Thiolato gold(i) complexes containing water-soluble phosphane ligands: A characterization of their chemical and biological properties

Article abstract of DOI:10.1039/c1dt10892a

A series of thiolate gold(i) derivatives bearing water soluble phosphanes - namely sodium triphenylphosphane monosulfonate (TPPMS), sodium triphenylphosphane trisulfonate (TPPTS), 1,3,5-triaza-7-phosphaadamantane (PTA) and 3,7-diacetyl-1,3,7-triaza-5-phos

Full text of DOI:10.1039/c1dt10892a

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PAPER
Thiolato gold(I) complexes containing water-soluble phosphane ligands: a
characterization of their chemical and biological properties†
Elena Vergara,a Elena Cerrada,a Catherine Clavel,b Angela Casini*b and Mariano Laguna*a
Received 12th May 2011, Accepted 8th August 2011
DOI: 10.1039/c1dt10892a
A series of thiolate gold(I) derivatives bearing water soluble phosphanes – namely sodium
triphenylphosphane monosulfonate (TPPMS), sodium triphenylphosphane trisulfonate (TPPTS),
1,3,5-triaza-7-phosphaadamantane (PTA) and 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane
(DAPTA) – is reported and the compounds studied for their luminescence properties in the solid state.
Two of these derivatives, [Au(SMe2pyrim)(PTA)] and [Au(SBenzoxazole)(DAPTA)], are also
structurally characterized by X-ray diffraction analysis. Strong antiproliferative effects are observed for
most of the compounds in the human ovarian carcinoma cell lines (A2780/S) and its cisplatin-resistant
variant (A2780/R), which depend on both the type of thiolate and phosphane ligands. ICP-MS studies
were also performed to evaluate the influence of the gold uptake on the cytotoxic potency of the
compounds.
reductase, cathepsins, protein tyrosine phosphatase, proteasome,
Introduction
and iodothyronine deiodinase.12,16–21 Recently, some of us reported
The use of gold complexes in medicine has been essentially centred
on the inhibition of the zinc finger protein poly(ADP-ribose)
in the treatment of rheumatoid arthritis.1,2 However, during the
last few decades many gold derivatives have been investigated for
their potential use as anticancer agents,1,3–7 mainly due to the
favourable properties of the gold centre and peculiar reactivity
with biomolecules with respect to classical Pt(II) anticancer drugs
known to possess DNA alkylating properties.8
Investigations on the cytotoxicity scores of gold(I) com-
plexes have been focused mainly on auranofin (1-thio-b-D-
glucopyranose-2,3,4,6-tetraacetato-S)(triethylphosphine) gold(I))
and its analogues, which present linear gold phosphane thiolate
structures.7 However, a variety of gold(III) derivatives have also
been tested as potential antitumor agents, including organogold
derivatives, complexes with polydentate ligands, gold porphyrins,
gold dithiocarbamates and dinuclear m-oxo derivatives.9–13 The
mechanisms of action of anticancer gold(I) and gold(III) complexes
appear in general to be DNA independent and essentially cisplatin-
unrelated.14 For example, it has been recognised that mitochondria
might be critical intracellular target involved in the antitumor
activity of the drugs.15–17 Moreover, several proteins playing
relevant functional roles in cells were proposed to represent
effective targets for cytotoxic gold compounds, such as thioredoxin
polymerase (PARP-1) by gold complexes.22 Interestingly, a recent
proteomic study by Messori et al. allowed the identification of
about 15 proteins showing significant changes in their respective
expression rates.23
Within this frame, we have described thiolate gold(I) deriva-
tives with the water soluble phosphanes PTA (1,3,5-triaza-7-
phosphaadamantane) and DAPTA (3,7-diacetyl-1,3,7-triaza-5-
phosphabicyclo[3.3.1]nonane), which display potent cytotoxicity
for ovarian, colon, renal and melanoma cancer cell lines in
comparison to cisplatin.24,25 In addition, some of these compounds
were screened for their inhibition of mammalian thioredoxin re-
ductases (TrxRs), crucial seleno-enzymes involved in the intracel-
lular redox balance.12 The results obtained showed that the gold(I)–
phosphane complexes efficiently inhibit cytosolic and mitochon-
drial TrxRs, similarly to auranofin and related compounds.25
Remarkably, an excellent correlation between cytotoxic properties
and TrxR inhibition was outlined.
With the aim of developing new water soluble cytotoxic
gold(I) derivatives with anticancer properties, we describe here
the synthesis of a series of thiolate gold(I) phosphane complexes
bearing sodium triphenylphosphane monosulfonate (TPPMS)
and sodium triphenylphosphane trisulfonate (TPPTS) ligands,
respectively (Scheme 1). Moreover, some previously reported
thiolate gold(I) complexes with PTA and DAPTA ligands,26 herein
further characterized via X-ray diffraction analysis, were also
studied for their luminescent properties in the solid state. We also
include here the preparation of the water soluble gold(I) derivatives
of auranofin bearing 1-thio-b-D-glucose tetraacetate and with the
PTA and DAPTA ligands replacing the triarylphosphane PEt3.
aDepartamento de Qu´ımica Inorga´nica, Instituto de S´ıntesis Qu´ımica y
Cata´lisis Homoge´nea (ISQCH)-C.S.I.C, Universidad de Zaragoza, 50009,
Zaragoza, (Spain). E-mail: mlaguna@unizar.es
bInstitut des Sciences et Inge´nierie Chimiques, Ecole Polytechnique Fede´rale
de Lausanne (EPFL), 1015, Lausanne, (Switzerland). E-mail: angela.
casini@epfl.ch
† CCDC reference numbers 790251 and 790252. For crystallographic data
in CIF or other electronic format see DOI: 10.1039/c1dt10892a
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Scheme 1 Preparation of thiolate–gold(I) compounds.
All the compounds have been tested for their antiproliferative
activities against human ovarian carcinoma cell lines, and addi-
tional cell uptake studies have been conducted in representative
cases by inductively coupled plasma mass spectrometry (ICP-MS)
to further investigate the differences observed in the cytotoxic
properties.
All the described gold compounds are highly water soluble with
values ranging from 90 to 480 g L-1. In the case of the TPPMS
derivatives, the solubilities are higher than that of the chloro–gold
precursors;29–32 conversely, lower values are found for the TPPTS
compounds compared with the starting material.
The crystalline structure of complex [Au(SMe2pyrim)(PTA)]
(3c) was determined by X-ray diffraction (Fig. 1), and found to
Results and discussion
The synthesis of thiolate gold(I) derivatives 1–8(a–d) with water
soluble phosphanes can be afforded by the addition of the
corresponding chloro gold(I) phosphane complex to a solution
of the thiol in the presence of a base, as depicted in Scheme 1.
The thiolate gold(I) complexes with sodium triphenylphosphane
monosulfonate (TPPMS, 1–7(a)) and trisulfonate (TPPTS, 1–6(b))
are isolated as pale yellow solids in good yields. All of them
1
show singlet resonances at about 37 ppm in their 31P{ H} NMR
spectra, displaced to a lower field compared with those of the free
1
phosphanes which appear at ca. -6 ppm in both cases. The H
NMR spectra display, apart from the absence of the S–H signal,
the characteristic phenyl signals of the phosphane and those due
to the thiolate, the latter being high field displaced in the case of
the TPPTS derivatives compared with those found in the TPPMS
complexes.
Interestingly, in recent years, several examples of carbohydrate
compounds have been developed for diverse medicinal appli-
cations, ranging from compounds with antibiotic, antiviral, or
fungicidal activity and anticancer compounds.27 Within this frame,
we have prepared two thioglucosetetraacetate Au(I) complexes,
namely 8c and 8d, with PTA and DAPTA ligands, respectively. In
fact, it has been shown that 1-thio-b-D-glucose 2,3,4,6-tetraacetate
is acting as a true substrate for the glucose active-transport system,
and can be used to increase the uptake of metal compounds.28
In general, the preparation and characterization of the thiolate
derivatives with PTA and DAPTA groups reported herein was
previously described by some of us,24,26 except that in the case of
the thioglucosetetraacetate thiolates (see Experimental).
Fig.
1
(a) Molecular structure of [Au(SMe2pyrim)(PTA)](3c). Se-
◦
#1
˚
lected bond lengths [A] and angles [ ]
x, -y+1/2, z: Au(1)–P(1)
2.235(3), Au(1)–S(1) 2.279(3), S(1)–C(5) 1.753(10), P(1)–C(8) 1.849(11),
P(1)–C(9) 1.862(9), P(1)–C(9)#1 1.862(9), P(1)–Au(1)–S(1) 172.50(11),
C(5)–S(1)–Au(1) 110.9(4). H are omitted for clarity. (b) View of the packing
in the unit cell of complex 3c.
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be similar to the previously described [Au(Spyrimidine)(PTA)].24
The structure displays a characteristic linear geometry around
the gold centre (P1–Au1–S1 of 172.50(11)◦). The main difference
between the two structures is the disposition of the molecules
in the cell. In the previously reported structure, the molecule
is associated with an additional unit related by symmetry in
a perpendicular rearrangement with Au ◊ ◊ ◊ Au interactions of
linear geometry around the metallic centre with a P1–Au1–S1
angle of 176.72(9)◦. To the best of our knowledge this is the
first gold DAPTA compound crystallographically characterised,
and only examples of palladium,26,33 platinum,24 tungsten and
chromium33 derivatives with DAPTA ligands have been described
previously. The P–C bond lengths in the DAPTA molecule are
˚
approximately 0.129 A longer than those observed in the free
phosphane,33 however the N(1)–C(13) of 1.365(12) and N(3)–
C(15) of 1.377(12) are almost identical compared to its parent.
The packing of the individual molecules (Fig. 2b) is influenced
˚
3.3536(7) A; however the structure of complex 3c consists of a
unique molecule with long intermolecular interactions (Au ◊ ◊ ◊ Au
˚
˚
of 4.154 A and Au ◊ ◊ ◊ S of 4.056 A) in a head-to-tail disposition
˚
˚
(Fig. 1b). Another difference is that the Au–P and Au–S distances
by long Au ◊ ◊ ◊ Au (4.151 A) and Au ◊ ◊ ◊ S (3.500 A) interactions
between molecules in a head-to-tail rearrangement, reinforced
by intermolecular C–H ◊ ◊ ◊ N interactions between the DAPTA
˚
(2.235(3) and 2.279(3) A, respectively) are shorter than those
described for [Au(Spyrimidine)(PTA)]24 (Au–P 2.2527(18), Au–
i
˚
˚
S 2.3085(18) A). Longer P–C distances in the PTA phosphane
molecules (C9–H9a ◊ ◊ ◊ N2 = 2.45 A, i : 1-x, y, z; C9 ◊ ◊ ◊ N2 =
◦
˚
˚
are also observed in 3c (1.857 A in average) compared with those
3.360(12) A and angle at H9a of 157 ). Additionally, intramolecu-
˚
found in [Au(Spyrimidine)(PTA)] (1.843 A on average).
lar C–H ◊ ◊ ◊ O interactions can be observed in the DAPTA molecule
˚
˚
The molecular structure of [Au(SBenzoxazole)(DAPTA)] (4d)
obtained by X-ray diffraction analysis (Fig. 2) displays a typical
(C9–H9a ◊ ◊ ◊ O3 = 2.25 A, C9 ◊ ◊ ◊ O3 = 2.703(11) A and angle
at H9a of 108◦ and C11–H11a ◊ ◊ ◊◦O2 = 2.29 A, C11 ◊ ◊ ◊ O2 =
˚
˚
2.723(13) A and angle at H11a of 106 ). The Au–P and Au–S bond
˚
lengths of 2.241(2) and 2.316(3) A, respectively, are in the same
range as those observed in gold–PTA34–37 and gold–PTA thiolate
derivatives.24,38
Both crystalline structures and the previously reported
[Au(Spyrimidine)(PTA)]24 display short interactions between the
metallic centres. This fact is very common in thiolate gold(I)
derivatives in the solid state. However, in the FAB/MS spectra,
only the corresponding molecular peaks are observed and no
additional peaks with higher molecular weight are detected, which
should be in accordance with the presence of monomers in
solution.
The presence of N atoms in the PTA molecule and in some of
the thiolate ligands provides the possibility of protonation upon
addition of acid. Thus, since such derivatives are water soluble,
we have examined sequential protonation of the compounds upon
addition of several equivalents of a water solution of HCl (0.05 M)
in an aqueous environment, by NMR spectroscopy, at room
temperature.
Representative results for [Au(Spyrim)(PTA)](1c) are collated
in Table 1. As can be observed, the 31P{ H} NMR signal is shifted
1
Fig. 2 (a) Molecular structure of ◦[Au(SBenzoxazole)(DAPTA)](4d).
to lower field as occurred in PTA complexes with Re39 and Ru,40
and it was reported as the decreasing on the net electron-donor
character of the protonated PTA. Similarly a deshielding effect
˚
Selected bond lengths [A] and angles [ ]: Au(1)–P(1) 2.241(2), Au(1)–S(1)
2.316(3), S(1)–C(1) 1.732(10), P(1)–C(9) 1.823(9), P(1)–C(10) 1.832(10),
P(1)–C(8) 1.842(10), P(1)–Au(1)–S(1) 176.72(9). H are omitted for clarity.
(b) View of the packing in the unit cell of complex 4d.
1
was also observed in the H NMR, being more pronounced on
1
Table 1 d (31P{ H} NMR), d (HA), d (HB) in NCHACHBN group and d (PCH2N) in 1H NMR after HCl addition to [Au(Spyrim)(PTA)](1c) in D2O
d (NCHACHBN)
1
HCl Eq.
d (HA)
4.38
4.39
4.40
4.42
4.44
4.51
d (HB)
4.46
4.47
4.48
4.49
4.51
4.58
d (PCH2N)
4.28
d (pyrimidine) [JHH/Hz]
d (31P{ H} NMR)
0
1
2
3
4
5
7.06(t) [4.8]
8.35(d) [5.1]
7.07(t) [4.8]
8.36(d) [5.1]
7.08(t) [4.8]
8.36(d) [5.1]
7.09(t) [4.8]
8.38(d) [5.1]
7.10(t) [4.8]
8.39(d) [5.1]
7.13(t) [4.8]
8.40(d) [5.1]
-45.4
4.29
-45.3
4.30
-44.9
4.31
-43.6
4.32
-42.9
4.34
-42.4
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Table 2 Excitation and emission data in the solid state
298 K
77 K
Compound
lexc (nm)
lemis (nm)
lexc (nm)
lemis (nm)
[Au(Spyrim)(PTA)](1c)
386
376
389
335
360
370
389
396
442
438
450
498
476
488
450
460
389
379
393
335
361
372
392
397
439
449
452
497
473
482
456
462
[Au(Spyrim)(DAPTA)](1d)
[Au(SMe2pyrim)(PTA)](3c)
[Au(SMe2pyrim)(DAPTA)](3d)
[Au(SBenzoxazole)(PTA)](4c)
[Au(SBenzoxazole)(DAPTA)](4d)
[Au(SGlucosetetracetate)(PTA)](8c)
[Au(SGlucosetetracetate)(DAPTA)] (8d)
the NCHACHBN (an AB system) than on the PCH2N protons,
in accordance with a protonation on the N atoms. A small lower
field shift was also detected in the corresponding signals of the
pyrimidine–thiolate ligand, indicative of the possible protonation
of the N atom of the thiolate ligand (Table 1).
are relatively long compared to those previously reported for
[Au(Spyrimidine)(PTA)]24 (3.3536(7) A, 1c in Table 2), while the
˚
emission values for the three compounds (1c, 3c and 4d) are very
close to each other and to the rest of the values depicted in Table 2.
As a consequence, these results provide support that the observed
luminescence originates from the S→Au charge transfer excitation
and there is no influence of the gold–gold distances.
The in vitro antiproliferative properties of the new Au(I)
complexes 1–7(a–b), 8c, and 8d, as well as those of the already
reported PTA and DAPTA derivatives (1d and 3–6(c–d)) were
evaluated against the human ovarian cancer cell line (A2780/S)
and its cisplatin-resistant variant (A2780/R) in comparison to
cisplatin and auranofin. The IC50 values (Table 3) were obtained
after 72 h drug exposure, using the well established MTT assay (see
Interestingly, some of the thiolate derivatives reported herein
display luminescence at room temperature and at 77 K in
the solid state (Table 2). As an example, the emission of
[Au(SMe2pyrim)(DAPTA)] (3d) is plotted in Fig. 3. All the
complexes reported in Table 2 emit intensively at 298 and 77 K.
The cooling produces small blue shifts (in the range 40–255 cm-1)
in the emission of 1c (155 cm-1), 3d (40 cm-1), 4c (133 cm-1) and 4d
(255 cm-1), and red shifts (range 52–293 cm-1) in the emission of 1d
(52 cm-1), 3c (99 cm-1), 8c (293 cm-1) and 8d (94 cm-1). The Stokes
shifts observed (> 3500 cm-1) point to a large distortion in the
excited state relative to the ground state, consistent with a charge
transfer transition. In the case of similar luminescent gold(I) PTA
derivatives with substituted benzenethiolate ligands,41 the emission
has been assigned to a metal to ligand charge transfer (LMCT),
with the excitation from an orbital associated with the sulfur to the
metal-based orbital of the excited state. It is well established that
the luminescence found in phosphane thiolate–gold(I) complexes
is significantly influenced by the nature of the thiolate,42 as
observed from the data reported in Table 2. In the case of the
above mentioned PTA thiolate derivatives,41 the presence of gold–
gold interactions affect the emission maxima, although there is no
correlation between such distance and the emission energy, and
only sometimes is the emission maximum shifted to lower energies.
Table 3 IC50 values of 1–8(a–d) against ovarian carcinoma cell lines
sensitive (A2780/S) or resistant to cisplatin (A2780/R), as compared with
cisplatin and auranofin
IC50 (mM)a
Compound
A2780/S A2780/R R.F.b
[Au(Spyrim)(TPPMS)](1a)
117
1
118
1
1.00
0.46
[Au(Spyrim)(TPPTS)](1b)
12.2 4.2 5.7 1.9
10.1 1.7 16.1 0.7 1.59
[Au(Spyrim)(DAPTA)](1d)
[Au(SMepyrim)(TPPMS)](2a)
[Au(SMepyrim)(TPPTS)](2b)
[Au(SMe2pyrim)(TPPMS)](3a)
[Au(SMe2pyrim)(TPPTS)](3b)
[Au(SMe2pyrim)(PTA)](3c)
2.9 0.7
0.7 0.1
19.8 0.8 16.5 0.6 0.83
2.5 0.2
0.7 0.3
1.0 0.1
0.6 0.1
0.4 0.2
0.20
0.57
1.1 0.8
0.8 0.5
1.2 0.4
0.44
1.14
1.20
0.64
[Au(SMe2pyrim)(DAPTA)](3d)
[Au(SBenzoxazole)(TPPMS)](4a)
[Au(SBenzoxazole)(TPPTS)](4b)
[Au(SBenzoxazole)(PTA)](4c)
[Au(SBenzoxazole)(DAPTA)](4d)
[Au(SBenzothiazol)(TPPMS)](5a)
[Au(SBenzothiazol)(TPPTS)](5b)
[Au(SBenzothiazol)(PTA)](5c)
[Au(SBenzothiazol)(DAPTA)](5d)
[Au(SBenzoimidazol)(TPPMS)](6a)
[Au(SBenzoimidazol)(TPPTS)](6b)
[Au(SBenzoimidazol)(PTA)](6c)
[Au(SBenzoimidazol)(DAPTA)](6d)
[Au(SThiazoline)(TPPMS)](7a)
[Au(SThiazoline)(PTA)](7c)
10.5 0.3 6.7 0.2
34.3 0.3 11.5 0.7 0.34
2.7 1.1 5.2 2.3 1.92
18.0 0.5 14.9 0.6 0.83
2.4 0.5
2.0 0.5
0.8 0.2
1.2 0.2
7.6 0.2
23.6 2.6 21.4 2.6 0.91
8.0 0.6
0.9 0.3
10.1 0.8 16.4 0.2 1.63
0.7 0.3
1.1 0.4
1.2 0.5
1.3 0.5
0.9 0.1
1.1 0.4
0.50
0.65
1.12
0.91
16.4 1.8 3.11
23.9 2.1 3.0
0.8 0.1 0.88
1.1 0.1
0.9 0.1
1.57
0.81
[Au(SThiazoline)(DAPTA)](7d)
[Au(SGlucosetetracetate)(PTA)](8c)
[Au(SGlucosetetracetate)(DAPTA)](8d) 7.5 0.6
cisplatin
10.1 1.8 34.3 1.6 2.98
27.1 1.2 3.61
4.3 0.5
18.2
1
4.23
1.2
Fig. 3 Excitation and emission spectra of [Au(SMe2pyrim)(DAPTA)](3d)
at room temperature (green) and at 77 K (blue).
auranofin
1.25 0.5 1.5 0.3
a Mean SE of at least three determinations. b R.F. ratio between IC50
values for A2780/R and A2780/S
From the analysis of the X-ray structures obtained for 3c and
4d, it can be noticed that the gold–gold distances of ca. 4.15 A
˚
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Experimental for details). Most of the thiolate gold(I) complexes
appear to be more cytotoxic than cisplatin and in some cases
even of auranofin. Cross-resistance profiles have been evaluated
by means of the resistance factor (R.F., see Table 3), which is
calculated as the ratio of IC50 values between both types of cell
lines. In most of the tested derivatives the R.F. values are lower
than that calculated for cisplatin, being even 20 times lower in the
best case (2a). These results support the hypothesis of a different
mechanism of action of the gold(I) derivatives with respect to
cisplatin. Indeed, none of the reported compounds seem to affect
plasmid DNA mobility studied by gel electrophoresis (data not
shown), confirming their poor reactivity with nucleic acids.
The antiproliferative properties of the screened compounds
seem to depend on both the type of thiolate and phosphane
ligands. Among the most effective complexes we observed were
those containing the SMepyrim and SMe2pyrim, as well as
Sbenzothiazol and SThiazoline ligands, with IC50 values ranging
from ca. 0.7 to 2.9 mM for the A2780/S cell line, and from 0.4
to 1.3 mM for the resistant cell line. In those cases the phosphane
moiety does not appear to have a major effect in modulating the
antiproliferative effects. Conversely, for the Spyrim, SBenzoxa-
zole and SBenzoimidazol containing compounds, the phosphane
groups can also alter the biological properties, as in the case of
compound 1a (bearing a TPPMS) which is 10 times less effective
than the TPPTS and DAPTA analogues 1b and 1d, respectively.
Additionally, no correlation between water solubility and
cytotoxicity can be inferred. In general, increased water solubility
does not correlate with a higher cytotoxic potency or vice versa.
Complexes with PTA and DAPTA, except the glucosetetraacetate
ones, are much less water soluble (values ranging from insoluble
to 51 g l-1) than the compounds with the sulphonated phosphanes
(values ranging from 85 to 440 g l-1). As an example, the water
insoluble derivatives 3c, 3d, 5c, 5d and 6d display IC50 values as
low as 2b, whose solubility is 340 g l-1, and as 7c and 7d, with much
more moderate solubility (42 and 51 g l-1, respectively).
in cytotoxic potency between the compounds. Indeed, auranofin
has the best cell uptake among the investigated Au(I) complexes
in both cell lines, corresponding to its higher cytotoxic effect.
Interestingly, the gold cell uptake for the thioglucose derivatives
8c and 8d seems not to account for the differences in the cytotoxic
potency of the two compounds towards both cancer cell lines. In
fact, the higher IC50 values of the two complexes for the cisplatin
resistant variant do not correspond to a lower Au uptake with
respect to the one recorded on the A2780/S cell line. Moreover, the
thioglucose analogues having similar IC50 values in the case of the
A2780/S cells do not manifest comparable cell uptake properties
in the same cell line (8c has a 2-3-fold reduced uptake relatively
to 8d). It is worth mentioning that application of carbohydrate–
metal complexes is an example of a targeted approach exploit-
ing the biochemical and metabolic functions of diverse sugars
in living organisms for transport and accumulation.27 Natural
carbohydrates and synthetic derivatives possess a manifold of
donors endowing them with the ability to coordinate metal centers
and providing some additional advantages over other ligands,
e.g., biocompatibility, non-toxicity, enantiomeric purity, water
solubility, and well-explored chemistry. Indeed, compounds 8c–
8d might exploit different cell uptake mechanisms, with respect to
the other complexes in the series, involving glucose transporters,
and therefore manifesting more complex cellular responses.
Conclusions
In this study, water soluble thiolate Au(I) complexes with phos-
phane ligands, namely TPPMS, TPPTS, PTA and DAPTA,
were synthesized and characterized for their chemical/structural
properties. Interestingly, the compounds present luminescence
properties in the solid state, which seems to be due to S→Au
charge transfer excitations. Addition of acid to a water solution
of some of the PTA thiolate derivatives reveals a decrease in the
nucleophilic character of the phosphane as result of its N-atom
protonation.
The compounds were also tested for their antiproliferative ac-
tivities on human ovarian cancer cell lines A2780/S and A2780/R
and resulted to exert a wide range of cytotoxic activities, mainly in
the low mM range. Among the best antiproliferative agents, those
containing SMepyrim and SMe2pyrim, as well as Sbenzothiazol
and SThiazoline ligands, reached the lowest IC50 values, in some
cases better than the reference drugs cisplatin and auranofin.
ICP-MS analysis of cell extracts out of A2780 cells pre-treated
with gold compounds, including auranofin, demonstrated that the
cytotoxicity is proportional to the gold uptake. However, in the
case of the thioglucose derivatives, the strongest antiproliferative
effects do not correspond to higher values of intracellular gold
concentration. In the latter case, the observed uptake might be
influenced by the affinity for the thioglucose moiety with the sugar
receptors of the cancer cells, therefore the sugar-based gold(I) com-
plexes manifest more complex correlations between cytotoxicity
and cell uptake with respect to the other phosphane derivatives.
In order to evaluate if the cell uptake of the compounds might
affect their cytotoxic properties, cell extracts from A2780/S and
A2780/R cancer cells treated with 1 mM of metal compound
for 3 h at 37 ◦C were analyzed by ICP-MS, as described in
the Experimental section. The obtained results for representative
compounds expressed as pmol Au/106 cells are reported in Table
4. Remarkably, the uptake of the poorly cytotoxic compound 1a
(IC50 ca. 100 mM) is ca. 3–4 times lower than in the case of the active
analogue 1b in each cell line, and about 10 fold less efficient than
in the case of auranofin, a fact that may account for the difference
Table 4 Cell uptake of Au(I) phosphane complexes in A2780/S and
A2780/R cancer cell lines after treatment with 1 mM metal compound
for 3 h
pmol Au/106 cellsa
Compound
A2780/S
A2780/R
1a [Au(Spyrim)(TPPMS)]
1b [Au(Spyrim)(TPPTS)]
8c [Au(SGlucosetetracetate)(PTA)]
8d [Au(SGlucosetetracetate)(DAPTA)]
auranofin
288 93
806 91
925 244
2587 522
2946 603
393 102
941 140
1102 259
1886 189
4638 1234
Experimental
General
NMR spectra were recorded on 400 MHz Bruker Avance spec-
trometer. Chemical shifts are quoted relative to external TMS
a Mean SE of at least three determinations.
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(1H) or 85% H3PO4 (31P); coupling constants are reported in
Hz. FAB mass spectra were measured on a Micromass Autospec
spectrometer in positive ion mode using NBA as matrix. IR
spectra were recorded as KBr or polyethylene disks on a Nicolet
Impact 410 or JASCO FTIR (far-IR) spectrometers. Steady-state
photoluminescence spectra were recorded with a Jobin–Yvon–
Horiba fluorolog FL-3-11 spectrometer using band pathways of
3 nm for both excitation and emission. Elemental analyses were
obtained in-house using a LECO CHNS-932 microanalyser. The
phosphanes PTA43 DAPTA,26,33 TPPMS,44 and [AuCl(PR¢3)]26,30
were prepared according to published methods. A sample of
TPPTS was kindly provided by European Oxo GmbH. Cisplatin
was purchased from Sigma–Aldrich and auranofin from Alexis
Biochemicals.
MeOD, 25 ◦C): d = 36.3 ppm. FAB MS: m/z 890 [M]+; elemental
analysis calcd. (%) for C23H17AuN2Na3O9PS4 (89◦0.56): C 31.02, H
1.92, N 3.15; found: C 31.21, H 1.78, N 2.90. S20 ,H2O: 344 g L-1
[Au(SMe2pyrim)(TPPMS)] (3a). 58% yield, pale yellow solid.
1H NMR (400 MHz, MeOD, 25 ◦C): d = 2.24 (s, 6H, CH3),
6.57 (s, 1H, pyrim-H4), 7.43–7.65 (m, 12H, o-, m- y p-Ph, o-
PhSO3Na (H6), m-PhSO3Na), 7,90–8,09 (m, 2H, o-PhSO3Na (H2),
◦
1
p-PhSO3Na). 31P{ H} NMR (162 MHz, MeOD, 25 C): d = 36.1
ppm. FAB MS: m/z 701 [M]+; elemental analysis calcd. (%) for
C24H21AuN2NaO3PS2 (70◦0.5): C 41.15, H 3.02, N 4.00; found: C
40.96, H 2.93, N 3.68. S20 ,H2O: 212 g L-1
1
[Au(SMe2pyrim)(TPPTS)] (3b). 89% yield, yellow solid. H
NMR (400 MHz, D2O, 25 ◦C): d = 2.19 (s, 6H, CH3), 6.62 (s,
1H, pyrim-H4), 7.50–7.74 (m, 6H, o-PhSO3Na (H6), m-PhSO3Na),
1
7.77–7.92 (m, 6H, o-PhSO3Na (H2), p-PhSO3Na) ppm. 31P{ H}
Synthesis of the [Au(SR)(PR¢3)] complexes
NMR (162 MHz, D2O, 25 ◦C): d = 36.2 ppm. FAB MS: m/z
905 [M]+; elemental analysis calcd. (%) for C24H19AuN2Na3O9PS4
(904.59): C 31.87, H 2.12, N 3.10; found: C 31.38, H 2.32, N 2.72.
To a solution of KOH (0.022 g, 0.385 mmol) in MeOH (ca.
10 mL) containing the thiol compound (0.308 mmol) was added
[AuCl(PR¢3)] (PR¢3 = TPPMS, TPPTS) (0.257 mmol). After stir-
ring the mixture for ca. 20 h at room temperature the solutions were
evaporated to dryness under vacuum and the residue extracted
in dichloromethane (3 ¥ 10 mL). The combined extracts were
passed through Celite and concentrated under vacuum to ca. 5 mL.
Addition of pentane or Et2O precipitated the products, which were
isolated by filtration and dried in air.
◦
S20 ,H2O: 480 g L-1
[Au(Sbenzoxazole)(TPPMS)] (4a). 69% yield, yellow solid. 1H
NMR (400 MHz, D2O, 25 ◦C): d = 7.08–7.17 (m, 2H, benzoxazol-
H2, HH3), 7.27–7.34 (m, 2H, benzoxazol-H1, HH4), 7.47–7.93 (m,
12H, o-, m- y p-Ph, o-PhSO3Na (H6), m-PhSO3Na), 7.93–8.10 (m,
1
2H, o-PhSO3Na (H2), p-PhSO3Na). 31P{ H} NMR (162 MHz,
◦
D2O, 25 C): d = 36.7 ppm. FAB MS: m/z 713 [M]+; elemental
Using this method the following complexes were prepared:
analysis calcd. (%) for C25H18AuNNaO4PS2 (711.47): C 31.87, H
1
[Au(Spyrim)(TPPMS)](1a). 49% yield, pale yellow solid. H
◦
2.12, N 3.10; found: C 41.87, H 2.01, N 1.54. S20 ,H2O: 90 g L-1
NMR (400 MHz, CDCl3, 25 ◦C): d = 7.01 (t, J = 4.8 Hz, 1H,
pyrim-H5), 7.38–7.65 (m, 12H, o-, m- y p-Ph, o-PhSO3Na (H6),
m-PhSO3Na), 7.91–8.10 (m, 2H, o-PhSO3Na (H2), p-PhSO3Na),
[Au(Sbenzoxazole)(TPPTS)] (4b). 85% yield, yellow solid. 1H
NMR (400 MHz, D2O, 25 ◦C): d = 7.01–7.06 (m, 2H, benzoxazol-
H2, H3), 7.11–7.26 (m, 2H, benzoxazol-H1, HH4), 7.36–7.52
(m, 6H, o-PhSO3Na (H6), m-PhSO3Na), 7.47–7.92 (m, 6H, o-
8.32 (d, J = 5.1 Hz◦, 2H, pyrim-H4, H6) ppm. 31P{ H} NMR (162
1
MHz, CDCl3, 25 C): d = 37.1 ppm. FAB MS: m/z 673 [M]+;
elemental analysis calcd. (%) for C22H17AuN2NaO3PS2 (672.44):
C 39.29, H 2.55, N 4.17; found: C 39.01, H 2.03, N 4.01. S20◦,H2O:
90 g L-1
1
PhSO3Na (H2), p-PhSO3Na). 31P{ H} NMR (162 MHz, D2O,
25 ◦C): d = 36.6 ppm. FAB MS: m/z 917 [M]+; elemental analysis
calcd. (%) for C25H16AuNNa3O10PS4 (915.56): C 32.80, H 1.76, N
◦
1.53; found: C 31.86, H 1.53, N 1.26. S20 ,H2O: 325 g L-1
1
[Au(Spyrim)(TPPTS)](1b). 72% yield, pale yellow solid. H
NMR (400 MHz, D2O, 25 ◦C): d = 6.89 (t, J = 4.8 Hz, 1H, pyrim-
H5) 7.46–7.65 (m, 6H, o-PhSO3Na (H6), m-PhSO3Na), 7.74–7.83
(m, 6H, o-PhSO3Na (H2), p-PhSO3Na), 8.22 (d, J = 5.1 Hz, 2H,
[Au(Sbenzothiazole)(TPPMS◦)] (5a). 68% yield, yellow solid.
1H NMR (400 MHz, D2O, 25 C): d = 7.20 (dt, J = 7.1/1.3Hz,
1H, benzothiaz-H3), 7.33 (dt, J = 7.1/1.3Hz, 1H, benzothiaz-H2),
7.45–7.70 (m, 13H, o-, m- y p-Ph, o-PhSO3Na (H6), m-PhSO3Na,
benzothiaz-H4), 7.73–8.10 (m, 3H, o-PhSO3Na (H2), p-PhSO3Na,
◦
pyrim-H4, H6) ppm. 31P{ H} NMR (162 MHz, D2O, 25 C): d =
36.7 ppm. FAB MS: m/z 877 [M]+; elemental analysis calcd. (%)
for C22H15AuN2Na3O9PS4 (876.53): C 30.15, H 1.72, N 3.20; found:
1
◦
1
benzothiaz-H1) ppm. 31P{ H} NMR (162 MHz, D2O, 25 C): d =
36.7 ppm. FAB MS: m/z 728 [M]+; elemental analysis calcd. (%)
for C25H18AuNNaO3PS3 (727.54): C 41.27, H 2.49, N 1.93; found:
◦
C 30.65, H 1.54, N 2.69. S20 , H2O: 102 g L-1
[Au(SMepyrim)(TPPMS)](2a).◦ 63% yield, pale yellow solid.
1H NMR (400 MHz, MeOD, 25 C): d = 2.54 (s, 3H, CH3), 7.21
(d, J = 5.3 Hz, 1H, pyrim-H5), 7.49–7.62 (m, 14H, o-, m- y p-Ph, o-
PhSO3Na (H6), m-PhSO3Na, o-PhSO3Na (H2), p-PhSO3Na), 8.29
◦
C 41.06, H 2.26, N 1.72. S20 ,H2O: 110 g L-1
[Au(Sbenzothiazole)(TPPTS)] (5b). 74% yield, yellow solid.
1H NMR (400 MHz, D2O, 25 ◦C): d = 7.19–7.29 (m, 2H,
benzothiaz-H3, HH2), 7.47–7.75 (m, 7H, o-PhSO3Na (H6),
m-PhSO3Na, benzothiaz-H4), 7.78–7.94 (m, 6H, o-PhSO3Na
(d,◦J = 5.0 Hz, 1H, pyrim-H6). 31P{ H} NMR (162 MHz, MeOD,
1
25 C): d = 36.1 ppm. FAB MS: m/z 687 [M]+; elemental analysis
1
calcd. (%) for C23H19AuN2NaO3PS2 (686.47): C 40.24, H 2.79, N
(H2), p-PhSO3Na, benzothiaz-H1). 31P{ H} NMR (162 MHz,
◦
◦
4.08; found: C 39.71, H 3.50, N 3.92. S20 ,H2O: 85g L-1
D2O, 25 C): d = 36.7 ppm. FAB MS: m/z 936 [M]+; ele-
mental analysis calcd. (%) for C25H16AuNNa3O9PS5 (931.63):
[Au(SMepyrim)(TPPTS)](2b)◦. 56% yield, pale yellow solid.
1H NMR (400 MHz, MeOD, 25 C): d = 2.34 (s, 3H, CH3), 7.05 (d,
J = 5.2 Hz, 1H, pyrim-H5), 7.36–7.52 (m, 6H, o-PhSO3Na (H6),
m-PhSO3Na), 7.72–7.80 (m, 6H, o-PhSO3Na (H2), p-PhSO3Na),
C25H16AuNNa3O9PS5 (931.63); found: C 32.50, H 1.75, N 2.03.
◦
S20 ,H2O: 440 g L-1
[Au(Sbenzimidazole)(TPPMS)] (6a◦). 60% yield, pale yellow
1
1
8.25 (d, J = 5.1 Hz, 1H, pyrim-H6). 31P{ H} NMR (162 MHz,
solid. H NMR (400 MHz, D2O, 25 C): d = 7.02–7.13 (m, 2H,
10932 | Dalton Trans., 2011, 40, 10927–10935
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benzimidaz-H2, H3), 7.18–7.33 (m, 2H, benzimidaz-H1, H4), 7.45–
7.70 (m, 12H, o-, m- y p-Ph, o-PhSO3Na (H6), m-PhSO3Na),
7.93–8.10 (m, 2H, o-PhSO3Na (H2), p-PhSO3Na), 11.96 (s, 1H,
Table 5 Crystal Data and Data Collection and Refinement for complexes
[Au(SMe2Pyrim)(PTA)] (3c) and [Au(SBenzoxazole)(DAPTA)] (4d)
3c
4d
NH). 31P{ H} NMR (162 MHz, D2O, 25 ◦C): d = 37.3 ppm.
1
FAB MS: m/z 712 [M]+; elemental analysis calcd. (%) for
Empirical formula
Mol wt
Color, habit
C12H19AuN5PS
493.32
Colourless, needle
Monoclinic, P21/m
10.735(5)
7.029(5)
13.608(5)
90
109.139(5)
90
970.1(9)
2
1.689
7.771
3.15 to 25.00
15722
1846 [R(int) = 0.0876]
0.0369
C16H22AuN4O4PS
594.37
C25H19AuN2NaO3PS2 (710.49): C 42.26, H 2.70, N 3.94; found:
Colourless, plate
◦
C 41.89, H 2.88, N 3.72. S20 ,H2O: 101 g L-1
¯
Space group
Triclinic, P1
˚
a (A)
6.0539(10)
10.5087(18)
15.828(3)
97.007(3)◦
96.101(3)
98.188(3)
981.4(3)
2
[Au(Sbenzimidazole)(TPPTS)] (6b). 85% yield, yellow solid.
1H NMR (400 MHz, D2O, 25 ◦C): d = 7.07–7.19 (m, 2H,
benzimidaz-H3, H2), 7.24–7.36 (m, 2H, benzimidaz-H1, H4), 7.39–
7.52 (m, 6H, o-PhSO3Na (H6), m-PhSO3Na), 7.72–7.80 (m, 6H,
˚
b (A)
˚
c (A)
a (◦)
b (◦)
g (◦)
1
o-PhSO3Na (H2), p-PhSO3Na), 12.20 (br. s, 1H, NH). 31P{ H}
3
˚
V(A)
NMR (162 MHz, D2O, 25 ◦C): d = 37.6 ppm. FAB MS: m/z
916 [M]+; elemental analysis calcd. (%) for C25H17AuN2Na3O9PS4
(914.58): C 32.83, H 1.87, N 3.06; found: C 33.42, H 1.70, N 2.75.
Z
D(calc) (g cm-3)
m (mm-1)
2.005
7.713
1.98 to 24.50
9236
3245 [R(int) = 0.0430]
0.0423
0.1111
1.055
q Range (deg)
N. data collected
N. unique data
R1a(F2> 2s(F2))
v R2b(all data)
Sc (all data)
◦
S20 ,H2O: 250 g L-1
1
[Au(Sthiazoline)(TPPMS)] (7a). 65% yield, yellow solid. H
NMR (400 MHz, D2O, 25 ◦C): d = 3.42 (t, J = 7.8 Hz, 2H,
thiazoline), 4.11 (t, J = 8.0 Hz, 2H, thiazoline) 7.35–7.64 (m,
12H, o-Ph, m-Ph, p-Ph, o-PhSO3Na (H6), m-PhSO3Na), 7.87–
0.0832
0.970
ꢀ
ꢀ
ꢀ
b
2
a R1(F) =
ꢀFo| - |Fcꢀ/
|Fo|. vR2(F2) = [ [v(Fo - Fc2)2]/
ꢀ
[w(Fo2)2]1/2, w-1 = [s (Fo2)+(aP)2+bP], where P = [max(Fo2, 0)+2Fc2)]/
2
1
8.02 (m, 2H, o-PhSO3Na (H2), p-PhSO3Na) ppm. 31P{ H} NMR
ꢀ
3. c S = [ [v(Fo - Fc2)2]/(n - p)]1/2, where n is the number of reflections
2
◦
(162 MHz, D2O, 25 C) d = 36.7 ppm. FAB MS: m/z 678 [M]+;
and p the number of refined parameters.
elemental analysis calcd. (%) for C21H16AuNNaO3PS3 (679.50): C
37.12, H 2.06, N 2.06; found: C 36.98, H 2.32, N 1.78. S20◦,H2O:
99 g L-1
X-ray diffraction analysis
Crystals suitable for X-ray diffraction were obtained by
slow diffusion of diethyl ether into ethanolic solutions.
Synthesis of [Au(Sglucosetetraacetate)(PR¢3)] (PR¢3 = PTA (8a)
and DAPTA (8b)) complexes
A
summary of the fundamental crystal and refinement
A mixture of [AuCl(PR¢3)] (0.26 mmol), 1-thio-b-D-glucose
tetraacetate (0.26 mmol) and K2CO3 (0.28 mmol) in EtOH
(10 mL) and water (10 mL) was stirred for ca. 18 h. The solvents
were removed under reduced pressure, and the solid residue was
extracted with acetone and filtered through Celite. The addition of
diethylether to the filtrate afforded the colourless product, which
was isolated by filtration and dried in air.
data of compounds [Au(SMe2pyrimidine)(PTA)] (3c) and
[Au(SBenzoxazole)(DAPTA)] (4d) is given in Table 5. The crystals
were mounted on glass fiber with inert oil and centred in a
Bruker-Smart CCD diffractometer with graphite-monochromated
˚
Mo-Ka (l = 0.7107 A) radiation for data collection. The
diffraction frames were integrated by using the SAINT45 package
and corrected for absorption with SADABS.46 The structure of
3c was solved by direct methods using SHELXS47 and 4d by
sir2002.48 Full-matrix least squares refinement was carried out
1
8a. Yield: 79%, colourless solid. H NMR (400 MHz, dmso-
◦
d6, 25 C): d = 1.90, 1.97, 1.99, 2.03 (s, 3H, Me), 3.88 (ddd, J =
9.8/4.5/2.3Hz, 1H, H5), 3.97 (dd, J = 12.4/2.4Hz, 1H, CH2),
4.09 (dd, J = 12.1/4.5Hz, 1H, CH2), 4.30 (s, 6H, NCH2P), 4.34,
4.53 (JAB = 12.8 Hz, 6H, NCH2N), 4.62 (t, J = 9.3 Hz, 1H, H2),
4.85 (t, J = 9.9 Hz, 1H, H4), 5.11 (t, 2H, J◦= 9.6 Hz, H1, H3)
using SHELXL minimizing w(F0 *-Fc )2. Hydrogen atoms were
included by using a riding model. In the case of 4d the hydrogens
of water molecules were not located. Weighted R factors (Rw) and
all goodness of fit S values are based on F2; conventional R factors
(R) are based on F. The PLATON SQUEEZE49,50 algorithm
was applied to 3c to model the diffuse contribution from a
highly disordered solvent of crystallisation to the electron density.
The complete crystallographic data have also been deposited
with the Cambridge Crystallographic Data Centre [Deposition
numbers are CCDC 790251 and 790252 for compounds 3c and 4d
respectively, see ESI†].
2
2
1
ppm. 31P{ H} NMR (162 MHz, dmso-d6, 25 C): d = -47.0 ppm.
C20H31AuN3O9PS (717.48): ◦calcd. C 33.48, H 4.35, N 5.86, found
C 33.34, H 4.25, N 6.09. S20 , H2O: 220 g L-1
1
8b. Yield: 79%, colourless solid. H NMR (400 MHz, D2O,
25 ◦C): d = 1.95, 1.98, 2.02, 2.05 (s, 3 H, Me), 2.06 (s, 6H, COMe),
3.84 (dt, J = 15.6/3.9 Hz, 1 H, H5), 3.95–4.00 (m, 2 H, NCH2P),
4.03 (s, 1 H, NCH2P), 4.11 (dd, J = 12.4, 1.6 Hz, 1 H, CH2), 4.2 (d,
1 H, J = 14.4 Hz, NCH2N), 4.30 (dd, J = 12.8/3.6 Hz, 1 H, CH2),
4.30–4.38 (m, 1 H, NCH2P), 4.80 (d, J = 10 Hz, 1 H, NCH2N),
4.84 (t, J = 9.2 Hz, 1 H, H2), 5.01 (d, J = 5.6 Hz, 1 H, NCH2P),
5.04 (t, J = 10 Hz, 1 H, H4), 5.15 (t, J = 9.2 Hz, 2 H, H1H3), 5.20 (d,
J = 9.2 Hz, 1H, NCH2N), 5.36 (dd, J = 16/8.4 Hz, 1 H, NCH2P),
Antiproliferative assays
Human A27807S and A2780/R cells were obtained from the
European Centre of Cell Cultures (ECACC, Salisbury, UK).
All cell culture reagents were obtained from Gibco-BRL (Basel,
Switzerland). Cells were grown in RPMI 1640 medium containing
10% fetal calf serum (FCS) and antibiotics. Stock solutions of the
complexes (in DMSO) were diluted in complete medium to the
1
5.54 (d, J = 14 Hz, 1H, NCH2N) ppm. 31P{ H} NMR (162 MHz,
D2O, 25 ◦C): d = -22.3 ppm. C23H35AuN3O11PS (789.54): calcd. C
34.99, H 4.47, N 5.32; found C 34.87, H 4.36, N 5.03. S20◦, H2O:
220 g L-1
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required concentration. DMSO at comparable concentrations did
not show any effects on cell cytotoxicity.
3 W. F. Kean and I. R. L. Kean, Inflammopharmacology, 2008, 16,
112.
4 E. R. T. Tieknik, Inflammopharmacology, 2008, 16, 138.
5 I. Kostova, Anti-Cancer Agents Med. Chem., 2006, 6, 19.
6 E. R. T. Tiekink, Crit. Rev. Oncol. Hematol., 2002, 42, 225.
7 L. Messori and G. Marcon, Metal Ions in Biological Systems. Gold
Complexes as antitumour agents, A Sigel and H Sigel (Eds), Marcel
Dekker Inc, New York, 2004.
8 A. Casini, C. Hartinger, C. Gabbiani, E. Mini, P. J. Dyson, B. K.
Keppler and L. Messori, J. Inorg. Biochem., 2008, 102, 564.
9 S. Nobili, E. Mini, I. Landini, C. Gabbiani, A. Casini and L. Messori,
Med. Res. Rew., 2010, 30, 550.
10 L. Ronconi and D. Fregona, Dalton Trans., 2009, 10670.
11 R. W. Y. Sun and C. M. Che, Coord. Chem. Rev., 2009, 253,
1682.
12 A. Bindoli, M. P. Rigobello, G. Scutari, C. Cabbiani, A. Casini and L.
Mesori, Coord. Chem. Rev., 2009, 253, 1692.
13 I. Ott, Coord. Chem. Rev., 2009, 253, 1670.
For cytotoxicity screening, cells were grown in 96-well cell
culture plates (Corning, NY) at a density of 25 ¥ 103 cells
per well. The culture medium was replaced with fresh medium
containing the complexes at concentrations varying from 0 to
20 mM, with an exposure time of 72 h. Thereafter, the medium was
replaced by fresh medium and cell survival was measured using
the MTT test as previously described. Briefly, 3-(4,5-dimethyl-
2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT, Merck) was
added at 250 mg mL-1 and incubation was continued for 2 h.
Then the cell culture supernatants were removed, the cell layer
was dissolved in DMSO, and absorbance at 540 nm was measured
in a 96-well multiwell-plate reader (iEMS Reader MF, Labsystems,
Bioconcept, Switzerland) and compared to the values of control
cells incubated in the absence of complexes. Experiments were
conducted in quadruplicate wells and repeated at least twice.
14 A. Casini, C. Hartinger, C. Gabbiani, E. Mini, P. J. Dyson, B. K.
Keppler and L. Messori, J. Inorg. Biochem., 2008, 102, 564.
15 K. M. Hindi, M. J. Panzner, C. A. Tessier, C. L. Cannon and W. J.
Youngs, Chem. Rev., 2009, 109, 3859.
16 M. J. McKeage, L. Maharaj and S. J. Berners-Price, Coord. Chem. Rev.,
2002, 232, 127.
Cell uptake studies and ICP-MS analysis
17 P. J. Barnard and S. J. Berners-Price, Coord. Chem. Rev., 2007, 251,
For evaluation of the cell uptake, cells were seeded in 6-well plates
and grown to approximately 70% confluency and incubated with
the corresponding metallodrug at 1 mM for 3 h. At the end of
the incubation period, cells were rinsed cells with 5 mL of PBS,
detached by adding 0.4 mL enzyme free cell dissociation solution
(Millipore, Switzerland) and collected by centrifugation. Cell lysis
was achieved using a freeze-thaw technique that was recently found
to be suitable for cell uptake studies.51 All samples were analyzed
for their protein content (to establish the number of cells per
sample) prior to ICP-MS determination using a BCA assay (Sigma
Aldrich, Switzerland). All samples were digested in ICP-MS grade
concentrated hydrochloric acid (Sigma Aldrich, Switzerland) for
3 h at room temperature and filled to a total volume of 8 ml with
ultrapure water. Indium was added as an internal standard at a
concentration of 0.5 ppb. Determinations of total metal contents
were achieved on an Elan DRC II ICP-MS instrument (Perkin
Elmer, Switzerland) equipped with a Meinhard nebulizer and
a cyclonic spray chamber. The ICP-MS instrument was tuned
daily using a solution provided by the manufacturer containing 1
ppb each of Mg, In, Ce, Ba, Pb and U. External standards were
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We thank the Ministerio de Ciencia e Innovacio´n (CTQ2008-
06716-CO3-01) for financial support. The authors wish also to
thank COST D39 action for stimulating discussion. AC thanks
the Swiss National Science Foundation (AMBIZIONE project
no. PZ00P2-121933) and the Swiss Confederation (Action COST
D39 - Accord de recherche - SER project no. C09.0027) for
financial support.
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