Naphthalimide gold(I) phosphine complexes as anticancer metallodrugs

Article abstract of DOI:10.1039/b912378d

Gold(I) phosphine complexes exhibit promising properties for anticancer drug development. Here we report on a series of gold(I) phosphine complexes containing a naphthalimide ligand. Strong antiproliferative effects were observed in MCF-7 breast cancer ce

Full text of DOI:10.1039/b912378d

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Naphthalimide gold(I) phosphine complexes as anticancer metallodrugs†
Christoph P. Bagowski,a Ya You,b Heike Scheffler,b Danielle H. Vlecken,a Daan J. Schmitza and Ingo Ott*c
Received 24th June 2009, Accepted 7th October 2009
First published as an Advance Article on the web 28th October 2009
DOI: 10.1039/b912378d
Gold(I) phosphine complexes exhibit promising properties for anticancer drug development. Here we
report on a series of gold(I) phosphine complexes containing a naphthalimide ligand. Strong
antiproliferative effects were observed in MCF-7 breast cancer cells as well as in HT-29 colon
carcinoma cells. The cellular and nuclear gold levels were increased compared to analogues, in which
the naphthalimide ligand was replaced by a chloro ligand. Compound 4a was selected for more detailed
biochemical and biological studies, which revealed solvent dependent fluorescence emission, uptake of
the compound into the organelles of tumor cells as well as antiangiogenic effects concerning
angiogenesis and tumor-induced angiogenesis in vivo. Antiangiogenic properties of 4a were observed in
two different zebrafish angiogenesis models, including a tumor-cell induced neovascularization assay.
Introduction
Platinum complexes have been playing an important role in
modern tumor chemotherapy for approximately three decades
but major problems, e.g. the occurrence of severe side effects, the
development of drug resistance or the limited applicability to many
widespread malignancies, have not yet been resolved.1
Current strategies to overcome these obstacles focus on the
optimisation of the clinical application of the existing platinum
Fig. 1 Gold(I) phosphine complexes.
drugs, e.g. the use of platinum compounds for breast cancer
therapy,2 or the development of novel transition metal complexes
containing improved organic ligands.3–7
ligands displaying pharmacological activity themselves could lead
An increasing number of reports describe gold complexes with
to new agents with the capability of addressing multiple tumor-
properties relevant for the design of novel anticancer therapeutics.
relevant targets. Accordingly, naphthalimides were chosen as a
Many derivatives trigger significant antiproliferative effects ac-
suitable class of bioactive ligands with significant cell growth
companied by the induction of apoptosis and antimitochondrial
effects. The enzyme thioredoxin reductase (TrxR), which is
involved in various tumor related biochemical pathways, has been
inhibiting properties.21–24
In fact, it was observed that the organic ligand in Au-Naphth-
1 was responsible for an accumulation of gold in the nuclei and
identified as probably being the most relevant molecular target for
gold complexes,8–10 and promising antiproliferative activity has
been reported for a variety of different gold compounds including
N-heterocyclic carbene species11,12 and gold(III) complexes.13–17
Recently, we described a novel antiproliferative gold(I) phos-
phine compound containing a naphthalimide ligand (Au-Naphth-
1, Fig. 1).18 The design of this compound is based on observations
on the lead compound auranofin, which had indicated that the
thiol-carbohydrate moiety of this metallodrug was exchanged
during cell experiments.19,20 Replacement of this ligand with
the triggering of antiangiogenic effects in developing zebrafish
embryos. Furthermore, the inhibition of thioredoxin reductase
by Au-Naphth-1 could be related to the presence of the gold
phosphine moiety.18
In another recent study we evaluated the biodistribution profile
of a series of chloro gold(I) phosphine derivatives.25 These studies
showed a rather low uptake of gold in the nuclei of tumor cells
for these kind of gold phosphine complexes and indicated that
larger, more lipophilic ligands at the phosphor center are able to
slightly increase the overall cellular uptake and biodistribution of
gold into the nuclei.
aDepartment of Integrative Zoology, Institute of Biology, University of
Leiden, 2333 AL Leiden, The Netherlands
In continuation of these studies we prepared a series of naph-
thalimide gold(I) phosphine complexes and evaluated their cy-
totoxic properties and biodistribution. Compounds with different
alkyl and aryl substituents on the phosphor were prepared in order
to evaluate the influence of these substituents on the bioactivity
and to shed more light on the general biological functions of gold
phosphine complexes with naphthalimide ligands. One compound
was selected for further studies concerning fluorescence moni-
toring and antiangiogenic properties (angiogenesis and tumor-
induced angiogenesis).
bInstitute of Pharmacy, Freie Universita¨t Berlin, Ko¨nigin-Luise-Str 2+4,
14195 Berlin, Germany
cInstitute of Pharmaceutical Chemistry, Technische Universita¨t Braun-
schweig, Beethovenstr. 55, 38106 Braunschweig, Germany. E-mail: ingo.ott@
tu-bs.de
† Electronic supplementary information (ESI) available: Studies on the
inhibition of angiogenesis in zebrafish embryos and reduction of tu-
mor cell-induced neovascularization in a zebrafish/tumor xenograft
angiogenesis assay by a non-metal naphthalimide derivative. See DOI:
10.1039/b912378d
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The Royal Society of Chemistry 2009
Dalton Trans., 2009, 10799–10805 | 10799
©

Results and discussion
Synthesis
The target compounds 1a–4a were prepared by a convenient re-
cently reported procedure starting from 4-mercaptonaphthalic an-
hydride (Fig. 2), and finally isolated by column chromatography.18
The structures were confirmed by 1H-NMR-spectroscopy, EI-MS
spectroscopy and elemental analyses.
Fig. 3 Cellular uptake of 5.0 mM gold(I) phosphine complexes into HT-29
and MCF-7 cells after 6 h; in some cases the error bars are hidden behind
the symbols; values of 1–4 are from ref. 25.
analogues 1a–4a was significantly increased compared to all
chloro analogues (1–4). This demonstrated that the organic thio-
naphthalimide ligand is a useful structure to obtain complexes
with enhanced cellular accumulation properties. Based on various
characteristics of tumor cells the cellular molar concentrations
can be estimated from the obtained cellular uptake values.26
Accordingly, the values for 1a–4a in HT-29 cells correlated to 227,
127, 162 and 104 mM, respectively. As the exposure concentration
was 5.0 mM in these experiments it can be concluded that the
cellular concentrations of 1a–4a exceeded the extracellular ones at
least 20 fold.
A very important observation of our previous study on 2
and 2a was that the naphthalimide ligand caused an increased
uptake of gold in the nuclei of tumor cells. Therefore, we also
investigated the gold levels of nuclei isolated from cells exposed to
5.0 mM of the different gold complexes for 6 h (Table 1). Under
these experimental conditions the nuclear biomass was strongly
decreased (presumably to early toxic effects) for 1a–4a making a
proper analysis impossible in most cases. However, where sufficient
amounts of nuclei could be obtained the respective gold levels of
1a–4a were strongly increased compared to 1–4 confirming the
previous result. Based on the elevated overall cellular uptake values
of 1a–4a it can be concluded that this strong accumulation of gold
in the nuclei is also a consequence of the higher cellular gold levels
caused by the presence of the naphthalimide ligand.
Fig. 2 Synthesis of 1a–4a, (a) 2-(dimethylamino)ethylamine, EtOH,
reflux heating, 6 h; (b) CH2Cl2, room temperature, 5 h.
Cell growth inhibitory effects, uptake into tumor cells and
into nuclei
The cell growth inhibitory effects of the naphthalimide gold(I)
phosphine target complexes 1a–4a were evaluated in comparison
to the respective chloro analogues 1–4 in HT-29 colon carcinoma
and MCF-7 breast cancer cells (see Table 1). Concerning the
trimethyl-, triethyl- and tri-tert-butyl analogues, the naphthalim-
ide containing complexes were approximately twice as active as the
chloro complexes whereas the activities of Cl-Au-P(Ph)3 (4) and
Naphth-Au-P(Ph)3 (4a) did not significantly differ. IC50 values for
1a to 4a were in the range of 1.1 to 3.7 mM, which is within the
range of the gold(I) phosphine lead compound auranofin (IC50
values of 2.6 mM in HT-29 cells26 and 1.1 mM in MCF-7 cells27),
the platinum anticancer drug cisplatin (IC50 values of 7.0 mM in
HT-29 cells and 2.0 mM in MCF-7 cells 28) and related non-metal
naphthalimides (IC50 values in the range of 1.9 mM to 4.6 mM22)
investigated under the same experimental conditions.
The cellular gold levels after 6 h exposure to 5 mM of 1–4
and 1a–4a were measured by electrothermal atomic absorption
spectroscopy and related to the present cell biomass (nmol Au per g
cellular protein values, Fig. 3). In good agreement with the results
of the proliferation experiments, the uptake of the naphthalimide
As this initial biological screening of 1a–4a did not reveal
substantial differences between the single complexes, we focused
on 4a in the following experiments, which is structurally most
different from the previously in more detail investigated 2a.18
Table 1 Cell growth inhibitory effects (IC50 values) and nuclear gold levels
(nmol Au per g nuclear protein) of gold(I) phosphine complexes in HT-29
and MCF-7 cells
Fluorescence spectroscopy and fluorescence microscopy of 4a
Many naphthalimide compounds exhibit strong luminescence
emission, which is environmentally sensitive concerning e.g. sol-
vent polarity or pH.22,29,30 In our previous study on 2a we reported
on a decrease in fluorescence intensity upon increasing solvent
polarity, which was accompanied by a red-shift and broadening
of the fluorescence emission maxima.18
A 3D-scan of 4a in DMSO showed an intensive maximum at
an excitation wavelength of 438 nm (Fig. 4, top). As expected the
intensity of this maximum was significantly enhanced in the apolar
solvent CHCl3 and polar media exhibited red-shifted, broadened
emission maxima (emission maxima of 4a for excitation at 438 nm:
CHCl3: 509–512 nm, DMSO: 530–538 nm, MeOH: 542–545 nm;
Fig. 4, bottom). In aqueous solvents such as phosphate buffered
IC50 HT-29 IC50 MCF-7 Nuclear Au HT-29 Nuclear Au MCF-7
(mM)
(mM)
(nmol Au/g)
(nmol Au/g)
0.6
0.9
0.8
1
2
3
4
5.2
5.3
5.2
4.2
3.9
3.2
3.1
2.6
1.8
1.3
1.1
3.7
21.6
62.4
84.3
n.d.
n.d.
109.4
1.9
1.3
3.0
2.1
0.4
25.9
39.3
0.9
0.1
14.2
17.0
181.8
57.6
0.6
0.0
1a 2.6
2a 2.6
3a 2.0
4a 3.2
—
—
—
—
0.4
0.2
1.6
0.7
55.1
300.3
330.8
194.2
0.1
137.8
0.8
43.4
9.1
294.5
n.d.: not detectable; —: no valid values obtained due to a strong reduction
in cell biomass. Values for 1–4 are from ref. 25.
10800 | Dalton Trans., 2009, 10799–10805
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©

MCF-7 cells exposed to a concentration of 4a below its IC50
value (2.0 mM) exhibited fluorescence emission from certain areas
of the cells (top sections of Fig. 5) related to an uptake of
the compound in cell organelles such as the nuclei. Exposure
to a concentration of 4a above its IC50 value (10 mM) led to
strong fluorescence emission roughly matching to the complete cell
monolayer (bottom sections of Fig. 5). At this concentration toxic
effects in the cells were already relevant as observed by a detaching
of cells from the well surface (Fig. 5, bottom left). In conclusion
these experiments indicate that 4a is preferentially biodistributed
into the nuclei and other cell organelles and that the uptake
strongly increases upon exposure to toxic concentrations, which
are supposedly accompanied by a breakdown of cell membrane
integrity.
Inhibition of angiogenesis in zebrafish embryos and reduction of
tumor cell-induced neovascularization in a zebrafish/tumor
xenograft angiogenesis assay
During our previous study on 2a we noted a significant inhibition
of angiogenesis in developing zebrafish embryos, which themselves
represent a convenient model for the in vivo study of angiogenic
processes.18,38,39
Accordingly, we evaluated the inhibition of blood vessel forma-
tion in the same assay for 4a and used 4 as a reference.
Animals exposed to 4 could not be distinguished from an
untreated control (data not shown) but animals exposed to 4a
for the indicated periods exhibited relevant damages in vessel
formation (as indicated by white arrows in Fig. 6) with 40–52%
of the embryos being affected (see Table 2). Antiangiogenic effects
can be observed in these experiments by an impaired or defective
formation of intersegmental vessels (IV), dorsal longitudinal
anastomic vessels (DLAV) and/or reduced subintestinal veins
(SIV).
Fig. 4 Fluorescence characteristics of 4a (5.0 mM), top: scan in DMSO,
bottom: fluorescence emission scans in various solvents at an excitation
wavelength of 438 nm.
These results are in good agreement with those of our recent
study on 2 and 2a, which had given comparable results.18 As chloro
gold phosphine compounds are not active in these experiments
in contrast to naphthalimide gold phosphine complexes it could
be concluded that the antiangiogenic effects are a consequence
of the presence of the heteroaromatic ligand. In order to study
this in more detail analogous experiments were performed on a
non-metal naphthalimide derivative, in which the gold phosphine
partial structure was replaced by an ethyl group, and indeed similar
results were observed (see ESI for details†).
saline pH 7.4 (PBS), 1 N HCl and 1 N NaOH no emission could
be detected. These effects can be related to the photoinduced
electron transfer (PET effect) between the side chain nitrogen and
the heterocyclic naphthalimide core. PET effects can be strongly
influenced by the polarity of the chemical environment.31,32
With the exception of the experiments in CHCl3, in which 2a had
shown a maximum at Ex./Em. 440/514–518 nm,18 the emission
maxima of 4a were approximately the same as those previously
reported for 2a indicating that the influence of the substituents on
the phosphine partial structure is negligible and the fluorescence
characteristics of the complexes are mainly determined by the
naphthalimide ligand.
Recently, a novel in vivo tumor xenograft angiogenesis as-
say (neovascularization assay) has been established in zebrafish
The described luminescence properties enable the evaluation of
cellular biodistribution by fluorescence microscopy. This method
offers the opportunity to study the location of the compounds in
living cells18,22,33,34 but has the disadvantage that it provides rather
qualitative information in contrast to the quantitative information
provided by other methods such as AAS26,35 or inductively
coupled plasma mass spectrometry (ICP-MS).36,37 Microscopic
images obtained with the compounds under study have to be
interpreted with caution based on the above described fluorescence
characteristics, which indicated that the complexes will be well
detectable in apolar cell environments (e.g. membranes) but could
be less visible or invisible in more polar locations such as the
cytosol.
Table 2 Inhibition of angiogenesis in developing zebrafish embryos
24 h
48 h
72 h
96 h
% defects
% defects
% defects
% defects/% alive
7
Control
0
0
0
40
42
0
0
0
52
49
0
0
0
52
50
0/91
0/90
3
4 0.01 mM
4 0.05 mM
4a 0.01 mM
4a 0.05 mM
2
0/89
9
2
1
1
5
6
51 /96
49 /90
3
3
2
2
Given are the percentages of embryos with defects in DLAV or/and SIV
formation after the indicated period and the percentages of embryos alive
after 96 h.
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©

Fig. 5 Microscopic pictures of MCF-7 cells exposed to 4a for 6 h; top left: 2.0 mM 4a, brightfield image, top right: 2.0 mM 4a, fluorescence image;
bottom left: 10 mM 4a, brightfield image; bottom right: 10 mM 4a, fluorescence image.
Table 3 Inhibition of tumor-cell induced neovascularization in develop-
when normalized to the solvent control (Table 3). Comparative
studies with the non-metal naphthalimide derivative mentioned
above again revealed comparable effects (see ESI†).
Taken together, these data show that compound 4a has antian-
giogenic properties and inhibits both, angiogenesis in developing
zebrafish embryos as well as tumor-cell induced angiogenesis.
These effects could be clearly related to the presence of the organic
naphthalimide ligand.
ing zebrafish embryos transplanted with human pancreatic cancer cells
4 (% positive)
4a (% positive)
4
4
0 mM (solvent control)
0.05 mM
0.1 mM
84
70
77
84
19
14
2
2
2
4
Given are the percentages of embryos with induced vessel formation at
24 h post injection of tumor cells. Three independent experiments were
performed and for each concentration and compound 100 embryos were
investigated. The assay was essentially performed as previously described.41
Non-transplanted zebrafish embryos do not show a similar formation of
microvasculature from the SIV (data not shown).
Conclusion
A series of thio-naphthalimide gold(I) phosphine complexes
(1a–4a) displayed promising cell growth inhibiting potencies.
Atomic absorption spectroscopy studies showed a stronger uptake
into the tumor cells as well as an enhanced accumulation of gold in
the nuclei of the cells for 1a–4a compared to the respective chloro
analogues 1–4. However, comparing the results obtained with
1a–4a it is obvious that the nature of the alkyl/aryl substituents
on the phosphor did not have a strong impact on bioactivity.
The triphenylphosphine analogue 4a was selected for more
detailed studies. Fluorescence spectroscopic and fluorescence
microscopic experiments indicated a significant influence of the
solvent polarity on the emission intensities and an uptake into
cell compartments including the nuclei. Additionally, 4a exhibited
strong antiangiogenic effects in developing zebrafish embryos con-
cerning “normal” angiogenesis and tumor-induced angiogenesis.
As the chloro analogue 4 was not active in this assay and a non-
metal naphthalimide derivative triggered similar effects, it can be
concluded that the antiangiogenic effects are a consequence of the
presence of the thio-naphthalimide ligand.
embryos.40,41 This novel model system allows us to follow the induc-
tion of blood vessel formation by xenotransplanted proangiogenic
human cancer cells in real-time in living zebrafish and enables the
quantification of neovascularization in vivo and in high-resolution.
In this study we used this tumor-cell induced angiogenesis model
in order to investigate the effects of 4 and 4a on the induction
of new blood vessels by transplanted human pancreatic tumour
cells (PaTu-8998T cells).42,43 Our results show inhibitory effects
of 4a on tumor-cell induced angiogenesis. 4a at two non-lethal
concentrations strongly inhibited the induction of new microvas-
culature in the zebrafish neovascularization assay when added to
the water containing the zebrafish embryos (Fig. 7 and Table 3).
A final concentration of 0.1 mM of compound 4a led to an 83%
reduction of tumor-induced angiogenesis when compared to the
solvent control DMSO (Table 3). In contrast, compound 4 at the
same final concentration of 0.1 mM showed minor effects and
led only to 9% reduction of tumor-induced neovascularization,
10802 | Dalton Trans., 2009, 10799–10805
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supposedly interacts with TrxR by covalent binding, which in
turn requires the removal of a coordinated ligand.8,19,20 For the
complexes described here it was observed that exposure of cells to
the naphthalimide containing derivatives 1a–4a yielded elevated
gold levels in the cells and in nuclei isolated from the cells. This
suggests on the one hand that the organic ligand transports gold
into the cells and nuclei and on the other hand that the gold–
sulfur bond can be considered to be sufficiently stable under in
vitro conditions (although it is very likely that the complexes
react with biomolecules over time and different gold species are
formed continuously). In this context it also should be noted that
previous studies on the interaction between 2a and a cysteine
containing model peptide had indicated that the phosphine ligand
is more stably bound to the gold central atom than the thiolate
ligand.18
Further studies on the evaluation of the biological pro-
file of these and structurally related gold metallodrugs are
ongoing.
Experimental
General
Chemicals and reagents were purchased from Sigma, Aldrich
or Fluka. 2a and 4-mercapto-1,8-naphthalic anhydride were
prepared as previously described.18 PBS: phosphate buffered saline
pH 7.4; NMR spectra were recorded on a 400 MHz spectrometer
(Avance/DPX 400, Bruker); elemental analysis: Perkin-Elmer
240 C; MS spectra: CH-/A-Varian MAT (70eV).
Fig. 6 Angiogenesis assay. Tg:fli-1/EGFP embryos were either treated
with compound 4 (a–d) or 4a (e–h). Pictures shown are examples of
embryos from 2 to 5 days post fertilization (dpf). The compounds were
added to the water containing 1 dpf dechorionated embryos at a final
concentration of 0.05 mM. In image b, an intersegmental vessel (IV), a
dorsal longitudinal anastomic vessel (DLAV) and the subintestinal vein
(SIV) are indicated by arrows. Arrows in e–h point to vessel defects in
the embryos treated with compound 4a. All fluorescence images of the
Tg:fli1/eGFP zebrafish were taken by laser scanning confocal microscopy.
Synthesis; general procedure
An amount of 100–120 mg 4-mercapto-1,8-naphthalic anhydride
was suspended in 15 mL of absolute ethanol, and 0.25 mL
(2.29 mmol) 2-(dimethylamino)ethylamine were added. The re-
sulting mixture was slowly heated and finally kept under reflux
heating for 6 h. The solvent was removed by evaporation, and
the remaining dark red oil was dried. The residue was dissolved
in 10 mL of absolute CH2Cl2, and 100–110 mg of 1, 3 or 4
and 0.2 g (1.45 mmol) of anhydrous K2CO3 were added. The
resulting mixture was stirred at room temperature for 5 h. The
solvent was evaporated and the product isolated by column
chromatography (stationary phase: silica, mobile phase: CH2Cl2
followed by CH2Cl2/MeOH 9/1).
Fig. 7 Tumor-induced angiogenesis. Shown are example pictures ob-
tained with the recently described neovascularization assay.41 Human
pancreatic tumor cells (PaTu-8998T cells) were transplanted close to the
developing subintestinal vein (SIV) into 48 hpf Tg:fli-1/EGFP embryos.
Immediately after transplantation, zebrafish embryos were either treated
with 4 (left image) or 4a (right image) and 24 h later investigated for the
formation of new vasculature from the SIV. Compound 4a showed strong
inhibition of tumor-induced angiogenesis, whereas 4 had a minor effect
both compared to the solvent control (see Table 3).
[N-(N¢,N¢-Dimethylaminoethyl)-1,8-naphthalimide-4-
sulfide](trimethyl-phosphine)gold(I) (1a)
116 mg (0.50 mmol) 4-mercapto-1,8-naphthalic anhydride; 103 mg
(0.33 mmol) 1, yield: 77 mg, red-orange crystals (0.13 mmol, 39%);
1H-NMR (CDCl3): (ppm) 1.66 (d, 9H, -P-CH3), 2.37 (s, 6H, -
N(CH3)2), 2.66 (t, 2H, 3J = 7.2 Hz, CH2), 4.31 (t, 2H, 3J = 7.2 Hz,
As the mode of molecular target interaction of gold phosphine
thiolate complexes is based on a loss of coordinated ligands and
the agents are therefore not completely stable during in vitro/
in vivo experiments, the nature of the active species of the studied
compounds (or related complexes already used as antirheumatic
drugs, respectively) is not completely clear at this stage.44 For
example, for auranofin it was reported that its thiolate ligand
was lost during cell culture experiments. Furthermore, this drug
3
3
CH2), 7.67 (dd, 1H, J = 7.3 Hz, 1H, J = 8.4 Hz, ArH6), 8.14
3
3
(d, 1H, J = 7.8 Hz, ArH3), 8.26 (d, 1H, J = 7.8 Hz, ArH2),
3
4
8.57 (dd, 1H, J = 7.3 Hz, J = 0.9 Hz ArH5), 9.04 (dd, 1H,
3J = 8.4 Hz, 4J = 0.9 Hz ArH7); MS (EI): m/z: 573.1 (M + H+);
elemental analyses (calc./found): C (39.87/40.04), H (4.23/4.42),
N (4.89/5.10).
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Dalton Trans., 2009, 10799–10805 | 10803
©

(tri–tert-Butylphosphine)[N-(N¢,N¢-dimethylaminoethyl)-1,8-
Fluorescence microscopy of tumor cells
naphthalimide-4-sulfide]gold(I) (3a)
Cells were grown in 6-well plates (Sarstedt) until at least 70% con-
fluency. The compounds were prepared freshly as stock solutions
in DMF and diluted with the cell culture medium to the final assay
concentrations (2.0 mM and 10 mM, 0.1% V/V DMF). The cell
culture medium was replaced with fresh medium containing 4a and
incubated for 6 h at 37 ◦C in a 5% CO2/95% air atmosphere. The
medium was removed, the cells were washed with PBS and finally
500 mL PBS was added to each well. Microscopy was performed
using a Axiovert 40 CFL microscope (Zeiss) equipped with a 50
103 mg (0.45 mmol) 4-mercapto-1,8-naphthalic anhydride; 108 mg
(0.25 mmol) 3, yield: 80 mg, red-orange crystals (0.11 mmol, 44%);
1H-NMR (CDCl3): (ppm) 1.56 (m, 27H, -C(CH3)3), 2.37 (s, 6H, -
N(CH3)2), 2.66 (t, 2H, 3J = 7.2 Hz, CH2), 4.31 (t, 2H, 3J = 7.2 Hz,
3
3
CH2), 7.66 (dd, 1H, J = 8.5 Hz, J = 7.3 Hz, ArH6), 8.19 (d,
3
3
1H, J = 7.9 Hz, ArH3), 8.24 (d, 1H, J = 7.9 Hz, ArH2), 8.56
3
4
3
(dd, 1H, J = 7.3 Hz, J = 1.2 Hz, ArH5), 9.08 (dd, 1H, J =
4
8.5 Hz, J = 1.2 Hz, ArH7); MS (EI): m/z: 699.2 (M + H+);
25
elemental analyses (calc./found): C (48.18/48.15), H (6.06/6.22),
N (4.01/3.90).
W mercury vapor short arc lamp and a Ex/Em 390 11/460 nm
filter.
Animal care and handling
[N-(N¢,N¢-Dimethylaminoethyl)-1,8-naphthalimide-4-
Transgenic zebrafish (Danio rerio) Tg(fli1:eGFP) were handled
in compliance with local animal care regulations and standard
protocols of the Netherlands. Fish were maintained at 28 ◦C in
aquaria with day/night light cycles (10 h dark versus 14 h light
periods). The fish were crossed in the afternoon and eggs were
harvested the next morning.
sulfide](triphenyl-phosphine)gold(I) (4a)
105 mg (0.46 mmol) 4-mercapto-1,8-naphthalic anhydride; 112 mg
(0.23 mmol) 4, yield: 96 mg, red-orange crystals (0.13 mmol, 56%);
1H-NMR (CDCl3): (ppm) 2.36 (s, 6H, -N(CH3)2), 2.65 (t, 2H,
3J = 7.2 Hz, CH2), 4.31 (t, 2H, 3J = 7.2 Hz, CH2), 7.45–7.57 (m,
14H, ArH), 7.65–7.70 (m, 2H, ArH), 8.25 (m, 2H, ArH2, ArH3),
8.58 (m, 1H, ArH5), 9.10 (m, 1H, ArH7); MS (EI): m/z: 759.2
(M + H+); elemental analyses (calc./found): C (53.83/53.92), H
(3.99/4.06), N (3.69/3.63).
Live imaging of angiogenesis
Substances were administered once to dechorionated (24 h post
fertilization) hpf embryos. For this purpose, complexes were
dissolved in DMF and the stock solutions were diluted (1 : 1000)
with the water containing the embryos to the final concentrations
of 0.01 and 0.05 mM. The control solutions contained the same
concentration of DMF (0.1%). The Biorad Confocal microscope
1024ES (Zeiss microscope) was used for investigating vessel defects
in the tranquilized (0.042 mg/mL tricaine (Sigma, St. Luis, MO))
Tg(fli1:eGFP) fish.
Antiproliferative effects
The antiproliferative effects in MCF-7 and HT-29 cells after
72 h (HT-29) or 96 h (MCF-7) exposure to the gold complexes
were evaluated according to recently described procedures.22,27 For
the experiments the compounds were prepared freshly as stock
solutions in DMF and diluted with the cell culture medium to the
final assay concentrations (0.1% V/V DMF). The IC50 value was
described as that concentration reducing proliferation of untreated
control cells by 50%.
Cell culture and staining of human pancreatic cancer cells42,43
PaTu8988T cells were cultured in DMEM high glucose, with
10% FCS and 1 : 100 Pen/Strep. After trypsinization, cells were
twice washed with 67% DPBS (GIBCO, Invitrogen), transferred
to 1.5 mL Eppendorf tubes and centrifuged for 5 min at 1500 rpm.
Cells were resuspended in 250 mL staining solution (67% DPBS
containing 4 ng/ml final concentration of CellTracker CM-Dil
(Invitrogen, Eugene, Oregon, USA). Cells were incubated at 37 ◦C
Uptake of gold into the cells and into the nuclei
Cells were grown until at least 70% confluency in 75 cm2 cell
culture flasks. The compounds were prepared freshly as stock
solutions in DMF and diluted with the cell culture medium to
a concentration of 5.0 mM (0.1% V/V DMF). The cell culture
medium was removed and replaced with fresh medium containing
the gold complexes. After 6 h exposure, the gold content of isolated
cells and isolated cell nuclei were measured by graphite furnace
atomic absorption spectroscopy and measured gold levels were
related to the protein contents of the cells and nuclei as described
in more detail recently.18,25,26
◦
for 4 min followed by a 15 min incubation period at 4 C. After
an initial washing step in 1.0 mL 100% FCS (Sigma) the cells were
twice washed with 67% DBPS.
Tumor-cell induced angiogenesis40,41
48 hpf zebrafish embryos were dechorionated and anesthesized
using 0.042 mg/mL Tricaine (Sigma, St. Luis, MO) and put
on a 1.8% agarose dish for injections. Cells were prepared as
described above, then suspended in 20 mL 12.0 mg/mL Matrigel
solution (Cultrex R Basement Membrane Extract, R and D
systems, Minneapolis, USA) and kept on ice for a short period
of time until injection. The CellTram Oil injector (Eppendorp,
Hamburg, Germany) was loaded by using a manual injector
(Eppendorf; Injectman NI2). An aliquot of 5 mL of the cell
suspension in Matrigel was loaded into an injection needle,
and embryos were injected as described in more detail in the
Fluorescence spectroscopy
All solvents used for the fluorimetric experiments were purged
with nitrogen prior to use. 4a was dissolved as a stock solution
(500 mM) in DMF, diluted 100 fold with the respective solvents
(CHCl3, DMF, DMSO, MeOH, PBS, 1 N NaOH and 1 N HCl)
and measured using a Hitachi F-4500 fluorescence spectrometer.
Only corrected spectra were taken.
10804 | Dalton Trans., 2009, 10799–10805
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The Royal Society of Chemistry 2009
©

literature.40,41 Complexes were dissolved in DMF and the stock
solutions were diluted (1 : 1000) with the water containing the
embryos to the final concentrations of 0.01 and 0.05 mM. The
control solutions contained the same concentration of DMF
(0.1%). After injection, embryos were incubated at 35 ◦C for 24 h.
Afterwards, embryos were checked and photographed using the
Biorad confocal microscope 1024ES (Zeiss microscope).
18 I. Ott, X. Qian, Y. Xu, D. H. W. Vlecken, I. J. Marques, D. Kubuat, J.
Will, W. S. Sheldrick, P. Jesse, A. Prokop and C. P. Bagowski, J. Med.
Chem., 2009, 52, 763.
19 R. M. Snyder, C. K. Mirabelli and S. T. Crooke, Biochem. Pharmacol.,
1986, 35, 923.
20 R. M. Snyder, C. K. Mirabelli and S. T. Crooke, Biochem. Pharmacol.,
1987, 36, 647.
21 M. F. Brana and A. Ramos, Curr. Med. Chem.: Anti-Cancer Agents,
2001, 1, 237.
22 I. Ott, Y. Xu, J. Liu, M. Kokoschka, M. Harlos, W. S. Sheldrick and
X. Qian, Bioorg. Med. Chem., 2008, 16, 7107.
23 H. Zhu, M. Huang, F. Yang, Y. Chen, Z. H. Miao, X. H. Qian, Y. F.
Xu, Y. X. Qin, H. B. Luo, X. Shen, M. Y. Geng, Y. J. Cai and J. Ding,
Mol. Cancer Ther., 2007, 6, 484.
24 A. Wu, Y. Xu and X. Qian, Bioorg. Med. Chem., 2009, 17, 592.
25 H. Scheffler, Y. Ya and I. Ott, Polyhedron, 2009,
DOI: 10.1016/j.poly.2009.06.007.
26 I. Ott, H. Scheffler and R. Gust, ChemMedChem, 2007, 2, 702.
27 I. Ott, T. Koch, H. Shorafa, Z. Bai, D. Poeckel, D. Steinhilber and R.
Gust, Org. Biomol. Chem., 2005, 3, 2282.
Acknowledgements
Financial support by DFG (Deutsche Forschungsgemeinschaft)
and GIF (German–Israeli Foundation) is gratefully acknowl-
edged. I. O. wants to thank Prof. R. Gust from Freie Universita¨t
Berlin for generous access to all facilities of his laboratories.
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