Synthesis and evaluation of 9-O-substituted berberine derivatives containing aza-aromatic terminal group as highly selective telomeric G-quadruplex stabilizing ligands

Article abstract of DOI:10.1016/j.bmcl.2009.05.030

A series of new 9-O-substituted berberine derivatives (4a-j) as telomeric quadruplex ligands was synthesized and evaluated. The results from biophysical and biochemical assay indicated that introducing of positive charged aza-aromatic terminal group into the side chain of 9-position of berberine significantly improved the binding ability with G-quadruplex, and exhibited the inhibitory effect on the hybridization and on telomerase activity. These derivatives showed excellent selectivity for telomeric G-quadruplex DNA over duplex.

Full text of DOI:10.1016/j.bmcl.2009.05.030

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3414–3417
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 9-O-substituted berberine derivatives containing
aza-aromatic terminal group as highly selective telomeric G-quadruplex
stabilizing ligands
Yan Ma, Tian-Miao Ou, Jia-Heng Tan, Jin-Qiang Hou, Shi-Liang Huang,
*
*
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 new 9-O-substituted berberine derivatives (4a–j) as telomeric quadruplex ligands was synthe-
sized and evaluated. The results from biophysical and biochemical assay indicated that introducing of
positive charged aza-aromatic terminal group into the side chain of 9-position of berberine significantly
improved the binding ability with G-quadruplex, and exhibited the inhibitory effect on the hybridization
and on telomerase activity. These derivatives showed excellent selectivity for telomeric G-quadruplex
DNA over duplex.
Received 14 March 2009
Revised 25 April 2009
Accepted 11 May 2009
Available online 14 May 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
G-Quadruplex
Berberine derivatives
Selectivity
Telomerase inhibitors
Telomerase can add telomere repeats onto chromosome ends
and plays key role in the regulation of length of telomeric DNA.
The activity of this enzyme is low in the somatic cells of normal tis-
sues while dramatically raises in most of human tumors (more
than 85%), which has made it as a highly selective target for anti-
O
O
O
O
N
Cl
N
Cl
O
O
N
O
O
1
–3
tumor drug design.
A valid strategy for telomerase inhibition is to stabilize the
higher-order quadruplex structures formed by telomeric G-rich se-
berberine
zh-4d
Figure 1. Structures of the berberine, 9-O-substitued derivative zh-4d.15
quence d[(TTAGGG)
alyzed by telomerase.
A few of small molecule ligands have been identified to stabilize
n
], which as a primer during the elongation cat-
4
,5
peared suitable for
Introduction of a side chain with terminal amino group in the 9-
position of the berberine could lead to significant increase stabil-
ization effect on telomeric G-quadruplex DNA.
p–p stacking interactions with a G-quartet.
6
–9
G-quadruplex structures and inhibit telomerase activity in vitro,
and several classes of these ligands have been pronounced effects
on cancer cell lines, such as growth inhibition and induction of cell
apoptosis and senescence.1
15,17
It could be
attributed to the terminal basic amino group in the side chain
strengthen the electrostatic interaction with the phosphate back-
bone of G-quadruplex DNA. And it was suggested that terminal
group of side chain should contribute much more for the interac-
tions of the derivatives with G-quadruplex DNA.
In present studies, in order to further improve the binding affin-
ity and selectivity with G-quadruplex DNA, we designed a new ser-
ies of 9-O-substituted berberine derivatives based on our previous
studies. We introduced a positive charged terminal aza-aromatic
group to increase the electrostatic interaction with the phosphate
0,11
Berberine, an alkaloid isolated from Chinese herbs, was initially
used as anti-microbial agent.1
2,13
Recently, berberine and some of
its derivatives have been reported as G-quadruplex stabilizing li-
1
4–18
gands.
Our previous studies on the interactions between G-quadruplex
DNA and berberine or its derivatives, such as zh-4d (Fig. 1),
+
showed that berberine with N -containing aromatic moiety ap-
backbone and p–p stacking interaction with the bases of the loops.
Molecular docking studies of the designed derivative 4f with telo-
*
Corresponding authors. Tel./fax: +86 20 39943055 (L.-Q.G.); tel./fax: +86 20
3
9943056 (Z.-S.H.).
meric G-quadruplex structure were performed by using AUTODOCK
E-mail addresses: cesglq@mail.sysu.edu.cn (L.-Q. Gu), ceshzs@mail.sysu.edu.cn,
1
9
huangzhishu@hotmail.com (Z.-S. Huang).
4.0 program (Fig. 2). The results indicated that the terminal
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.030

Y. Ma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3414–3417
3415
The stabilization ability of the compounds to DNA was evalu-
O
O
2
3
ated using a FRET melting assay. Two different oligomers were
used here, including human telomeric G4 F21T and F10T which
could self-associate a hairpin structure. To identify the competitive
affinity of the compounds on G-quadruplex DNA to duplex DNA, a
N
Cl
24,25
system containing a duplex oligomer ds26 was also applied.
The
m
DT values of the F21T G-quadruplex treated with the ber-
O
O
N
berine and its derivatives listed in Table 1. As shown in Table 1, the
derivative 4f had the most distinct binding affinity with G-quadru-
plex (
D
T
m
= 25 °C), and the
D
T
m
value of 4a–4e and 4i are closely
value
around 20 °C, while derivatives 4g and 4j showed lower
DT
m
of 10–13 °C. The data showed derivatives 4a–j possessed much
stronger stabilizing ability on the telomeric G-quadruplex than
N
berberine which inducing the
m
DT of 4 °C. Especially, some com-
pounds, such as 4b, 4d, and 4f even excelled zh-4d that was the
1
5
4
f
best ligand of the series in our previous work. The results demon-
strated that introducing of the positive charged aza-aromatic
group made more contribution to the interaction with G- quadru-
plex structure than those neutral terminal groups (4g, 4j, zh-4d).
The electronic-donoring effect of the terminal group, such as the
derivatives 4d with methyl substituent and 4f with N, N-dimethyl-
amino substituent could help the binding ability. In addition, the
length of the side chain played a slight favorable role in this inter-
action according to the data from 4a, 4b, and 4c. Surprisingly, the
Figure 2. The interaction between 4f and G-quadruplex created by PyMOL.5 (view
onto the plane of the 3 surface of human G-quadruplex complex with 4f).
0
+
aza-aromatic group N -containing in the side chain fitted well with
grooves of G-quadruplex, and the derivative has much higher bind-
ing affinity with G-quadruplex compared to berberine and its
derivative zh-4d, the free energies of binding were ꢀ8.66, ꢀ7.49,
and ꢀ5.69 kcal/mol for 4f, zh-4d, and berberine, respectively.
The synthetic pathway for 9-O-substituted berberine deriva-
tives 4a–j was shown in Scheme 1. The key intermediates, 9-bro-
moalkyl berberine 3a–c were prepared according to the reported
The final products 4a–j were prepared by nucleo-
philico-aromatic displacement of 3a–c with pyridine analogs and
exchanged with anion in 47–70% yield.
The interaction of compounds with telomeric DNA was identi-
fied by Circular Dichroism (CD) spectroscopy (see Supplementary
data). Upon addition of compound 4f to HTG21 in buffer containing
KCl, the CD spectra changed with a disappearance of the small po-
sitive band at 270 nm, indicating the possible destruction of the
parallel structure of the mixed G-quadruplex conformations, while
the positive band at 290 nm increased and the negative band
Table 1
G-Quadruplex FRET and binding constants (K
b
, M 1
ꢀ
)
1
5,20
procedure.
a
b
c
ꢁ 10ꢀ6 (Mꢀ1
Compd
D
T
m
(°C)
D
T
m
(°C)
K
b
)
4
4
4c
a
b
18
21
20
21
20
25
10
17
20
13
20
4
0.5
0.5
0
0.5
0
0
0
0
0
7.3
8.1
8.1
7.3
7.8
4d
4
4
4
4
e
f
g
h
12
6.2
8.9
7.5
6.5
7.5
2.0
4i
4j
0
1
1
zh-4dd
ber
around 260 nm appeared, suggesting the formation of an antipar-
allel structure according to the previous studies.2
1,22
The CD spec-
tra of HTG21 titrated with 4f in the absence of metal ion was found
that the positive peak at 295 nm increased significantly, and a neg-
ative peak at 265 nm emerged, which indicated that 4f had the
ability to promote the formation of antiparallel G-quadruplex
structure. Other derivatives exhibited the similar results to that
of 4f.
Data by berberine derivatives with G-quadruplex.
a
D
T
m
values of 0.2
lM F21T incubated in the presence of KCl 60 mM, compound
2
2
.0
l
D
M.
b
T
m
values of 0.2
l
m
M F10T incubated in the presence of KCl 60 mM, compound
a,b
.0
lM.
D
T
m
= T
(DNA + ligand) ꢀ T
m
(DNA).
T
m
(F21T) = 58 °C,
T
m
(F10T) = 60 °C.
c
ꢀ1
Binding constants (K
zh-4d was reported as the best ligand during that series in our previous study.
b
, M ) by Fluorescence titration.
d
15
4
5
O
O
O
O
O
O
O
N
Cl
N
Br
N
Cl
N C7l
O
1
i
ii
iii
iv
9
O
O
OH
O
O
O
Br
O
O
R
1
3
n
n
1
1
1
2
3
4a-j
4
a
4b
4c
4d
4e
4f
4g
4h
4i
4j
n
3
5
6
3
3
3
3
3
3
3
R
Scheme 1. Reagents and conditions: (i) 190 °C, 20–30 mmHg, 15 min; (ii) Br(CH
)
2 n
3
Br, CH CN, 60 °C, 3 h; (iii) pyridine analogs, DMSO, 90 °C; (iv) Dowex anion exchange resin.

3
416
Y. Ma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3414–3417
stability of derivative 4i with more bulky end group was equivalent
to those with smaller groups (4d, 4e), which indicated the quino-
linium group also could be easily accommodated in quadruplex
grooves or loops.
m
neutral ones. The data were also correlated to DT values in Table
1, which indicated the stability of human telomeric G-quadruplex
induced by berberine derivatives was an important factor for
inhibiting the gene amplification.
Moreover, the derivatives had a weak effect on the thermal sta-
Telomerase inhibition was identified using telomerase repeat
amplification protocol (TRAP) assay. The derivatives at certain con-
centrations were added into the telomerase reaction mixture con-
taining extract from cracked MCF-7 breast carcinoma cell lines and
the inhibitory concentrations by half ( IC50) values of these com-
pounds were listed in Table 2. It was found the inhibitory effects on
bility of the duplex DNA (Table 1,
m
DT <1 °C), implied that the
derivatives were not the typical duplex DNA ligands. In the compe-
tition experiments (Fig. 3), the derivatives showed good selectivity
for G-quadruplex versus 50 times excess of duplex DNA, which
indicated that these derivatives could selectively bind to the quad-
ruplex over duplex. Both results of the high binding affinity and
good selectivity verified our original design that the more multiple
Tel
telomerase activity of derivatives were significantly enhanced
Tel
compared with berberine ( IC50 = 70 lM), which were in line with
interactions, such as
trostatic interactions with the phosphate backbone of grooves, and
stacking interactions and hydrophobic forces of terminal
group with the nucleotide of loops (adenine or thymine) would im-
prove the binding affinity and selectivity.
p
–
p
stacking interaction with G-quartet, elec-
the current thinking that a stronger ligand of the G-quadruplex
was also a good inhibitor of telomerase activity.
p–p
In summary, a series of 9-O-substituted berberine derivatives
containing aza-aromatic terminal group (4a–j) was designed and
semi-synthesized. The interaction of derivatives with the human
telomeric G-quadruplex DNA had been intensively evaluated by
CD spectroscopy, fluorescence spectroscopy, molecular modeling,
FRET-melting assay, PCR stop assay and TRAP assay. The CD results
indicated the derivatives were capable of inducing the formation of
antiparallel G-quadruplex and the results from fluorescence meth-
od, classical and competitive FRET melting assays, clearly showed
that derivatives with positive charged aza-aromatic terminal group
had high binding affinity and superior selectivity because of possi-
ble multiple interactions with G-quartet, grooves and loops of G-
quadruplex DNA. The results of structure–activity relationships
The binding affinities of berberine derivatives to HTG21 were
also investigated by fluorescence titration assay. The DNA binding
constant K
b
obtained by Scatchard analysis was presented in Table
1
8
1.
All derivatives had higher DNA binding affinities compared
with berberine. It was found derivative 4f had the highest affini-
ties, which was correlated with the data from FRET-melting assay.
Derivatives 4g and 4j with neutral terminal group had higher bind-
ing affinity than berberine, but weaker than those with positive
charged terminal group (4a–f, 4h, 4i). This suggested that the
introduction of the side chain could remarkably increased binding
affinity, and the positive charge on the end of side chain should
play an important role in the interaction with G-quadruplex.
The induction of biologically relevant G-quadruplex formation
in the HTG21 by berberine derivatives was investigated by PCR
stop assay. After the PCR procedure and gel separation, the concen-
trations for inhibition of amplification by 50% (IC50) were calcu-
lated and listed in Table 2. Derivatives with positive charged
terminal group (4a–f, 4h, 4i) showed inhibitory effect on the
hybridization of HTG21 at lower concentration than those with
+
(SAR) study indicated that the central N -containing chromophore
ring and the terminal positive charged aza-aromatic substituent
might be important for G-quadruplex binding activity and selectiv-
ity, while the bond length of linker played slight role once the units
were more than three.
Acknowledgments
We thank the National Nature Science Foundation of China
(
20772159, 90813011, U0832005), the NSFC/RGC joint Research
Scheme (30731160006) for financial support of this study.
2
2
1
1
5
0
5
0
5
0
ds26 0 μM
ds26 3 μM
ds26 10 μM
Supplementary data
Supplementary data (synthetic procedure, structural analysis
data, activities assay methods, and molecular modeling method)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.05.030.
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