Stabilization of G-quadruplex DNA with platinum(II) Schiff base complexes: Luminescent probe and down-regulation of c-myc oncogene expression

Article abstract of DOI:10.1002/chem.200901943

The interactions of a series of platinum(II) Schiff base complexes with c-myc G-quadruplex DNA were studied. Complex [PtL^1a] (1a; H ₂L^1a= N,N'-bis(salicylidene)-4,5-methoxy-1,2- phenylenediamine) can moderately inhibit c-m

Full text of DOI:10.1002/chem.200901943

DOI: 10.1002/chem.200901943
Stabilization of G-Quadruplex DNA with Platinum(II) Schiff Base
Complexes: Luminescent Probe and Down-Regulation of c-myc Oncogene
Expression
Peng Wu,[a] Dik-Lung Ma,[a] Chung-Hang Leung,[a] Siu-Cheong Yan,[a] Nianyong Zhu,[a]
R. Abagyan,[b] and Chi-Ming Che*[a]
Abstract: The interactions of a series
of platinum(II) Schiff base complexes
with c-myc G-quadruplex DNA were
studied. Complex [PtL1a] (1a; H2L1a =
N,N’-bis(salicylidene)-4,5-methoxy-1,2-
phenylenediamine) can moderately in-
hibit c-myc gene promoter activity in a
cell-free system through stabilizing the
G-quadruplex structure and can inhibit
c-myc oncogene expression in cultured
cells. The interaction between 1a and
G-quadruplex DNA has been exam-
ined by 1H NMR spectroscopy. By
using computer-aided structure-based
drug design for hit-to-lead optimiza-
tion, an in silico G-quadruplex DNA
model has been constructed for dock-
ing-based virtual screening to develop
new platinum(II) Schiff base com-
plexes with improved inhibitory activi-
ties. Complex [PtL3] (3; H2L3 =N,N’-
bis{4-[1-(2-propylpiperidine)oxy]salicy-
lidene}-4,5-methoxy-1,2-phenylenedia-
mine) has been identified with a top
score in the virtual screening. This
complex was subsequently prepared
and experimentally tested in vitro for
its ability to stabilize or induce the for-
mation of the c-myc G-quadruplex.
The inhibitory activity of 3 (IC50 =
4.4 mm) is tenfold more than that of 1a.
The interaction between 1a or 3 with
c-myc G-quadruplex DNA has been
examined by absorption titration, emis-
sion titration, molecular modeling, and
NMR titration experiments, thus re-
vealing that both 1a and 3 bind c-myc
G-quadruplex DNA through an exter-
nal end-stacking mode at the 3ꢀ termi-
nal face of the G-quadruplex. Such
binding of G-quadruplex DNA with 3
is accompanied by up to an eightfold
increase in the intensity of photolumi-
nescence at lmax =652 nm. Complex 3
also effectively down-regulated the ex-
pression of c-myc in human hepatocar-
cinoma cells.
Keywords: G-quadruplex DNA
·
luminescent probes · onogenes ·
platinum · Schiff bases
Introduction
quadruplex sequences,[4–8] the human oncogene c-myc plays
a nontrivial role in many cellular events, and the overexpres-
sion of this oncogene is linked to cellular proliferation in
malignant tumors.[9] In principle, the transcription of c-myc
is mainly controlled by the nuclear hypersensitivity element
III1 (NHE III1), which is upstream of the P1 promoter of c-
myc.[10–14] Thus, the inhibition of the transcription of c-myc
by inducing and/or stabilizing G-quadruplex formation is a
promising strategy for developing efficacious anticancer
therapies. Planar aromatic molecules, such as cationic por-
phyrins (e.g., TMPyP4 and Se2SAP),[15,16] perylenes,[17] and
the natural products telomestatin,[18] quindolines,[19] and ber-
berine,[19] are known to bind to and stabilize G-quadruplex
DNA in the NHE III1 sequence and, in some cases, to sup-
press c-myc transcription in cancer cell lines.
G-quadruplex DNA is putatively present in pivotal genomic
regions, such as telomeres, oncogene promoters, and most
growth control genes.[1,2] Among many of these promoter
[a] Dr. P. Wu, Dr. D.-L. Ma, Dr. C.-H. Leung, Dr. S.-C. Yan, Dr. N. Zhu,
Prof. Dr. C.-M. Che
Department of Chemistry and Open Laboratory of Chemical Biology
Institute of Molecular Technology for Drug Discovery and Synthesis
The University of Hong Kong, Pokfulam Road, Hong Kong (China)
Fax : (+852)2857-1586
E-mail: cmche@hku.hk
[b] Prof. R. Abagyan
Skaggs School of Pharmacy & Pharmaceutical Sciences
University of California, San Diego
Over the past decades, a number of metal complexes de-
veloped by various research groups have been reported to
be anticancer and antitumor active.[20,21] Lippard, Che,
9500 Gilman Drive, MC 0747 La Jolla, CA 92037-0747 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901943.
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McMillin, Lowe, and others have demonstrated that both
In this context, we employed the polymerase stop assay
and reverse transcriptase-polymerase chain reaction (RT-
PCR) assay of c-myc expression for the initial screening of
platinum(II) Schiff base complexes that may stabilize G-
quadruplex DNA. Among the complexes examined, [PtL1a]
(1a; H2L1a =N,N’-bis(salicylidene)-4,5-methoxy-1,2-phenyle-
nediamine; Scheme 1) moderately inhibited c-myc gene pro-
moter activity in a cell-free system through stabilizing c-myc
G-quadruplex DNA and inhibited c-myc expression in cul-
tured cells. Thus, this platinum(II) complex was a vital initial
lead for optimization through docking-based virtual screen-
ing to develop new platinum(II) salphen complexes with im-
proved inhibitory activities. To develop a high-throughput
screening platform for compounds that bind to G-quadru-
plex DNA, a model of the intramolecular G-quadruplex
loop isomer of NHE III1 was constructed by using the X-ray
[Pt
[Pt
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
n=2) can bind to DNA and that some of these complexes
display promising anticancer properties.[22] Barton and co-
workers pioneered the studies on the interactions of DNA
with octahedral d6 metal complexes, such as complexes of
ruthenium(II) and rhodiumACTHNURGTNE(NUG III). By the judicious choice of
auxiliary ligands, octahedral d6 metal complexes can be de-
veloped into good metallointercalators of double-stranded
DNA.[23] Despite extensive work on the interactions of
metal complexes with helical DNA duplexes, related studies
with higher-order secondary DNA structures are still in the
rudimentary stages.[24] A manganese
ACTHNUTRGNEUNG(III) porphyrin devel-
oped by Pratviel and co-workers bound G-quadruplex DNA
with 10000-fold selectivity over duplex DNA and inhibited
telomerase
with
IC50 =
580 nm.[24i] Neidle and co-work-
ers reported that square-planar
nickel(II) salphen (H2salphen=
N,N’-bis(salicylidene)-1,2-phe-
nylenediamne) complexes act
as potent telomerase inhibitors
through the stabilization of G-
quadruplex DNA.[24e,n] In view
of these findings, we conceive
that platinum(II) complexes
with Schiff base ligands can be
used in the design of G-quadru-
plex DNA binders for the in-
hibition of c-myc transcription
activity because 1) the PtII ion
is substitutionally inert and
hence its complexes are kineti-
cally more stable in solution,
2) the planar coordination ge-
ometry of a PtII center together
with the use of p-conjugated
Schiff base ligands would lead
to compounds that stack on the
face of guanine quartet of the
G-quadruplex, 3) Schiff base li-
gands are easily structurally
modified, and 4) the PtII cation
can locate in the centre of the
quartet (i.e., the surface of the
G-quadruplex ion channel).
Also, platinum(II) complexes
containing p-conjugated ligands
usually display photolumines-
cence properties that can be
strongly affected by subtle
changes in their local environ-
ment and can therefore be ap-
plied to develop luminescent
probes for G-quadruplex DNA
in the P1 promoter of c-myc.
Scheme 1. Structures of complexes 1–3.
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C.-M. Che et al.
crystal structure of the intramolecular human telomeric G-
quadruplex DNA (Protein Data Bank (PDB) code:
1KF1).[25] The aforementioned intramolecular G-quadruplex
loop isomer model of NHE III1 has previously been utilized
to reveal quindoline complexes to have a salutary effect on
stabilizing the G-quadruplex structure in c-myc.[19] We
report herein that the platinum(II) salphen complex with
amine side chains at the periphery of Schiff base ligand 3 is
one of the top-scoring complexes found through in silico vir-
tual-ligand screening. Even at micromolar concentrations,
this ligand can stabilize or induce G-quadruplex formation
and down-regulate c-myc in HepG2 cancer cells through in-
hibition of c-myc transcription.
spectively). A moderately intense band is observed at lmax =
482 nm (eꢀ1.7ꢂ104 molꢁ1 dm3 cmꢁ1), with a shoulder at
lmax =520 nm (see Figure S1 in the Supporting Information).
According to previous reports,[26a–d] the absorption bands at
l=372–390 nm are assigned to intraligand transition(s)
(ILs), in which the band at l=482 nm is assigned to mixed
metal-to-ligand charge-transfer (1MLCT) [Pt(d5)!p*-
AHCTUNGTRENNUNG
(salphen)] and ligand-to-ligand charge-transfer (1LLCT)
[(phenoxide)!p*
ACHTUNGTRENUN(NG imine)] transitions. The absorption spec-
tra of complexes 1a–d and 2a–d are similar to that of 3 in
the spectral region l=280–400 nm, and all these complexes
display intense absorption bands between l=400 and
500 nm (see Figures S2–S9 in the Supporting Information).
The absorption spectra of 1–3 are sensitive to the solvent.
For example, the absorption maximum of 1a in CH2Cl2 is
similar to that in CH3CN (lmax =370 and 363 nm, respective-
ly), but the lower-energy broad absorption band (and the
shoulder (sh) in particular) is red-shifted to low energy in
low-polarity solvents; for example, lmax =487 (518 (sh)), 488
(525 (sh)), 495 (538 (sh)), 497 (550 (sh)) nm in MeOH,
CH3CN, CH2Cl2, and benzene, respectively. Such negative
solvatochromic shifts reveal that the ground state is more
polar than the excited state. This phenomenon has been re-
ported for related platinum(II) Schiff base complexes such
as [Pt(8-quinolinol)2].[26e,f] The UV/Vis absorption spectra of
1a (20 mm) in various solvents are given in the Supporting
Information (Figure S2) and the UV/Vis absorption data of
1–3 are summarized in Table 1.
Results
The Schiff base ligands were prepared according to previous
reports (see the Experimental Section). The reactions of 1,2-
diamine with substituted salicylaldehydes in ethanol gave
salphen or N,N’-bis(salicylidene)ethylenediaminato (salen)
ligands, which were recrystallized from ethanol. Treatment
of K2PtCl4 with Schiff base ligands in acetonitrile (with di-
methyl sulfoxide (DMSO)) followed by subsequent purifica-
tion gave complexes 1–3 (see Scheme S1 in the Supporting
Information). The structure of 1c was established by an X-
ray crystallographic study (Figure 1). The bond angles and
Excitation of 3 (50 mm in CH3CN) at l=372 nm gives an
emission at lmax =626 nm (see Figure S10 in the Supporting
Information). Emission lifetimes are t=1.40–4.60 ms and
emission quantum yields are around f=0.01 in CH3CN. The
emission maxima of complexes 1a–d in CH3CN are at lmax
=
638 (f=0.05, t=1.90 ms), 600 (f=0.22, t=5.60 ms), 606
(f=0.10, t=4.30 ms), and 595 nm (f=0.13, t=6.60 ms), re-
spectively. Complexes 2a,b in CH3CN display emission
maxima at lmax =536 (f=0.13, t=3.10 ms) and 522 nm (f=
0.25, t=7.60 ms), respectively (see Figures S11–S18 in the
Supporting Information). Complexes 2c,d are weakly emis-
sive in CH3CN or CH2Cl2. When compared with absorption
data, the emission maxima of 1–3 are less sensitive to sol-
vent effects. For example, the emission maximum of 3 shifts
slightly from lmax =626 nm in CH3CN to lmax =628 nm in
CH2Cl2 (see Figure S10 in the Supporting Information). The
photophysical data of 1–3 are summarized in Table 1.
Figure 1. X-ray crystal structure of 1c.
Polymerase stop assay: The induction and/or stabilization of
a biologically relevant G-quadruplex by complexes 1 and 2
were examined.[16,18,19a,27] It has been reported that G4A1
distances are comparable to those reported for other plati-
num(II) Schiff base complexes.[26b] Based on UV/Vis absorp-
tion and NMR spectroscopic measurements, the platinu-
m(II) Schiff base complexes are stable in aqueous solutions
for 72 h at room temperature.
oligomer
(5’-TGGGGAGGGTGGGGAGGGTGGG-
GAAGG-3’) could form an intramolecular G-quadruplex
structure in the presence of 100 mm KCl. A polymerase stop
assay can evaluate the stabilization of intramolecular G-
quadruplex structures in the presence of platinum(II) Schiff
base complexes. The formation of a G-quadruplex structure
would prohibit DNA hybridization and the subsequent poly-
merase chain reaction (PCR). The relative binding affinities
Spectroscopic properties: The UV/Vis absorption spectrum
of 3 in CH2Cl2 shows two intense absorption bands at lmax
=
372 and 390 nm (eꢀ4.8ꢂ104 and 3.3ꢂ104 molꢁ1 dm3 cmꢁ1, re-
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Investigation of Luminescent Platinum(II) Schiff Base Complexes
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Table 1. Photophysical data of 1–3 in various solvents at 298 K.[a]
Complex
Medium
T [K]
labs [nm] (e [ꢂ104 molꢁ1 dm3 cmꢁ1])
lem(max) [nm] (t [ms]; Fem)
1a
C6H6
CH2Cl2
MeCN
MeOH
C6H6
CH2Cl2
MeCN
MeOH
C6H6
CH2Cl2
MeCN
MeOH
C6H6
CH2Cl2
MeCN
C6H6
CH2Cl2
MeCN
MeOH/CH2Cl2 (10:1)
C6H6
CH2Cl2
MeCN
MeOH
C6H6
CH2Cl2
MeCN
MeOH/CH2Cl2 (10:1)
C6H6
CH2Cl2
MeCN
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
298
321 (1.7), 370 (3.6), 392 (4.3), 497 (1.1), 550 (0.9)
320 (1.8), 370 (3.8), 386 (4.2), 495 (1.0), 538 (0.8)
316 (1.6), 363 (3.1), 379 (3.4), 488 (0.5), 525 (sh, 0.6)
315 (1.3), 362 (2.4), 379 (2.6), 487 (0.7), 518 (sh, 0.6)
340 (1.5), 388 (3.3), 403 (2.9), 460 (4.7), 491 (5.3),
336 (1.3), 387 (3.3), 405 (3.4), 459 (5.0), 489 (6.1)
331 (1.1), 383 (2.9), 397 (2.9), 453 (4.2), 482 (5.0)
329 (1.1), 383 (3.3), 398 (3.5), 455 (5.0), 482 (5.8)
351 (1.4), 402 (2.5), 425 (2.9), 469 (4.1), 500 (5.2)
346 (1.0), 402 (2.0, sh), 424 (2.6), 469 (3.3), 500 (4.4)
341 (1.0), 400 (2.3, sh), 417 (2.8), 463 (3.6), 491 (4.7)
339 (1.0), 398 (2.4, sh), 417 (2.9), 463 (3.8), 491 (4.7)
350 (1.0), 425 (2.0), 464 (2.8), 493 (3.5)
639 (2.3; 0.07)
639 (2.1; 0.06)
638 (1.9; 0.05)
638 (1.4; 0.03)
600 (7.7; 0.31)
600 (6.1; 0.23)
600 (5.6; 0.22)
600 (4.8; 0.16)
608 (7.6; 0.21)
608 (5.4; 0.15)
606 (4.3; 0.10)
605 (4.4; 0.10)
596 (7.5; 0.21)
596 (6.9; 0.20)
595 (6.6; 0.13)
543 (3.3; 0.16)
542 (3.1; 0.15)
536 (3.1; 0.13)
535 (3.0; 0.13)
531 (8.1; 0.32)
523 (8.0; 0.30)
522 (7.6; 0.25)
521 (6.9; 0.22)
576 (6.2; 0.08)
575 (6.1; 0.08)
571 (6.0; 0.08)
568 (6.0; 0.07)
577 (6.2; 0.08)
576 (6.0; 0.08)
574 (4.7; 0.07)
573 (4.2, 0.07)
628 (4.6; 0.01)
626 (3.1; 0.01)
626 (1.4; <0.01)
n.d.
1b
1c
1d
2a
345ACTHNGUTERNN(UG 0.8), 425 (2.0), 463 (2.7), 493 (3.5)
340 (0.9), 419 (2.1), 458 (2.9), 487 (3.5)
288 (1.4), 364 (3.2), 380 (1.7, sh), 488 (0.01, sh)
285 (1.2), 359 (2.9), 382 (1.6, sh), 486 (0.01, sh)
285 (0.8), 347 (3.0), 388 (0.95, sh), 478 (0.01, sh)
282 (0.75), 345 (3.1), 395 (1.0, sh), 478 (0.01, sh)
345 (0.6, sh), 373 (1.1), 406 (0.4), 485 (0.01, sh)
343 (0.6), 367 (1.1), 399 (0.5, sh), 487 (0.01, sh)
340 (0.6, sh), 361 (1.0), 395 (0.7, sh), 488 (0.01, sh)
339 (0.6), 363 (0.82), 394 (0.5, sh), 485 (0.01, sh)
306 (0.4), 322 (0.40, sh), 426 (0.24), 488 (0.03)
304 (0.4), 315 (0.4, sh), 424 (0.23), 486 (0.02)
304 (0.4), 310 (0.4, sh), 421 (0.21), 485 (0.02)
295 (0.4), 309 (04, sh), 417 (0.28), 485 (0.01)
287 (1.5), 341 (0.43, sh), 427 (0.22), 489 (0.02)
280 (1.7), 341 (0.51, sh), 426 (0.31), 489 (0.02)
277 (1.7), 338 (0.52, sh), 421 (0.30), 488 (0.03)
275 (1.1), 332 (0.38, sh), 417 (0.23), 488 (0.03)
314 (1.9), 328 (1.8), 372 (4.8), 390 (3.3), 453 (1.7), 482 (1.7)
309 (1.9), 324 (1.8), 369 (3.3), 383 (3.0), 448 (1.6), 476 (1.6)
309 (1.9) 324 (1.8), 368 (3.1), 383 (2.9), 446 (1.6), 474 (1.5)
n.d.
2b
2c
2d
3
MeOH/CH2Cl2 (10:1)
CH2Cl2
MeCN
MeOH
C6H6
[a] n.d.=not determined due to poor solubility; sh=shoulder.
of the platinum(II) complexes to the intramolecular G-
quadruplex structure were determined by incubating fixed
concentrations of each platinum(II) complex with a DNA
template containing the G4A1 c-myc sequence in the pres-
ence of Taq polymerase. In the presence of a platinum(II)
Schiff base complex, the single-stranded G4A1 sequence
could be induced into a G-quadruplex structure that blocked
hybridization with a complementary strand overlapping the
last G repeat. Also, introduction of the platinum(II) com-
plex could stabilize the preformed G4A1-quadruplex struc-
ture. In either case, PCR would be inhibited, and the final
43-base pair (bp) double-stranded DNA PCR product
would not be detected. The addition of complexes 1a–d and
2a,b at a concentration of 50 mm resulted in a decrease in
the intensity of the band that corresponds to the 43-bp
double-stranded PCR product in all cases. The highest de-
crease in the formation of the PCR product was attained
with complexes 1a–d (Figure 2). The induction or stabiliza-
tion of G4A1 G-quadruplex formation by 2c,d could not be
determined due to their low solubility in buffer solution.
To confirm the impact of platinum(II) Schiff base com-
plexes on G4A1 G-quadruplex stabilization, a parallel ex-
Figure 2. Effect of 1 and 2 (50 mm) on G4A1 G-quadruplex stabilization
by the polymerase stop assay.
periment that used oligomer G4A2 (5’-TGGGGAGGGTG-
GAAAGGGTGGGGAAGG-3’) was performed. The G4A2
sequence is unable to form a G-quadruplex structure, and
no inhibition was observed under the same reaction condi-
tions.
Inhibition of the expression of c-myc in cancer cells: To
evaluate the ability of platinum(II) Schiff base complexes to
inhibit c-myc transcription in cancer cells, RT-PCR assays
were performed.[19a] Complex 1a was the strongest inhibitor
of c-myc expression. At a concentration of 50 mm, this com-
plex significantly decreased the mRNA level of c-myc in
HepG2 cells (human hepatocarcinoma; Figure 3). This
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C.-M. Che et al.
Figure 3. a) Expression of c-myc in HepG2 cells treated with 1 and 2 by
RT-PCR. b) Percentage of c-myc activity for 1 and 2.
Figure 4. NMR titration (600 MHz, 278C) of the intramolecular
G4A3 quadruplex with 1a at various ratios of [1a]/[G4A3 quadruplex] in
H2O/D2O (90:10) with KCl (150 mm), KH2PO4 (25 mm), and EDTA
(1 mm; pH 7.0). The imino proton resonances of the residues in the G-
tetrad were assigned on the basis of previous data.[28]
result suggests that 1a can suppress the transcription of c-
myc through interaction with the NHE III1 upstream of the
P1 promoter of c-myc. Because 1a was the most potent
complex against c-myc transcription, it was used in subse-
quent studies to elucidate the interaction between plati-
num(II) Schiff base complexes and the G-quadruplex.
NMR spectroscopic experiments: To elucidate the binding
mode of 1a, we conducted a number of H NMR spectro-
Molecular modeling: We performed molecular modeling of
the binding of 1a on the model of the NHE III1 intramolec-
ular G-quadruplex loop isomer to further investigate the
mode of binding. Hurley and co-workers showed that the
biologically relevant parallel c-myc G-quadruplexes can
exist as a mixture of four loop isomers (see Figure S19 in
the Supporting Information) with predominance of the 1:2:1
and 2:1:1 isomers.[29a] Independent work by Patel and co-
workers that employed high-field NMR spectroscopic analy-
sis also showed the predominance of the 1:2:1 loop isomer
in the c-myc parallel G-quadruplex.[29b] Because neither
NMR spectroscopic nor X-ray crystallographic information
of the NHE III1 1:2:1 loop isomer is available, a model was
built from the known, closely related X-ray crystal structure
of the human intramolecular telomeric G-quadruplex DNA.
1
scopic experiments in phosphate buffer solution (H2O/D2O
(90:10) with KCl (150 mm), KH2PO4 (25 mm), and ethylene-
diaminetetraacetic acid (EDTA; 1 mm), pH 7.0). An intra-
molecular
G4A3quadruplex[28]
(5’-TGAGGGTGGI-
GAGGGTGGGGAAGG-3’) with clearly assigned G-tetrad
imino proton signals[28] was used for these studies. Upon the
addition of 1a to a solution of the G4A3quadruplex in the
phosphate buffer (1.0 mm) to obtain a [1a]/[G4A3 qua-
druplex] ratio of 1:1, the imino proton resonances of the res-
idues in the G-tetrad were shifted upfield (Figure 4). At a
ratio of 1:0 for [1a]/[G4A3 quadruplex], the G6 and G20
imino proton signals experienced the largest shift, thus re-
vealing that 1a binds close to the 3ꢀ terminal face of the G-
quadruplex DNA. The changes in chemical shifts are similar
to those displayed by daunomycin, which is known to bind
to G-quadruplexes.[28] We favor the model that 1a binds ex-
ternally to the stacks of guanine quartets within a quadru-
plex, rather than intercalating between the stacks.
A
truncated 18-bp sequence (i.e., 5’-AGGGTGGG-
GAGGGTGGGG-3’) was used for this study because nucle-
otides G2–G5 in the G4A1-c-myc sequence 5’-
TGGGGAGGGTGGGGAGGGTGGGGAAGG-3’ are not
involved in the G-quartet structure. The modeling study mir-
rors the general trend that 1a binds to the G-quadruplex
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Investigation of Luminescent Platinum(II) Schiff Base Complexes
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with
a
calculated binding energy of approximately
discovery of derivatives of 1a that exhibit greater specificity
and selectivity toward the G-quadruplex structure of c-myc.
By using the above approach and with 1a as the structural
template, 60 new platinum(II) Schiff base complexes con-
taining different amine side chains were virtually screened
(see Figure S20 in the Supporting Information). The top-
scoring platinum(II) complex was evaluated for its effect on
stabilizing G-quadruplex DNA. The platinum(II) complex
was docked to a grid representation of the receptor and a
score was assigned to each complex that reflected the quali-
ty of the interaction according to the iterated conditional
modes (ICM) method (Molsoft).[30] Analysis of the confor-
mational energy of all of the platinum(II) Schiff base com-
plexes revealed that the side chains can bury deeply in the
G-quadruplex DNA grooves, thus making several electro-
static and hydrogen-bonding contacts. However, the n=2
side chain is generally superior to the n=1, 3, 4, and 5 side
chains (see Figure S20 in the Supporting Information). The
top-scoring molecule identified through virtual screening
ꢁ50 kcalmolꢁ1 and is stacked at the ends of G-quadruplex
at the GT-quadruplex terminus, close to the 3ꢀ terminal face
of the G-quadruplex. This model is consistent with that de-
rived from the results of the NMR spectroscopic experi-
ments described in previous section. In addition, the unfav-
orable binding energies of approximately +30 kcalmolꢁ1,
calculated for the intercalation binding sites, suggest that
the interactions between 1a and G-quadruplex DNA should
not be intercalative in nature (see Table S1 in the Support-
ing Information). As such, 1a is an useful scaffold for devel-
oping new platinum(II) analogues with improved inhibitory
activities against c-myc transcription.
Lead optimization: The encouraging results obtained from
preliminary screening suggested the use of 1a as an appro-
priate starting scaffold for hit-to-lead optimization through
virtual screening. To give new lead derivatives with im-
proved potency and specificity against c-myc transcription,
we adopted a structure-guided parallel synthesis. In this ap-
proach, the Schiff base is modified on the basis of receptor
structural information, and the mode of binding of the plati-
num(II) complex with the G-quadruplex DNA serves as a
template for lead optimization (Figure 5). Molecular model-
ing permits rapid screening of multiple analogues, thus facil-
itating preselection of a subset of furnished analogues with
favorable binding affinities. This approach can expedite the
was [PtII
ACTHNUTRGNEUNG(N,N’-bis{4-[1-(2-propylpiperidine)oxy]salicylidene}-
4,5-methoxy-1,2-phenylenediamine] (3; Figure 6). This com-
plex was subsequently prepared and tested experimentally
on its ability to inhibit the amplification in the promoter
region of c-myc with the polymerase stop assay. Notably, 3
promoted/stabilized the formation of the G-quadruplex to
the greatest extent with a tenfold boost in inhibition activity
(IC50 =4.4 mm) over the initial hit complex 1a (see Fig-
ure S21 in the Supporting Information).
Figure 5. Schematic diagram of the a) side and b) top views of the inter-
actions of 1a with the G-quadruplex structure of c-myc. The G-quadru-
plex and 1a are shown in ribbon and space-filling representations, respec-
tively.
Figure 6. Schematic diagram of the a) side and b) top views of the inter-
actions of 3 with the G-quadruplex structure of c-myc. The G-quadruplex
and 3 are shown in ribbon and space-filling representations, respectively.
Chem. Eur. J. 2009, 15, 13008 – 13021
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C.-M. Che et al.
Absorption titration: The ability of the top-scoring complex
3 to interact with the intramolecular G-quadruplex DNA of
c-myc was studied by absorption titration experiments. An
intramolecular G-quadruplex c-myc was prepared by incu-
bating a G4A1 oligonucleotide in tris(hydroxymethyl)ami-
nomethane (Tris)/KCl buffer, which was heated to 958C for
10 minutes and cooled to room temperature overnight. The
expected secondary DNA structure was confirmed by a pos-
itive CD peak at l=262 nm and a negative CD peak at ap-
proximately l=240 nm. Isosbestic spectral changes and hy-
pochromism effects were observed in the UV/Vis absorption
spectral changes (Figure 7). This hypochromic phenomenon
Dosage-dependent inhibition of cancer-cell growth and c-
myc expression: To evaluate the dose response of platinu-
m(II) Schiff base complexes on the inhibition of cancer-cell
growth, proliferation assays were performed. By using an
MTT assay, the cytotoxicities of 1a, 1c, 3, and cisplatin
against HeLa, HepG2, SUNE1, and NCI-H460 cancer-cell
lines were determined in vitro, whereas the cytotoxicity
toward normal cells was evaluated with the CCD-19Lu
normal lung fibroblast cell line. The IC50 value of the com-
plexes after incubation for 48 h are on the order: 1a>3ꢀ
cisplatin@1c (Table 2). Notably, the cytotoxicity of 1a
against cancer-cell lines (IC50 = ꢀ1 mm) is approximately
tenfold higher than cisplatin.
Complex 3 is moderately cyto-
toxic with IC50 values of 8–
14 mm. Importantly, this com-
plex is approximately tenfold
less cytotoxic (IC50 =85 mm)
toward normal lung fibroblast
cells. The treatment of HepG2
with 25 mm
3 decreased the
level of c-myc expression
(Figure 8). The transcription of
c-myc is significantly sup-
pressed in the presence of
100 mm 3. We have thus demon-
strated that 3 can down-regu-
late the expression of c-myc at
the RNA level in a dose-depen-
dent manner in HepG2 cells.
Emission titration: The design
of
efficacious
luminescent
probes for G-quadruplexes is of
considerable interest in bio-
chemistry. For this reason, emis-
Figure 7. UV/Vis spectra of 3 (25 mm) in Tris/KCl buffer (KCl (100 mm), Tris/HCl (10 mm); pH 7.5) with in-
creasing amount of oligonucleotide G4A1 at 20.08C ([G4A1 quadruplex]/[Pt]=0–20:1). Inset: plot of D/Deap
versus D.
Table 2. Cytotoxicities of 1a, 1c, 3, and cisplatin toward four carcinoma cell lines.[a]
is assigned to the strong inter-
action between the electronic
states of the platinum(II) com-
plex and DNA bases. By using
the Scatchard equation,[31] the
Complex
IC50 [mm]
HeLa
HepG2
SUNE-1
NCI-H460
CCD-19 Lu
1a
1c
3
1.28ꢂ0.33
86ꢂ5.3
1.09ꢂ0.14
>100
1.04ꢂ0.21
>100
4.80ꢂ0.56
>100
46.11ꢂ0.53
>100
85.04ꢂ0.20
12.1ꢂ0.43
8.07ꢂ0.11
14.04ꢂ0.23
11.90ꢂ0.26
cisplatin
12.5ꢂ1.1
11.5ꢂ0.12
13.6ꢂ0.32
16.5ꢂ0.82
85.2ꢂ0.36
plot of D/Deap versus
D
(Figure 7, inset) revealed the
binding constant K of 3 with
the G-quadruplex to be K=
[a] HeLa=human cervix epithelioid carcinoma, HepG2=human hepatocellular carcinoma, SUNE-1=human
nasopharyngeal carcinoma, NCI-H460=human non-small cell lung cancer, and CCD-19Lu=normal human
lung fibroblast.
1.72ꢂ0.26ꢂ105 dm3 molꢁ1
at
20.08C. The K values for 1 and
2 were similarly determined to be approximately K=
105 dm3 molꢁ1. To examine the binding specificity, we em-
ployed a duplex G4A1 oligonucleotide as a control. Binding
of 3 to G-quadruplex DNA is favored by approximately ten-
fold compared to non-quadruplex double-stranded DNA
molecules (K=1.91ꢂ1.10ꢂ104 dm3 molꢁ1 for double-strand-
ed DNA).
sion titration experiments were conducted to determine the
ability of the platinum(II) Schiff base complexes to detect
G-quadruplex structures. Complex 3 is weakly emissive in
aqueous Tris/KCl buffer solution at 20.08C. Upon addition
of the G4A1 quadruplex, an intense emission at lmax
=
652 nm is observed (Figure 9). The emission intensity in-
creases with the concentration of the G-quadruplex and is
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Investigation of Luminescent Platinum(II) Schiff Base Complexes
FULL PAPER
Figure 8. Above: the expression of c-myc in HepG2 cells treated with 3
by RT-PCR. Below: percentage of c-myc mRNA level (lower panel).
saturated at a [G-quadruplex]/[3] ratio of ꢃ20:1. The emis-
sion intensity at l=652 nm is enhanced by up to eightfold.
Figure 9. a) Emission spectral traces of 3 (50 mm) in Tris/KCl buffer with
increasing concentration of the G-quadruplex at 20.08C. b) Photographs
of 3 in the absence and the presence of the G-quadruplex.
Discussion
Overexpression of the c-myc oncogene is linked to increased
cellular proliferation and inhibition of differentiation. Thus,
c-myc is implicated in the development of human and
animal malignancies, including carcinomas of the breast,
colon, cervix, small-cell lung cancer, osteosarcomas, glioblas-
tomas, and myeloid leukemias.[1–9] Therefore, the inhibition
of c-myc gene transcription by inducing G-quadruplex for-
mation and/or stabilizing the G-quadruplex structure can be
targeted, which can be used in the design of new anticancer
therapeutic agents. There have already been reports of or-
ganic compounds as telomerase inhibitors that bind to and
stabilize G-quadruplexes, thus resulting in the down-regula-
tion of c-myc.[17–20] Herein, a class of phosphorescent plati-
num(II) complexes containing p-conjugated Schiff base li-
gands with amine side chains were found through computer-
aided structure-based drug design. This class of platinum(II)
Schiff base complexes are structurally similar to the report-
ed planar organic molecules that are known to bind to G-
quadruplexes.
minor groove of DNA. The work by Smith and co-workers
showed that dendritic arrays of spermine ligands with inher-
ent repetitive branded structures offer a useful scaffold for
organizing multiple ligands on an accessible surface to ach-
ieve high nonspecific minor-groove DNA binding affinity.[32]
However, the poor water solubility of complexes 2c,d limits
their applications in DNA binding studies.
Based on the results of the initial preliminary screening
by using the polymerase stop assay and RT-PCR assay, com-
plex 1a emerged as an appealing scaffold for optimization
through docking-based virtual screening to improve inhibi-
tory activities. Subsequently, we designed and prepared plat-
inum(II) Schiff base complexes with the following character-
istics: 1) a rigid flat Schiff base mainframe that promotes p–
p stacking for binding to a G-quadruplex and 2) amine side
chains attached to the Schiff base ligand for interactions
with G-quadruplex loops and grooves.
Eight platinum(II) Schiff base complexes were prepared
for initial screening to induce and/or stabilize the c-myc G-
quadruplex structures and inhibit c-myc expression by using
polymerase stop assays and RT-PCR assays. The platinu-
m(II) complexes 2c,d containing dendritic ligands were pre-
pared for their ability to simultaneously interact with the
Specificity for G-quadruplex DNA of the c-myc sequence
over duplex DNA: We studied the interaction of the plati-
num(II) Schiff base complexes with the G-quadruplex oligo-
nucleotide from the promoter region of the oncogene c-myc
by UV/Vis absorption and emission titration experiments.
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C.-M. Che et al.
The results show that the platinum(II) Schiff base com-
plexes can bind to G-qudruplex DNA with binding constants
of approximately K=105 dm3 molꢁ1. By comparing the
K values, complex 3 has a nearly tenfold higher binding af-
finity toward the intramolecular c-myc G-quadruplex DNA
from oligonucleotide G4A1 than for non-quadruplex
double-stranded DNA molecules consisting of complemen-
tary G4A1 oligonucleotides. This significant level of selectiv-
ity for c-myc G-quadruplex DNA over duplex DNA vali-
dates the results of the computer-aided design of the Schiff
base ligands with hydrogen-bonding donor/acceptor amine
side chains for effective stabilization of G-quadruplex struc-
tures.
G-quadruplex)). Initially, various orientations of 3 were ob-
tained that differ in orientation of the core with respect to
the loops of the quadruplex. The energy of these structures
was minimized, and the most stable structure that showed
the best interaction energy and optimum interactions be-
tween 3 and the G-tetrad was selected. A similar external
end-stacking binding mode has also been reported in a
recent crystal-structure analysis of disubstituted acridine de-
rivatives and a dimeric Oxytricha quadruplex.[33] Neidle and
co-workers have reported X-ray fiber diffraction data of the
cocomplex formed by interaction of a 1,4-disubstituted ami-
doanthraquinone (BSU-1071) with a synthetic [dACHTNUGTRENUNG(TGGGT)]4
sequence, thus revealing an external end-stacking binding
mode.[34]
Hit-to-lead optimization through computer-aided virtual
screening: In this study, we identified that complex 3 dis-
plays selectivity for the c-myc G-quadruplex over double-
stranded DNA. Molecular modeling was used to rationalize
the selectivity of 3. Our initial lead complex 1a was shown
to bind to the c-myc G-quadruplex, thus leading to inhibi-
tion of c-myc gene promoter activity and repression of c-
myc transcription. Computer-aided molecular docking of the
interactions of a series of platinum(II) Schiff base complexes
with the c-myc G-quadruplex revealed that these platinu-
m(II) complexes have optimal stacking interactions at one
end of the G-quadruplex structure. The platinum(II) ion in
these complexes is positioned at the center of the quartet
(i.e., the surface of the G-quadruplex ion channel), which
can provide additional electrostatic stabilization. As report-
ed previously, the strength of the interaction between planar
p-conjugated molecules and G-quadruplex DNA can be in-
creased considerably by attaching side chains to the planar
core of the molecule.[19,20,24e,l] We have examined the effect
of the length of the aliphatic spacer on the binding proper-
ties of platinum(II) Schiff base complexes by molecular
docking. The n=2 side chain is generally superior to the n=
1, 3, 4, and 5 side chains (see Figure 6 and Figure S20 in the
Supporting Information).
It has been reported that the biologically relevant G-
quadruplexes of NHE III1 exist as a dynamic mixture of
four different loop isomers, and all four isomers contribute
to the stability of the silencer element in NHE III1 of the c-
myc promoter, thus functioning as transcriptional repressor
elements. The 1:2:1 form is the major isomer,[29] but because
neither NMR spectroscopic nor X-ray crystallographic infor-
mation is available for the 1:2:1 loop isomer of NHE III1,
the model was built from the known, closely related X-ray
crystal structure of the human intramolecular telomeric G-
quadruplex DNA as previously described.[19a]
In addition, the unfavorable binding energies of approxi-
mately 43 kcalmolꢁ1 calculated for the intercalation binding
sites reveal that the interaction between 3 and G-quadruplex
DNA is not intercalative in nature. All these results can be
rationalized by a molecular modeling study on the interac-
tions of 3 with the intercalation sites of G-quadruplex DNA,
thus revealing that the core of molecule 3 and the long side
chains are too large for intercalation.
Inhibition of c-myc gene promoter activity in a cell-free
system and its expression in cultured cells: The specific
binding of complexes 1a–d, 2b, and 3 with intramolecular
G-quadruplex structures in the promoter region prevents
DNA hybridization and subsequent Taq polymerase-mediat-
ed DNA extension, the inhibition of which can be used as a
measure of the stabilization of G-quadruplex structures. Re-
sults from the polymerase stop assays reveal that the con-
centration-dependent inhibition of DNA amplification by
complex 3 is due to stabilization of the G-quadruplex struc-
ture formed in the DNA templates containing the G4A1 c-
myc sequence. For each platinum(II) complex examined,
there is significantly greater decrease in the amount of the
PCR product upon increasing the concentration of the com-
plex. Complex 3 displays the strongest inhibition (IC50 =
4.4 mm) and is of comparable potency to quindoline com-
pounds (IC50 = ꢀ6–36 mm) and reported 9-N-substituted ber-
berine compounds (IC50 =2–7.1 mm).[19] Compared to 1a,
complex 3 displayed a tenfold increase in potency toward
the c-myc G-quadruplex (Figure 3). The most potent c-myc
G-quadruplex stabilizer that has been reported is Se2SAP,
with IC50 =0.03 mm. However, Se2SAP only moderately in-
hibits down-regulation of c-myc in cancer cells.[16] It is possi-
ble that Se2SAP is not taken up into the cells due to its rela-
tively large, expanded porphyrin scaffold.
To verify the observed effects of 3 on the c-myc promoter
G-quadruplex, we examined whether treatment of HepG2
cells with 3 could interfere with c-myc expression. The re-
sults indicate that 3 decreases c-myc expression at the RNA
level in HepG2 cells. In contrast, the initial hit complex 1a
displayed a decreased effect in all of the cell-free and cell-
based assays compared with 3. Complex 3 has an extended
p-conjugated planar organic ligand that contains hydrogen-
bonding donor/acceptor amine side chains. These amine side
On the basis of NMR titration experiments, we hypothe-
sized that 1a or 3 binds to this G-quadruplex and is stacked
on the ends of the G-quadruplex at the GT-quadruplex ter-
minus, which is close to the 3ꢀ terminal face of the G-quad-
ruplex. This proposed model is supported by a molecular
modeling study of the binding interaction between 3 and in-
tramolecular G-quadruplex DNA (with a favorable calculat-
ed binding energy of ꢁ65.88 kcalmolꢁ1 (i.e., intramolecular
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Investigation of Luminescent Platinum(II) Schiff Base Complexes
FULL PAPER
tilled water. Further dilutions to working concentrations were made with
double-distilled water.
chains are needed for specific G-quadruplex binding, as the
Schiff base ligand alone promotes or stabilizes the formation
of an intramolecular G-quadruplex to a much lesser extent,
as confirmed by the results of the UV/Vis absorption titra-
tion experiment and polymerase stop assays.
Physical measurements: Absorption spectra were recorded on a Perkin–
Elmer Lambda 19 UV/Vis spectrophotometer. Emission spectra were re-
corded on a SPEX Fluorolog-2 Model fluorescence spectrophotometer.
Positive-ion FAB mass spectra were recorded on a Finnigan MAT95
mass spectrometer.
For G-quadruplex binding molecules to act as c-myc on-
cogene regulators, the former must display a high selectivity
for G-quadruplex DNA over duplex DNA and a low inhibi-
tion concentration against c-myc transcription. Based on the
RT-PCR assays, complex 3 decreased the transcription of c-
myc in HepG2 cells at 25 mm and that transcription was sig-
nificantly repressed at 100 mm. We also used an MTT assay
to determine cytotoxicity against tumor cells and normal
cells. Complex 3 displays moderate, but significant, cytotox-
icity against cancerous cells (IC50 =8–14 mm) but is tenfold
less cytotoxic (IC50 =85 mm) toward normal lung fibroblast
cells. This selectivity makes it possible for 3 to effectively in-
hibit c-myc gene promoter activity at concentrations that do
not have a significant toxic effect upon normal cells. As an
aside, we note that complex 1a is highly cytotoxic against
cancer cells (IC50 =1 mm), whereas the effective concentra-
tion needed to inhibit c-myc transcription is approximately
50 mm, thus revealing that the primary cellular target of 1a
may not be the c-myc G-quadruplex.
N,N’-Bis(salicylidene)-4,5-methoxy-1,2-phenylenediamine (H2L1a): Salicy-
laldehyde (0.4 g, 3.3 mmol) in ethanol (5 mL) was added dropwise to 4,5-
dimethoxy-o-phenylenediamine (0.22 g, 1.3 mmol) in ethanol (20 mL) at
room temperature. The reaction mixture was gradually heated to 608C
and maintained at this temperature for 4 h. Upon removal of the solvent
and cooling, a yellow precipitate was collected and recrystallized from
ethanol to give the product (0.2 g, 40%). 1H NMR (CDCl3, 300 MHz):
d=13.15 (s, 2H), 8.60 (s, 2H), 7.39–7.35 (m, 4H), 7.05 (d, 2H), 6.94 (d,
2H), 6.80 (s, 2H), 3.97 ppm (s, 6H); MS (FAB, +ve): m/z: 376 [M]+.
AHCTUNGTRENNUNG
[PtL1a] (1a): Sodium acetate (0.1 g, 0.27 mmol) was added to H2L1a
(0.1 g, 0.27 mmol) dissolved in MeCN (20 mL), and the solution was
stirred for 5 min. K2PtCl4 (0.11 g, 0.27 mmol) and DMSO (1 mL) were
added to the reaction mixture, which was heated to 408C and stirred
overnight. After cooling to room temperature, the solvent was removed
under vacuum. A yellow precipitate was collected and dissolved in
CH2Cl2 (10 mL). The organic layer was washed with water (3ꢂ10 mL),
dried over Na2SO4, and concentrated to give a red solid (61 mg, 40%).
1H NMR (CD3CN, 400 MHz): d=8.99 (s, 2H), 7.77 (s, 2H), 7.61 (s, 2H),
7.58 (t, 2H), 7.15 (d, 2H), 6.82 (t, 2H), 3.99 ppm (s, 6H); MS (FAB,
+ve): m/z: 570 [M]+; elemental analysis calcd (%) for C22H18N2O4Pt: C
46.40, H 3.19, N 4.92; found: C 46.39, H 3.20, N 4.90.
N,N’-Bis(4-diethylaminosalicylidene)-1,2-phenylenediamine (H2L1b): 4-
(Diethylamino)salicylaldehyde (1 g, 5.2 mmol) in ethanol (10 mL) was
added dropwise to 1,2-phenylenediamine (0.56 g, 5.2 mmol) in ethanol
(40 mL) at room temperature. The mixture was gradually heated to 608C
and maintained at this temperature for 4 h. Upon removal of the solvent
and cooling, a yellow precipitate was collected and recrystallized from
ethanol to give the product (1.95 g, 82%). 1H NMR (CDCl3, 300 MHz):
d=13.52 (s, 2H), 8.39 (s, 2H), 7.16 (d, 2H), 7.00 (d, 2H), 6.75 (d, 2H),
6.25 (d, 2H), 6.20 (s, 2H), 3.38 (q, 8H), 1.20 ppm (t, 12H); 13C NMR
(CDCl3, 300 MHz): d=165.0, 161.2, 152.3, 142.8, 134.1, 126.5, 119.6,
109.9, 104.1, 98.6, 45.0, 13.1 ppm; MS (FAB, +ve): m/z: 459 [M]+; ele-
mental analysis calcd (%) for C28H34N4O2: C 73.33, H 7.47, N 12.22;
found: C 72.76, H 7.48, N 12.81.
Conclusions
In summary, a series of platinum(II) complexes containing
Schiff base ligands have been prepared and characterized,
including X-ray crystallography studies. One of the plati-
num(II) Schiff base complexes is a promising initial lead
compound that shows selective binding to the c-myc pro-
moter G-quadruplex over duplex DNA. This complex was
demonstrated to inhibit c-myc gene promoter activity in a
cell-free system and c-myc expression in cultured cells. Sub-
sequent computer-aided hit-to-lead optimization furnished a
new platinum(II) Schiff base complex with amine side
chains that displays superior c-myc regulator properties, pre-
sumably through stabilization of the G-quadruplex DNA
structure in the NHE III1 sequence.
AHCTUNGTRENNUNG
[PtL1b] (1b): Sodium acetate (0.33 g, 4.0 mmol) was added to H2L1b
(1.0 g, 2.0 mmol) dissolved in MeCN (50 mL), and the solution was
stirred for 5 min. K2PtCl4 (0.84 g, 2.0 mmol) and DMSO (5 mL) were
added to the reaction mixture, which was heated to 408C and stirred
overnight. After cooling to room temperature, the solvent was removed
under vacuum. A yellow precipitate was collected and dissolved in
CH2Cl2 (10 mL). The organic layer was washed with water (3ꢂ10 mL),
dried over Na2SO4, and concentrated to give a red solid (0.93 g, 71%).
1H NMR (CDCl3, 300 MHz): d=8.33 (s, 2H), 7.70 (s, 2H), 7.25 (d, 2H),
7.05 (s, 2H), 6.53 (s, 2H), 6.22 (d, 2H), 3.40 (q, 8H), 1.23 ppm (t, 12H);
13C NMR (CDCl3, 300 MHz): d=167.2, 153.8, 145.6, 145.3, 136.6, 125.8,
114.8, 114.3, 105.5, 101.1, 45.1, 13.3 ppm; MS (FAB, +ve): m/z: 651 [M]+;
elemental analysis calcd (%) for C28H32N4O2Pt: C 51.61, H 4.95, N 8.60;
found: C 51.60, H 4.91, N 8.51.
Experimental Section
Materials:
GAGGGTGGGGAAGG-3’;
GAAAGGGTGGGGAAGG-3’;
DNA
oligomers
(G4A1=5’-TGGGGAGGGTGGG-
G4A2=5’-TGGGGAGGGTG-
G4A3=5’-TGAGGGTGGI-
N,N’-Bis(4-diethylaminosalicylidene)-4,5-dichloro-1,2-phenylenediamine
(H2L1c): 4-(Diethylamino)salicylaldehyde (2 g, 10 mmol) in ethanol
(10 mL) was added dropwise to 4,5-dichloro-o-phenylenediamine (0.92 g,
5.2 mmol) in ethanol (40 mL) at room temperature. The mixture was
gradually heated to 608C and maintained at this temperature for 4 h.
Upon removal of the solvent and cooling, a yellow precipitate was col-
lected and recrystallized from ethanol to give the crude product (0.86 g,
35%). 1H NMR (CDCl3, 300 MHz): d=13.43 (s, 2H), 8.33 (s, 2H), 7.24
(s, 2H), 7.12 (d, 2H), 6.20 (d, 2H), 6.15 (s, 2H), 3.39 (q, 8H), 1.21 ppm
(t, 12H); 13C NMR (CDCl3, 300 MHz): d=164.9, 161.5, 152.7, 142.5,
134.5, 129.3, 120.7, 109.7, 104.5, 98.3, 45.1, 13.1 ppm; MS (FAB, +ve): m/
z: 527 [M]+.
GAGGGTGGGGAAGG-3’; c-myc S=5’-GTTAGCTTCACCAACAG-
GAACTATG-3’; c-myc A=5’-ATACAGTCCTGGATGATGATGTTTT-
3’;
b-actin S=5’-GCGTCTGGACCTGGCTGGCCGGGAC-3ꢀ
b-
actin A=5’-AAGGAAGGCTGGAAGAGTGCCTCAG-3’) were ob-
tained from Tech Dragon Limited (Carlsbad, CA). Unless otherwise
stated, spectroscopic titration experiments were performed in 10 mm Tris/
HCl (pH 7.4) containing 100 mm KCl. SuperScript II Reverse Transcrip-
tase was purchased from Invitrogen (Hong Kong). Taq DNA polymerase
and the total RNA isolation kit (RNeasy mini kit) were purchased from
QIAGEN (Valencia, CA). Stock solutions of all the platinum(II) com-
plexes (10 mm) were made in dimethyl sulfoxide (DMSO) or double-dis-
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13017

C.-M. Che et al.
A
6.04 (s, 2H), 3.35 (dd, 8H), 1.39 (s, 12H), 1.16 ppm (t, 12H); 13C NMR
(CDCl3, 300 MHz): d=169.81, 159.80, 152.67, 134.00, 108.65, 103.46,
99.41, 63.73, 44.89, 23.39, 13.18 ppm; MS (FAB, +ve): m/z: 467 [M+1]+;
elemental analysis calcd (%) for C28H42N4O2: C 72.07, H 9.07, N 12.01;
found: C 71.37, H 9.08, N 11.81.
(0.36 g, 0.7 mmol) dissolved in MeCN (10 mL), and the solution was
stirred for 5 min. K2PtCl4 (0.29 g, 0.7 mmol) and DMSO (1 mL) were
added to the reaction mixture, which was heated to 408C and stirred
overnight. After cooling to room temperature, the solvent was removed
under vacuum. A yellow precipitate was collected and dissolved in
CH2Cl2 (10 mL). The organic layer was washed with water (3ꢂ10 mL),
dried over Na2SO4, and concentrated to give a red solid (0.22 g, 42.7%).
1H NMR (CDCl3, 300 MHz): d=8.25 (s, 2H), 7.85 (s, 2H), 7.29 (d, 2H),
6.54 (s, 2H), 6.29 (d, 2H), 3.39 (q, 8H), 1.23 ppm (t, 12H); MS (FAB,
+ve): m/z: 720 [M]+; elemental analysis calcd (%) for C28H30Cl2N4O2Pt:
C 46.67, H 4.20, N 7.78; found: C 46.60, H 4.21, N 7.71.
N,N’-Bis(4-diethylaminosalicylidene)-2,3-pyridylenediamine (H2L1d): 4-
(Diethylamino)salicylaldehyde (1 g, 5.2 mmol) in ethanol (5 mL) was
added dropwise to 2,3-diaminopyridine (0.28 g, 2.6 mmol) in ethanol
(5 mL) at room temperature. The reaction mixture was gradually heated
to 608C and maintained at this temperature for 4 h. Upon removal of the
solvent and cooling, a yellow precipitate was collected and recrystallized
from ethanol to give the product (0.38 g, 32%). 1H NMR (CDCl3,
300 MHz): d=12.92 (s, 2H), 8.34 (s, 2H), 7.91 (d, 1H), 7.16–7.11 (m,
3H), 6.67 (m, 1H), 6.25~6.15 (m, 4H), 3.41 (q, 8H), 1.21 ppm (t, 12H);
MS (FAB, +ve): m/z: 459 [M]+.
AHCTUNGTRENNUNG
[PtL2b] (2b): Sodium acetate (0.034 g, 0.42 mmol) was added to H2L2b
(0.1 g, 0.21 mmol) dissolved in MeCN (10 mL), and the solution was
stirred for 5 min. K2PtCl4 (0.085 g, 0.21 mmol) and DMSO (1 mL) were
added to the reaction mixture, which was heated to 408C and stirred
overnight. After cooling to room temperature, the solvent was removed
under vacuum. A yellow precipitate was dissolved in CH2Cl2 (10 mL).
The organic layer was washed with water (3ꢂ10 mL) dried over Na2SO4,
and concentrated to give
a
yellow solid (84.2 mg, 61%). 1H NMR
(CDCl3, 300 MHz): d=7.78 (s, 2H), 7.08 (d, 2H), 6.46 (s, 2H), 6.08 (d,
2H), 3.33 (dd, 8H), 1.41 (s, 12H), 1.17 ppm (t, 12H); 13C NMR (CDCl3,
300 MHz): d=165.46, 152.78, 149.54, 135.15, 114.03, 108.60, 104.73, 74.91,
44.92, 24.89, 13.21 ppm; MS (FAB, +ve): m/z: 659 [M]+; elemental anal-
ysis calcd (%) for C28H40N4O2Pt: C 50.98, H 6.11, N 8.49; found: C 50.90,
H 6.19, N 8.41.
General procedure for the synthesis of dendritic benzyl alcohols: A mix-
ture of the appropriate dendritic benzyl bromide (2.00 equiv), 3,5-dihy-
droxybenzyl alcohol (1.00 equiv), dried potassium carbonate (2.50 equiv),
and [18]crown-6 (0.2 equiv) in dry acetone was heated to reflux and
stirred vigorously under nitrogen for 48 h. The reaction mixture was al-
lowed to cool and evaporated to dryness under reduced pressure. The
residue was partitioned between water and CH2Cl2 and the aqueous layer
was extracted with CH2Cl2. The combined organic layers were dried and
evaporated to dryness. The crude product was crystallized from ethyl ace-
tate/hexane.
ACHTUNGTRENNUNG
[PtL1d] (1d): Sodium acetate (0.16 g, 2.0 mmol) was added to H2L1d
(0.43 g, 0.94 mmol) dissolved in MeCN (20 mL), and the solution was
stirred for 5 min. K2PtCl4 (0.39 g, 0.94 mmol) and DMSO (2 mL) were
added to the reaction mixture, which was heated to 408C and overnight.
After cooling to room temperature, the solvent was removed under
vacuum. A yellow precipitate was collected and dissolved in CH2Cl2
(10 mL). The organic layer was washed with water (3ꢂ10 mL), dried
over Na2SO4, and concentrated to give a red solid (77.2 mg, 12.6%).
1H NMR (CDCl3, 300 MHz): d=9.19 (s, 1H), 8.40 (s, 1H), 8.23 (d, 1H),
8.07 (d, 1H), 7.44 (d, 1H), 7.28 (d, 1H), 7.06 (m, 1H), 6.57 (s, 2H), 6.30
(d, 2H), 3.42 (q, 8H), 1.23 ppm (t, 12H); MS (FAB, +ve): m/z: 653 [M]+;
elemental analysis calcd (%) for C27H31N5O2Pt: C 49.69, H 4.79, N 10.73;
found: C 50.09, H 4.81, N 10.78.
N,N’-Bis(4-diethylaminosalicylidene)-1,2-ethylenediamine (H2L2a): 4-(Di-
ethylamino)salicylaldehyde (1 g, 5.2 mmol) in ethanol (5 mL) was added
dropwise to 1,2-diaminoethane (0.16 g, 2.6 mmol) in ethanol (5 mL) at
room temperature. The reaction mixture was gradually heated to 608C
and maintained at this temperature for 4 h. Upon removal of the solvent
and cooling, a yellow precipitate was collected and recrystallized from
ethanol to give the product (0.86 g, 81%). 1H NMR (CDCl3, 300 MHz):
d=12.72 (s, 2H), 8.02 (s, 2H), 6.95 (d, 2H), 6.13 (d, 2H), 6.09 (s, 2H),
3.76 (s, 4H), 3.34 (dd, 8H), 1.16 ppm (t, 12H); 13C NMR (CDCl3,
300 MHz): d=165.15, 164.76, 154.98, 133.37, 108.75, 103.46, 98.58, 58.66,
44.86, 13.12 ppm; MS (FAB, +ve): m/z: 411 [M]+; elemental analysis
calcd (%) for C24H34N4O2: C 70.21, H 8.35, N 13.65; found: C 69.99, H
8.34, N 13.52.
(Denron-G1)OH: Prepared from benzyl bromide (90%). 1H NMR
(CDCl3, 300 MHz): d=7.34–7.43 (m, 10H), 6.68 (s, 2H), 6.62 (s, 1H),
5.04 (s, 4H), 4.62 ppm (d, 2H); MS (FAB, +ve): m/z: 320 [Mꢁ1]+.
(Denron-G2)OH: Prepared from [Denron-G1]-Br (67.2%). 1H NMR
(CDCl3, 300 MHz): d=7.33–7.42 (m, 20H), 6.68 (s, 4H), 6.60 (s, 2H),
6.59 (s, 2H), 6.56 (s, 1H), 5.03 (s, 8H), 4.97 (s, 4H), 4.6 ppm (d, 2H); MS
(FAB, +ve): m/z: 745 [M+1]+.
General procedure for the synthesis of dendritic benzyl bromides: Tri-
phenylphosphine (1.25 equiv) was added to a mixture of the appropriate
dendritic benzyl alcohol (1.00 equiv) and carbon tetrabromide (1.25
equiv) in dry THF, and the reaction mixture was stirred in a nitrogen at-
mosphere for 20 min. The reaction mixture was poured into water and
extracted with CH2Cl2. The combined extracts were dried and evaporated
to dryness. The crude product was crystallized from ethyl acetate/hexane.
(Denron-G1)Br: Prepared from [Denron-G1]-OH (89.2%). 1H NMR
(CDCl3, 300 MHz): d=7.34–7.43 (m, 10H), 6.64 (s, 2H), 6.55 (s, 1H),
5.03 (s, 4H), 4.41 ppm (d, 2H); MS (FAB, +ve): m/z: 384 [M]+.
(Denron-G2)Br: Prepared from [Denron-G2]-OH (75.1%). 1H NMR
(CDCl3, 300 MHz): d=7.33–7.40 (m, 20H), 6.66 (s, 4H), 6.62 (s, 2H),
6.61 (s, 2H), 6.57 (s, 1H), 5.03 (s, 8H), 4.36 (s, 4H), 4.40 ppm (d, 2H);
MS (FAB, +ve): m/z: 809 [M+2]+.
ACHTUNGTRENNUNG
[PtL2a] (2a): Sodium acetate (0.02 g, 0.24 mmol) was added to H2L2a
(0.05 g, 0.12 mmol) dissolved in MeCN (10 mL), and the solution was
stirred for 5 min. K2PtCl4 (0.05 g, 0.12 mmol) and DMSO (1 mL) were
added to the reaction mixture, which was heated to 408C and stirred
overnight. After cooling to room temperature, the solvent was removed
under vacuum. A yellow precipitate was collected and dissolved in
CH2Cl2 (10 mL). The organic layer was washed with water (3ꢂ10 mL),
dried over Na2SO4, and concentrated to give a yellow solid (14 mg,
(Denron-G1)salicylic aldehyde: K2CO3 (1.38 g, 0.01 mol) and KI (16.6 g,
0.1 mol) were added subsequently to a solution of 2,5-dihydroxybenzylal-
dehyde (1.38 g, 0.01 mol) and [Denron-G1]-Br (3.85 g, 0.01 mol) in ace-
tone. The suspension was heated to reflux for 6 h. After cooling to room
temperature, CH2Cl2, and H2O were added, the layers were separated,
and the aqueous layer was extracted. After drying over MgSO4, the sol-
vent was evaporated under vacuum and the resulting residue was purified
23%). H NMR (CDCl3, 300 MHz): d=7.75 (s, 2H), 7.07 (d, 2H), 6.46 (s,
2H), 6.09 (d, 2H), 3.64 (s, 4H), 3.33 (dd, 8H), 1.16 ppm (t, 12H); MS
(FAB, +ve): m/z: 603 [M]+; elemental analysis calcd (%) for
C24H32N4O2Pt: C 47.76, H 5.34, N 9.28; found: C 47.67, H 5.38, N 9.31.
by column chromatography (hexane/ethyl acetate=3:1) to afford
a
yellow solid (1.33 g, 30%). 1H NMR (CDCl3, 300 MHz): d=7.31–7.42
(m, 10H), 7.19 (m, 2H), 6.84 (d, 1H), 6.65 (s, 2H), 6.57 (s, 1H), 5.03 (s,
4H), 4.93 ppm (s, 2H).
N,N’-Bis(4-diethylaminosalicylidene)-1,1,2,2-tetramethylethylenediamine
(H2L2b): 4-(Diethylamino)salicylaldehyde (0.51 g, 2.6 mmol) in ethanol
(5 mL) was added dropwise to 2,3-diamino-2,3-dimethylbutane (0.16 g,
1.3 mmol) in ethanol (5 mL) at room temperature. The reaction mixture
was gradually heated to 608C and maintained at this temperature for 4 h.
Upon removal of the solvent and cooling, a yellow precipitate was col-
lected and recrystallized from ethanol to give the product (0.34 g, 56%).
1H NMR (CDCl3, 300 MHz): d=7.93 (s, 2H), 6.92 (d 2H), 6.12 (d, 2H),
(Denron-G2)salicylic aldehyde: K2CO3 (0.15 g, 1.1 mmol) and KI (1.6 g,
0.01 mol) were subsequently added to a solution of 2,5-dihydroxybenzy-
laldehyde (0.15 g, 1.1 mmol) and [Denron-G2]-Br (0.88 g, 1.1 mmol) in
acetone and the suspension was heated to reflux for 6 h. After cooling to
room temperature, CH2Cl2 and H2O were added, the layers were separat-
ed, and the aqueous layer was extracted. After drying over MgSO4, the
13018
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 13008 – 13021

Investigation of Luminescent Platinum(II) Schiff Base Complexes
FULL PAPER
solvent was evaporated under vacuum and the resulting residue was puri-
fied with column chromatography (hexane/ethyl acetate=3:1) to afford a
yellow solid (0.272 g, 63.1%). 1H NMR (CDCl3, 300 MHz): d=7.32–7.38
(m, 20H), 7.21 (m, 2H), 6.82 (d, 1H), 6.66 (s, 6H), 6.55 (s, 3H), 5.00 (s,
8H), 4.94 (s, 4H), 4.41 ppm (d, 2H).
ACHTUNGTRENNUNG
N,N’-BisACHTUNGTRENNUNG[(G-1)dendritic salicylidene]-1,1,2,2-tetramethylethylenediamine]
(H2L2c): A solution of (G-1)dendritic salicylic aldehyde (0.6 g, 1.4 mmol)
and 2,3-diamino-2,3-dimethylbutane (83 mg, 0.7 mmol) in ethanol
(15 mL) was heated to reflux for 5 h. The solvent was evaporated and the
residue was redissolved in diethyl ether. After the organic layer was
washed with H2O and brine and dried over MgSO4, the solvent was
evaporated and the residue was purified by column chromatography
(hexane/CH2Cl2 =2:1) to afford the product (0.24 g, 36%). 1H NMR
(CDCl3, 300 MHz): d=8.30 (s, 2H), 7.33–7.40 (m, 20H), 7.21 (s, 2H),
concentrated to give
a
red solid (0.1 g, 51%). 1H NMR (CDCl3,
300 MHz): d=8.50 (s, 2H), 7.23 (d, 2H), 7.08 (s, 2H), 6.71 (s, 2H), 6.29
(d, 2H), 4.11 (t, 4H), 3.73 (s, 6H), 2.80 (t, 4H), 2.53 (t, 8H), 1.60 (m,
8H), 1.46 ppm (m, 4H); MS (FAB, +ve): m/z: 824 [M]+; elemental anal-
ysis calcd (%) for C36H44N4O6Pt: C 52.48, H 5.38, N 6.80; found: C 52.44,
H 5.38, N 6.78.
Polymerase stop assay: The polymerase stop assay was performed by
using a modified protocol of the previously reported method.[19a] The re-
actions were performed in 1x PCR buffer, containing each pair of oligo-
mers (G4A1 or G4A2; 10 mmol), deoxynucleotide triphosphate (dNTP;
0.16 mm), Taq polymerase (2.5 U), and increasing concentrations of the
platinum(II) complex (from 1.25 to 50 mm). The reaction mixtures were
incubated in a thermocycler under the following cycling conditions: 948C
for 3 min followed by 30 cycles of 948C for 30 s, 588C for 30 s, and 728C
for 30 s. The amplified products were resolved on 15% nondenaturing
polyacrylamide gels in 1ꢂtris/borate/EDTA (TBE) buffer and silver
stained.
6.96 (d, 2H), 6.88ACHTUNGTRENNUNG(s, 2H), 6.81ACHTUNRTGENN(GUN s, 4H), 6.57 (d, 2H), 5.03 (s, 8H), 4.97ACTHNUGTREN(NGUN s,
4H), 1.26 ppm (s, 12H); MS (FAB, +ve): m/z: 962 [M+1]+; elemental
analysis calcd (%) for C62H60N2O8: C 77.48, H 6.29, N 2.91; found: C
75.51, H 6.35, N 3.23.
ACHTUNGTRENNUNG
[PtL2c] (2c): K2PtCl4 (37 mg, 0.09 mmol) and NaOAc (15 mg, 0.18 mmol)
were added to a solution of H2L2c (85.5 mg, 0.09 mmol) in MeCN (5 mL).
DMSO (1 mL) was added to the reaction mixture, which was heated to
408C and stirred overnight. The solvent was evaporated and the residue
was dissolved in CH2Cl2 (10 mL). After the organic layer was washed
with H2O and brine and dried over MgSO4, the solvent was evaporated
and the residue was purified by column chromatography (hexane/
CH2Cl2 =1:1) to afford a yellow solid (14 mg, 13.6%). 1H NMR (CDCl3,
300 MHz): d=8.16 (s, 2H), 7.33–7.42 (m, 20H), 7.22 (s, 2H), 7.10 (d,
NMR spectroscopic experiments: The NMR titration experiment was
conducted in phosphate buffer (H2O/D2O (90:10) with KCl (150 mm),
KH2PO4 (25 mm), and EDTA (1 mm); pH 7.0). G4A3-quadruplex DNA
in buffer was titrated with a platinum(II) complex solution (10 mm in
[D6]DMSO). The spectra were recorded on 600-MHz Bruker spectrome-
ters at 258C.
2H), 6.91ACHTUNGTRENNUNG(s, 2H), 6.75 (s, 4H), 6.60 (d, 2H), 5.03 (s, 8H,), 4.92 (s, 4H),
1.26 ppm (s, 12H); MS (FAB, +ve): m/z: 1152 [Mꢁ1]+; elemental analy-
sis calcd (%) for C62H58N2O8Pt: C 64.52, H 5.06, N 2.43; found: C 64.44,
H 5.04, N 2.40.
Molecular modeling: A model study on the stacking interactions between
1a/3 and G-quadruplex DNA was performed by using Gaussian 03.[36]
The platinum complex was optimized by using DFT calculations with a
LanL2MB basis set.[35] The optimized structure of the platinum complex
was used to do the docking. Molecular docking was performed by using
the ICM-Pro 3.4–8a program (Molsoft).[30] According to the ICM
method, the molecular system was described by using internal coordi-
nates as variables. Energy calculations were based on the ECEPP/3 force
field with a distance-dependent dielectric constant. The biased probabili-
ty Monte Carlo (BPMC) minimization procedure was used for global-
energy optimization. The BPMC global-energy-optimization method con-
sists of 1) a random conformation change of the free variables according
to a predefined continuous probability distribution; 2) local-energy mini-
mization of analytical differentiable terms; 3) calculation of the complete
energy including nondifferentiable terms such as entropy and solvation
energy; 4) acceptance or rejection of the total energy based on the Met-
ropolis criterion and return to step (1). The binding between 1a/3 and
DNA was evaluated by binding energy, including grid energy, continuum
electrostatic, and entropy terms. The initial models of loop isomers A
and B were built from the X-ray crystal structures of human intramolecu-
lar telmoeric G quadruplex (PDB code: 1 KF1),[25] according to a previ-
ously reported procedure. Hydrogen and missing heavy atoms were
added to the receptor structure followed by local minimization by using
the conjugate gradient algorithm and analytical derivatives in the internal
coordinates. In the docking analysis, the binding site was assigned across
the entire structure of the DNA molecule. The ICM docking was per-
formed to find the most favorable orientation. The resulting trajectories
of the complex between 1a/3 and G-quadruplex DNA were energy mini-
mized, and the interaction energies were computed.
N,N’-BisACHTUNGTRENNUNG[(G-2)dendritic salicylidene]-1,1,2,2-tetramethylethylenediamine]
(H2L2d): A solution of (G-2)dendritic salicylic aldehyde (0.28 g, 0.32 mol)
and 2,3-diamino-2,3-dimethylbutane (20 mg, 0.16 mmol) in EtOH (5 mL)
was heated to reflux for 5 h. The solvent was evaporated and the residue
was dissolved in diethyl ether. After the organic layer was washed with
H2O and brine and dried over MgSO4, the solvent was evaporated and
the residue was purified by column chromatography (hexane/CH2Cl2 =
1:1) to afford the product (30 mg, 10.4%). 1H NMR (CDCl3, 300 MHz):
d=8.20 (s, 2H), 7.26–7.38 (m, 40H), 7.08 (d, 2H), 6.68 (m, 4H), 6.66 (m,
10H), 6.56 (m, 8H), 5.01 (s, 16H), 4.96 (s, 8H), 4.63 (s, 4H), 1.25 ppm (s,
12H); MS (FAB, +ve): m/z: 1811 [M+1]+; elemental analysis calcd (%)
for C118H108N2O16: C 78.30, H 6.01, N 1.55; found: C 78.13, H 6.23, N
1.53.
ACHTUNGTRENNUNG
[PtL2d] (2d): K2PtCl4 (42 mg, 0.1 mmol) and NaOAc (16 mg, 0.2 mmol)
were added to a solution of H2L2d (0.24 g, 0.1 mmol) in MeCN (5 mL).
DMSO (1 mL) was added to the reaction mixture, which was heated to
408C and stirred overnight. The solvent was evaporated and the residue
was dissolved in CH2Cl2 (10 mL). After the organic layer was washed
with H2O and brine and dried over MgSO4, the solvent was evaporated
and the residue was purified by column chromatography (hexane/
CH2Cl2 =1:1) to afford
a
yellow product (83.2 mg, 32%). 1H NMR
(CDCl3, 300 MHz): d=8.30 (s, 2H), 7.60–7.26 (m, 40H), 7.09 (d, 2H),
6.92 (s, 2H), 6.76 (m, 2H), 6.67 (m, 12H), 6.57 (m, 6H), 5.91 (m, 16H),
4.84 (s, 8H), 4.54 (s, 4H), 1.26 ppm (s, 12H); MS (FAB, +ve): m/z: 2002
[M]+; elemental analysis calcd (%) for C118H106N2O16Pt·2H2O: C 69.50,
H 5.44, N 1.37; found: C 69.49, H 5.40, N 1.35.
N,N’-Bis{4-[1-(2-propylpiperidine)oxy]salicylidene}-4,5-methoxy-1,2-phe-
nylenediamine (H2L3): 4-[1-(2-Propylpiperidine)oxy]salicylaldehyde (1 g,
4.0 mmol) in ethanol (5 mL) was added to 4,5-dimethoxy-o-phenylenedi-
amine (0.34 g, 2.0 mmol) in ethanol (20 mL) at room temperature. The
reaction mixture was gradually heated to 608C and maintained at this
temperature for 4 h. Upon removal of the solvent and cooling, a yellow
precipitate was collected and recrystallized from ethanol to give the
product (0.58 g, 46%). 1H NMR (CDCl3, 300 MHz): d=13.75 (s, 2H),
8.47 (s, 2H), 7.03 (d, 2H), 6.76 (s, 2H), 6.40–6.35 (m, 4H), 4.12 (t, 4H),
3.84 (d, 6H), 2.76 (t, 4H), 2.49 (t, 8H), 1.58 (m, 8H), 1.44 ppm (m, 4H);
MS (FAB, +ve): m/z: 630 [M]+.
Absorption titration: Solutions of the platinum(II) complexes (50 mm)
were prepared in Tris/HCl buffer (10 mm; pH 7.4) containing KCl buffer
(100 mm), and aliquots of a millimolar stock solution of G4A1 DNA in
Tris/KCl buffer (0–500 mm) were added. Absorption spectra were record-
ed in the spectral range l=200–600 nm after equilibration at 20.08C for
10 min.
Cell line: Human normal lung fibroblast (CCD-19Lu), human hepatocar-
cinoma (HepG2), human large-cell lung carcinoma (NCI-H460), and
human cervical cancer (HeLa) were obtained from the American Type
Chem. Eur. J. 2009, 15, 13008 – 13021
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
13019

C.-M. Che et al.
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RNA extraction: Total RNA was extracted using the RNeasy mini kit.
Briefly, 5ꢂ105 HepG2 cells were seeded in six-well plates and treated
with the indicated concentrations of metal complexes after 24 h. The
drug-treated and untreated cells were harvested after 24 h. The cell pel-
lets were lysed and RNA was extracted according to the manufacturerꢀs
instruction and eluted in RNase-free water (50 mL).
RT-PCR: Reverse transcription was carried out by incubating RNA
(1 mg), random primers (100 ng), and dNTPs (0.5 mm) at 658C for 5 min,
followed by a further incubation with SuperScript II Reverse Transcrip-
tase (200 U) in 1ꢂfirst-strand buffer at 428C for 50 min. PCR amplifica-
tions were performed in a reaction containing cDNA (1 mL), dNTPs
(200 mm), Taq DNA polymerase (2.5 U; Qiagen), 1ꢂPCR buffer, and an
aliquot of each primer (0.5 mm). PCR was performed for 22 cycles: 958C
for 15 s, 588C for 30 s, and 728C for 30 s. The samples were heated at
958C for 5 min before the first cycle, and the extension time was length-
ened to 5 min during the last cycle. The PCR products were size fractio-
nated on a 1.3% agarose gel.
Cytotoxicity test (MTT Assay): The cells were seeded in a 96-well flat-
bottomed microplate at 8000 cellswellꢁ1 in growth medium solution
(100 mL; fetal bovine serum (10%), l-glutamine (1%), and Penicillin
streptomycin (1%) in RPMI-1640 medium or minimum essential
medium). Each of the platinum(II) complexes and cisplatin (positive con-
trol) were dissolved in DMSO and mixed with the growth medium (final
concentration: ꢄ1% DMSO). Serial dilution of each complex was added
to each well. The microplate was incubated at 378C in CO2/air (95:5) in a
humidified incubator for 48 h. After incubation, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrasodium bromide (MTT) (10 mL, 5 mgmLꢁ1) was
added to each well. The microplate was reincubated at 378C in 5% CO2
for 4 h. Solubilization solution (100 mL; sodium dodecyl sulphate (SDS;
10%) in HCl (0.01m)) was added to each well. The microplate was left in
an incubator for 24 h. The absorbance at l=580 nm was measured by a
microplate reader. The IC50 values of the platinum(II) complexes (the
concentration that is required to reduce the absorbance by 50% relative
to the controls) were determined by the dose dependence of the surviv-
ing cells after exposure to the metal complexes for 48 h.
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Acknowledgements
This work was supported by the Area of Excellence Scheme established
under the University Grants Committee (HKSAR, China (AoE/P-10/01),
the University of Hong Kong (University Development Fund), and The
Chinese Academy of Sciences—Croucher Foundation Funding Scheme
For Joint Laboratories.
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Investigation of Luminescent Platinum(II) Schiff Base Complexes
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