Gold(I) complexes derived from alkynyloxy-substituted anthraquinones: Syntheses, luminescence, preliminary cytotoxicity, and cell imaging studies

Article abstract of DOI:10.1021/om300475y

A series of mono- and dimetallic Au(I) triphenylphosphine complexes derived from 1,2-, 1,4-, and 1,8-dialkynyloxyanthraquinone have been prepared. The photophysical and cytotoxic behavior of the ligands and complexes have been explored, with all of the complexes showing both appreciable cytotoxicity against the MCF-7 carcinoma cell line and useful room-temperature anthraquinone-based visible luminescence, which allowed their successful application as fluorophores in cell imaging microscopy. The implications of the photophysical and toxicological properties for the design and investigation of gold-based anticancer agents are discussed.

Full text of DOI:10.1021/om300475y

Article
pubs.acs.org/Organometallics
Gold(I) Complexes Derived from Alkynyloxy-Substituted
Anthraquinones: Syntheses, Luminescence, Preliminary Cytotoxicity,
and Cell Imaging Studies
Rebeca G. Balasingham,† Catrin F. Williams,‡ Huw J. Mottram,§ Michael P. Coogan,*,†
and Simon J. A. Pope*,†
†School of Chemistry, Cardiff University, Main Building, Cardiff CF10 3AT, Cymru/Wales
‡School of Biosciences, Cardiff University, Main Building, Cardiff CF10 3AT, Cymru/Wales
§School of Pharmacy, Cardiff University, Redwood Building, Cardiff CF10 3NB, Cymru/Wales
S
* Supporting Information
ABSTRACT: A series of mono- and dimetallic Au(I) triphenylphosphine complexes
derived from 1,2-, 1,4-, and 1,8-dialkynyloxyanthraquinone have been prepared. The
photophysical and cytotoxic behavior of the ligands and complexes have been explored,
with all of the complexes showing both appreciable cytotoxicity against the MCF-7
carcinoma cell line and useful room-temperature anthraquinone-based visible lumines-
cence, which allowed their successful application as fluorophores in cell imaging
microscopy. The implications of the photophysical and toxicological properties for the
design and investigation of gold-based anticancer agents are discussed.
Recent studies on a gold complex incorporating a tethered
luminescent naphthalimide chromophore have shown DNA
intercalation and anticancer activity:8 the complexes demon-
strate a general enhancement in activity compared to [Cl-Au-
(PEt3)], probably due to improved uptake by nuclei.
INTRODUCTION
■
The therapeutic potential of gold(I) complexes has been
demonstrated in many studies, and there are a number of
gold(I) species that have been applied in therapy, notably
“auranofin”, an antiarthritic that has recently shown promise in
other areas, including anti-HIV.1 In fact, “chrysotherapy” has a
centuries-old tradition, but it is only very recently that, in line
with the rise of applications of organometallic species in biology
and medicine, there has been interest in the applications of
organogold species. In fact, a small range of cyclometalated
Au(III)-η1 aryl complexes derived from dimethylbenzylamine2
showed early promise in the 1990s, but then the area was
largely dormant until much more recently. In 2006 the first
reports appeared of Au(I) NHC complexes as potential
antitumor agents,3 and in 2009 a study4 of the cytotoxicity of
Au(I) alkynyls was reported. These reports followed from
recent investigations of the cytotoxicity of Au(I) thiolate and
phosphine complexes, which have indicated a mode of action
involving interference with mitochondria, triggering release of
cytochrome c, and inducing apoptosis.5 While the mode of
action is not fully understood, it may involve binding of gold
ions to selenocysteine residues of thioredoxin reductase (Trx-
R), in line with the high affinity of gold for selenium donors.6 It
is interesting, however, that a cell imaging study with one of the
Au(I)NHC complexes3 designed to target mitochondria, which
had been reengineered for luminescence, showed not
mitochondrial localization, but entrainment in lysosomes.7
However, a study of a range of Au(I) alkynyls that showed
moderate to high cytotoxicity also studied cellular distribution
by fluorescence imaging, showing that the complex was
distributed throughout the cells, rather than being limited to
one area.9 These last two reports combine the potential
therapeutic properties of gold complexes with another well-
known property of certain gold complexes, their luminescence.
There are a wide range of mechanisms by which gold
complexes can show luminescence, including gold−gold
interactions in either the solid state or multinuclear systems10
and in particular many interesting phenomena associated with
phosphorescence from gold(I) alkynyls, which have been
widely investigated.11 Given their apparent apoptosis induction
and potentially useful luminescence, applications of Au(I)
alkynyls in combined studies aimed at using fluorescent cell
imaging to inform cytotoxicity studies became attractive,
particularly if these properties can be coupled with a vector
of known biological utility, as a starting point for their design.
Special Issue: Organometallics in Biology and Medicine
Received: May 30, 2012
Published: August 3, 2012
© 2012 American Chemical Society
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The biological and pharmaceutical science of anthraquinones
has received significant attention, with derivatives showing a
breadth of applicability (e.g., antibiotics and antitumor
activity):12 the hydroxy-anthraquinone unit has been identified
as a key biologically active site in antitumor anthracyclines.13
Hydroxy-anthraquinone is a naturally occurring genotoxic
compound found in medicinal plants, including Rubia
tinctorium L.;13b,c its increased commercialization exploits an
antimicrobial effect.13a The antitumor activity of anthraqui-
nones is thought to be due to the intercalation of the planar
ring system between the base pairs of the DNA,13c resulting in
an inhibition of DNA transcription and replication.13c The
extraction from Scutia myrtina has yielded three anthrone-
anthraquinone derivatives, a new bisanthrone-anthraquinone,
and the known aloesaponairn I,14 which displayed antiprolifer-
ative (cancer/antiangiogenic) and moderate antimalarial
activities. Rhubarb root contains the natural anthraquinones
aloe-emodin, chrysophanol, physicon, and rhein,15 derivatives
that are based on a 1,8-dihydroxyanthraquinone core: studies
have shown that different substitution patterns can affect the
antiangiogenic activity of a given anthraquinone.
In an optical context, our previous studies have shown that
picolyl-functionalized anthraquinone chromophores (L) can be
incorporated into luminescent complexes based on fac-
[Re(CO)3(bpy)(L)]+, giving species that are generally dual
emissive from both anthraquinone-centered (actually charge
transfer dominated) and 3MLCT excited states.16 Amido-
functionalized anthraquinone chromophores can also be
utilized as chromophoric antennae for near-IR emitting
lanthanide complexes,17 advantageously facilitating visible
light sensitization. Such species have also been shown to bind
to DNA, with the affinity being determined by the substitution
pattern of the anthraquinone (1,4- vs 1,5-diamido).18
Figure 1. Key to structures of the ligands and (inset) gold complexes.
and L7 under the same conditions produced a mixture of mono-
(i.e., L2 and L6) and dipropargylated products (using either
sodium or potassium carbonate), which were each isolated in
poor yields. Increasing the reaction time by an additional 48 h
(from a standard 72 h for previous examples) resulted in a 5-
fold increase in yield for the dialkynyl species (L3 3 days 5.6%, 5
days 29%; L7 3 days 14.3%, 5 days 40%). It is likely that the low
solubility of the resultant phenoxides, coupled with the
enhanced reactivity of the dianions over the monoanions, is
responsible for the sluggish dipropargylation. That is, the
mono- and diphenoxides were both observed to be of low
solubility, but it is likely that the dianionic species formed by
double deprotonation of the starting material is highly reactive
and is rapidly propargylated, either on the surface of the
precipitate or by rapid exchange of the small quantity in
solution. Upon monopropargylation, however, the remaining
monoanionic species is apparently far less reactive and is still of
low solubility, leading to a slow reaction. The syntheses of
mono- and dialkynes based upon 1,5-dihydroxy anthraquinone
(L4 and L5) were attempted; however, due to the poor yields
obtained (L4, 4% and L5, 3%) under the conditions most
suitable for the other isomers, this route was abandoned as
impractical. The anions derived from 1,5-dihydroxyanthraqui-
none appeared to be the least soluble of all, and it is likely that
the low yields merely reflect an extremely slow reaction. In all
cases the conversion of the hydroxyanthraquinone to the
desired alkynoxy products was initially indicated by the
The dual advantages offered by the optical and biological
functionality of anthraquinone units have led to their limited
use in imaging studies. The ability of anthraquinone-tethered
platinum(II) complexes to intercalate with DNA19a has led to
enhanced cellular accumulation in cancer cell (model)
spheroids19b when compared to cisplatin. Uptake was tracked
within the spheroid, via the fluorescent anthraquinone moiety,
using confocal microscopy.
Therefore, the aims of this study were to investigate the
potential application of gold(I) alkynyls derived from hydroxy-
substituted anthraquinone fluorophores, with a view to
exploiting the biological properties and photophysical charac-
teristics of the respective component parts.
RESULTS AND DISCUSSION
■
Synthesis of Ligands. Anthraquinone-derived ligands L1−7
were synthesized by the base-mediated reaction of the relevant
hydroxyanthraquinone with propargyl bromine (Figure 1).
Both the reaction time and the base used had an influence on
the nature and yield of the products formed. Literature reports
the use of potassium carbonate as the base,20 and this was
successful for the 1,2-dipropargyl species, L1, which was formed
in moderate (30−40%) yield as a single product. However, this
route was less successful for the ligands derived from the 1,4-
and 1,8-dihydroxyanthraquinone isomers L2, L3, L6, and L7,
which required the improved solubility of sodium carbonate to
give reasonable yields. The situation was more complex in the
specific cases of the dialkynes: although L1 was formed in
reasonable yield (30−40%) as a single product with two
propargyl units appended to the core, attempts to generate L3
appearance of a signal in the H NMR spectra characteristic
1
of the methylene unit of the propargyl ether at 4.5−5.0 ppm
with the characteristic propargyl coupling (d, JHH ≈ 2.3 Hz),
which has shifted to higher frequency than the bromide starting
material, along with the distinctive triplet (JHH ≈ 2.3 Hz) for
the acetylenic proton at 2.0−2.5 ppm.
Synthesis of Complexes. With a series of mono- and
dipropargylated dihydroxyanthraquinones available a study of
their coordination chemistry with gold(I) was undertaken.
Following a variation on standard procedures, each of the
ligands was treated with a slight excess of the standard gold
precursor [Cl-Au-PPh3]21 in the dark, in the presence of
potassium ethoxide, itself generated in situ from potassium tert-
butoxide in ethanol. The monometallic products remained in
solution and were easily isolated by filtration from the KCl
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Table 1. Solution (MeCN, 10−5 M) Absorption and
Emission (λex = 345 nm) Data for the Ligands and
Complexes
byproduct and then evaporation; the dimetallic complexes
precipitated from the reaction mixture and thus were isolated
by filtration, redissolution in dichloromethane, refiltration, and
evaporation of solvent. Each complex was purified by
crystallization, using vapor diffusion of diethyl ether into a
concentrated THF solution. It was observed that prolonged
exposure to chlorinated solvents, especially chloroform,
resulted in the breakdown of the complex and the recovery
of the gold starting material [Cl-Au-PPh3]. In each case
formation of the desired product complexes was initially
confirmed by 1H NMR spectroscopy, in which disappearance of
the acetylenic triplet (and a shift to higher frequency of the
propargylic singlet) indicated terminal coordination of gold.
31P{1H} NMR spectra for the complexes also confirmed
formation of a σ-bonded terminal Au(I) alkynyl triphenylphos-
phine species, with singlet resonances at around +42 ppm, in
the region expected from literature precedent.11 Mass
spectrometry was not useful in these cases, as each of the
complexes gave only a peak at 721 amu. This has been reported
by Schmidbaur et al.22 and observed previously by ourselves23
and has been attributed to the ligand dissociation and then
recombination to form [Au(PPh3)2]+; attribution of this
phenomenon was confirmed by a lack of a characteristic
resonance for [Au(PPh3)2]+ in the 31P{1H} NMR spectra.24
Solid-state IR spectroscopy studies were also carried out, which
showed subtle changes in the ν(CC) in comparison to the
corresponding free ligands. The ligands display an IR stretching
band typical of terminal aliphatic alkynes at 2120 cm−1, whereas
the complexes display a weaker intensity IR absorption band at
around 2132 cm−1.
compound
absorption/nm
257, 369
emission/nm
L1
398
[L1-(Au-PPh3)2]
272, 375
406, 538, 566
408, 540 br
425, 522
487
L2
270, 326, 432
[L2-Au-PPh3]
L3
[L3-(Au-PPh3)2]
L7
[L7-(Au-PPh3)2]
272, 324 sh, 448
268 sh, 302 sh, 394
266 sh, 322 sh, 406
254, 278 sh, 378
257, 275 sh, 382
415, 435
505
491
Figure 3. Molecular structure of [L7-(Au-PPh3)2] (ORTEP,26 50%
Single-Crystal X-ray Diffraction Studies. Diffusion of
diethyl ether into a concentrated chloroform solution, over a
period of 24 h, of [L7-(Au-PPh3)2] yielded pale orange crystals
suitable for X-ray diffraction studies. The crystal solved in space
group P21/n and was refined using the SHELX suite of software
to give confirmation of the structure of the expected molecule:
two molecules of chloroform solvent per asymmetric unit
enclosed within the crystal lattice (Figure 2).25 Tables of bond
angles and lengths are summarized in Table 1 (Supporting
Information). The structure overall shows a nonplanar
anthraquinone unit with the central ring distorted to give an
open-book conformation (Figure 3) with an angle between the
probability) showing curvature of the anthraquinone unit.
two terminal rings of approximately 161°. The two
propargyloxy-gold-phosphine units are in a cis arrangement,
overlapping one face of the anthraquinone, with the aromatic
rings of the triphenyl phosphines proximate, but without any
distinct close contacts indicating strong intramolecular π-
stacking. However, examination of the packing of the
asymmetric units reveals intramolecular close contacts (closer
than the sum of the van der Waals radii) between adjacent
molecules involving both the phenyl units of the phosphine
ligands and the polarized anthraquinone rings. The cis
arrangement therefore seems to be favored by the combination
of weak intra- and stronger intermolecular π-stacking of the
aromatic units of the phosphine ligands and, in particular, by
facilitating the overlap of anthraquinone rings from adjacent
molecules. The quinone oxygen cis to the propargyloxy units is
distorted out of the plane of the C−C vector containing the
two quinoid carbons, with a C−C−O angle of 155° (Figure 4).
́
This oxygen is weakly hydrogen bonded (2.236 Å) to the
hydrogen of a chloroform solvent of crystallization, but it is
likely that the distortion is caused by electronic repulsion from
the 1,8 dioxygens. The two-coordinate gold atoms are
essentially linear, and the Au−C and Au−P distances lie within
the range of literature values.11
UV−Vis Absorption and Luminescence Properties of
the Ligands and Complexes. The UV−visible absorption
properties of the ligands comprise IL π−π* (both aryl and
alkynyl) transitions of <310 nm, with a broadened lowest
energy contribution (Figure 5) expected to comprise significant
charge transfer character arising from O(alkoxy)-to-quinone
transitions,27,28 with L2 displaying the greatest bathochromic
shift in the visible region. When compared to the relevant free
Figure 2. Molecular structure of [L7-(Au-PPh3)2] (ORTEP,26 50%
probability). Solvent molecules are omitted for clarity.
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disubstituted species with intermediate to lower relative
energies. In this series, the comparison of L1 with L2 revealed
an additional, lower energy emission peak at ca. 550 nm for the
latter. The dual emissive nature of related dihydroxy-
anthraquinone species30,31 has been attributed to the
tautomerized 1,10-keto form of the ligand (versus the 9,10-
keto form); dual emission actually arises from the excited-state
intramolecular proton transfer between the 4-hydroxy sub-
stituent and the neighboring carbonyl. However, the 1,10-keto
form usually absorbs and emits at lower energy,31 and therefore
it may be that for L2 (and [L2-Au-PPh3]) the higher energy
emission peak at 409 nm is actually due to the 9,10-keto form
and that tautomerization accounts for the dual emission.
The luminescence properties (λex = 340−400 nm; aerated
MeCN) of the complexes are again dictated by the nature of
the appended anthraquinone chromophore (Table 1), with no
clear evidence for long-lived MAuLalkyneCT phosphorescence.
The spectra showed that for dual-emissive [L2-Au-PPh3], the
relative intensities of the peaks were sensitive to the
complexation of Au(I) and the nature of the solvent.28,29
Nonpolar solvents (CHCl3) promote the long-wavelength
form, whereas in polar protic solvent the emission profile was
dominated by the shorter wavelength fluorescence. The
emission behavior of this type of anthraquinone chromophore
is notoriously sensitive to solvent,31,32 and H-bonding can
provide very effective pathways for excited-state quenching.27
The emission profile of [L3-(Au-PPh3)2] was unusually broad
and ill-defined, with a lowest energy maximum at 524 nm. In
contrast, the spectral profile for [L7-(Au-PPh3)2] showed a
single emission peak at 491 nm (Figure 6), which was subtly
Figure 4. Molecular structure of [L7-(Au-PPh3)2] (ORTEP,26 50%
probability) showing distortion of the quinoid oxygens.
Figure 5. UV−vis absorption (MeCN) spectra comparing L6 (solid
line) and L7 (dashed line).
ligands, the absorption spectra of the complexes principally
reveal increased absorption coefficients, <270 nm, correspond-
ing to the presence of multiple phenyl chromophores
(associated with the “Au-PPh3” units). Of course, Au-alkynyl
species have been shown to possess metal-to-alkynyl charge
transfer (MLCT) bands, and these are presumed to contribute
to the overlapping spectral profile at 300−400 nm.29 However,
the broad, unstructured visible absorption characteristics of the
complexes can be wholly attributed to the appended
anthraquinone chromophores in each case; very minor shifts
are noted for these bands upon coordination of Au(I) to the
alkyne termini. It is also noteworthy that within this series the
1,4-disubstituted anthraquinone species L2 and [L2-Au-PPh3]
possessed the largest bathochromic shift for the charge transfer
band.
In terms of the luminescence properties, the visible emission
spectra (λex = 340−400 nm; aerated MeCN) for these
anthraquinone species are quite broad in appearance, with
some examples (e.g., L3) revealing poorly resolved vibronic
features. The broadness of the emission profiles is again
consistent with a charge transfer dominated excited state,27,30,31
and this assignment, together with the emission energies,
compares quite well to the visible emission characteristics
shown by related parent hydroxy-anthraquinones. In accord-
ance with related work on amino- and alkoxy-substituted
anthraquinones,16,29,30 the emission properties of this series of
alkoxy derivatives also show pronounced variations according
to substituent position. The emission from the 1,2-anthraqui-
none is at the shortest wavelength, with the 1,4- and 1,8-
Figure 6. Excitation (left) and emission (right) spectra for [L7-(Au-
PPh3)2].
hypsochromically shifted relative to the free ligand, L7. It is
worth considering the severe out-of-plane distortion revealed
by the solid-state structure of [L7-(Au-PPh3)2]: such a
distortion would be expected to perturb the energetics of the
O(alkoxy)-to-quinone charge transfer excited state. Time-
resolved measurements revealed that the luminescence lifetimes
associated with the ligands are comparable to previous reports
relating to donor-functionalized anthraquinone chromo-
phores:31 the emission was short-lived (<5 ns) and indicative
of fluorescence in all cases. For the complexes, the lifetimes are
also similarly short, which is again attributed to the fluorescence
of the anthraquinone chromophore; there was no evidence for
heavy atom assisted phosphorescence from the complexes.
Cytotoxicity Studies. A preliminary study of the
cytotoxicity of some selected ligands (L2, L3, and L7) and the
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corresponding complexes ([L2-Au-PPh3], [L3-(Au-PPh3)2], and
[L7-(Au-PPh3)2]) was undertaken using the MTT assay with
four different cancer cell lines: MCF7 (breast adenocarcinoma),
A549 (lung adenocarcinoma), PC3 (prostate adenocarcinoma),
and LOVO (colon adenocarcinoma). For the MTT assay, the
compounds were initially dissolved in DMSO, and doses of 0.1,
1, 10, and 100 μM were tested to analyze the activity of
different concentrations and then compared to a control
medium with no treatment. The approximate IC50 values for
these compounds are shown in Table 2.
of the intrinsic membrane permeancy of the species. The cells
were washed free of excess agent, then allowed to warm to
ambient temperature to allow energy-dependent intracellular
distribution pathways to re-emerge, while blocking energy-
dependent uptake, before examination by confocal laser
microscopy. In all cases the excitation wavelength was 405
nm and the images were collected between 530 and 580 nm,
correlating to the low-energy tail of the anthraquinone
emission, and thus well separated from endogenous auto-
fluorescence. The dialkynyl ligand L3 showed (Figure 7) good
a
Table 2. Cytotoxicity of Ligands and Complexes
ligand/complex
IC50 MCF7
IC50 PC3
IC50 A549
IC50 Lovo
L2
80
5
>100
50
>100
100
>100
100
70
[L2-Au-PPh3]
L3
>100
5
>100
50
>100
>100
>100
>100
[L3-(Au-PPh3)2]
L7
[L7-(Au-PPh3)2]
80
>100
5
>100
>100
>100
>100
a
MTT assay: full experimental details are provided in the Supporting
Figure 7. Images of MCF-7 cells incubated with L3 (100 μg mL−1, 4
°C, 30 min). Excited at 405 nm, acquired 530−580 nm.
Information.
It is clear from the data presented in Table 2 that the gold(I)
complexes are dramatically more toxic than the corresponding
free ligands, which is gratifying, as it demonstrates that the
observed cytotoxicity is a result of the chemistry associated with
the gold alkynyl unit, rather than the biological properties of
the anthraquinones. Thus, the anthraquinone, which is useful in
this context for both luminescence and the potential to direct
the localization of the complexes in cells, has not dominated the
cellular behavior. Also obvious was that all of the complexes
were considerably more cytotoxic to MCF7s than the other cell
lines tested, although the cytotoxicities are not in themselves
particularly low, even in MCF7s (cf. [AuCl(PPh3)]33 IC50 ≈ 2.6
and cisplatin IC50 = 2.0 μM). It is noteworthy that the
complexes show much greater toxicity in MCF7s, a cell line
known to have a higher mitochondrial mass than many other
immortal cell lines, which are often deficient in mitochondrial
activity in line with the “Warburg effect”,34 given that the
mitochondria are believed to be the key intracellular target of
gold agents.5 Interestingly, this series of complexes appear to be
much less cytotoxic than related Au(I) tertiary phosphine
species incorporating biologically inspired mercapto-pteridine
ligands.35 Having established that the complexes displayed
toxicity toward MCF7s, and in light of earlier imaging studies
that indicated lysosomal entrapment for some gold agents,
attention subsequently turned to the study of cellular
distribution by fluorescence microscopy.
Live Cell Imaging Studies. Having established the
photophysical properties of ligands and complexes and
undertaken a study of their cytotoxicity in a variety of cell
lines, their cellular uptake and localization in MCF-7 cells (one
of the cell lines used in the toxicity study that had shown the
most sensitivity to the complexes) was investigated. For the
imaging studies the dialkynyl ligand, L3, and examples of both
mono- and dimetallic gold complexes, [L2-Au-PPh3] and [L3-
(Au-PPh3)2], were selected for investigation, as these had
shown promising cytotoxicity and/or were of interest as a
ligand−complex comparison. The incubations were carried out
at 4 °C, at which temperature energy-dependent uptake
processes, such as endocytosis, are inhibited in order to avoid
entrainment in endosomes and in order to allow a comparison
uptake, with >80% of the population showing fluorophore-
based emission and possessing a healthy cell morphology. The
emission was reasonable, although not particularly bright, and
was detected throughout the cytoplasm, with little or no
nuclear localization. Although there appeared to be certain “hot
spots” of luminescence in individual cells, the localization was
clearly not specific to a particular organelle, but was throughout
a variety of cytoplasmic structures.
The monogold complex [L2-Au-PPh3] was again taken up by
>80% of the cell population, and again apparently healthy cell
morphology was retained (Figure 8). In this case the
Figure 8. Images of MCF-7 cells incubated with [L2-Au-PPh3] (100
μg mL−1, 4 °C, 30 min). Excited at 405 nm, acquired 530−580 nm.
luminescence was considerably increased, which is likely to
indicate better uptake as a result of the enhanced lipophilicity of
[L2-Au-PPh3] compared to L2 upon inclusion of the triphenyl
phosphine units. Again the luminescence originated from the
cytoplasm, with little or no nuclear localization, and with
certain bright spots indicating concentrations in certain
organelles. Although these bright spots were more distinct
than for the free ligand, the pattern of distribution may favor
certain organelles. [L2-Au-PPh3] had clearly accumulated a
significant concentration across the entire cytoplasm, including
all organelles, which constitutes a significant proportion of the
volume of the cytoplasm.
The dimetallic gold complex [L3-(Au-PPh3)2] showed (as
qualitatively judged by luminescence intensity) a similar uptake
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to the mononuclear complex [L2-Au-PPh3], which was
unexpected from the additional lipophilicity of an additional
triphenyl phosphine unit. The pattern of distribution (Figure 9)
able to access organelles within the cytoplasm, in line with the
suggested mode of gold cytotoxicity that involves mitochondrial
inhibition.
EXPERIMENTAL SECTION
■
Cytotoxicity Assessment via MTT Assay. The cytotoxicity of
the complexes was assessed using the colorimetic and quantitative
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay, first reported by Mosmann et al.36 Quantification was achieved
using a multiwell scanning spectrophotometer and is reported as an
IC50 value.33
Method for Cytotoxicity Analysis. Antitumor evaluation in
MCF7, LoVo, A549, and PC3 cell lines was performed by the MTT
assay. Compounds were prepared as 0.1−100 mM stock solutions
dissolved in DMSO and stored at −20 °C. Cells were seeded into 96-
well microtiter plates at a density of 5 × 103 cells per well and allowed
24 h to adhere. Decimal compound dilutions were prepared in
medium immediately prior to each assay (final concentration 0.1−100
μM). Experimental medium was DMEM + 10% FCS (PC3 and Lovo)
or RPMI + 10% heat-inactivated FCS (A549 and MCF7). Following
96 h compound exposure at 37 °C and 5% CO2, MTT reagent (Sigma
Aldrich) was added to each well (final concentration 0.5 mg/mL).
Incubation at 37 °C for 4 h allowed reduction of MTT by viable cells
to an insoluble formazan product. MTT was removed and formazan
solubilized by addition of 10% Triton X-100 in phosphate buffer saline
(PBS). Absorbance was read on a Tecan Sunrise spectrophotometer at
540 nm as a measure of cell viability; thus inhibition relative to control
was determined (IC50).
Figure 9. Images of MCF-7 cells incubated with [L3-(Au-PPh3)2] (100
μg mL−1, 4 °C, 30 min), excited at 405 nm, acquired 530−580 nm
showing: (A) cytoplasmic distribution (overlaid luminescence and
transmitted light); (B) appearance of vacuoles upon irradiation
(transmitted light only); (C and D) photobleaching (luminescence
only).
was also similar, with general cytoplasmic staining being
apparent, with little or no nuclear localization, and with bright
spots. It is possible that the sheer steric bulk of the complex
impedes uptake by passive diffusion in line with Fick’s law.
However, intensity may be misleading in this case, due to
photobleaching (see additional comment below). It was notable
that, in addition to more severe photobleaching than was
observed with L2 and monomeric complex [L2-Au-PPh3], the
cells showed apparent toxic effects upon irradiation, with large
vacuoles appearing in the cells, which characterize membrane
damage. It is possible that the phenomena of photobleaching
and cell damage are linked, as photobleaching usually involves
the generation of toxic species (e.g., oxygen radicals) through
the reaction of the lumophore excited state with material in the
environment. The unusually broad emission maximum of this
complex, noted above, may indicate properties of the excited
state that render photochemical reaction with the medium, and
thus the generation of toxic species, more rapid than with the
other complexes/ligands. While it is noted that this complex
showed the broadest range of activity and lowest IC50 in the
cytotoxicity studies described above, it should also be noted
that at the outset of the imaging experiment the cells treated
with [L3-(Au-PPh3)2] appeared healthy (the time scales of the
experiments are different) and the vacuoles appeared only upon
irradiation, so while it is possible that the cytotoxicity and
apparent phototoxicity are linked, this cannot be assumed.
Human Cell Incubation and Confocal Microscopy. Human
adenocarcinoma cells (MCF-7), obtained from the European
Collection of Cell Cultures (Porton Down, Wiltshire, UK), were
maintained in Hepes modified minimum essential medium supple-
mented with 10% fetal bovine serum, penicillin, and streptomycin.
Cells were detached from the plastic flask using trypsin-EDTA solution
and suspended in an excess volume of growth medium. The
homogeneous cell suspension was then distributed into 1 mL aliquots,
with each aliquot being subjected to incubation with a different
lumophore. Lumophores were initially dissolved in DMSO (5 mg/
mL) before being added to the cell suspensions (final concentration
100 μg mL−1) before incubation at 4 °C for 30 min. Cells were finally
washed three times in PBS (pH 7.2), removing lumophore from the
medium, then harvested by centrifugation (5 min, 800g) and mounted
on a slide for imaging. Preparations were viewed using a Leica TCS
SP2 AOBS confocal laser microscope using a 63× or 100× objective,
with excitation at 405 nm and detection at 530−580 nm.
Data Collection and Processing. Diffraction data for [L7-(Au-
PPh3)2] were collected on a Bruker-Nonius diffractometer equipped
with a KappaCCD detector (Mo Kα radiation, λ = 0.71075 Å) from
fine-focus sealed tube source equipped with a graphite monochroma-
tor at 100 K. Data collection was performed using COLLECT (Nonius
BV, 1997−2001) with cell refinement and data reduction using
Denzo/Scalepack.
CONCLUSIONS
■
The ligands L1−3 and L6,7, which were prepared in useful
quantity by functionalizing various hydroxyanthraquinone
derivatives with propargylic groups, revealed luminescence
properties attributable to the various isomeric forms of the
substituted anthraquinone chromophoric cores. Upon for-
mation of the gold complexes, changes in the emission profiles
are observed, but there is no indication of triplet emission as a
result of incorporating the heavy atom. It appears that the
dominant features of the emission spectra are, in all cases,
attributable to the anthraquinone-based processes, rather than
transitions involving substantial metal character. The ligands
generally show low cytotoxicity, while in all cases the complexes
are considerably more toxic to one of the cell lines at least
(MCF-7) and in some cases to more of the lines, indicative of a
gold-dependent cytotoxicity. Cellular imaging utilizing the
anthraquinone-based fluorescence showed that the agents are
Structure Analysis and Refinement. The structure was solved
by direct methods using SHELXS-9725 and was completed by iterative
cycles of ΔF-syntheses and full-matrix least-squares refinement. All
non-H atoms were refined anisotropically, and difference Fourier
syntheses were employed in positioning idealized hydrogen atoms,
which were allowed to ride on their parent C atoms. Disordered
solvent molecules were modeled using partial occupancy. All
refinements were against F2 and used SHELXL-97. Figures were
created using the ORTEP3 software package.26
Crystal data for [L7-(Au-PPh3)2]: C58H38Au2Cl6O4P2, M = 1467.46,
yellow plate, 0.35 × 0.30 × 0.04 mm3, monoclinic, space group P21/n
(No. 14), a = 18.4561(9) Å, b = 11.6603(5) Å, c = 26.6072(7) Å, β =
98.614(2)°, V = 5661.4(4) Å3, Z = 4, Dc = 1.722 g/cm3, F000 = 2832,
KappaCCD, Mo Kα radiation, λ = 0.71073 Å, T = 293(2) K, 2θmax
=
55.1°, 12 698 reflections collected, 12 698 unique (Rint = 0.0000). Final
GooF = 1.021, R1 = 0.0871, wR2 = 0.2013, R indices based on 6193
reflections with I >2(I) (refinement on F2), 686 parameters, 63
5840
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Organometallics
Article
restraints. Lp and absorption corrections were applied; μ = 5.561
mm−1.
ArH), 7.48−7.52 (1H, m, ArH), 7.62−7.70 (1H, m, ArH), 7.7−7.82
(2H, m, ArH), 7.98−8.05 (1H, m, ArH) ppm. 13C{1H} NMR (CDCl3,
101 MHz, 298 K): δC 57.3, 77.6, 119.0, 121.2, 121.8, 123.5, 132.9,
135.8, 138.1, 163.4 (CO not observed) ppm. ES MS m/z: 239.21 [M
− C3H3]+. HR MS (ES+): found m/z 279.0652; [C17H11O4]+ requires
279.0652. IR (Nujol) ν: 2112 (CC), 1664 (CO), 1634 (CO), 1583
(CO) cm−1. UV−vis (ε/M−1 cm−1) (MeCN) λmax: 254 (24 300), 408
(12 000) nm.
All reactions were performed with the use of vacuum line and
Schlenk techniques. Reagents were commercial grade and were used
without further purification. 1H and 13C{1H} NMR spectra were
recorded on an NMR-FT Bruker 400 MHz or Joel Eclipse 300 MHz
spectrometer and recorded in CDCl3. 1H and 13C{1H} NMR chemical
shifts (δ) were determined relative to residual solvent peaks with
digital locking; 31P{1H} chemical shifts (δ) were determined relative to
H3PO4 and are given in ppm. Low-resolution mass spectra were
obtained by the staff at Cardiff University. High-resolution mass
spectra were carried out at the EPSRC National Mass Spectrometry
Service at Swansea University. UV−vis studies were performed on a
Jasco V-570 spectrophotometer as MeCN solutions (1 × 10−5 M).
Photophysical data were obtained on a JobinYvon−Horiba Fluorolog
spectrometer fitted with a JY TBX picosecond photodetection module
as MeCN solutions. Emission spectra were uncorrected, and excitation
spectra were instrument corrected. The pulsed source was a Nano-
LED configured for 372 nm output operating at either 500 kHz or 1
MHz. Luminescence lifetime profiles were obtained using the
JobinYvon−Horiba FluoroHub single photon counting module, and
the data fits yielded the lifetime values using the provided DAS6
deconvolution software.
Synthesis of L5. The reaction was performed as for L4, but the
reaction mixture was heated at reflux for three days with an extra
portion of potassium carbonate in an attempt to produce the
dialkylated species. The filtrate was concentrated under reduced
1
pressure to yield an orange solid (yield: 0.012 g, 22%). H NMR
3
(CDCl3, 400 MHz, 298 K): δH 2.48 (2H, t, JHH = 2.3 Hz, CH), 4.82
(4H, d, 3JHH = 2.3 Hz, CH2), 7.37 (2H, d, 3JHH = 8.4 Hz, ArH), 7.61−
3
7.67 (2H, ArH), 7.91 (2H, d, JHH = 7.7 Hz, ArH) ppm.
Synthesis of L6. The reaction was performed as for L1 except using
1,8-dihydroxyanthraquinone (0.500 g, 2.08 mmol). Column chroma-
tography was used to isolate the monoalkylated product as an orange
1
solid (yield: 0.078 g, 13%). H NMR (CDCl3, 400 MHz, 298 K): δH
2.52 (1H, t, 3JHH = 2.36 Hz, CH), 4.91 (2H, d, 3JHH = 2.36 Hz, CH2),
3
3
7.2 (1H, d, JHH = 9.43 Hz, ArH), 7.42 (1H, d, JHH = 9.3 Hz, ArH),
7.49−7.54 (1H, m, ArH), 7.57−7.65 (2H, m, ArH), 7.94 (1H, d, 3JHH
= 8.7 Hz, ArH) ppm. 13C{1H} NMR (CDCl3, 101 MHz, 298 K): δC
57.2, 77.3, 118.9, 120.5, 121.2, 124.8, 135.8, 138.1, 161.0, 163.9 (CO
not observed) ppm. MS (ES) m/z: 252 [M − C2H3]+. HR MS (ES+):
found m/z 279.0651; [C17H10O4]+ requires 279.0652. IR (Nujol) ν:
2034 (CC), 1675 (CO), 1643 (CO), 1584 (CO) cm−1. UV−vis (ε/
M−1 cm−1) (MeCN) λmax: 254 (24 300), 408 (12 000) nm.
Synthesis of Ligands. Synthesis of L1. To an excess of K2CO3 in
acetone (40 mL) were added alizarin (0.500 g, 2.08 mmol) and
propargyl bromide (0.320 mL, 4.37 mmol). The reaction mixture was
heated at reflux for three days and then filtered. The filtrate was
concentrated under reduced pressure to yield a light brown solid
(yield: 0.260 g, 29%). 1H NMR (CDCl3, 400 MHz, 298 K): δH 2.48−
2.53 (1H, m, CH), 2.58−2.62 (1H, m, CH), 4.84−4.96 (4H, m, CH2),
7.42 (1H, d, 3JHH = 6.1 Hz, ArH), 7.73−7.85 (2H, m, ArH), 8.18−8.30
(3H, m, ArH) ppm. 13C{1H} NMR (CDCl3, 101 MHz, 298 K): δC
56.9, 61.0, 75.2, 77.2, 118.0, 118.4, 125.6, 126.8, 127.4, 127.9, 128.2,
132.9, 133.7, 133.8, 134.1, 135.0, 147.3, 157.1, 182.3, 182.6 ppm. MS
(ES+) m/z: 316.01 [M + H]+. HR MS (ES+): found m/z 317.0812;
[C20H13O4]+ requires 317.0808. IR (Nujol) ν: 2129 (CC), 1673
(CO), 1568 (CO) cm −1. UV−vis (ε/M−1 cm −1) (MeCN) λmax: 257
(47 000), 369 (11 000) nm.
Synthesis of L7. The reaction was performed as for L6, but the
reaction mixture was heated at reflux for five days to produce the
dialkylated product. The filtrate was concentrated under reduced
1
pressure to yield an orange solid (yield: 0.354 g, 40%). H NMR
(CDCl3, 400 MHz, 298 K): δH 2.52 (2H, t, 3JHH = 2.34 Hz, CH), 4.88
(4H, d, 3JHH = 2.35 Hz, CH2), 7.42 (2H, d, 3JHH = 8.3 Hz, ArH), 7.51
3
3
(2H, app. t, JHH = 8.0 Hz, ArH), 7.82 (2H, d, JHH = 7.8 Hz, ArH)
ppm. 13C{1H} NMR (CDCl3, 101 MHz, 298 K): δC 57.3, 77.5, 120.4,
121.1, 133.8, 134.5, 157.2 (CO not observed) ppm. MS (ES) m/z:
277.04 [M − C3H3]+. HR MS (ES+): found m/z 316.0707;
[C20H12O4]+ requires 316.0730. IR (Nujol) ν: 2130 (CC), 1671
(CO), 1658 (CO), 1585 (CO) cm−1. UV−vis (ε/M−1 cm−1) (MeCN)
λmax: 254 (29 200), 374 (10 400) nm.
Synthesis of L2. The reaction was performed as for L1, except using
1,4-dihydroxyanthraquinone (0.500 g, 2.08 mmol). Column chroma-
tography (silica; CH2Cl2) was used to remove the dialkylated
byproduct, and the desired monoalkylated product was isolated as
1
an orange solid (yield: 0.085 g, 15%). H NMR (CDCl3, 400 MHz,
Synthesis of Complexes. Synthesis of [L1-(Au-PPh3)2]. To a
round-bottom flask wrapped in Al foil were added L1 (0.022 g, 0.07
mmol), [Cl-Au-PPh3] (0.072 g, 0.15 mmol), and KOtBu (0.011 g, 0.1
mmol) in ethanol (2 mL), and the mixture was stirred under nitrogen
for 12 h. The solution was concentrated, redissolved in chloroform,
and filtered. The filtrate was concentrated in vacuo to produce a yellow
3
3
298 K): δH 2.49 (1H, t, JHH = 2.4 Hz, CH), 4.73 (2H, d, JHH = 2.4
Hz, CH2), 7.12−7.16 (1H, m, ArH), 7.37−7.41 (1H, m, ArH), 7.63−
7.81 (2H, m, ArH), 8.13−8.24 (2H, m, ArH) ppm. 13C{1H} NMR
(CDCl3, 101 MHz, 298 K): δC 58.4, 77.8, 115.5, 121.5, 126.1, 126.5,
126.5, 127.5, 128.1, 128.2, 133.6, 134.9, 135.0, 151.8, 158.7, 181.8,
188.9 ppm. ES MS m/z: 278 [M]+, 240 [M − C3H2]+. HR MS (ES+):
found m/z 278.0574; [C17H10O4]+ requires 278.0574. IR (Nujol) ν:
2115 (CC), 1666 (CO), 1663 (CO), 1593 (CO) cm−1. UV−vis (ε/
M−1 cm −1) (MeCN) λmax: 270 (27 000), 326 (5500), 432 (14 000)
nm.
1
solid (yield: 0.043 g, 48%). H NMR (CDCl3, 400 MHz, 298 K): δH
4.99−5.07 (4H, overlapping m, 2 × CH2), 7.31−7.58 (32H, m, ArH),
7.61−7.66 (1H, m, ArH), 8.11−8.31 (3H, m, ArH) ppm. 31P{1H}
NMR (CDCl3, 202.4 MHz, 298 K): δP +42.46 ppm. MS (ES+) m/z:
721.15 [Au(PPh3)2]+. IR (Nujol) ν: 2184 (CC), 1720 (CO) cm−1.
UV−vis (ε/M−1 cm−1) (MeCN) λmax: 273 (55 000), 375 (14 000) nm.
Synthesis of [L2-Au-PPh3]. To a round-bottom flask wrapped in Al
foil were added L2 (0.020 g, 0.074 mmol), [Cl-Au-PPh3] (0.040 g,
0.080 mmol), and KOtBu (0.009 g, 0.80 mmol) in ethanol (2 mL),
and the mixture was stirred under nitrogen for 12 h. Workup was like
that for [L1-(Au-PPh3)2] (yield: 0.015 g, 28%). 1H NMR (CDCl3, 400
MHz, 298 K): δH 5.01 (2H, s, CH2), 7.16−7.23 (2H, m, ArH), 7.3−
7.42 (15H, m, ArH), 7.62−7.79 (2H, m, ArH) 8.12−8.19 (2H, m,
ArH) ppm. 31P{1H} NMR (CDCl3, 202.4 MHz, 298 K): δP +42.4
ppm. MS (ES+) m/z: 721.15 [Au(PPh3)2]+. IR (Nujol) ν: 2133 (CC),
1665 (CO), 1664 (CO), 1593 (CO) cm−1. UV−vis (ε/M−1 cm−1)
(MeCN) λmax: 272 (24 400), 448 (10 600) nm. Anal. Calcd (%) for
AuPC35H24O4(MeCN)(H2O)4: C, 52.31; H, 4.15; N, 1.65. Found
(%): C, 52.87; H, 4.05; N, 1.08.
Synthesis of L3. The reaction was performed as for L2, but the
reaction mixture was heated at reflux for five days to produce the
dialkylated product. The filtrate was concentrated under reduced
1
pressure to yield an orange solid (yield: 0.205 g, 23%). H NMR
3
(CDCl3, 400 MHz, 298 K): δH 2.52 (2H, t, JHH = 2.4 Hz, CH), 4.79
3
(4H, d, JHH = 2.4 Hz, CH2), 7.41 (2H, s, ArH), 7.59−7.62 (2H, m,
ArH), 8.06−8.12 (2H, m, ArH) ppm. 13C{1H} NMR (CDCl3, 101
MHz, 298 K): δC 58.3, 77.4, 123.8, 126.6, 133.6, 155.8 (CO not
observed) ppm. ES MS m/z: 316 [M]+ 277.05 [M − C3H3]+. HR MS
(ES+): found m/z 316.0746; [C20H12O4]+ requires 316.0730. IR
(Nujol) ν: 2119 (CC), 1671 (CO), 1582 (CO) cm−1. UV−vis (ε/M−1
cm−1) (MeCN) λmax: 275 (26 500), 302 (12 000), 394 (10 000) nm.
Synthesis of L4. The reaction was performed as for L1, except using
1,5-dihydroxyanthraquinone (0.500 g, 2.08 mmol). Column chroma-
tography was used to isolate the monoalkylated species as an orange
Synthesis of [L3-(Au-PPh3)2]. The reaction was performed as for
1
solid (yield: 0.025 g, 4%). H NMR (CDCl3, 400 MHz, 298 K): δH
[L1-(Au-PPh3)2] except using L3 (0.016 g, 0.101 mmol), giving the
1
2.52−2.60 (1H, m, CH), 4.89−4.95 (2H, m, CH2), 7.21−7.28 (1H, m,
product as an orange solid (yield: 0.022 g, 36%). H NMR (CDCl3,
5841
dx.doi.org/10.1021/om300475y | Organometallics 2012, 31, 5835−5843

Organometallics
Article
400 MHz, 298 K): δH 5.01 (4H, s, CH2), 7.23−7.45 (30H, m, ArH),
7.48−7.53 (2H, m, ArH), 7.68 (2H, s, ArH), 8.01−8.20 (2H, m, ArH)
ppm. 31P{1H} NMR (CDCl3, 202.4 MHz, 298 K): δP +42.46 ppm. MS
(ES+) m/z: 721.15 [Au(PPh3)2]+. IR (Nujol) ν: 2021 (CC), 1898
(CO), 1666 (CO) cm−1. UV−vis (ε/M−1 cm −1) (MeCN) λmax: 250
(12 700), 406 (18 00) nm. An al. Calcd (%) for
Au2P2O4C56H40(H2O)18: C, 43.20; H, 4.92. Found (%): C, 42.75;
H, 4.11.
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Synthesis of [L7-(Au-PPh3)2]. The reaction was performed as for
[L1-(Au-PPh3)2] except using L7 (0.010 g, 0.030 mmol), giving the
1
product as an orange solid (yield: 0.032 g, 50%). H NMR (CDCl3,
300 MHz, 298 K): δH 5.08 (4H, s, CH2), 7.33−7.52 (30H, m, ArH),
3
7.63 (2H, app. t, JHH = 7.96 Hz, ArH), 7.72−7.79 (2H, m, ArH),
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(Nujol) ν: 2239 (CC), 2129 (CC), 1667 (CO), 1584 (CO) cm−1.
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ASSOCIATED CONTENT
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: cooganmp@cf.ac.uk; popesj@cardiff.ac.uk.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(16) Jones, J. E.; Kariuki, B. M.; Ward, B. D.; Pope, S. J. A. Dalton
Trans. 2011, 40, 3498.
We thank Cardiff University and EPSRC for financial support
and the staff of the EPSRC Mass Spectrometry National Service
(University of Swansea) for providing MS data.
(17) Jones, J. E.; Pope, S. J. A. Dalton Trans. 2009, 8421.
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Buurma, N. J.; Pope, S. J. A. Chem. Commun. 2011, 47, 3374.
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Hambley, T. W. Chem. Commun. 2009, 2673. (b) Alderden, R. A.;
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(20) Burchell, T. J.; Jennings, M. C.; Puddephatt, R. J. Inorg. Chim.
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