S.-C.; Gu, L.-Q. J. Med. Chem. 2007, 50,
1465.
12. Bates, P.; Mergny, J. L.; Yang, D. EMBO Rep. 2007, 8, 1003.
13. Qin, Y.; Pang, J. Y.; Chen, W. H.; Zhao, Z. Z.; Liu, L.; Jiang, Z. H. Chem. Biodivers.
2007, 4, 481.
23. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
24. Sanner, M. F. J. Mol. Graphics Modell. 1999, 17, 57.