A gold(I) phosphine complex containing a naphthalimide ligand functions as a TrxR inhibiting antiproliferative agent and angiogenesis inhibitor

Article abstract of DOI:10.1021/jm8012135

The novel luminescent gold(I) complex [N-(N'N'-dimethylaminoethyl)-1,8- naphthalimide-4-sulflde](trieth- ylphosphine)gold(I) was prepared and investigated for its primary biological properties. Cell culture experiments revealed strong antiproliferative ef

Full text of DOI:10.1021/jm8012135

J. Med. Chem. 2009, 52, 763–770
763
A Gold(I) Phosphine Complex Containing a Naphthalimide Ligand Functions as a TrxR
Inhibiting Antiproliferative Agent and Angiogenesis Inhibitor
Ingo Ott,*,†,‡ Xuhong Qian,‡ Yufang Xu,‡ Danielle H. W. Vlecken,§ Ines J. Marques,§ Dominic Kubutat,| Joanna Will,|
William S. Sheldrick,| Patrick Jesse, Aram Prokop, and Christoph P. Bagowski§
Institute of Pharmacy, Freie UniVersita¨t Berlin, Ko¨nigin-Luise-Strasse 2+4, 14195 Berlin, Germany, Shanghai Key Laboratory of
Chemical Biology, East China UniVersity of Science and Technology, Meilong Road 130, Shanghai 200237, China, Department of
IntegratiVe Zoology, Institute of Biology, UniVersity of Leiden, AL Leiden, The Netherlands, Lehrstuhl fu¨r Analytische Chemie,
Ruhr-UniVersita¨t Bochum, 44780 Bochum, Germany, and Department of Pediatric Oncology/Hematology, UniVersity Medical Center
Charite´ Berlin, D-13353 Berlin, Germany
ReceiVed September 25, 2008
The novel luminescent gold(I) complex [N-(N′,N′-dimethylaminoethyl)-1,8-naphthalimide-4-sulfide](trieth-
ylphosphine)gold(I) was prepared and investigated for its primary biological properties. Cell culture
experiments revealed strong antiproliferative effects and induction of apoptosis via mitochondrial pathways.
Biodistribution studies by fluorescence microscopy and atomic absorption spectroscopy showed the uptake
into cell organelles, an accumulation in the nuclei of tumor cells, and a homogeneous distribution in zebrafish
embryos. In vivo monitoring of vascularisation in developing zebrafish embryos revealed a significant anti-
angiogenic potency of the complex. Mechanistic experiments indicated that the inhibition of thioredoxin
reductase (based on the covalent binding of a gold triethylphosphine fragment) might be involved in the
pharmacodynamic behavior of this novel gold species.
Introduction
The vast majority of todays marketed drugs are organic
compounds not containing metal atoms. However, the continuing
success story of the platinum anticancer drugs (e.g., cisplatin;
see Figure 1) and more recently the introduction of various non-
platinum agents into clinical trials (e.g., the antitumor ruthenium
complex NAMI-A or the ferrocenium compound ferroquine1
for the treatment of malaria) demonstrate that there is a
Figure 1. Various metallodrugs.
considerable potential for the development of novel metallo-
drugs, which may offer interesting pharmacological properties
Scheme 1. Drug Development Strategy
based on different chemical and geometrical features compared
to “organic drugs”.2-4
Among novel non-platinum based antitumor agents, gold
complexes have recently gained attention because of their strong
antiproliferative effects. Furthermore, a strong inhibition of the
enzyme thioredoxin reductase (TrxRa), which is involved in
tumor cell proliferation, has been noted for many derivatives
and an antimitochondrial mode of action for these complexes
has been proposed.5,6
The lead structure for antitumor-active gold complexes is
auranofin (see Scheme 1), which is characterized by its gold(I)
central atom as well as a triethylphosphine and a carbohydrate
ligand. The mode of action of auranofin and other related gold
complexes (e.g., the chloro analogue Et3PAuCl) is most likely
* To whom correspondence should be addressed. Address: Institute of
Pharmacy, Freie Universita¨t Berlin, Ko¨nigin-Luise-Strasse 2+4, 14195
Berlin, Germany. Phone: +49 30 8385 6209. Fax: +49 8385 6906. E-mail:
ottingo@zedat.fu-berlin.de.
†
Freie Universita¨t Berlin.
East China University of Science and Technology.
University of Leiden.
Ruhr-Universita¨t Bochum.
‡
§
based on a covalent interaction with disulfide reductases (such
as TrxR) under loss of the ligands (for a structural study on the
interaction of a gold phosphole complex with the enzyme
glutathione reductase, see ref 7), indicating that the active species
is the gold ion itself and the ligands are more relevant for the
biodistribution and kinetic properties of the agents.
|
University Medical Center Charite´ Berlin.
a Abbreviations: AAS, atomic absorption spectroscopy; DLAV, dorsal
longitudinal anastomotic vessels; dpf, days postfertilization; hpf, hours
postfertilization; IV, intersegmental vessels; MMP, mitochondrial membrane
potential; PET, photoinduced electron transfer; SIV, subintestinal vein; TrxR,
thioredoxin reductase.
10.1021/jm8012135 CCC: $40.75
2009 American Chemical Society
Published on Web 01/05/2009

764 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Scheme 2. Synthesis of the Target Compounda
Ott et al.
a (a) Na2S, DMF, room temp., 6 h; (b) 2-(dimethylamino)ethylamine, EtOH, reflux heating, 6 h; (c) Et3PAuCl, CH2Cl2, room temp, 5 h.
Figure 2. Left: 3D-contour plot of 0.5 µM 3 in CHCl3. Right: emission spectra (excitation, 440 nm) of 5 µM 3 in various solvents: (a) CHCl3, (b)
DMF, (c) DMSO, (d) MeOH, (e) PBS.
Consequently, the nature of the ligands attached to the gold
central atom is a valuable parameter for drug design. Previous
structure-activity relationship studies on linear, two coordinated
gold(I) complexes indicated that the presence of the phosphine
ligand is important for the biological potency of the complexes,
leaving the carbohydrate ligand of auranofin (or the chlorine
ligand of Et3PAuCl) as the first choice for structural variations.8
In order to create an agent with biological properties beyond
those resulting from the presence of the central gold atom, the
addition of an antiproliferative compound as ligand and retention
of the triethylphosphine moiety were considered (as outlined
in more detail in Scheme 1). We chose N-(N′,N′-dimethylami-
noethyl)-4-mercapto-1,8-napthalimide, a ligand that contains the
relevant pharmacophores of the naphthalimide class of antitumor
agents: a heterocyclic naphthalimide core for DNA intercalation
and a side chain containing a protonable nitrogen, which enables
an initial contact to the DNA phosphate backbone.9 As many
naphthalimide derivatives exhibit interesting fluorescence prop-
erties, this ligand supposedly also provides an improved
bioimaging of the compounds during in vitro and in vivo
example, the fluorescence emission intensity was found to be
significantly influenced by the chemical environment concerning
solvent polarity, pH value, or presence of metal ions.10-17
Regarding metal ions, enhancement of the signal intensity was
also observed depending on the nature of the respective metal.
Interestingly, for gold naphthalene complexes room temperature
phosphorescence from the naphthalene chromophore has been
reported recently.18 In this context it was of interest to
characterize the basic fluorescence properties of 3.
Figure 2 (left) shows a contour plot obtained with 3 in CHCl3.
Significant fluorescence emission was observed with a broad
maximum at Ex/Em 440/516 nm. Emission spectra recorded in
solvents with differing polarity (see Figure 2, right) showed that
the strongest signal intensities were present in CHCl3 whereas
in solvents with higher polarity (such as DMSO) the emission
was significantly decreased and almost disappeared in aqueous
buffer solution (PBS, pH 7.4). In 1 N NaOH or 1 N HCl no
emission signals could be recorded (data not shown). Besides
this lowered intensity, a red shift of the maxima and broadening
of the spectra were found in the more polar media compared to
the measurements in CHCl3 (emission maxima for excitation
at 440 nm: CHCl3, 514-518 nm; DMF, 521-528 nm; DMSO,
530-535 nm; MeOH, 542-546 nm).
10-17
examinations.
This report describes the preparation and primary biological
evaluation of the resulting gold complex [N-(N′,N′-dimethyl-
aminoethyl)-1,8-naphthalimide-4-sulfide](triethylphosphine)-
gold(I).
The described effects can be attributed to an influence of the
solvent polarity on the photoinduced electron transfer (PET)
between the side chain nitrogen and the aromatic naphthalimide
moiety. In solvents with higher polarity the PET can be
hampered because of an interaction of solvent molecules with
the free electron pair of the side chain nitrogen.
Chemistry
The target compound 3 was prepared by a three-step synthetic
procedure (see Scheme 2). Commercially available 4-bromo-
1,8-naphthalic anhydride 1 was reacted with an excess of Na2S
to yield 4-mercapto-1,8-naphthalic anhydride 2, which was
converted to a naphthalimide intermediate by reflux heating with
2-(dimethylamino)ethylamine. Finally, the gold phosphine
complex 3 was obtained by reaction of the crude intermediate
with chloro(triethylphosphine)gold(I) and isolated by column
chromatography. The structure was confirmed by 1H NMR, ESI,
and elemental analysis.
Antiproliferative Effects and Biodistribution
The antiproliferative effects of 3 were evaluated in HT-29
colon carcinoma and MCF-7 breast cancer cells. As a gold
phosphine containing reference, Et3PAuCl was used. IC50 values
obtained with 3 were slightly lower than those obtained with
Et3PAuCl in both cell lines (HT-29 cells, 5.3 ( 1.9 µM for
Et3PAuCl and 2.6 ( 0.4 µM for 3; MCF-7 cells, 3.2 ( 1.3 µM
for Et3PAuCl and 1.3 ( 0.7 µM for 3), indicating that the
replacement of the chlorine of Et3PAuCl by the naphthalimide
Interesting fluorescence properties of certain naphthalimide
derivates have been described in a broad range of reports. For

Gold(I) Phosphine Complex
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 765
Figure 3. Microscopic images (40-fold enlargement) of cells incubated with 5 µM 3 for 6 h: (top left) bright-field image of HT-29 cells; (top right)
bright-field image of MCF-7 cells; (bottom left) luminescence image of HT-29 cells; (bottom right) luminescence image of MCF-7 cells. For
MCF-7 cells an area showing increased luminescence colocalized with morphological cell surface changes is indicated by orange arrows.
Table 1. Uptake into the Nuclei of Tumor Cells after 24 h of Exposure
ligand (as realized in 3) offers a suitable strategy to obtain
Expressed as (nmol of Gold)/(mg of Nuclear Protein)
bioactive compounds. For auranofin similar activity had been
HT-29
MCF-7
noted in the same assay during previous investigations (IC50
values of 2.6 µM in HT-2919 and 1.1 µM in MCF-720 cells).
On the basis of the above-described fluorescence character-
istics, the cellular distribution of 3 could be studied by
fluorescence microscopy (see Figure 3). For both HT-29 and
MCF-7 cells exposed to 3, significant blue emission was
observed. Interestingly, the luminescence was not spread all over
the cells and appeared to be located in specific areas within the
cells. In MCF-7 cells the emission was found to be colocalized
with cell surface areas showing significant morphological
alterations.
Due to the mentioned effects of the solvent polarity on the
fluorescence intensity (see Figure 2, right), it can be assumed
that 3 is not detectable in the aqueous cytosolic parts of the
cells but will show strong emission if located within the apolar
membranes of cell organelles or chemical environment of
medium polarity. Thus, the observed distribution of fluorescence
emission can be attributed to the uptake of 3 in specific cell
compartments.
In this context it is of interest to note that the biodistribution
of a luminescent gold(I) N-heterocyclic carbene complex into
the lysosomes has been reported recently and an enhanced
uptake of various gold(I) complexes into mitochondria has been
determined.21-23
On the basis of its low detection limits and high analytical
selectivity, atomic absorption spectroscopy (AAS) provides a
convenient tool for quantitative cellular biodistribution studies
of metal complexes.19,24,25 In the present study it was of interest
to compare the biodistribution of Et3PAuCl, which cannot be
detected by fluorescence microscopy, with that of 3.
Therefore, we isolated the nuclei of HT-29 and MCF-7 cells
and investigated them for their gold content by AAS (see Table
1). After incubation with Et3PAuCl the nuclei of both cell lines
contained only a small amount of gold. Upon exposure to 3 the
nuclear gold concentration was increased strongly in comparison
to the results obtained with Et3PAuCl (approximately 5-fold for
Et3PAuCl
3
0.076 ( 0.012
0.350 ( 0.099
0.052 ( 0.004
1.399 ( 0.132
HT-29 cells and approximately 25-fold for MCF-7 cells). The
nuclear metal concentration resulting from exposure to 3 was
high in comparison to other metallodrugs investigated in the
same assay (e.g., up to 0.22 nmol/mg for a cobalt nucleoside
derivative in MCF-7 cells26 or certain cobalt peptide nucleic
acid bioconjugates in HT-29 cells25). These data clearly
demonstrate that the naphthalimide ligand, which itself repre-
sents a DNA targeting structure,9 is able to transport consider-
able amounts of the gold phosphine moiety into the nuclei,
making the DNA a reasonable biological target for 3.
Interaction with TrxR and Induction of Apoptosis
The inhibition of the enzyme thioredoxin reductase had to
be considered as another potential mechanism responsible for
the antiproliferative effects of 3. Thus, the inhibitory potentials
of Et3PAuCl and 3 were studied using isolated rat liver TrxR
by the DTNB (dithiobisnitrobenzoic acid) reduction assay. This
assay makes use of the fact that TrxR reduces the disulfide bonds
of DTNB with formation of 5-thionitrobenzoic acid, which can
be detected photometrically. In fact both agents inhibited the
activity of the enzyme with low EC50 values (0.14 ( 0.04 µM
for Et3PAuCl and 0.27 ( 0.01 µM for 3), confirming TrxR as
a possible biological target.
As mentioned above, the mechanism of action of gold
complexes is reportedly based on a covalent binding to cysteine
or selenocysteine residues of the active site of TrxR. In order
to evalute the possiblity of this mechanism for the gold
complexes under study, we performed hyphenated LC/ESI
tandem mass spectrometry experiments on the cysteine contain-
ing model peptide Ala-Gly-Cys-Val-Gly-Ala-Gly-Leu-Ile-Lys
incubated with 3 or Et3PAuCl for 24 h at 37 °C.
The mass spectra for the resulting reaction mixtures both
contained the molecular ion at m/z ) 1202 at high relative

766 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Ott et al.
Figure 4. MS/MS spectrum of the molecular ion [peptide + Au]+ at 1084.3 generated by neutral loss of triethylphosphine from the adduct [peptide
+ Au(PEt3)]+ (m/z ) 1202.5).
abundance for the peptide adduct of the {(PEt3)Au}+ fragment
(see Figure 4). This indicates that a covalent binding mechanism
under loss of the respective non-phosphine ligand could be
relevant for both of the gold complexes. Cysteine was confirmed
as the gold(I) binding site by analysis of the molecular ion at
m/z ) 1084.3 following its formation by neutral loss of the
triethylphosphine ligand from the original peptide adduct. Seven
b+ ions (b3+-b9+) and five y+ ions (y5+-y9+) can be identified
in the MS/MS spectrum of the adduct ion [peptide + Au]+ (see
Figure 4).
The inhibition of TrxR by gold compounds such as 3
reportedly triggers antimitochondrial and apoptotic effects.6,23,27,28
As the investigation of these parameters makes use of assays
requiring a flow cytometric analysis, which leads to less reliable
results with detached adherent growing cells, suspension grow-
ing BJAB lymphoma cells were used for these studies (see
Figure 5). Initially, the antiproliferative potency of 3 in these
cells was confirmed (>50% reduction of cell number in
concentrations between 2.5 and 10 µM; see Figure 5a).
The antiproliferative effect was accompanied by a strong
induction of apoptosis (>40% DNA fragmentation; see Figure
5b) with the majority of the cells in the late stage of apoptosis
(see Figure 5c). No significant necrosis was observed showing
that the cell death was not caused by unspecific (physical)
effects.
of angiogenesis.30,31 According to this fact and motivated by
reported antiangiogenic properties of other metal anticancer
drugs (e.g., the ruthenium complex NAMI-A32 or the hexacar-
bonyldicobalt complex Co-ASS33), we investigated the angio-
genesis inhibiting properties of Et3PAuCl and 3 by monitoring
the blood vessel formation in developing zebrafish (Danio rerio)
embryos.
Zebrafish embryos offer an established in vivo model for the
study of angiogenesis and vascular development.32-36 For the
experiments we used the transgenic zebrafish line, Tg:fli1/eGFP,
which exhibits a fluorescent green vasculature (based on the
expression of the green fluorescence protein under an early
endothelial promoter) and therefore allows microscopic live
imaging of angiogenesis.37
The biodistribution of 3 in the embryos was evaluated 1 day
after application by use of fluorescence microscopy (see Figure
6) and confirmed an efficient uptake and distribution of
luminescence throughout the whole organism. At this stage of
the embryo development most of the intersegmental vessels have
been lumenized and possess active circulation.
We further determined for the zebrafish embryos the maxi-
mum nonlethal dose (maximum tolerated dose, MTD) for
Et3PAuCl and 3. For both compounds the MTD was 0.1 µM.
This concentration was the maximum dosage used for the
angiogenesis experiments.
As depicted in Figure 7, treatment with 3 induced significant
anti-angiogenic effects as could be observed by the impaired
formation and absence of intersegmental vessels (IV), of dorsal
longitudinal anastomotic vessels (DLAV), and reduced sub-
intestinal veins (SIV) at 4 and 5 days postfertilization (dpf).
Table 2 lists the details on the observed vascularisation damages.
At exposure to 0.1 µM 3, 91% of the embryos showed vessel
defects. In contrast Et3PAuCl induced only marginal effects on
angiogenesis. Only 4% of the embryos exhibited impaired vessel
formation upon treatment with 0.1 µM Et3PAuCl.
The measurement of the mitochondrial permeability transition
reflects cells with a lower mitochondrial membrane potential
(MMP) undergoing apoptosis via the mitochondrial pathway.
Experiments concerning this effect showed a concentration
dependent increase of cells with impaired mitochondrial perme-
ability transition (see Figure 5d), which is in line with several
reports on the antimitochondrial effects of various gold
complexes.23,27,29
Inhibition of Angiogenesis in Zebrafish Embryos
TrxR contributes to tumor growth and development not only
by the inhibition of apoptotic events but also by the stimulation
The activity of 3 and the inactivity of Et3PAuCl indicate that
the observed angiogenesis inhibiting effects are in this case most

Gold(I) Phosphine Complex
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 767
Figure 5. Effects of 3 in BJAB cells: (a) proliferation after 24 h: (b) DNA fragmentation after 72 h; (c) cells in apoptotic/necrotic stage after 72 h;
(d) impairment of mitochondrial membrane potential (MMP) after 48 h.
probably not related to TrxR inhibition or the gold phosphine
moiety and are rather a consequence of the naphthalimide ligand
of 3. Anti-angiogenic properties of naphthalimides have not been
reported so far, and to the best of our knowledge 3 represents
the first gold complex and the first naphthalimide for which
angiogenesis inhibiting properties have been shown.
Further studies on the pharmacodynamics and pharmacoki-
netics of 3 and related gold complexes are ongoing.
Experimental Section
General. Chemicals and reagents were purchased from Sigma,
Aldrich, or Fluka. Chloro(triethylphosphine)gold(I) (Et3PAuCl) was
obtained from Sigma. PBS (phosphate buffered saline), pH 7.4,
was used. Compounds were freshly dissolved as stock solutions in
dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) prior
to the experiments and diluted with the respective cell culture media
or buffer during the assay procedures. Unless stated otherwise, the
final concentration of the DMF or DMSO vehicle was 0.1% (V/
V). NMR spectra were recorded on a 400 MHz spectrometer or a
500 MHz NMR spectrometer (Bruker). Elemental analysis was done
with a Perkin-Elmer 240 C, and MS spectra were collected on an
HP-1100 LC-MS spectrometer.
Conclusions
The gold(I) phosphine complex 3 displayed significant
antiproliferative effects in cultured tumor cells. Biodistribution
experiments by fluorescence microscopy showed an efficient
uptake of 3 into specific cell compartments, and AAS experi-
ments confirmed an elevated uptake of 3 into the nuclei. As
the non-naphthalimide Et3PAuCl showed a much lower nuclear
uptake, it may be speculated that the naphthalimide ligand of 3
might be a useful vector to facilitate transport of metals into
the nucleus. Studies on the mode of action indicated that the
inhibition of TrxR and induction of apoptosis via the mitochon-
drial pathway also play major roles in the pharmacodynamics
of this novel gold antitumor agent. For a cysteine containing
model peptide the covalent binding of 3 and Et3PAuCl to the
cysteine side residue after loss of the respective non-phosphine
ligand could be confirmed.
Interestingly, 3 exhibited significant anti-angiogenic effects
in developing zebrafish embryos in contrast to Et3PAuCl, which
was almost inactive in this assay. Consequently, the anti-
angiogenic properties can be attributed to the naphthalimide
ligand of 3, which is not present in Et3PAuCl.
The above-described results reveal a rather complex phar-
macological profile of 3 and indicate the presence of multiple
biological targets, of which nuclear (DNA) and mitochondrial
(TrxR) macromolecules may have high relevance. The mech-
anism underlying the observed anti-angiogenic effects remains
to be clarified.
Synthesis. [N-(N′,N′-Dimethylaminoethyl)-1,8-naphthalimide-
4-sulfide](triethylphosphine)gold(I) (3). An amount of 320 mg
(1.39 mmol) of 4-mercapto-1,8-naphthalic anhydride 2 was sus-
pended in 30 mL of absolute ethanol, and 0.55 mL (5.0 mmol)
2-(dimethylamino)ethylamine were added. The resulting 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 25 mL of CH2Cl2,
and 250 mg of chloro(triethylphosphine)gold(I) (0.71 mmol) and
0.21 g (1.52 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 and CH2Cl2/MeOH
10/1). Yield: 107 mg (0.174 mmol, 25%) of red-orange crystals.
1H NMR (CDCl3): (ppm) 1.26 (m, 9H, CH3), 1.92 (m, 6H, CH2),
2.37 (s, 6H, N(CH3)2), 2.65 (t, 2H, J ) 7.2 Hz, CH2), 4.31 (t, 2H,
J ) 7.2 Hz, CH2), 7.67 (dd, 1H, J ) 7.8 Hz, J ) 8.0 Hz, ArH6),
8.16 (d, 1H, J ) 7.8 Hz, ArH3), 8.26 (d, 1H, J ) 7.8 Hz, ArH2),
8.57 (m, 1H, ArH5), 9.06 (m, 1H, ArH7). HRMS: 615.15 g/mol
(M + H+). Anal. (C22H30AuN2O2PS) C, H, N.

768 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Ott et al.
Figure 7. Scanning laser confocal microscopy images of zebrafish
embryos exposed to Et3PAuCl (0.1 µM) or 3 (0.1 µM). Fishes were
treated only once, at 1 dpf with the respective drug. Pictures show
zebrafish larvae at 4 and 5 dpf, as indicated. (a) Et3PAuCl 4dpf: no
vessel defects were detectable, which is similar to solvent control (DMF)
treated fishes (data not shown). Examples of DLAV, IV, and SIV are
marked by white arrows. b) An example of a zebrafish embryo treated
with 0.1 µM 3 is shown at 4dpf. Damages of DLAV, IV, and SIV are
marked with white arrows. (c) A Et3PAuCl treated embryo at 5 dpf:
no vessel defects and no difference to the solvent control (DMF) treated
fishes were observed (data not shown). (d) An example of a 5dpf
zebrafish embryo treated with 3 shows severe damages of DLAV and
IV (marked with white arrows).
Figure 6. Dual contrast inverted microscopy images of zebrafish
embryos exposed to 0.1 µM Et3PAuCl or 0.1 µM 3. An example of a
2 days postfertilization (2dpf) embryo treated with 0.1 µM of 3 for
24 h is shown (a-d): (a) bright-field image; (b) luminescence image
showing the blue emission of 3 (in the small window a untreated control
embryo at 2dpf is depicted showing autofluorescence of the yolk but
no signal in the embryo); (c) luminescence image showing the green
emission of the green fluorescence protein; (d) overlay of (b) and (c).
centrifugation (1500 rpm, 5 min) pellets were resuspended in 300
µL of RSB-1 (0.01 M Tris-HCl, 0.01 M NaCl, 1.5 mM MgCl2, pH
7.4) and left for 10 min in an ice bath. The swollen cells were
centrifuged (2000 rpm, 5 min), resuspended in 300 µL of RSB-2
(RSB-1 containing each 0.3% v/v Nonidet-P40 and sodium des-
oxycholate) and homogenized by 10-15 up/down-pushes in a 1
mL syringe with needle. The homogenate was centrifuged at 2500
rpm for 5 min, and the resulting crude nuclei were taken up in 150
µL of 0.25 M sucrose containing 3 mM CaCl2. The suspension
was underlayed with 150 µL of 0.88 M sucrose and centrifuged 10
min at 2500 rpm. The nuclei pellets were stored at -20 °C or
immediately dissolved in 500-1000 µL of water and disrupted by
use of a sonotrode. The gold content of the samples was determined
by graphite furnace atomic absorption spectrometry according to a
recently published procedure and the protein content by the method
of Bradford.19 Results are expressed as the mean of two independent
experiments as nmol of gold per milligram of nuclear protein.
TrxR Inhibition. Commercially available rat liver TrxR (Sigma)
was used to determine TrxR inhibition by the compounds. The assay
was performed according to the manufacturer’s instructions (Sigma
product information sheet T9698) with appropriate modifications.
Initially, the TrxR rat liver solution was diluted with potassium
phosphate buffer, pH 7.0. To 25 µL aliquots of this solution (each
containing approximately 0.18 units of the enyzme) each 25 µL of
potassium phosphate buffer pH 7.0, containing the compounds or
vehicle without compound (control) were added, and the resulting
solutions were incubated for 1 h at 37 °C with moderate shaking.
The solutions were transferred quantitatively to 96-well plates, and
each 250 µL of reaction mixture (10 mL of reaction mixture
consisted of 1.0 mL of 1.0 M potassium phosphate buffer, pH 7.0,
0.20 mL of 500 mM EDTA solution pH 7.5, 0.80 mL of 63 mM
DTNB in ethanol, 0.10 mL of 20 mg/mL bovine serum albumine,
0.05 mL of 48 mM NADPH and 7.85 mL of water) were added.
To correct for nonenzymatic product formation, 50 µL of 1.0 M
potassium phosphate buffer, pH 7.0, and 250 µL of reaction mixture
were processed simultaneously (blank value). After proper mixing
Fluorimetric Scans. All solvents used for the fluorimetric
experiments were purged with nitrogen prior to use. Compound 3
was dissolved as a stock solution (50 or 500 µM) in DMF, diluted
100-fold with the respective solvents (CHCl3, DMF, DMSO, PBS,
1 N NaOH, and 1 N HCl) and measured using a Hitachi F-4500
fluorescence spectrometer. Only corrected spectra were taken.
Antiproliferative Effects. The antiproliferative effects in MCF-7
and HT-29 cells after 72 h (HT-29) or 96 h (MCF-7) of exposure
to Et3PAuCl and 3 were evaluated according to recently described
procedures.19,20 The IC50 value was described as that concentration
reducing proliferation of untreated control cells by 50%.
Fluorescence Microscopy of Tumor Cells. Cells were grown
in six-well plates (Sarstedt) until at least 70% confluency. The cell
culture medium was replaced with fresh medium containing the
compounds in a concentration of 5.0 µM (0.1% v/v DMSO) and
incubated for 6 h at 37 °C in a 5% CO2/95% air atmosphere. The
medium was removed, and the cells were washed with PBS. Finally
an amount of 500 µL of PBS was added to each well. Microscopy
was performed using an Axiovert 40 CFL microscope (Zeiss)
equipped with a 50 W mercury vapor short arc lamp and a Ex/Em
390 ( 11/460 ( 25 nm filter.
Gold Content of Nuclei. The nuclei of the tumor cells were
isolatedaccordingtoadescribedprocedurewithsomemodifications.25,38
Cells were grown in 175 cm2 cell culture flasks until at least 70%
confluency. The medium was removed and replaced with 10 mL
of medium containing 5.0 µM drug. After 24 h of incubation at 37
°C in a humidified atmosphere, the drug containing medium was
removed, cells were trypsinized, resuspended in 10 mL of cell
culture medium, and isolated by centrifugation (1500 rpm, 5 min),
and 0.5-1.0 mL of 0.9% NaCl solution were added. After

Gold(I) Phosphine Complex
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 769
Table 2. Effects on Angiogenesisa
compd [concn]
total embryos (with defects) [% with defects]
DLAV
DLAV + IV
DLAV + SIV
DLAV + IV + SIV
3 [0.1 µM ]
200 (181) [91%]
200 (101) [51%]
200 (7) [4%]
200 (4) [2%]
200 (0)
32
14
4
3
0
25
22
3
1
0
19
16
0
0
0
105
49
0
0
0
3 [0.05 µM]
Et3PAuCl [0.1 µM ]
Et3PAuCl [0.05 µM]
DMF
a Vessel defects were tested in live 4 dpf TG:fli1/eGFP fishes by confocal microscopy. Given is the total number of fishes tested versus the number of
fishes with vessel defects and the percentage of fishes with vessel defects. Three independent experiments were combined with similar results. Fishes were
viable and showed no significant mortality at 0.1 µM Et3PAuCl and 0.1 µM 3, which was for both compounds the maximal tolerated dose. The solvent
(DMF) was used at 0.1% v/v as control. The number of embryos with defects in the different vessels and in the combinations shown is listed. Defects only
in IV or only in SIV and in the combination of both were not found.
the formation of 5-thionitrobenzol was monitored in a microplate
reader (Flashscan, AnalytikJena AG) at 412 nm in 12 s intervals
for 4 min. The absorbance of the blank was subtracted from that
of the control and treated wells. The increase in 5-thionitrobenzol
concentration over time followed a linear trend (r2 g 0.99), and
the enzymatic activities were calculated as the slopes (increase in
absorbance per second) thereof. The EC50 values were calculated
as the concentration of compound decreasing the enzymatic activity
of the untreated control by 50%.
Mass Spectrometry. ESI-MS and MS/MS spectra were collected
on a Finnigan LCQ mass spectrometer (Thermo Electron Corp.,
San Jose, CA), which was operated in the positive ion mode with
a capillary temperature of 200 °C and a spray voltage of 1.8 kV.
Samples were prepared by incubating the model peptide AGCV-
GAGLIK with complexes Et3PAuCl and 3 at a 1:5 molar ratio for
24 h at 37 °C. Delivery to the mass spectrometer was performed
by a syringe pump operating at a flow rate of 1.0 µL/min. The
relative collision energy for collision-induced dissociation was set
at 30%.
Determination of Cell Concentration and Cell Viability of
BJAB Cells. Cell viability was determined by CASY cell counter
+ analyzer system of Innovatis (Bielefeld, Germany). Settings were
specifically defined for the requirements of the used cells. With
this system the cell concentration is analyzed simultaneously in
three different size ranges; cell debris, dead cells, and viable cells
were determined in one measurement. Cells were seeded at a density
of 1 × 105 cells/mL and treated with different concentrations of
the agents; nontreated cells served as controls. After 24 h of
incubation, cells were resuspended properly and 100 µL of each
well were diluted in 10 mL of CASYton (ready-to-use isotonic
saline solution) for an immediate automated count of the cells.
Measurement of DNA Fragmentation. Apoptotic cell death
was determined by a modified cell cycle analysis, which detects
DNA fragmentation on the single cell level as described.39,40 BJAB
cells were seeded at a density of 1 × 105 cells/mL and treated with
different concentrations of 3. After 72 h of incubation at 37 °C,
cells were collected by centrifugation at 1500 rpm for 5 min, washed
with PBS at 4 °C, and fixed in PBS/2% (v/v) formaldehyde on ice
for 30 min. After fixation, cells were pelleted, incubated with
ethanol/PBS (2:1, v/v) for 15 min, pelleted, and resuspended in
PBS containing 40 µg/mL RNase A. RNA was digested for 30
min at 37 °C. Afterward cells were pelleted again and finally
resuspended in PBS containing 50 µg/mL propidium iodide. Nuclear
DNA fragmentation was quantified by flow cytometric determina-
tion of hypodiploid DNA. Data were collected and analyzed using
a FACScan (Becton Dickinson, Heidelberg, Germany) equipped
with the CELLQuest software. Data are given in % hypoploidy
(subG1), which reflects the number of apoptotic cells.
twice with ice-cold PBS and then resuspended in binding buffer
(10 mM N-(2-hydroxyethyl)piperazine-N′-3(propanesulfonicacid)
(HEPES)/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) at a
concentration of 1 × 105 cells/mL. Next, 10 µL of annexin-V-FITC
(BD Pharmingen, Heidelberg, Germany) and 10 µL of 50 µg/mL
PI (Sigma-Aldrich, Taufkirchen, Germany) were added to the cells.
Analyses were performed on a FACScan (Becton Dickinson,
Heidelberg, Germany) using the CellQuest analysis software.
Measurement of the Mitochondrial Permeability Transi-
tion. After incubation for 48 h with different concentrations of 3,
cells were collected by centrifugation at 1500 rpm at 4 °C for 5
min. Mitochondrial permeability transition was then determined by
staining cells with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-
dazolylcarbocyanine iodide (JC-1, Molecular Probes). Then 1 ×
105 cells were resuspended in 500 µL of phenol red-free RPMI
1640 without supplements and JC-1 was added to give a final
concentration of 2.5 µg/mL (38 µM). The cells were incubated for
30 min at 37 °C with moderate shaking. Control cells were likewise
incubated in the absence of JC-1 dye. The cells were harvested by
centrifugation at 1500 rpm at 4 °C for 5 min, washed with ice-
cold PBS, and resuspended in 200 µL of PBS at 4 °C. Mitochondrial
permeability transition was then quantified by flow cytometric
determination of cells with decreased fluorescence, i.e., with
mitochondria displaying a lower membrane potential. Data were
collected and analyzed using a FACScan (Becton Dickinson,
Heidelberg, Germany) equipped with the CELL Quest software.
Data are given in % cells with low ∆Ψm, which reflects the number
of cells undergoing mitochondrial apoptosis.
Animal Care. The transgenic zebrafish (Danio rerio) line
Tg(fli1:eGFP) was used for the angiogenesis experiments, and fishes
were handled in compliance with local animal care regulations and
standard protocols of The Netherlands. Fishes were kept at 28 °C
in aquaria with standard day/night light cycles. Substances were
administered once to dechorionated 24 hpf embryos by adding stock
solutions of the compounds into the water.
Live Imaging. Confocal pictures were taken with the Biorad
Confocal microscope 1024ES (Zeiss microscope) combined with
a krypton/argon laser, or with the dual laser scanning confocal
microscope Leica DM IRBE (Leica). Dual inverted contrast (DIC)
microscopy pictures were taken using an Axioplan 2 microscope
with an AxioCam MR5 camera (Carl Zeiss).
Acknowledgment. Financial support by DFG-Deutsche
Forschungsgemeinschaft (Project FOR-630), BMBFsBundes-
ministerium fu¨r Bildung and Forschung (Project CHN 08/007),
and the Portuguese Foundation for Science and Technology
(Grant SFRH/BD/27262/2006) is gratefully acknowledged. We
are also grateful for technical support by Heike Scheffler.
Annexin-V-Propidium Iodide Binding Assay. Cell death was
determined by staining cells with annexin-V-FITC and counter-
staining with propidium iodide (PI). During apoptosis, the phos-
pholipid phosphatidylserine is exposed to the outer leaflet of the
plasma membrane.41,42 Annexin-V-FITC then binds to phosphati-
dylserine leading to an increase of the fluorescence. On the other
hand, PI is excluded from cells with intact membranes. PI positivity
is therefore a sign of cell necrosis, whereas cells that are annexin-
V-FITC positive but PI negative are generally defined as apop-
totic.43 For the annexin-V-PI assay, 1 × 105 cells were washed
Supporting Information Available: Elemental analysis data of
3 and the synthetic procedure of 2. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Dive, D.; Biot, C. Ferrocene Conjugates of Chloroquine and Other
Antimalarials: The Development of Ferroquine, a New Antimalarial.
ChemMedChem 2007, 3, 383–391.

770 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Ott et al.
(2) Ott, I.; Gust, R. Non Platinum Metal Complexes as Anti-Cancer Drugs.
Arch. Pharm. Chem. Life Sci. 2007, 340, 117–126.
(3) Bruijnincx, P. C. A.; Sadler, P. J. New Trends for Metal Complexes
with Anticancer Activity. Curr. Opin. Chem. Biol. 2008, 12, 197–
206.
(23) Rackham, O.; Nichols, S. J.; Leedman, P. J.; Berners-Price, S. J.;
Filipovska, A. A Gold(I) Phosphine Complex Selectively Induces
Apoptosis in Breast Cancer Cells: Implications for Anticancer
Therapeutics Targeted to Mitochondria. Biochem. Pharmacol. 2007,
74, 992–1002.
(24) Ott, I.; Scharwitz, M.; Scheffler, H.; Sheldrick, W. S.; Gust, R. Atomic
Absorption Spectrometric Determination of the Iridium Content in
Tumor Cells Exposed to an Iridium Metallodrug. J. Pharm. Biomed.
Anal. 2008, 47, 938–942.
(25) Kirin, S. I.; Ott, I.; Gust, R.; Mier, W.; Weyhermu¨ller, T.; Metzler-
Nolte, N. Cellular Uptake Quantification of Metalated Peptide and
Peptide Nucleic Acid Bioconjugates by Atomic Absorption Spectros-
copy. Angew. Chem., Int. Ed. 2008, 47, 955–958.
(26) Sergeant, C. D.; Ott, I.; Sniady, A.; Meneni, S.; Gust, R.; Rheingold,
A. L.; Dembinski, R. Metallo-nucleosides: Synthesis and Biological
Evaluation of Hexacarbonyldicobalt 5-Alkynyl-2′-deoxyuridines. Org.
Biomol. Chem. 2008, 6, 73–80.
(27) Marzano, C.; Gandin, V.; Folda, A.; Scutari, G.; Bindoli, A.; Rigobello,
M. P. Inhibition of Thioredoxin Reductase by Auranofin Induces
Apoptosis in Cisplatin-Resistant Human Ovarian Cancer Cells. Free
Radical Biol. Med. 2007, 42, 872–881.
(28) Rigobello, M. P.; Messori, L.; Marcon, G.; Cinellu, M. A.; Bragadin,
M.; Folda, A.; Scutari, G.; Bindoli, A. Mitochondrial Thioredoxin
Reductase Inhibition by Gold(I) Compounds and Concurrent Stimula-
tion of Permeability Transition and Release of Cytochrome C. J. Inorg.
Biochem 2004, 98, 1634–1641.
(29) Rush, G. F.; Smith, P. F.; Hoke, G. D.; Alberts, D. W.; Snyder, R. M.;
Mirabelli, C. K. The Mechanism of Acute Cytotoxicity of Trieth-
ylphosphine Gold(I) Complexes. II. Triethylphosphine Gold Chloride-
induced Alterations in Mitochondrial Function. Toxicol. Appl. Phar-
macol. 1987, 90, 391–400.
(30) Streicher, K. L.; Sylte, M. J.; Johnson, S. E.; Sordillo, L. M.
Thioredoxin Reductase Regulates Angiogenesis by Increasing Endot-
helial Cell-Derived Vascular Endothelial Growth Factor. Nutr. Cancer
2004, 50, 221–231.
(31) Powis, G.; Kirkpatrick, D. L. Thioredoxin Signaling as a Target For
Cancer Therapy. Curr. Opin. Pharmacol. 2007, 7, 392–397.
(32) Vacca, A.; Bruno, M.; Boccarelli, A.; Coluccia, M.; Ribatti, D.;
Bergamo, A.; Garbisa, S.; Sartor, L.; Sava, G. Inhibition of Endothelial
Cell Functions and of Angiogenesis by the Metastasis Inhibitor NAMI-
A. Br. J. Cancer 2002, 86, 993–998.
(33) Ott, I.; Kircher, B.; Bagowski, C. P.; Vlecken, D. H. W.; Ott, E. B.;
Will, J.; Bensdorf, K.; Sheldrick, W. S.; Gust, R. Modulation of the
Biological Properties of Aspirin by Bioorganometallic Derivatisation.
Angew. Chem., Int. Ed., DOI: 10.1002/ange.200803347.
(34) Norrby, K. In Vivo Models of Angiogenesis. J. Cell. Mol. Med. 2006,
10, 588–612.
(35) Habeck, H.; Odenthal, J.; Walderich, B.; Maischein, H.; Schulte-
Merker, S.; Tu¨bingen 2000 Screen Consortium. Analysis of a Zebrafish
VEGF Receptor Mutant Reveals Specific Disruption of Angiogenesis.
Curr. Biol. 2002, 12, 1404–1412.
(36) Isenberg, J. S.; Jia, Y.; Field, L.; Ridnour, L. A.; Sparatore, A.; Del
Soldato, P.; Sowers, A. L.; Yeh, G. C.; Moody, T. W.; Wink, D. A.;
Ramchandran, R.; Roberts, D. D. Modulation of Angiogenesis by
Dithiolethione Modified NSAIDs and Valproic Acid. Br. J. Pharmacol.
2007, 151, 142–151.
(37) Lawson, N. D.; Weinstein, B. M. In Vivo Imaging of Embryonic
Vascular Development Using Transgenic Zebrafish. DeV. Biol. 2002,
248, 307–318.
(38) Ott, I.; Schmidt, K.; Kircher, B.; Schumacher, P.; Wiglenda, T.; Gust,
R. Antitumor-Active Cobalt-Alkyne Complexes Derived from Ace-
tylsalicylic Acid: Studies on the Mode of Drug Action. J. Med. Chem.
2005, 48, 622–629.
(39) Nicoletti, I.; Migliorati, G.; Pagliacci, M. G.; Grignani, F.; Riccardi,
C. A Rapid and Simple Method for Measuring Thymocyte Apoptosis
by Propidium Iodide Staining and Flow Cytometry. J. Immunol.
Methods 1991, 139, 271–279.
(40) Riccardi, C.; Nicoletti, I. Analysis of Apoptosis by Propidium Iodide
Staining and Flow Cytometry. Nat. Protoc. 2006, 1, 1458–1461.
(41) Fadok, V. A.; Xue, D.; Henson, P. If Phosphatidylserine Is the Death
Knell, a New Phosphatidylserine-Specific Receptor Is the Bellringer.
Cell Death Differ. 2001, 8, 582–587.
(42) Schlegel, R. A.; Williamson, P. Phosphatidylserine, a Death Knell.
Cell Death Differ. 2001, 8, 551–563.
(4) Hambley, T. W. Metal-Based Therapeutics. Science 2007, 318, 1392–
1393.
(5) Gromer, S.; Arscott, L. D.; Williams, C. H.; Schirmer, R. H.; Becker,
K. Human Placenta Thioredoxin Reductase. Isolation of the Sele-
noenzyme, Steady State Kinetics, and Inhibition by Therapeutic Gold
Compounds. J. Biol. Chem. 1998, 273, 20096–200101.
(6) Barnard, P. J.; Berners-Price, S. J. Targeting the Mitochondrial Cell
Death Pathway with Gold Compounds. Coord. Chem. ReV. 2007, 251,
1889–1902.
(7) Urig, S.; Fritz-Wolf, K.; Reau, R.; Herold-Mende, C.; Toth, K.;
Davioud-Charvet, E.; Becker, K. Undressing of Phosphine Gold(I)
Complexes as Irreversible Inhibitors of Human Disulfide Reductases.
Angew. Chem., Int. Ed. 2006, 45, 1881–1886.
(8) Mirabelli, C. K.; Johnson, R. K.; Hill, D. T.; Faucette, L. F.; Girard,
G. R.; Kuo, G. Y.; Sung, C. M.; Crooke, S. T. Correlation of the in
Vitro Cytotoxic and in Vivo Antitumor Activities of Gold(I) Coordina-
tion Complexes. J. Med. Chem. 1986, 29, 218–223.
(9) Brana, M. F.; Ramos, A. Naphthalimides as Anti-Cancer Agents:
Synthesis and Biological Activity. Curr. Med. Chem.: Anti-Cancer
Agents 2001, 1, 237–255.
(10) Ott, I.; Xu, Y.; Liu, J.; Kokoschka, M.; Harlos, M.; Sheldrick, W. S.;
Qian, X. Sulfur-Substituted Naphthalimides as Photoactivatable
Anticancer Agents: DNA Interaction, Fluorescence Imaging, and
Phototoxic Effects in Cultured Tumor Cells. Bioorg. Med. Chem. 2008,
16, 7107–7116.
(11) Cao, H.; Chang, V.; Hernandez, R.; Hagy, M. D. Matrix Screening of
Substituted N-Aryl-1,8-naphthalimides Reveals New Dual Fluorescent
Dyes and Unusually Bright Pyridine Derivatives. J. Org. Chem. 2005,
70, 4929–4934.
(12) Pardo, A.; Poyato, J. M. L.; Martin, E. Photophysical Properties of
1,8-Naphthalimide Derivatives. J. Photochem. 1987, 36, 323–329.
(13) Ramachandram, B.; Saroja, G.; Sankaran, N. B.; Samanta, A.
Unusually High Fluorescence Enhancement of Some 1,8-Naphthal-
imide Derivatives Induced by Transition Metal Salts. J. Phys. Chem.
B 2000, 104, 11824–11832.
(14) Zhang, Z.; Wu, D.; Guo, X.; Qian, X.; Lu, Z.; Xu, Q.; Yang, Y.;
Duan, L.; He, Y.; Feng, Z. Visible Study of Mercuric Ion and Its
Conjugate in Living Cells of Mammals and Plants. Chem. Res. Toxicol.
2005, 18, 1814–1820.
(15) Gan, J.; Tian, H.; Wang, Z.; Chen, K.; Hill, J.; Lane, P. A.; Rahn,
M. D.; Fox, A. M.; Bradley, D. D. C. Synthesis and Luminescence
Properties of Novel Ferrocene-Naphthalimides Dyads. J. Organomet.
Chem. 2002, 645, 168–175.
(16) Guo, X.; Qian, X.; Jia, L. A Highly Selective and Sensitive Fluorescent
Chemosensor for Hg2+ in Neutral Buffer Aqueous Solution. J. Am.
Chem. Soc. 2004, 126, 2272–2273.
(17) Xu, Z.; Qian, X.; Cui, J. Colorimetric and Ratiometric Fluorescent
Chemosensor with a Large Red-Shift in Emission: Cu(II)-Only Sensing
by Deprotonation of Secondary Amines as Receptor Conjugated to
Naphthalimide Fluorophore. Org. Lett. 2005, 7, 3029–3032.
(18) Osawa, M.; Hoshino, M.; Hashizume, D. Photoluminescent Properties
and Molecular Structures of [NaphAu(PPh3)] and [µ-Naph
{Au(PPh3)}2]ClO4 (Naph ) 2-naphthyl). Dalton Trans. 2008, 2248–
2252.
(19) Ott, I.; Scheffler, H.; Gust, R. Development of a Method for the
Quantification of the Molar Gold Concentration in Tumour Cells
Exposed to Gold-Containing Drugs. ChemMedChem 2007, 2, 702–
707.
(20) Ott, I.; Koch, T.; Shorafa, H.; Bai, Z.; Poeckel, D.; Steinhilber, D.;
Gust, R. Synthesis, Cytotoxicity, Cellular Uptake and Influence on
Eicosanoid Metabolism of Cobalt-Alkyne Modified Fructoses in
Comparison to Auranofin and the Cytotoxic COX Inhibitor Co-ASS.
Org. Biomol. Chem. 2005, 3, 2282–2286.
(21) Barnard, P. J.; Wedlock, L. E.; Baker, M. V.; Berners-Price, S. J.;
Joyce, D. A.; Skelton, B. W.; Steer, J. H. Luminescence Studies of
the Intracellular Distribution of a Dinuclear Gold(I) N-Heterocyclic
Carbene. Angew. Chem. 2006, 118, 6112–6116.
(22) Liu, J. J.; Galettis, P.; Farr, A.; Maharaj, L.; Samarasinha, H.;
McGechan, A. C.; Baguley, B. C.; Bowen, R. J.; Berners-Price, S. J.;
McKeage, M. J. In Vitro Antitumour and Hepatotoxicity Profiles of
Au(I) and Ag(I) Bidentate Pyridyl Phosphine Complexes and Relation-
ships to Cellular Uptake. J. Inorg. Biochem. 2008, 102, 303–310.
(43) Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutellingsperger, C.
A Novel Assay for Apoptosis. J. Immunol. Methods 1995, 184, 39–51.
JM8012135