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www.rsc.org/chemicalscience EDGE ARTICLE
Luminescent cyclometalated platinum(II) complexes containing
N-heterocyclic carbene ligands with potent in vitro and in vivo anti-cancer
properties accumulate in cytoplasmic structures of cancer cells†
Raymond Wai-Yin Sun,‡ Andy Lok-Fung Chow,‡ Xiao-Hua Li, Jessie Jing Yan, Stephen Sin-Yin Chui
and Chi-Ming Che*
Received 26th November 2010, Accepted 21st December 2010
Published on 21 January 2011. Downloaded on 14/12/2016 13:41:29.

DOI: 10.1039/c0sc00593b

Contrary to most platinum-based anti-cancer agents which target DNA, coordination of


N-heterocyclic carbene (NHC) ligands to cyclometalated platinum(II) complexes confers these
luminescent complexes to other cellular target(s). The strong Pt–Ccarbene bond(s) renders the
platinum(II) complexes to display unique photophysical properties and enhanced stability against
biological reduction and ligand exchange reactions. The platinum complexes described in this work are
highly cytotoxic and display high specificity to cancerous cells. Among them,
[(C^N^N)PtII(N,N0 -nBu2NHC)]PF6 (1a, where HC^N^N ¼ 6-phenyl-2,20 -bipyridine) with a lipophilic
carbon chain on the carbene ligand induces apoptosis in cancer cells, demonstrates an enhancing
synergistic effect with cisplatin in vitro, and displays potent in vivo activities using nude mice models. As
this complex is strongly emissive, its cellular localization can be traced using emission microscopy. In
contrast to common platinum-based anti-cancer agents, 1a does not accumulate in the vicinity of DNA
but preferentially accumulates in cytoplasmic structures including sites where active survivin, an
inhibitor of apoptosis (IAP), is located. In vitro, 1a significantly inhibits the expression of survivin,
activates poly(ADP-ribose) polymerase (PARP) and induces apoptosis in cancer cells. Given the ease of
structural modification of NHC ligand to alter the overall biological activities, these
[(C^N^N)PtII(NHC)]+ complexes having unique photophysical properties provide an entry to a new
class of potential anti-cancer drug leads.

Introduction complexes including that of platinum, palladium, gold, rhodium,


silver and copper have been reported to display extraordinary
Since the reports on the preparation and isolation of metal practical applicability especially in the area of catalysis.2 Metal
N-heterocyclic carbene (NHC) complexes derived from azolium complexes of NHC ligands are reminiscent of metal-phosphine
compounds respectively in 1962 and in 1991,1 various NHC complexes,3 but they offer additional advantages including high

Department of Chemistry and Open Laboratory of Chemical Biology of Fig. S9, degassed fluid emission of 1a and 1c; Fig. S10, emission
the Institute of Molecular Technology for Drug Discovery and spectra of 1f; Fig. S11, emission spectra of 1b; Fig. S12, emission
Synthesis, The University of Hong Kong, Pokfulam Road, Hong Kong, spectra of 1f in different solvents; Fig. S13, excitation and emission
China. E-mail: cmche@hku.hk; Fax: +852-2857 1586 spectra of 2a; Fig. S14, excitation and emission spectra of 3a;
† Electronic supplementary information (ESI) available: Detailed Fig. S15, excitation and emission spectra of 1f; Fig. S16, excitation
synthetic procedure and characterization of the precursors and and emission spectra of 2b; Fig. S17, emission spectra of 1f at
platinum(II) complexes; experimental procedures for emission and different concentrations; Fig. S18, cytotoxicity profiles of 1a;
lifetime measurements, single crystal X-ray diffraction, cell culture, Fig. S19, tube-formation assay of 1a; Fig. S20, wound-healing
cytotoxicity evaluation, tube-formation assay, wound-healing assay of 1a; Fig. S21, absorption titration of 1a with ctDNA;
assay, in vivo anti-cancer study, detection of cellular localization of Fig. S22, gel-mobility shift assay of 1a; Fig. S23, viscosity
1a, survivin enzyme immunometric assay, western blotting, measurement of 1a; Fig. S24, topoisomerase I assay of 1a; Table
absorption titration, gel-mobility-shift assay, viscosity S1; calculated lipophilicity of the carbene ligands; Table S2, crystal
measurement and topoisomerase I assay. Fig. S1, Schematic data of 1a, 1c and 1f; Table S3, selected bond distances and bond
drawings of the precursors; Fig. S2, UV-vis spectrum of 1a; angles of 1a, 1c and 1f; Table S4, UV-vis absorption and emission
Fig. S3, molecular diagram of 1f; Fig. S4, VT-NMR spectroscopic data of 1c. CCDC reference numbers 756500, 756501 and 756623.
measurement of 1a; Fig. S5, VT-NMR spectroscopic measurement For ESI and crystallographic data in CIF or other electronic
of 1c; Fig. S6, VT-NMR spectroscopic measurement of 1a; Fig. S7, format see DOI: 10.1039/c0sc00593b
UV-vis spectra of 1b, 2a and 3a; Fig. S8, UV-vis spectra of 1c; ‡ These authors contributed equally to this work.

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thermal stability and stability towards air and moisture,4 and the
coordination of carbene ligand to metal ion can be achieved
under mild experimental conditions.5 Additionally, functionality
and lipophilicity of the carbene ligands could be systematically
modified. In literature, studies on metal-NHC complexes have
mainly been focused on their catalytic properties. Yet, their
biological activities and therapeutic properties, particularly that
of platinum-NHC complexes,6a remain largely unexplored.6
The discovery of cisplatin has stimulated worldwide efforts to
develop new anti-cancer platinum complexes.7 Cisplatin (Plati-
nol), carboplatin (Paraplatin), and recently oxaliplatin have
received worldwide approval for clinical uses.8 However, clinical
studies revealed that the elevated level of biological reductant
glutathione (GSH) in some cancer cells could deactivate these
platinum complexes subsequently leading to the generation of
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drug resistance.9a To circumvent this problem, it is of paramount


importance to identify new compounds which are stable and
display different modes of anti-cancer action. Cyclometalated
platinum(II) complexes containing tridentate p-conjugated
organic ligands have been receiving a surge of interest for their
application in light-emitting devices and chemical sensors.10
These complexes display rich and diverse photoluminescent
properties that are sensitively affected by local medium. The
planar motifs of these platinum(II) complexes could insert
between two adjacent DNA base pairs through non-covalent Fig. 1 [(C^N^N)PtII(NHC)]+ complexes.
ligand–ligand p–p stacking interactions, thus rendering them as
DNA metallointercalators instead of covalently cross-linking to X-Ray crystallography
the DNA base pairs.11 Extensive studies have revealed that Crystals of 1a, 1c and 1f (Fig. 2) were obtained by slow diffusion
various platinum(II) intercalators display promising in vitro and of diethyl ether into CH3CN solutions, and crystallographic
in vivo anti-cancer activities.12 To synergize the advantages of the data of 1a, 1c and 1f (Table S2, ESI) and bond angles/distances
NHC ligands (ease in structural modification, capability of
forming stable metal complexes and their relative non-toxic
nature) and of the cyclometalated platinum(II) complexes
(luminescent properties and DNA intercalation), we developed
[(C^N^N)PtII(NHC)]+ complexes which display intriguing pho-
toluminescent properties as well as anti-cancer activities.

Results
Synthesis, characterization and photophysical properties
The [(C^N^N)PtII(NHC)]+ complexes (Fig. 1) were prepared
from precursors P1–P10 having a dynamic range of lipophilicity
(Fig. S1 and Table S1, Electronic Supporting Information†).13
These complexes were characterized by FAB-MS, 1H NMR and
13
C NMR spectroscopies, and elemental analyses. They display
a good solubility (>10 mg mL1) in dichloromethane (CH2Cl2),
methanol (CH3OH), acetonitrile (CH3CN), and dimethyl sulf-
oxide (DMSO), but are less soluble (<0.5 mg mL1) in water.
They are stable at room temperature for at least one month as
confirmed by NMR spectroscopy. Using 1a as an example, we
examined the stability of these complexes against reduction/
substitution by glutathione (GSH) by means of UV-visible
spectrophotometry. We found that treating 1a with GSH at 2
mM in PBS/DMSO (19 : 1) did not cause significant spectral
changes (<5%) upon standing the solution for 72 h (Fig. S2,
ESI†). In contrast, cisplatin has been reported to be highly Fig. 2 Perspective views of the X-ray crystal structures of the cations of
unstable in the presence of glutathione.9 1a (upper left), 1c (upper right), and 1f (bottom).

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(Table S3, ESI) are given in the Supporting Information.† A Table 1 UV-visible absorption data of 1a–1f, 2a, 2b, 3a and 3b (2  105
distorted square-planar geometry is revealed from the trans mol dm3 in CH3CN)
C–Pt–N angles of 160.5(3) in 1a and of 159.5(3) in 1c. The Complex labs/nm (3/dm3 mol1 cm1)
NHC ligands in both 1a and 1c are perpendicular to the
[(C^N^N)PtII]+ plane with an angle of 93.0 . Intermolecular 1a 270 (60300), 337 (12700), 354
Pt–Pt distances are 8.87 and 8.65 A  for 1a and 1c, respectively. (11600), 368 (5120), 392 (2730),
430 (1520), 485 (1030)
Complex 1f has two NHC planes at torsion angles of 103.17 and 1b 255 (49500), 266 (52900), 316
107.81 from the C^N^N planes. This complex displays intra- (17200), 330 (19000), 337
molecular interaction between the two [(C^N^N)PtII]+ planes as (20700), 353 (19100), 369 (9060),
 and the p–p separation
revealed by the Pt–Pt distance of 3.536 A 392 (3720), 484 (330)
 (Fig. S3, 1c 251 (38600), 255 (39800), 266
between two [(C^N^N)PtII]+ planes of 3.435 and 3.495 A (43300), 312 (13000), 335
ESI†). The Pt–Ccarbene distances in 1a and 1c are 1.984(7) and (15000), 352 (13700), 394 (2600),
 respectively, while 1f has two Pt–Ccarbene distances of
2.016(9) A, 429 (1250), 485 (750)
 1d 252 (34900), 255 (35900), 266
1.968(11) and 1.974(12) A. (37600), 312 (11800), 337
(13600), 352 (12600), 392 (2480),
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429 (1220), 485 (640)


Variable-temperature 1H NMR spectroscopic measurements 1e 254 (49600), 265 (48400), 334
(18200), 354 (15800), 384 (5750),
As revealed from X-ray crystallography, the NHC ligand 418 (3540), 485 (1060)
spatially restrains the mononuclear [(C^N^N)PtII(NHC)]+ com- 1f 255 (38100), 319 (16300), 331
plexes from intermolecular p–p and Pt–Pt interactions. These (16600), 355 (9480), 364 (5570),
380 (6210), 420 (3530), 464
interactions are crucial to the photophysical properties and
(1680), 485 (670)
notably the luminescent properties of these platinum(II) 2a 245 (30500), 287 (36000), 358
complexes. The 1H-NMR spectra of 1a (Fig. S4, ESI) and 1c (14700), 425 (2260), 471 (1290)
(Fig. S5, ESI) in CD3CN at temperature between 233 K and 2b 283 (33300), 322 (16700), 356
(10300), 420 (2850), 487 (1330)
333 K are given in the Supporting Information.† In brief, the 3a 241 (20100), 264 (19400), 287
spectra reveal no significant changes in both structure and (21900), 319 (14800), 355 (8100),
intermolecular interaction(s). Conversely, the 1H-NMR spec- 395 (2090), 433 (870)
trum of 1f in CD3CN is temperature-dependent (Fig. S6, ESI†). 3b 246 (52700), 287 (40300), 320
(25400), 355 (13900), 376 (5850)
For an example, there is a cluster of NMR signals at d ¼ 6.50 at
333 K, whereas these signals are resolved into three sets of
doublet when the temperature is lowered to 233 K.
(Fig. 4) and 1b–1d show well-resolved vibronic structured emis-
sion bands (lex ¼ 340 nm) with emission lmax being insensitive
Absorption and emission spectroscopy
to the complex concentration. For both the mononuclear com-
The UV-visible spectral data of [(C^N^N)PtII(NHC)]+ complexes plexes 1a and 1c, there is no significant change in the emission
are listed in Table 1. The absorption spectra of the mononuclear energies with complex concentration in the range of 1  106 to
complexes 1a–1d (Fig. 3, upper) show intense intraligand tran- 1  104 mol dm3 (Fig. S9, ESI†). There is also no significant
sitions (IL) in the 245–375 nm spectral region, and singlet metal- change in emission energy upon changing the solvent polarity,
to-ligand charge transfer (1MLCT) transitions Pt(5d) / C^N^N while both s0 and F decrease as the polarity of the solvent
(p*) at 375–400 nm.14a When comparing these absorption spectra increases (non-emissive in DMF).
with that of [(C^N^N)PtIICl], substitution of the Cl by NHC The binuclear complexes 1e, 1f, 2b and 3b are emissive both in
ligand leads to blue-shift of 1MLCT transition(s). For complexes solid state and in degassed CH3CN. (Table 2). At room
1b, 2a and 3a, all of which have the same N,N0 -nPr2NHC ligand, temperature, the emission lmax of complexes 1f, 2b and 3b in
the absorption spectra of 2a and 3a are red shifted from that of solutions, all of which have a C1 spacer (methyl linker) on the
1b (Fig. S7, ESI†). It should be noted that although 1e is bridging carbene ligand, are significantly red-shifted compared
a binuclear complex, its absorption spectrum (Fig. 3, lower) is to that of the mononuclear complexes 1b, 2a and 3a, respectively.
similar to those of the mononuclear complexes 1a–1d. However, In contrast, complex 1e which has a C3 spacer (propyl linker)
the absorption spectrum of the binuclear complex 1f, which has displays photophysical properties similar to that of the mono-
a shorter bridging ligand, is red shifted from that of 1a–1d at l > nuclear counterparts in the context of emission energies and s0
370 nm. in solutions. In solid state, 1f shows a broad emission band at
Using 1c as an example, solvatochromism of the 1MLCT 594 nm with a lifetime of 2.1 ms at room temperature. Upon
absorption band of the mononuclear [(C^N^N)PtII(NHC)]+ cooling to 77 K, the band width of the emission decreases and
complexes at around 350 nm in different solvents is observed, the emission is resolved into two peak maxima at 572 nm and
and details of the spectral data are given in the Supporting 615 nm. The emission lmax of 1f (Fig. S10, ESI†) in both solid
Information (Table S4 and Fig. S8†). state (lem ¼ 592 nm) and in CH3CN (lem ¼ 619 nm) are signif-
The mononuclear complexes, 1a–1d, 2a and 3a, are emissive in icantly red-shifted from its mononuclear counterpart 1b (solid:
solid state and in degassed CH3CN with emission lmax at 545 to lem ¼ 553 nm, CH3CN: lem ¼ 546 nm, Fig. S11, ESI†).
546 nm, emission lifetime (s0) of 0.6 to 2.9 ms, and emission Regarding the solvent effect, there is no significant change in the
quantum yield (F) of 0.087 to 0.23 (Table 2). Complexes 1a emission energy upon changing the solvent polarity from

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The emissions of 1a, 1f, 2a–2b and 3a–3b in frozen CH3CN


solutions at 77 K have been studied. The emissions of the
complexes 1a (Fig. 4), 2a (Fig. S13, ESI†) and 3a (Fig. S14,
ESI†) in 77 K CH3CN solutions are insensitive to the complex
concentration from 106 to 104 mol dm3. Vibronically
structured emission bands with lem 527–570 nm and vibra-
tional spacing of ca. 1200 cm1 attributed to the skeletal
stretching of the tridentate HC^N^N ligand were recorded.
The emission lmax of the binuclear complex 1f in 77 K CH3CN
solution is at 634 nm which is red-shifted from that at 298 K
(Fig. S15, ESI†); another binuclear complex 2b also displays
red-shifted emission band at 625 nm (cf., RT: 610 nm) in frozen
CH3CN (Fig. S16, ESI†). The emission properties of all of
the [(C^N^N)PtII(NHC)]+ complexes in glassy solutions
(MeOH–EtOH–DMF ¼ 5 : 5 : 1) at 77 K have also been
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studied (Table 1). The emission of 1f is sensitive to the complex


concentration (106–104 mol dm3, Fig. S17, ESI†). This
complex displays red-shifted emission band with lmax at 621
nm, 617 nm and 612 nm in glassy solution at complex
concentration of 104 mol dm3, 105 mol dm3 and 106 mol
dm3, respectively.

Fig. 3 UV-Visible absorption spectra of 1a–1d (upper) and 1e–1f (lower,


2  105 mol dm3) in CH3CN at 298 K.

CH3CN to CH3OH, acetone, CH2Cl2 and tetrahydrofuran


(Fig. S12, ESI†), but the emission lifetime at 617 nm varies from
1.6 ms to 0.2 ms and emission quantum yield decreases from 0.074
to 0.014 when the solvent is changed from CH2Cl2 to CH3OH.
Such solvatochromic behavior of the metal-metal-to-ligand
charge transfer excited state (3MMLCT) emissions of the dinu-
clear PtII complexes is similar to that of the 3MLCT emission of
the mononuclear complex 1c.14b The emission of 3b in CH3CN is
at an energy similar to that of 3a (3a: 546 nm and 3b: 540 nm; Fig. 4 Emission spectra of 1a in CH3CN at 298 K and 77 K (2  105
Table 2). mol dm3), lex ¼ 340 nm (normalized intensities).

Table 2 Emission data of 1a–1f, 2a, 2b, 3a and 3b

Degassed solution
(CH3CN; 298 K)
lem [nm] (s0 [ms]); F Solid state emission Solid state emission Glassy emission
Complex (quantum yield)a (298 K) lem [nm] (s0 [ms]) b (77 K) lem [nm] (s0 [ms]) b (77 K) lem [nm] (s0 [ms]) c

1a 545 (1.2); 0.23 511, 538 (max, 0.7), 569 524 (max, 7.4), 560 516 (max, 75), 552, 595
1b 546 (1.1); 0.19 527, 553 (max, 0.7), 590 542 (max, 5.0), 581 515 (max, 71), 552, 590
1c 545 (0.9); 0.12 510 (max, 0.6), 542, 582 520 (max, 6.2), 540, 552, 590 515 (max, 64), 551, 595
1d 546 (0.6); 0.087 596 (max, 0.6), 670 564 (max, 5.7), 607, 669 514 (max, 74), 552, 591
1e 546 (0.8); 0.051 574 (1.4) 535 (max, 7.5), 578 456, 487, 516 (max, 59), 549
1f 619 (1.3); 0.056 592 (2.1) 572, 615 (max, 8.2) 525, 617 (max, 251)
2a 546 (1.2); 0.13 557 (1.9) 548 (max, 6.9), 568 521 (max, 112), 557, 602
2b 542, 610 (max, 0.8), 660; 0.031 620 (0.9) 637 (4.6) 511 (max, 232), 539, 620
3a 546 (2.9); 0.11 554 (2.1) 540 (max, 5.8), 580 518 (max, 108),554, 596
3b 540 (max, 0.6), 631, 663; 0.032 605 (0.8) 485 (max, 3.9), 566, 670 513 (max, 101), 547. 583
a
Complexes 1a–1f, 2a, 2b, 3a and 3b were excited at 340 nm. b The emission data were measured by excitation at 350 nm. c The glassy emission data were
measured in a concentration of 2  105 mol dm3 in a MeOH–EtOH–DMF mixture (5 : 5 : 1, v/v).

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Cytotoxicity tests (MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5- (IC50 ¼ 0.49 mM) and 3a (IC50 ¼ 0.27 mM) were more cytotoxic
tetrazolium bromide) Assay) towards HepG2 cells when compared with 1b (IC50 ¼ 1.1 mM).
In addition to the cytotoxicity evaluation, the anti-angiogenic
Since the discovery of the clinically-used cisplatin for anti-cancer
and anti-metastatic activities of 1a have been examined.16 By
treatment, a variety of platinum(II) complexes have been
means of the tube-formation assay on MS1 cells and wound-
identified to display promising anti-cancer activities.7 In this
healing assay on HeLa cells, there was no apparent anti-angio-
study, we examined the in vitro anti-cancer properties of
genic (Fig. S19, ESI†) and anti-metastatic (Fig. S20, ESI†)
[(C^N^N)PtII(NHC)]+ towards human cancer cell lines of
activities of 1a at its sub-toxic concentration.
cervical epithelioid carcinoma (HeLa), hepatocellular carcinoma
(HepG2), and nasopharyngeal carcinoma (SUNE1), and
a normal cell derived human lung fibroblast cell line (CCD-19Lu)
In vivo anti-cancer study
by means of MTT assay.15 Using 1a as an example, the IC50
values (dose required to inhibit 50% cellular growth) were In addition to the promising in vitro anti-cancer activity, the in
determined from the dose-dependence of surviving cancer cells vivo anti-cancer activity of 1a was examined. The experiments
after cellular exposure to 1a for 48 h (Fig. S18, ESI†). Complex were conducted at PearL Materia Medica Development
1a was found to display promising anti-cancer activity towards (Shenzhen) Limited and performed with approval from the
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these three cancer cell lines (IC50 ¼ 0.057 – 0.77 mM), and Committee on the Use of Live Animals for Teaching and
specifically displays higher cytotoxic potency towards HeLa cells. Research. NCI-H460 non-small lung carcinoma cells were first
Notably, this complex is less cytotoxic to the normal-derived implanted into nude mice. When the tumors were approximately
human cell line (CCD-19Lu) with IC50 value of 11.6 mM, which is 50 mm3 in size, nude mice were randomly separated into four
232 folds higher than that towards HeLa cells. For comparison, groups to receive treatment of twice-a-week intraperitoneal
the cytotoxicity of cisplatin was also examined. We found that 1a injection of 20% PET vehicle control (20% PET ¼ 12% poly-
displays 300-fold higher potency toward HeLa than cisplatin. ethylene glycol 400; 6% ethanol; 2% Tween 20; 80% phosphate-
Cisplatin is also less cytotoxic toward CCD-19Lu cells, as its IC50 buffered saline), 1a at 1 mg kg1, 1a at 3 mg kg1 or the positive
value was found to be >100 mM. In addition, 1a was found to control cyclophosphamide at 30 mg kg1. After 28 days, the mice
display a synergistic anti-cancer effect with cisplatin. With the were sacrificed and the tumors were taken out and their weights
co-incubation of cisplatin at fixed concentrations of 0.5 and were measured. Results showed that injection of 1a at 3 mg kg1
5 mM, the IC50 value of 1a against HeLa cells shifted from and cyclophosphamide at 30 mg kg1 significantly inhibited the
0.057 mM, to 0.035 and 0.021 mM, respectively. NCI-H460 tumor growth by 55  11% (0.78  0.19 g) and 56 
The cytotoxicity and IC50 values of other [(C^N^N)PtII- 6% (0.76  0.10 g), respectively (Fig. 5), whereas treatment of 1a
(NHC)]+ complexes including 1b–1f were determined in a similar at 1 mg kg1 was not effective. Importantly, 1a did not cause
manner, and the results are summarized in Table 3. The death of mice, and regular body-weight measurement showed
[(C^N^N)PtII(NHC)]+ complexes are more cytotoxic than
cisplatin; the IC50 of 1c and 1d with NHC ligand having two
N-CH2CH3 and N-CH3 groups, respectively, are at least 31 and
45 fold higher than that of cisplatin towards HeLa. Although the
two dinuclear complexes 1e and 1f both show higher potency
than cisplatin, they are less cytotoxic than the mononuclear
complexes 1a–1d (Table 3). Besides 1a, the cytotoxicities of other
platinum complexes towards normal human lung fibroblast cell
line of CCD-19Lu were also examined. All of these complexes
were found to display lower cytotoxicity to this cell line.
Using human hepatocellular carcinoma cell line (HepG2) as
a model, complexes 1b, 2a and 3a were chosen for examining the
effect of ligand variation on the cytotoxicity via increasing the
lipophilicity of the tridentate C^N^N ligand. We found that 2a

Table 3 Cytotoxic IC50 values (mM) of 1a–1f in human carcinoma cell


lines of HeLa, HepG2, SUNE1 and in a normal derived human lung
fibroblast cell line (CCD-19Lu)

Complex HeLa HepG2 SUNE1 CCD-19Lu

1a 0.057 0.77 0.14 11.6


1b 0.052 1.1 0.16 4.3
1c 0.48 1.3 0.32 2.1
1d 0.33 0.31 0.51 5.7
1e 3.9 7.1 5.6 27
Fig. 5 (A) Statistical representation of the tumor weights and (B)
1f 8.0 9.4 6.4 40
Cisplatin 15 15 2.4 >100 photographs showing the reduction of NCI-H460 tumor in size upon
treatment of 1a in nude mice models.

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that mice receiving 1a (32.6  5.1 g) had no significant weight loss


compared to that in the vehicle control group (34.5  4.7 g).

Localization in cancer cells


In view of the favorable emission properties of the cyclo-
metalated [(C^N^N)PtII(NHC)]+ complexes, HeLa cells treated
with 1a (1 mM) for 1 h were imaged by fluorescence microscope
(Fig. 6, left column).17 To ascertain the site of cellular localiza-
tion, the 1a-treated cells were co-stained with other known
fluorescent dyes including the mitotrackerTM (for mitochondria
and cytoplasmic structure), Hoechst 3342 (for DNA) and lyso-
trackerTM (for lysosomes) (Fig. 6, middle column). We found that
majority of 1a can be co-localized with mitotracker (Fig. 6, right
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column). In contrast, 1a did not co-localize with the DNA binder


Hoechst 33342 and the lysosome binder lysotrackerTM.

Inhibition of survivin and activations of caspases and poly(ADP-


ribose) polymerase
Survivin, an inhibitor of apoptosis (IAP), is selectively expressed
in most human cancers and is associated with tumor progression
in patients.18 Importantly, survivin expression has been reported
at low or non-detectable levels in normal tissue, rendering this
protein to serve as an important target for anti-cancer treat-
ment.19 As described in the previous section, 1a was found to
accumulate in the cytoplasmic structure of cancer cells where
activated survivin is located. By means of a survivin enzyme
immunometric assay,20 treatment of 1a in HeLa cells time-
(24 and 48 h) and dose- (0.1 and 1 mM) dependently inhibited the
survivin expression (Fig. 7A). We further confirmed the expres-
sion of survivin by Western blotting (Fig. 7B). In concomitance
with the survivin inhibition, 1a was able to activate caspase-3,
and could activate poly(ADP-ribose) polymerase (PARP) in
HeLa cells in a time- and dose-depending manner (Fig. 7C).

Fig. 7 Inhibition of the (A) activity and (B) expression of survivin, and
(C) activation of caspase 3 and PARP-1 in HeLa cells treated with 1a.

Interaction with double-stranded DNA


Platination of DNA is widely believed to account for the anti-
cancer effect of cisplatin as well as various platinum(II)
complexes. Yet in our study, we found that 1a preferentially
accumulates in the cytoplasmic structure of the cells instead of
covalently binding to nuclear DNA (Fig. 6). In cell-free condi-
tions, 1a was found to display poor binding affinity to double
stranded DNA. The interaction between 1a and calf-thymus
DNA (ctDNA) in PBS/DMSO (19 : 1) solution was examined by
means of UV-visible absorption titration experiment. Isosbestic
Fig. 6 Fluorescent microscopic examination of 1a in the same batch of spectral changes and hyperchromicity (15%) were observed
HeLa cells either co-incubated with mitotrackerTM (top), Hoechst 33342 for the electronic transitions of the [(C^N^N)PtII]+ moiety at
(middle) or lysotrackerTM (bottom), showing that the majority of 1a can 280–320 nm upon addition of ctDNA to a solution of 1a
be co-localized with mitotracker. (Fig. S21, ESI†). The binding constant (Kb) of 1a toward ctDNA

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was determined, from the plot of [ctDNA]/D3ap versus [ctDNA], intramolecular conformational change25 with much closer
to be 4.8  103 dm3 mol1.21 intramolecular contact between two [(C^N^N)PtII]+ planes at low
A gel-mobility-shift assay was employed to examine the temperature, thus accounting for the different emission behav-
intercalating property of 1a.22 A 100-bp DNA ladder treated with iors when compared to the mononuclear complexes 1a and 1c.
1a or ethidium bromide (DNA intercalator) was resolved by At l < 400 nm, the absorption spectra of the [(C^N^N)PtII-
agarose-gel electrophoresis (Fig. S22, ESI†). Only samples that (NHC)]+ complexes are dominated by intense 1IL transitions;
contained EB (lanes B and C) or 1a (lanes D–G) exhibited there are less intense absorption bands with 3 values of 300–
a tailing effect, which can be accounted for by the elongation of 3900 dm3 mol1cm1 in the 400–500 nm spectral region. It should
DNA resulting from the intercalation of EB or 1a with DNA. In be noted that [PtII(C^N^N)Cl], the parental precursor for the
contrast, DNA treated with vehicle control (lanes A and H) did [(C^N^N)PtII(NHC)]+ complexes, displays a much lower molar
not reveal the apparent tailing effect. By viscosity analysis,22 we absorption coefficient in the spectral region of 400–500 nm.24
further confirmed that 1a could act as a DNA metal- With reference to previous work on binuclear platinum(II)
lointercalator. Addition of either EB or 1a increases the viscosity complexes bearing p-conjugated C- and N-donor ligands having
of the DNA by increasing its hydrodynamic length (Fig. S23, metal–metal and ligand–ligand interactions,24,26 the absorption
ESI†). In contrast, Hoechst 33342 (minor groove binder) and the bands of 1f and 2b at 410–490 nm are tentatively assigned to
Published on 21 January 2011. Downloaded on 14/12/2016 13:41:29.

vehicle control failed to lengthen the DNA and did not cause any metal-metal-to-ligand charge transfer 1MMLCT 1[ds* / s (p*)]
changes in DNA viscosity. and intraligand 1[s*(p) / s (p*)] transitions.24,26 Although 1e is
In addition to the interaction with double stranded DNA, the a binuclear complex, its absorption spectrum is similar to those
inhibitory activity of 1a on topoisomerase I (TopoI), a DNA of the mononuclear complexes 1a–1d. Thus, the longer carbon
binding protein which catalyzes topological changes in DNA by chain of the bridging carbene ligand in 1e renders larger spatial
the formation of DNA strand breaks, was examined.23 TopoI separation between two [(C^N^N)PtII]+ units resulting in the
induced formation of nicked or relaxed supercoiled forms of latter behaving as two independent non-interacting cations.
tertiary DNA structure which could be inhibited in the presence With reference to previous work on degassed CH3CN solu-
of TopoI poison camptothecin (CPT, Fig. S24, ESI†). However, tions of [(C^N^N)PtII(PPh3)]+,24 the structureless emission bands
co-incubation of 1a at concentrations up to 1 mM with TopoI of 1a–1d at 545 nm (RT) are assigned to excited states with mixed
3
could not inhibit the protein activity since both the nicked- and MLCT and 3IL characters. Their emission quantum yields of
relaxed forms of the supercoiled DNA were still detected. 0.087 to 0.23 are higher than that of [(C^N^N)PtII(PPh3)]+ (cf., F
¼ 0.062) in CH3CN under similar conditions. The emission
energies of 1f and 2b are significantly red-shifted from their
Discussion
corresponding mononuclear complexes 1b and 2a, revealing that
In this work, the cyclometalated platinum(II) complexes bearing the intramolecular Pt–Pt and p–p interactions in 1f and 2b
N-heterocyclic carbene (NHC) ligand [(C^N^N)PtII(NHC)]+ are account for the low lying 3MMLCT excited states.24 On the other
lipophilic cations having a range of lipophilicity attributed to the hand, for 1f and 2b in 77 K-frozen CH3CN solutions and at
different substituents on NHC or [C^N^N] ligands. X-ray higher concentrations (>104 mol dm3), the observed red-shifted
crystal structures revealed that the Pt–Ccarbene distances of emissions are tentatively ascribed to excimeric intraligand excited
[(C^N^N)PtII(NHC)]+ complexes are shorter than that of Pt–L states27 arising from weak p-stacking interactions between the
distances in related [PtII(C^N^N)L]n+ complexes (where L ¼ C^N^N ligands.
halide, phosphine or amine).14,24,26 The strongly coordinating tri- A strategy in the design of anti-cancer agents is to view the
dentate C^N^N and carbene ligands account for the stability of [(C^N^N)PtII(NHC)]+ complexes as planar lipophilic cations.
the platinum(II) complexes against GSH reduction/substitution in The use of planar p-conjugated organic cations to target mito-
solutions. The stability under physiological conditions could be chondria in cancer treatment has previously been proposed.28a As
a critical factor for new drug design since elevated cellular GSH compared to the clinically-used platinum drugs cisplatin, car-
level has been implicated in cisplatin-resistant cancer cells boplatin and oxaliplatin, the [(C^N^N)PtII(NHC)]+ complexes
presumably through sequestration of cisplatin.9 are stabilized by the tridentate C^N^N and NHC ligands,
Variable temperature-1H NMR experiments showed that there rendering them to have a much higher kinetic stability in phys-
is no close intermolecular interaction between the [(C^N^N)PtII- iological conditions.
(NHC)]+ cations at temperature from 233 K to 333 K. As We have examined the cytotoxicity of the [(C^N^N)PtII-
revealed from the X-ray crystal structures of 1a and 1c, the (NHC)]+ complexes toward a panel of cancer cell lines by means
orientation of the NHC ligand disfavors the approach of two of MTT assay.15 These complexes, notably 1a, were found to
[(C^N^N)PtII]+ planes in close proximity, accounting for the exhibit potent in vitro anti-cancer activities and display higher
absence of intermolecular Pt–Pt and p–p interactions. Thus, potencies than the clinically-used cisplatin. Complex 1a showed
these mononuclear complexes have a low tendency to aggregate high specificity to fast-growing HeLa cells as revealed from the
in solutions and this is important as aggregation of the plati- smallest cytotoxic IC50 value toward this cell line. This complex
num(II) complexes could impede the entrance to cancerous cells. also displays synergistic effect with cisplatin as indicated from
In contrast, the short bridging carbene ligand of 1f confines the the change in the IC50 value in the presence of a fixed concen-
two [(C^N^N)PtII]+ planes in close proximity rendering intra- tration of cisplatin, implying that 1a and cisplatin exert different
molecular Pt–Pt interactions feasible. Together with the changes anti-cancer mechanism(s). Complex 1a is relatively less cytotoxic
in chemical shifts of 1H NMR signals and emission properties to the human cell line derived by normal lung fibroblast cells
at various temperature, we reckon that 1f displays fluxional (CCD-19Lu), rendering this complex to have safety therapeutic

734 | Chem. Sci., 2011, 2, 728–736 This journal is ª The Royal Society of Chemistry 2011
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windows (at ranges of dose which is cytotoxic to cancer cells synthesized and characterized. These complexes are strongly
only). Furthermore, by means of tube-formation and wound- emissive and stable under physiological conditions. Among the
healing assays, we found that 1a displays no apparent anti- complexes examined, 1a is the most potent anti-cancer agent with
angiogenic and anti-metastatic activity at its sub-cytotoxic cytotoxic activity higher than that of the clinically-used cisplatin
concentrations, suggesting that the anti-cancer activity of 1a under in vitro conditions and could significantly inhibit tumor
is dominated by its cytotoxic property. When comparing the growth in the nude mice model. Complex 1a preferentially
cytotoxicity values of 1a–1d, we found that lengthening the accumulates in cytoplasmic structures, and could act as a survi-
hydrophobic carbon chain of NHC ligand (e.g., 1a and 1b) vin suppressant in triggering activation of poly(ADP-ribose)
confers higher lipophilicity to the complex thus facilitating the polymerase (PARP) and hence apoptosis in cancer cells. It is
cellular uptake of the metal complex by cancer cells.28b The in vivo conceived that this class of anti-cancer platinum(II) complexes
anti-cancer activity of 1a was examined. Complex 1a could [(C^N^N)PtII(NHC)]+ may complement the clinically used
significantly inhibit tumor growth in vivo with no significant cisplatin by offering new modes of mechanism.
reduction in body weight of the examined mice, and no induction of
acute toxicity to the nude mice. These promising in vivo data Acknowledgements
warrant evaluation of the anti-cancer efficacy of 1a.
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Due to their strong emission properties, the localization of the We acknowledge support from the ITF-Tier 2 project (ITS/134/
[(C^N^N)PtII(NHC)]+ complexes in cancer cells can be traced 09FP) administrated by Innovation and Technology Commis-
using emission microscopy. Using 1a as an example, we found sion (HKSAR, China), and the Areas of Excellence Program
that this complex mainly co-localized with mitotracker, revealing (AoE/P-10/01) administrated by University Grants Council
that 1a could accumulate in the cytoplasmic structures. In (HKSAR, China). We thank Drs J.-S. Huang and C.-N. Lok for
contrast, this complex did not co-localize with the known fluo- their helpful discussion to this project.
rescent DNA binder and we found that DNA (using calf thymus
DNA as an example) does not quench the emission of 1a (data Notes and references
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736 | Chem. Sci., 2011, 2, 728–736 This journal is ª The Royal Society of Chemistry 2011