Gold(I) complexes with alkylated PTA (1,3,5-triaza-7-phosphaadamantane) phosphanes as anticancer metallodrugs

Article abstract of DOI:10.1016/j.ejmech.2014.04.001

New stable thiolate gold(I) derivatives containing the alkylated phosphanes [PTA-CH₂Ph]Br and [PTA-CH₂COOMe]Br derived from 1,3,5-triaza-7-phosphaadamantane (PTA) have been prepared by different routes of synthesis. By the use of bas

Full text of DOI:10.1016/j.ejmech.2014.04.001

European Journal of Medicinal Chemistry 79 (2014) 164e172
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Gold(I) complexes with alkylated PTA (1,3,5-triaza-7-
phosphaadamantane) phosphanes as anticancer metallodrugs
Elena García-Moreno a, Sonia Gascón b, Elena Atrián-Blasco a, Ma Jesus Rodriguez-Yoldi b,
,
Elena Cerrada a, Mariano Laguna a
*
a Departamento de Química Inorgánica, Instituto de Síntesis Química y Catálisis Homogénea, Universidad de Zaragoza-C.S.I.C., E-50009 Zaragoza, Spain
b Departamento de Farmacología y Fisiología, Unidad de Fisiología, Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, CIBERobn, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
New stable thiolate gold(I) derivatives containing the alkylated phosphanes [PTAeCH2Ph]Br and [PTA
eCH2COOMe]Br derived from 1,3,5-triaza-7-phosphaadamantane (PTA) have been prepared by different
routes of synthesis. By the use of basic media to deprotonate the corresponding thiol in the former and
by transmetallation reactions from tin (IV) complexes, in the later, thus avoiding side reactions on the
phosphane. Strong antiproliferative effects are observed for most of the compounds, including the
chloro- and bromo precursors with the series of phosphanes derived from PTA, in human colon cancer
cell lines (Caco-2, PD7 and TC7 clones). Apoptosis-induced cell death is found for all compounds, being
the thiolate derivatives with [PTAeCH2Ph]Br the most effective, as shown by an annexin-V/propidium
iodide double-staining assay.
Received 27 January 2014
Received in revised form
13 March 2014
Accepted 1 April 2014
Available online 1 April 2014
Keywords:
Gold
Thiolate
Water soluble
PTA
Ó 2014 Elsevier Masson SAS. All rights reserved.
Antitumor properties
1. Introduction
research on gold derivatives for cancer chemotherapy. Thus, over
the last decades different type of gold-based drugs candidates have
Medicinal properties of gold compounds have been exploited
from ancient cultures such as India, Egypt and China [1]. Although
the rational use of gold in medicine, began in the early 1920s, with
the discovery of inhibitory properties of gold cyanide in Tubercle
bacillus. From this moment, several diseases have been treated
with gold derivatives, including tuberculosis [2] and arthritis
rheumatoid [3]. Several thiolate gold(I) complexes have been used
clinically in the arthritis rheumatoid treatment, namely aurothio-
glucose (solganol), aurothiomalate (myocrisin) [4,5] and triethyl-
phosphine gold(I) tetraacetothioglucose (auranofin) [6,7]. In the
last few decades, these derivatives and related ones have been
tested against HIV for the treatment of AIDS [8,9], acute forms of
asthma chronic (corticosteroid-dependent asthma), pemphigus (an
autoimmune disease of the skin) [10], in treatment of malaria [11],
Chagas disease [12] and cancer [13e15].
been reviewed, where different kind of ligands have been
employed: phosphanes, bischelating phosphanes, thiolates, di-
thiocarbamates, porphyrines or N-heterocyclic carbenes [14,15,19e
28].
It has been shown in gold(I) bisphosphanes, that the presence of
high lipophilic character, results in severe toxicity to heart, liver and
lung, as a consequence of the non-selective concentration of
compounds into mitochondria of both carcinogenic and healthy
cells [29e31]. Additionally, in similar gold(I) bisphosphane de-
rivatives, a direct relationship between hepatotoxicity and lip-
ophilicity has been found [32]. Consequently, the selectivity of such
kind of derivatives for cancer over normal tissue could be improved
by optimizing their lipophilic-hydrophilic balance, with the
appropriate ligands [33e36]. For this reason, a balanced relation-
ship between lipophilic and hydrophilic character is an important
parameter in optimizing biodistribution, activity and selectivity of
the drugs. Accordingly, water solubility of the drugs could provide
such balanced relationship. The use of water-soluble phosphanes
can lead to the synthesis of soluble or partially soluble complexes in
water. Thus, we have previously described some water-soluble
thiolate [37e40] or alkyne [41,42] gold(I) derivatives with the
phosphanes PTA (1,3,5-triaza-7-phosphaadamantane), DAPTA (3,7-
In the mid-1980s auranofin was found to show strong anti-
proliferation potency against cancer cells in vitro [16e18], as well as
having limited in vivo antitumor activity, being only active against
P388 leukemia cancer [18]. These studies have triggered an intense
* Corresponding author.
E-mail address: mlaguna@unizar.es (M. Laguna).
diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane),
sodium
http://dx.doi.org/10.1016/j.ejmech.2014.04.001
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

E. García-Moreno et al. / European Journal of Medicinal Chemistry 79 (2014) 164e172
165
triphenylphosphane monosulfonate (TPPMS) or sodium triphe-
nylphosphane trisulfonate (TPPTS), which in most of the cases have
shown from moderate to excellent antiproliferative properties.
In addition, we have recently shown that the hydrophilicity of
the phosphane ligand can be enhanced by alkylation of the PTA
molecule, giving rise to new phosphanes with greater water solu-
bilities compared with the free PTA and their corresponding water-
soluble gold(I) compounds [43,44]. Within this frame, here we
describe the synthesis of new thiolate gold(I) derivatives with some
of these phosphanes derived from PTA. These complexes and their
corresponding precursors, previously described by some of us [43],
have been screened for their antitumor activity against human
colon cancer cell lines Caco-2, as well as their apoptotic activity
evaluation and their implication in the cell cycle progression.
similar intensities are observed in their 31P{1H} NMR spectra and
multiple signals can be detected in the 1H NMR spectra. The same
results are obtained independently of the basic media (KOMe/
MeOH, NaOMe/MeOH, NEt3/CH2Cl2 or Na2CO3/CH2Cl2). It has been
recently published that PTA derivatives such as [PTAeCH2COOMe]
Br and [PTA-Me2](TfO)2 react in the presence of the base NaOH or
KOH to give the new phosphanes [PTAeCH2COOH]Br in the former,
consequently of the methyl ester transformation to carboxylic acid
[44] and [PTA-Me2] (dmoPTA) (3,7-dimethyl-1,3,7-triaza-5-
phosphabicyclo[3.3.1]nonane) in the later, result of the elimina-
tion of the methylene group located between both NMe units
[45,46]. In our case, the presence of a more complicated pattern
than the expected in the 1H NMR spectra and two resonances in the
31P{1H} NMR experiments could be explained by similar side re-
actions of the phosphane in the basic media.
Since the synthesis of thiolate complexes with the [PTAe
CH2COOMe]Br phosphane proved problematical, we decided to use
an alternative route. We have focused in tin(IV) derivatives as
transmetallating agents, since it has been previously shown their
ability to transfer dithiolate ligands to other metals under mild
conditions [47e50]. Therefore the corresponding tin thiolates,
synthesized as previously described [51], were then reacted with
the bromo derivative 1b giving rise to the new thiolate gold(I)
complexes [Au(SR)(PTAeCH2COOMe)]Br (SR ¼ Spy, 3b; Spyrim; 4b,
SMepyrim; 5b and SMe2pyrim, 6b) (Scheme 1, iii) as pure samples.
These compounds exhibit similar 1H NMR spectra than those
observed in the related thiolates 3e6a, with the main difference in
the corresponding signals to the PTA phosphane. However a
different behavior is detected in the singlets measured in the 31P
{1H} NMR spectra, inasmuch as the resonances of the thiolates
derived from [PTAeCH2COOMe]Br are low field displaced
compared with the bromo gold (I) starting material 1b
2. Results and discussion
2.1. Synthesis and characterization
The conventional route for the synthesis of thiolate gold(I) de-
rivatives consists of the addition of the corresponding halogen gold
phosphane complex to a solution of the thiol in the presence of a
base. The use of [AuBr(PTAeCH2Ph)]Br (1a) leads to the isolation of
the air stable solids with the formula [Au(SR)(PTAeCH2Ph)]Br
(SR ¼ Spy, 3a; Spyrim; 4a, SMepyrim; 5a and SMe2pyrim, 6a)
(Scheme 1, ii) in good yields. All of them show the characteristic
signals of the thiolates and those due to the [PTAeCH2Ph]Br
phosphane in their 1H NMR spectra, apart from the absence of the
SeH resonance from the starting thiol. The 31P{1H} NMR displays a
low-field displaced singlet compared to those in the free phos-
phane, but at higher field with respect to the starting material
[AuBr(PTAeCH2Ph)]Br.
(d
¼ ꢀ41.6 ppm), whilst upfield shifts are observed in the thiolates
with [PTAeCH2Ph]Br in comparison with [AuBr(PTAeCH2Ph)]Br
(1a) (
¼ ꢀ39 ppm).
Treatment of [AuX(PTAeCH2COOMe)]X (X ¼ Br, 1b or Cl, 1b0)
with the thiolates in the same conditions gave mixtures of com-
plexes, as deducted by NMR experiments. Thus, two singlets with
d
Scheme 1. Preparation of gold(I) derivatives with [PTA-R]X phosphanes.

166
E. García-Moreno et al. / European Journal of Medicinal Chemistry 79 (2014) 164e172
The MALDIþ spectra for all the new thiolate gold(I) complexes
those with alkyl substituents in the pyrimidine ring. Similar hy-
drophilic character is found in all the compounds with the most
water soluble phosphane [PTAeCH2COOMe]Br, with negative
values of log D7.4 ranging from ꢀ0.26 to ꢀ0.51.
with both alkylated PTA phosphanes display molecular ion peaks
identified as the corresponding cation.
These new thiolate compounds resulted poorly soluble in water,
with the best values ranging from 7 to 21 g Lꢀ1. Curiously, although
the PTA with the ester group [PTAeCH2COOMe]Br, that is six times
more water-soluble than the PTA, gave lower solubilities in the final
complexes than those measured in the related with PTA [40].
2.3. Biological studies
The precursors chloro-, bromo gold(I) derivatives (1aed) with
the phosphanes derived from PTA ([PTA-R]X,
R
¼
CH2Ph,
CH2COOMe, CH2CN, CH2COOH), the chloro gold(I) bisphosphanes
(2aeb), depicted in Scheme 1, and the corresponding thiolate gold
complexes with the phosphanes [PTAeCH2Ph]Br (3e6a) and [PTAe
CH2COOMe]Br (3e6b) were screened for their cytotoxic properties
against human colon cancer cell lines Caco-2 (clones Caco-2/PD7,
from early passage, and Caco-2/TC7, from late passage), in com-
parison to cisplatin and auranofin under the same experimental
conditions. Cells were exposed to the metallic complexes for 72 h.
The IC50 values (Table 1) were calculated by using the colorimetric
mitochondrial function-based MTT viability assay (see
experimental).
2.2. Lipophilicity
As stated in the introduction, a balanced relationship between
lipophilicity and hydrophilicity would be important in drug de-
livery process. We have measured such relationship in terms of log
D7.4 (n-octanol/water partition coefficient under physiological
conditions) (Table 1) of the new thiolate derivatives and the chloro
or bromo precursors depicted in Scheme 1. Complexes [AuCl(PTAe
CH2CN)]Br (1c) and [AuCl(PTAeCH2COOH)]Br (1d) are the most
hydrophilic derivatives with log D7.4 values next to ꢀ1 (ꢀ1.04
and ꢀ0.81 respectively). However, the rest of the compounds
display a balanced relationship between their lipophilic and hy-
drophilic character, with log D7.4 values ranging from ꢀ0.5
and þ0.18.
All the tested compounds showed comparable cytotoxicity to
that observed in auranofin, but markedly lower values than those
found for cisplatin. In general, exposure of Caco-2/PD7 cell lines to
increasing concentrations (0e20
mM) of the complexes with the
There is no direct relationship between the water solubility of
the phosphane and the log D7.4 value of the corresponding gold
compounds since the highest hydrophilicity is not found in the
complexes with the most water-soluble phosphane ([PTAe
CH2COOMe]Br). Compounds [AuCl(PTAeCH2CN)]Br (1c) and
[AuCl(PTAeCH2COOH)]Br (1d) display the highest values of log
D7.4, ꢀ1.04 and ꢀ0.81 respectively, and the solubility in water of the
PTA derivatives are up to 12 and 2.5 times lower than that found in
[PTAeCH2COOMe]Br, respectively. Accurately, these derivatives
display the highest solubility in water of all complexes described
herein (see Ref. [43]). Comparing the thiolate gold compounds,
bearing the less soluble phosphane [PTAeCH2Ph]Br, the liphophi-
licities vary with the nature of the thiolate, being more hydrophilic
phosphane [PTAeCH2Ph]Br led to lower IC50 values (1.75e
4.67
9.86
m
M) than the measured for Caco-2/TC7 cell lines (3.95e
mM), except in the complexes [AuCl(PTAeCH2Ph)2]Br2 (2a) and
[Au(SMepyrim)(PTAeCH2Ph)]Br (5a). However, the most water-
soluble phosphane [PTAeCH2COOMe]Br gives alternatively the
lowest IC50 values in both cell lines and depending on the halogen,
being lower for PD7 clones in the chloro compounds.
Remarkably, [Au(Spyrim)(PTAeCH2Ph)]Br (4a) showed the
highest cytotoxicity in both clones with low IC50 values in PD7 and
TC7 cell lines (2.49 and 3.95 mM respectively), similar to auranofin
and besides being 15 and 11.5 times lower than those of cisplatin. A
major activity is found in all the thiolate complexes 3e6a/b with
the phosphanes [PTAeCH2Ph]Br and [PTAeCH2COOMe]Br in com-
parison with the halogen precursors or the bis-phosphane
[AuCl(PTAeCH2Ph)2]Br2 (2a) and [AuCl(PTAeCH2COOMe)2]Br2
(2b).
The free phosphanes were much less effective in decreasing
cancer viability over both colon cancer cell lines, with IC50 values
higher than 50 mM in all the cases and even higher than 250 mM for
Table 1
IC50 values in Caco-2 cell lines and distribution coefficients of 1e6 complexes, aur-
anofin and cisplatin.
Compound
Log D7.4 IC50 (m
M)a
Caco-2/PD7
Caco-2/TC7
the phosphane [PTACH2Ph]Br in Caco-2/PD7 cell line, confirming
that the gold center was necessary to obtain bioactive derivatives.
The ability of these compounds to induce apoptosis was tested
by incubating both cancer cell lines Caco-2 with the gold de-
rivatives 1e6, cisplatin and auranofin for 72 h, using the annexin-V/
PI double staining assay. As summarized in Table 2 and Fig. 1
compounds [AuCl(PTAeCH2CN)]Br (1c), [Au(Spyrim)(PTAe
CH2Ph)]Br (4a) and [Au(SMepyrim)(PTAeCH2Ph)]Br (5a) are the
most effective with practically no live cells after 72 h of treatment
in both cell lines. These derivatives are able to induce cell death by
an apoptotic pathway, as derived from the higher amounts of early
and late apoptosis observed in comparison to DMSO for both cancer
cell lines (PD7 and TC7). In addition, the lack of significant propi-
dium iodide uptake indicates that neither complex induces necrotic
cell death after 72 h. One important difference is found in complex
4a, that exhibits the highest amount of late-stage apoptosis
compared to 1c and 5a (61.2% versus 36.8 and 22.6%, respectively)
in Caco-2/PD7 cells and similar amounts in early and late-stage
apoptosis in Caco-2/TC7 clone (approx. 47%). The remaining thio-
late derivatives [Au(Spy)(PTAeCH2Ph)]Br (3a) and [Au(SMe2pyr-
im)(PTAeCH2Ph)]Br (6a) differ from 4a and 5a mainly in the
population of live cells in both clones, giving higher amounts (40.3%
[PTAeCH2Ph]Br
91 ꢁ 8
4.67 ꢁ 0.02
4.4 ꢁ 0.29
77 ꢁ 5
260 ꢁ 12
9.86 ꢁ 0.75
6.88 ꢁ 0.6
54 ꢁ 3
[AuBr(PTAeCH2Ph)]Br (1a)
[AuCl(PTAeCH2Ph)]Cl (1a0)
[PTAeCH2COOMe]Br
[AuBr(PTAeCH2COOMe)]Br (1b)
[AuCl(PTAeCH2COOMe)]Cl (1b0)
[PTAeCH2CN]Br
ꢀ0.14
ꢀ0.34
ꢀ0.40
ꢀ0.40
14.20 ꢁ 0.81
4.26 ꢁ 0.95
87 ꢁ 9
2.11 ꢁ 0.69
11.80 ꢁ 1.1
52 ꢁ 5
[AuCl(PTAeCH2CN)]Br (1c)
[PTAeCH2COOH]Br
ꢀ1.04
4.51 ꢁ 1.44
58 ꢁ 4
3.04 ꢁ 0.85
66 ꢁ 3
[AuCl(PTAeCH2COOH)]Br (1d)
[AuCl(PTAeCH2Ph)2]Br2 (2a)
[AuCl(PTAeCH2COOMe)2]Br2 (2b)
[Au(Spy)(PTAeCH2Ph)]Br (3a)
[Au(Spyrim)(PTAeCH2Ph)]Br (4a)
[Au(SMepyrim)(PTAeCH2Ph)]Br (5a)
ꢀ0.81
ꢀ0.01
ꢀ0.01
0.15
0.18
ꢀ0.55
6.79 ꢁ 1.17
14.74 ꢁ 0.22
0.93 ꢁ 0.20
8.48 ꢁ 1.9
5.53 ꢁ 0.0045 10.17 ꢁ 0.94
4.07 ꢁ 0.55
2.49 ꢁ 0.02
6.63 ꢁ 0.69
1.75 ꢁ 0.34
9.35 ꢁ 2.1
4.16 ꢁ 1.7
8.11 ꢁ 1.8
5.05 ꢁ 0.05
3.95 ꢁ 0.04
5.56 ꢁ 0.02
7.5 ꢁ 1.5
[Au(SMe2pyrim)(PTAeCH2Ph)]Br (6a) ꢀ0.48
[Au(Spy)(PTAeCH2COOMe)]Br (3b)
ꢀ0.30
7.67 ꢁ 1.5
2.8 ꢁ 1.0
[Au(Spyrim)(PTAeCH2COOMe)]Br (4b) ꢀ0.26
[Au(SMepyrim)
ꢀ0.51
5.55 ꢁ 1.4
(PTAeCH2COOMe)]Br (5b)
[Au(SMe2pyrim)
(PTAeCH2COOMe)]Br (6b)
Auranofin
ꢀ0.46
4.63 ꢁ 1.3
6.37 ꢁ 1.1
1.8 ꢁ 0.1
2.1 ꢁ 0.4
Cisplatin
37.24 ꢁ 5.15
45.6 ꢁ 8.08
a
Mean ꢁ SD of at least three determinations.

E. García-Moreno et al. / European Journal of Medicinal Chemistry 79 (2014) 164e172
167
Table 2
Summary of the effects of treating PD7 and PC7 cell lines with the metallic compounds 1e6, cisplatin and auranofin after 72 h. [AuBr(PTAeCH2Ph)]Br (1a), [AuCl(PTAeCH2Ph)]
Cl (1a0), [AuBr(PTAeCH2COOMe)]Br (1b), [AuCl(PTAeCH2COOMe)]Cl (1b0), [AuCl(PTAeCH2CN)]Br (1c), [AuCl(PTAeCH2COOH)]Br (1d), [AuCl(PTAeCH2Ph)2]Br2 (2a),
[AuCl(PTAeCH2COOMe)2]Br2 (2b), [Au(Spy)(PTAeCH2Ph)]Br (3a), [Au(Spyrim)(PTAeCH2Ph)]Br (4a), [Au(SMepyrim)(PTAeCH2Ph)]Br (5a), [Au(SMe2pyrim)(PTAeCH2Ph)]Br
(6a), [Au(Spy)(PTAeCH2COOMe)]Br (3b), [Au(Spyrim)(PTAeCH2COOMe)]Br (4b), [Au(SMepyrim)(PTAeCH2COOMe)]Br (5b), [Au(SMe2pyrim)(PTAeCH2COOMe)]Br (6b).
Complex
PD7
TC7
Live (%)
Early apoptosis
Late apoptosis
Necrosis (%)
Live (%)
Early apoptosis
Late apoptosis
Necrosis (%)
1a
52.4
53.3
59.8
58.1
5.0
75.4
14.3
31.3
40.3
0.5
17.3
14.1
6.6
14.0
55.0
8.6
4.41
31.5
15.2
37.4
77.0
45.8
6.8
21.4
18.4
17.5
19.8
36.8
9.7
43.7
36.7
37.0
61.2
22.6
19.2
8.9
7.8
16.5
9.8
22.4
32.2
5.8
8.9
14.2
16.2
8.1
3.2
6.3
0.6
0.5
7.5
0.9
0.0
0.4
4.1
5.5
6.4
5.2
14.6
22.7
1.1
72.2
69.3
76.7
77.9
4.4
87.7
12.0
59.8
45.9
1.5
6.7
26.0
3.0
2.8
79.6
4.9
47.5
20.7
13.3
47.2
34.1
43.1
7.2
3.8
9.6
7.2
3.3
18.3
2.7
17.0
8.7
9.9
4.1
19.4
10.5
6.1
0.3
3.3
0.9
0.8
4.0
3.4
0.1
0.4
7.2
7.2
1.7
3.1
6.0
0.2
3.5
1a0
1b
1b0
13.2
15.7
4.0
39.6
18.7
36.8
47.9
35.3
6.8
7.7
6.2
7.8
6.8
1c
1d
2a
2b
3a
4a
5a
6a
3b
4b
5b
6b
Cisplatin
Auranofin
DMSO
0.3
0.5
34.7
80.2
81.1
66.7
80.1
55.4
37.4
89.9
49.7
77.9
82.2
80.9
83.0
82.0
0.0
5.7
10.4
4.9
7.6
7.7
8.8
81.5
5.7
3.2
88.1
and 34.7% in PD7; 45.9% and 49.7% in TC7, respectively) to those
measured in 4a and 5a. Induction to apoptosis is also observed in
these thiolate compounds as well as in the bis-phosphane de-
particular case of the most active complex: [Au(Spyrim)(PTAe
CH2Ph)]Br (4a) and compared to cisplatin (see Supporting
Information). Treatment with increasing amounts of the gold
complex does not affect the mobility of the plasmid, conversely
with the observed in cisplatin. The lack of reactivity with DNA is
comparable to that observed for auranofin and in previously re-
ported gold(I) derivatives [41,52].
rivatives
[AuCl(PTAeCH2Ph)2]Br2
(2a)
and
[AuCl(PTAe
CH2COOMe)2]Br2 (2b), along with no significant necrotic popula-
tion. Less effectiveness can be deduced in the chloro or bromo-gold
derivatives 1a, 1a0, 1b, 1b0, 1d and the thiolate 3e6b with [(PTAe
CH2COOMe)]Br that behave similarly to cisplatin where high
amount of live population remains after 72 h of treatment in
addition to a moderate percentage of apoptotic cells.
To evaluate a possible influence of the gold derivatives on the
cell cycle, we analyzed the cell cycle progression and DNA frag-
mentation in surviving cells, following drug exposure by PI staining
and flow cytometric analyses in both cancer cell lines after 72 h of
treatment with complexes 1e6, cisplatin and auranofin. Complexes
[AuCl(PTAeCH2CN)]Br (1c), [AuCl(PTAeCH2Ph)2]Br2 (2a), [Au(S-
pyrim)(PTAeCH2Ph)]Br (4a), [Au(SMepyrim)(PTAeCH2Ph)]Br (5a)
and auranofin led to a markedly perturbed flow cytometry profile,
most likely due to extensive cell death. Accurately these derivatives
give the lowest percentages for live cells after 72 h of complex
treatment (see Table 2 and Fig. 1). Fig. 2 shows a histogram of Caco-
2/PD7 and Caco-2/TC7 cells treated with DMSO and the complexes
in which it was possible to determine the percentages of G1-, S- and
G2-phase cells.
Finally, the stability of [Au(Spyrim)(PTAeCH2Ph)]Br (4a) against
both cancer cell lines under physiological conditions was analyzed
by absorption spectroscopy (Fig. 3). A solution suitable for spec-
trophotometric analysis was prepared by diluting a DMSO mother
solution of the complex with aqueous buffer PBS. Complex 4a is
characterized by an intense band at 290 nm, which remains un-
changed over 24 h of the study under incubation of the solution at
37 ꢂC. Neither band shifts nor additional bands at around 550 nm,
characteristic of metallic gold formation, are detected during the
experiment, which clearly showed that this complex is essentially
stable for at least 24 h in PBS solution at 37 ꢂC. In addition a control
NMR experiment was performed under buffer conditions (PBS/d6-
DMSO/D2O solution) (see supporting information for more details).
The addition of the buffer solution in water gives the released of the
phosphane [(PTAeCH2Ph)]Br, which is immediately oxidized, as
evidenced by the 31P{1H} NMR with the appearance of the corre-
sponding signal of the oxide. The signals observed in the 1H NMR
remain intact after 24 h in PBS solution. The dissociation of the
phosphane (PEt3) and subsequent oxidation is also observed in
auranofin when is added to blood, as a consequence of its reaction
with cysteine-34 residue present in the serum albumin [14]. The
predisposition of complex 4a to dissociate the phosphane under
physiological conditions could facilitate the following reactions
with biomolecules thus justifying its high antitumor activity.
In general, the mechanism of biological action of gold de-
rivatives is still not fully elucidated. Although new protein targets
for these compounds are emerging, for instance zinc-finger pro-
teins, aquaporins, carbonic anhydrases among others [53e55], the
activity of gold compounds is often associated with the inhibition of
the seleno-enzyme thioredoxin reductase (TrxR) [20,56e59]. This
inhibition can disturb mitochondrial function and generate
elevated ROS levels, leading to a decrease in the mitochondrial
membrane potential [60]. We have previously shown the inhibition
Treatment of Caco-2/PD7 cells with complexes 1a, 1a0, 1b, 1b0,
3a, 6a, 3e6b and cisplatin did not significantly affect the cell cycle
after 72 h incubation, where the majority of the cells are in the G1
and S-phases. Only [AuCl(PTAeCH2COOMe)2]Br2 (2b) displayed a
variation worthy of mention in the treatment with Caco-2/PD7 cells
with an appreciable increase in the population of S-phase with a
decrease in the G1- and G2-phases. A slight increase in the popu-
lation in the G1-phase, resulting in a concomitant decrease in the S-
phase is detected in complexes 1a, 1a0 and 1b after incubation with
TC7 clone, that could prevent the entry of cells into the subsequent
S and G2/M phases. This fact could be in accordance with the
highest values of necrotic population measured in these derivatives
(8.9e16.2%, Table 2).
To evaluate the reactivity of the gold derivatives with DNA, we
monitored their influence on pIRES2-EGFP (5308 pb) plasmid DNA,
following incubation at 37 ꢂC, by agarose gel electrophoresis, in the

168
E. García-Moreno et al. / European Journal of Medicinal Chemistry 79 (2014) 164e172
Fig. 1. Quantitative flow cytometry analyses using propidium iodide (PI) uptake and annexin-V staining in PD7 and TC7 colon cancer cells treated with DMSO, 20
1e6, cisplatin and auranofin after 72 h.
mM in complexes
of cytosolic and mitochondrial thioredoxin reductases by related
thiolate derivatives with PTA [39]. Although additional studies will
be conducted on the new thiolate compounds, we can imagine a
similar behavior to the found in the reported derivatives.
more efficiently, giving to low amounts of live population and
high percentages of apoptotic cells after 72 h of treatment. A
balanced relationship between hidrophilicity and lipophilicity is
found in all the complexes with the phosphanes [PTAeCH2Ph]Br
and [PTAeCH2COOMe]Br, however the use of [PTAeCH2CN]Br or
[PTAeCH2COOH]Br led to more hydrophilic character, with log
D7.4 next to ꢀ1. Complex [Au(Spyrim)(PTAeCH2Ph)]Br (4a), which
resulted the most effective in both cell lines, is essentially stable
under PBS buffer solution at 37 ꢂC as deduced from absorption
spectroscopy. Since this complex did not damage DNA and dis-
played predisposition to dissociate the phosphane under PBS
buffer solution, its in vitro effectiveness might be due to in-
teractions with critical proteins/enzymes in keeping with other
gold complexes.
3. Conclusions
Two different routes of synthesis of thiolate gold(I) with water
soluble phosphanes derived from PTA [PTAeCH2Ph]Br and [PTAe
CH2COOMe]Br are included in the paper. The conventional way by
deprotonating the thiol in basic media led to mixtures in the case
of [PTAeCH2COOMe]Br due to side reactions, which is avoided by
using transmetallating reactions starting from tin(IV) thiolates.
These new thiolates, their corresponding chloro- and bromo-
precursors and chloro gold(I) derivatives with similar phosphanes
have been tested for their antiproliferative activity against the
human colon cancer cell line Caco-2 (PD7 and TC7 clones). All the
compounds showed comparable cytotoxicity to auranofin and
remarkably better than cisplatin. Although all the thiolate gold(I)
derivatives appeared to be the most effective complexes, only
those bearing the phosphane [PTAeCH2Ph]Br induced apoptosis
4. Experimental section
4.1. General procedures
1H, 31P{1H} (161.97 MHz) and 13C{1H} (100.62 or 75.4 MHz) NMR
spectra were recorded on 400 MHz or 300 MHz Bruker Avance

E. García-Moreno et al. / European Journal of Medicinal Chemistry 79 (2014) 164e172
169
spectrometers. Chemical shifts are quoted in ppm relative to
external TMS (1H, 13C) or 85% H3PO4 31P); coupling constants are
(
reported in hertz. MALDI mass spectra were measured on a
Micromass Autospec spectrometer in positive ion mode using
trans-2-[3-(4-tertbutylphenyl)-2-methyl-2-propenylidene]maloni-
trile (DCTB) as the matrix. IR spectra were recorded on a Perkine
Elmer Spectrum 100 FTIR (far-IR) spectrophotometer. Elemental
analyses were obtained in-house using a LECO CHNS-932 micro-
analyzer. The gold derivatives [AuCl(PTA-R)]X (R ¼ eCH2Ph (1a,a0),
eCH2COOMe (1b,b0), eCH2CN (1c), eCH2COOH (1d); X ¼ Br or Cl)
were prepared as published elsewhere [44]. The thiolato tin(IV)
[SnMe2(SR)] (R ¼ compounds) were prepared as similar previously
described complexes [51,61].
4.2. Synthesis
Fig. 3. Hydrolysis profile of complex [Au(Spyrim)(PTAeCH2Ph)]Br (4a) under phos-
phate buffer 10 mM pH 7.4. Spectra were recorded at different times over 24 h at 37 ꢂC.
4.2.1. General synthesis of [Au(SR)(PTAeCH2Ph)]Br (SR ¼ Spy, 3a;
Spyrim, 4a; SMepyrim, 5a; SMe2pyrim, 6a)
To a solution of KOH (0.55 mmol) in MeOH (ca.10 mL) containing
the thiol compound (0.5 mmol) was added [AuBr(PTAeCH2Ph)]Br
(0.5 mmol). After stirring the mixture for ca. 12 h at room temper-
ature the solutions were evaporated under vacuum to dryness and
the residue extracted in dichloromethane (3 ꢃ 10 mL). The com-
bined extracts were passed through Celite and concentrated under
vacuum to ca. 5 mL. Addition of Et2O allowed the precipitation of the
products, which were isolated by filtration and dried in air.
The following complexes were prepared using this method.
4.2.1.1. [Au(Spy)(PTAeCH2Ph)]Br (3a). Pale yellow solid in 68%
yield. 1H RMN (400 MHz, dmso-d6, 25 ꢂC):
d
¼ 8.09 (d, 1H,
JHH ¼ 4 Hz, py), 7.59e7.54 (m, 5H, Ph), 7.37e7.35 (m, 2H, py), 6.87 (t,
JHH ¼ 5.6 Hz, 1H, py), 5.18 and 4.97 (AB system, 4H, JAB ¼ 11.6 Hz,
NCH2N), 4.7e4.56 (m, 3H, PCH2N þ NCH2N), 4.43e4.24 (m, 2H,
PCH2N), 4.26 (s, 2H, CH2Ph), 4.16e4.07 (m, 3H, PCH2N þ NCH2N).
31P{1H} RMN (dmso-d6):
(18%). I.R.:
d
: ꢀ40.1 ppm. MALDI MS: m/z 556 [M]þ
n
(SeAu): 551 cmꢀ1. C18H23AuBrN4O2PS (634.04): C
Fig. 2. Cell-cycle analysis after treatment with the metal complexes for 72 h. Cell cycle and DNA fragmentation were determined by propidium iodide staining, showing the
corresponding percentages of G1-, S- and G2-phase cells.

170
E. García-Moreno et al. / European Journal of Medicinal Chemistry 79 (2014) 164e172
ꢂ
ꢂ
,
34.03, H 3.65, N 8.82; found: C 33.76, H 3.38, N 9.12. S25 (H2O):
(617.25): C 27.24, H 3.43, N 9.08; found: C 27.16, H 3.41, N 9.42. S25
H2O < 0.1 g Lꢀ1
6.76 g Lꢀ1
.
.
4.2.1.2. [Au(Spyrim)(PTAeCH2Ph)]Br (4a). Yellow solid in 86% yield.
1H NMR (400 MHz, MeOD, 25 ꢂC):
4.2.2.2. [Au(Spyrim)(PTAeCH2COOMe)]Br (4b). Pale yellow solid in
83% yield. 1H RMN (400 MHz, dmso-d6, 25 ꢂC):
¼ 8.35 (d, 2H, JHe
d
¼ 8.34 (d, 2H, JHH ¼ 4.8 Hz,
d
pyrim), 7.59e7.54 (m, 5H, Ph), 7.02 (t, 1H, JHH ¼ 4.9 Hz, pyrim), 5.14
and 4.94 (AB system, 4H, JAB ¼ 12 Hz, NCH2N), 4.67 (d, 2H;
JHH ¼ 13.7 Hz, NCH2N), 4.48 (d, JHH ¼ 13.2 Hz, 2H, PCH2N), 4.38 (s,
2H, CH2Ph), 4.18e4.08 (m, 2H, PCH2N), 4.00e3.95 (m, 2H, PCH2N).
¼ 4.6 Hz, pyrim), 6.98 (t, 1H, pyrim), 5.45 and 5.30 (AB system, 4H,
H
JAB ¼ 11.2 Hz, NCH2N), 5.06 (s, 2H, NCH2N); 4.66e4.63 (m, 1H,
NCH2P), 4.46e4.34 (m, 5H, NCH2P), 4.21 (s, 2H, CH2COOMe), 3.79 (s,
3H, COOMe). 31P{1H} RMN (dmso-d6):
d
: ꢀ36.8(s) ppm. 13C{1H}
31P{1H} NMR MeOD:
(75 MHz, dmso-d6):
d
(ppm) ¼ ꢀ66.13 (s) ppm. 13C{1H} NMR
NMR (75.4 MHz, dmso-d6):
pyrim), 80.52 (s, NCH2N), 69.03 (d, JPC ¼ 5 Hz, NCH2P), 59.64 (s,
CH2COOMe), 54.41 (s, NCH2N), 53.6 (s, Me), 48.13 (d, JPC ¼ 15 Hz,
d
¼ 164.55 (s, C]O), 157.6, 116.39 (s,
d
¼ 133.48, 130.70, 130.34, 129.5 (Ph); 156.82
(Spyrim); 79.8 (s, NCH2N); 69.4 (s, NCH2N); 55.7 (d, JPC ¼ 25.87 Hz,
PCH2N); 46.0 (d, JPC ¼ 15.67 Hz, PCH2N, 2C); 49.3 (s, CH2Ph). MALDI
NCH2P). I.R.: n
(C]O): 1745 cmꢀ1. MALDI MS (m/z): 539 [M]þ (15).
MS (m/z): 556 [M]þ (20%). IR
n
(SeAu): 567 cmꢀ1. C17H22AuBrN5PS
C
13H20AuBrN5O2PS (618.24): C 25.26, ꢀH1 3.26, N 11.33; found: C
ꢂ
ꢂ
(636.3): C 32.09, H 3.48, N 11.01; found: C 32.16, H 3.31, N 11.40. S25
,
25.05, H 3.21, N 11.27. S25 , H2O: 21 g L .
H2O < 0.1 g Lꢀ1
.
4.2.2.3. [Au(SMepyrim)(PTAeCH2COOMe)]Br (5b). Pale yellow solid
in 80% yield. 1H RMN (400 MHz, dmso-d6, 25 ꢂC):
¼ 8.18 (d, 1H, JHe
4.2.1.3. [Au(SMepyrim)(PTAeCH2Ph)]Br (5a). Yellow solid in 75%
yield. 1H NMR (400 MHz, dmso-d6, 25 ꢂC):
JHH ¼ 6.8 Hz, pyrim), 7.38e7.22 (m, 5H, Ph), 6.73 (d, 1H, JHH ¼ 6.8 Hz,
pyrim), 4.92 and 4.82 (AB system, JAB ¼ 16 Hz, 4H, NCH2N), 4.52 and
4.31 (AB system, 2H, JAB ¼ 16.4 Hz, NCH2N), 4.39e4.36 (m, 1H,
NCH2P), 4.21 (s, 2H, CH2Ph), 3.99e3.90 (m, 2H, NCH2P), 3.79e3.65
d
d
¼ 8.12 (d, 1H,
¼ 4 Hz, pyrim), 6.86 (d, 1H, JHeH ¼ 3.9 Hz pyrim), 5.44 and 5.33 (AB
H
system, 4H, JAB ¼ 11.2 Hz, NCH2N), 5.17e5.05 (m, 2H, NCH2P), 4.57e
4.42 (m, 6H, NCH2N þ NCH2P), 4.22 (s, 2H, CH2COOMe), 3.78 (s, 3H,
COOMe), 2.28 (s, 3H, SMepyrim). 31P{1H} RMN (dmso-d6):
d
¼ ꢀ31.3(s) ppm. 13C{1H} NMR (75.4 MHz, dmso-d6):
d
¼ 164.5(s,
(m, 3H, NCH2P). 31P{1H} NMR MeOD:
NMR (100.72 MHz, dmso-d6):
d
¼ ꢀ47.8 (s) ppm. 13C{1H}
C]O), 125.8, 117 (s, pyrim), 80.5 (s, NCH2N), 69.0 (s, NCH2N), 59.5 (s,
CH2COOMe), 54.2 (d, JPC ¼ 18 Hz NCH2P), 53.5 (s, Me), 47.8 (d,
JPC ¼ 20 Hz, NCH2P), 26.3 (s, Mepyrim). MALDI MS (m/z): 553 [M]þ
d
(ppm) ¼ 166.15 (s, SMepyrim),
129.39, 128.95, 128.48, 127.74 (Ph), 114.46 (s, SMepyrim), 80.1 (s,
NCH2N), 70.56 (s, NCH2N), 55.45 (d, JPC ¼ 26 Hz, PCH2N), 46.0 (s,
Me), 45.6 (d, JPC ¼ 26.4 Hz, PCH2N, 2C). MALDI MS (m/z): 570 [M]þ
(18). I.R.:
n
(C]O): 1745 cmꢀ1. C14H22AuBrN5O2PS (632.27): C 26.59,
ꢀ1
ꢂ
H 3.51, N 11.08; found: C 26.26, H 3.31, N 11.07. S25 , H2O: 15 g L
.
(40%). IR
n
(SeAu): 571 cmꢀ1 C18H24AuBrN5PS (650.3): C 33.24,ꢀH1
ꢂ
3.72, N 10.77; found: C 33.16, H 3.45, N 10.72. S25 , H2O < 0.1 g L
.
4.2.2.4. [Au(SMe2pyrim)(PTAeCH2COOMe)]Br (6b). Pale yellow
solid in 74% yield. 1H RMN (400 MHz, dmso-d6, 25 ꢂC):
d
¼ 6.75 (s,
4.2.1.4. [Au(SMe2pyrim)(PTAeCH2Ph)]Br (6a). Yellow solid in 75%
yield, 1H NMR (400 MHz, dmso-d6, 25 ꢂC):
1H, pyrim), 5.40 and 5.26 (AB system, 4H, JAB ¼ 11.2 Hz, NCH2N),
5.02 (s, 2H, NCH2P), 4.63e4.60 (m, 1H, NCH2N), 4.44e4.37 (m, 5H,
NCH2N þ NCH2P), 4.17 (s, 2H, CH2COOMe), 3.79 (s, 3H, COOMe),
d
¼ 7.62e7.55 (m, 5H,
Ph), 6.96 (s, 1H, pyrim), 5.29 and 4.04 (AB system, 4H, JAB ¼ 12 Hz,
NCH2N), 5.24e5.22 (m, 2H, NCH2N), 4.90 (d, 2H, J ¼ 11.6 Hz, PCH2N),
4.8e4.79 (m, 2H, PCH2N), 4.67e4.64 (m, 2H, PCH2N), 4.26 (s, 2H,
2.22 (s, 6H, Mepyrim).31P{1H} RMN (dmso-d6):
d
: ꢀ36.5 (s, br) ppm.
13C{1H} NMR (75.4 MHz, dmso-d6):
d
¼ 164.5 (s, C]O), 118.6 (s,
CH2Ph), 2.72 (s, 6H, 2Me). 31P{1H} NMR (dmso-d6):
d
¼ ꢀ42.7 (s)
pyrim), 80.39 (s, NCH2N), 69.0 (s, NCH2N), 59.57 (s, CH2COOMe),
54.3 (d, JPC ¼ 11 Hz, NCH2P), 53.9 (s, Me), 48.2 (d, JPC ¼ 16.5 Hz,
ppm. 3C{1H} NMR (100.72 MHz, dmso-d6):
d
(ppm) ¼ 128.9, 128.14,
121.24 (Ph), 115.57 (s, SMe2pyrim), 79.9 (s, NCH2N), 56.3 (d,
JPC ¼ 25 Hz, PCH2N), 49.5 (s, Me), 47.5 (d, JPC ¼ 26 Hz, PCH2N). MALDI
NCH2P), 24.4 (s, Mepyrim). MALDI MS (m/z): 567 [M]þ (17). IR
n(C]
O): 1745 cmꢀ1. C15H24AuBrN5O2PS (646.29): C 27.88,ꢀH1 3.74, N
MS (m/z): 559 [M]þ (40%). IR
n
(SeAu): 567 cmꢀ1. C19H26AuBrN5PS
10.84; found: C 27.66, H 3.71, N 10.72. S25 , H2O: 10 g L
.
ꢂ
(664.35): C 34.35, H 3.94, N 10.54; found: C 33.96, H 3.79, N 10.75.
ꢀ1
ꢂ
S25 , H2O < 0.1 g L
.
4.3. Distribution coefficients (log D7.4)
4.2.2. General synthesis of [Au(SR)(PTAeCH2COOMe)]Br (SR ¼ Spy,
3b; Spyrim, 4b; SMepyrim, 5b; SMe2pyrim, 6b)
The n-octanol-water partition coefficients of the complexes
were determined as previously reported [62] using a shake-flask
method. PBS buffered distilled water (100 mL, phosphate buffer
To a solution of [AuBr(PTAeCH2CO2Me)]Br (0.5 mmol) in MeOH
(ca. 10 mL) under argon atmosphere [Sn(SR)2Me2] (0.25 mmol) was
added. After stirring the mixture for ca. 4 h at room temperature
the solutions were concentrated under vacuum. Addition of Et2O
allowed the precipitation of the products, which were isolated by
filtration and dried in air.
½PO43ꢀꢄ ¼ 10
M, [NaCl] ¼ 0.15 M, pH 7.4) and n-octanol (100 mL)
m
were saturated for 72 h. 1 mg of the complexes was mixed in 1 mL
of aqueous and organic phase, respectively for 10 min. The resultant
emulsion was centrifuged to separate the phases. The concentra-
tion of the compound in each phase was determined using UV
absorbance spectroscopy. Log D7.4 was defined as log{[com-
pound(organic)]/[compound(aqueous)]}.
Using this method the following complexes were prepared:
4.2.2.1. [Au(Spy)(PTAeCH2COOMe)]Br (3b). Pale yellow solid in 65%
yield. 1H RMN (400 MHz, dmso-d6, 25 ꢂC):
d
¼ 7.93 (s, br, 1H, Spy),
4.4. In vitro assays
7.42 (t, 1H, JHeH ¼ 8 Hz, Spy), 7.35 (d, 1H, JHeH ¼ 8.6 Hz, Spy), 6.85 (t,
JHeH ¼ 6 Hz, 1H, Spy), 5.40 and 5.26 (AB system, 4H, JAB ¼ 11.2 Hz,
NCH2N), 5.01 (s, 2H, NCH2P), 4.62e4.58 (m, 1H, NCH2N), 4.41e4.20
(m, 5H, NCH2P þ NCH2N), 4.17 (s, 2H, CH2COOMe), 3.79 (s, 3H,
4.4.1. Cell viability assay
Human Caco-2 cell line PD7 and TC7 clones were kindly pro-
vided by Dr. Edith Brot-Laroche (Université Pierre et Marie Curie-
Paris 6, UMR S 872, Les Cordeliers (France)). Caco-2 cells were
maintained in a humidified atmosphere of 5% CO2 at 37 ꢂC. Cells
(passages 50e80) were grown in Dulbecco’s Modified Eagles me-
dium (DMEM) (Gibco Invitrogen, Paisley, UK) supplemented with
20% fetal bovine serum (FBS), 1% non essential amino acids, 1%
COOMe). 31P{1H} RMN (dmso-d6):
d
¼ ꢀ38.8 (s,br) ppm. 13C{1H}
NMR (75.4 MHz, dmso-d6):
137.37 (s, Spy), 80.4 (s, NCH2N), 59.59 (s, CH2COOMe), 54.35 (s,
NCH2P), 53.56 (s, Me), 48.09 (d, JPC ¼ 14 Hz, NCH2P). MALDI MS: m/z
d
¼ 164.55 (s, C]O), 129.7, 132.90,
537[M]þ (100). I.R.:
n . C14H21AuBrN4O2PS
(C]O): 1744 cmꢀ1

E. García-Moreno et al. / European Journal of Medicinal Chemistry 79 (2014) 164e172
171
penicillin (1000 U/ml), 1% streptomycin (1000
amphotericin (250 U/ml). Experiments were performed 24 h post-
seeding.
m
g/mL) and 1%
1 ꢃ 10ꢀ4 M, and the sample was analyzed spectrophometrically
over 24 h at 37 ꢂC.
Stock solutions of the complexes (saline solution or DMSO) were
diluted in complete medium to the required concentration. DMSO
at similar concentrations did not show any effects on cytotoxicity.
For cytotoxicity screening experiments, cells were seeded in 96-
well plates at a density of 4 ꢃ 103 cells/well. The culture medium
was replaced with fresh medium (without FBS) containing the
Acknowledgments
Authors thank to Centro de Investigación Biomédica de Aragón
(CIBA), España for the technical assistance: http://www.ics.aragon.
es and to Torrecid S.A. for a generous donation of H[AuCl4].
complexes at concentrations varying from 0 to 20
mM, with an
Appendix A. Supplementary data
exposure time of 72 h. Thereafter, the cell survivals were measured
using the MTT test as previously described [63]. The assay is
dependent on the cellular reduction of 3-(4,5-dimethyl-2-
thiazoyl)-2,5-diphenyltetrazolium bromide (MTT, Merck) by the
mitochondrial dehydrogenase of viable cells to a blue formazan
product which can be measured spectrophotometrically. Following
appropriate incubation of cells, with or without the metallic com-
plexes, MTT was added to each well in an amount equal to 10% of
the culture volume and gentle stirring in a gyratory shaker which
enhances dissolution and incubation was continued 37 ꢂC for 4 h.
Thereafter the medium and MTT are removed and DMSO is added
to each well. At the end, the results are obtained by measuring
absorbance with a scanning multiwell spectrophotometer (BIOTEX
SINERGY HT SIAFRTD) at wavelength of 560/670 nm and compared
to the values of control cells incubated in the absence of complexes.
Experiments were conducted in quadruplicate wells and repeated
at least three times.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.001.
References
[1] S.P. Fricker, Gold Bulletin 29 (1996) 53e60.
[2] C. Orvig, M.J. Abrams, Chemical Reviews 99 (1999) 2201e2203.
[3] E.R.T. Tiekink, Gold Bulletin 36 (2003) 117e124.
[4] J.E. Pope, P. Hong, B.E. Koehler, Journal of Rheumatology 29 (2002) 255e260.
[5] A.J. Lehman, J.M. Esdaile, A.V. Klinkhoff, E. Grant, A. Fitzgerald, J. Canvin,
Arthritis & Rheumatism 52 (2005) 1360e1370.
[6] B.M. Sutton, E. McGusty, D.T. Waltz, M.J. Di Martino, Journal of Medicinal
Chemistry 15 (1972) 1095e1098.
[7] B.M. Sutton, Gold Bulletin 19 (1986) 15e16.
[8] T. Okada, B.K. Patterson, S.Q. Ye, M.E. Gurney, Virology 192 (1993) 631e642.
[9] K. Yamaguchi, H. Ushijima, M. Hisano, Y. Inoue, T. Shimamura, T. Hirano,
W.E.G. Muller, Microbiology and Immunology 45 (2001) 549e555.
[10] R.E. Thomas, R.A. Papandrea, Medical Journal of Australia 158 (1993) 720.
[11] M. Navarro, H. Perez, R.A. Sanchez-Delgado, Journal of Medicinal Chemistry 40
(1997) 1937e1939.
[12] M. Navarro, E.J. Cisneros-Fajardo, T. Lehmann, R.A. Sanchez-Delgado,
R. Atencio, P. Silva, R. Lira, J.A. Urbina, Inorganic Chemistry 40 (2001) 6879e
6884.
[13] M.J. McKeage, L. Maharaj, S.J. Berners-Price, Coordination Chemistry Reviews
232 (2002) 127e135.
4.4.2. Measurements of apoptosis
Human Caco-2 cell line PD7 and TC7 clones were exposed for
72 h with 20 mM of the metallic compounds, collected and stained
[14] S. Nobili, E. Mini, I. Landini, C. Gabbiani, A. Casini, L. Messori, Medicinal
Research Reviews 30 (2010) 550e580.
[15] I. Ott, Coordination Chemistry Reviews 253 (2009) 1670e1681.
[16] T.M. Simon, D.H. Kunishima, G.J. Vibert, A. Lorber, Cancer Research 41 (1981)
94e97.
[17] T.M. Simon, D.H. Kunishima, G.J. Vibert, A. Lorber, Cancer 44 (1979) 1965e
1975.
[18] C.K. Mirabelli, R.K. Johnson, C.-M. Sung, L.F. Faucette, K. Murihead, S.T. Crooke,
Cancer Research 45 (1985) 32e39.
[19] C.F. Shaw, Chemical Reviews 99 (1999) 2589e2600.
[20] S.J. Berners-Price, A. Filipovska, Metallomics 3 (2011) 863e873.
[21] C.-M. Che, R.W.-Y. Sun, C.B. Ko, N. Zhu, H. Sun, Chemical Communications
(2003) 1718.
[22] E.R.T. Tiekink, Inflammopharmacology 16 (2008) 138e142.
[23] W.F. Kean, I.R.L. Kean, Inflammopharmacology 16 (2008) 112e125.
[24] V. Milacic, D. Fregona, Q.P. Dou, Histology and Histopathology 23 (2008) 101e
108.
with Annexin V-FTIC according to manufacturer’s recommenda-
tion. A negative control was prepared by unreacted cells, that was
used to define the basal level of apoptotic and necrotic or dead cells.
After incubation, cells were transferred to flow-cytometry tubes
and washed twice with temperate phosphate-buffered saline (PBS)
and resuspended in 100
Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), 5
Annexin V-FITC and 5 L of PI to each 100 L of cell suspension.
After incubation for 15 min at room temperature in the dark, 400
m
L Annexin V binding buffer (10 mM
mL of the
m
m
mL
of 1X Annexin binding buffer were added and analyzed by flow
cytometry within 1 h. The signal intensity was measured using a
FACSARIA BD and analyzed using FASCDIVA BD.
[25] R.W.-Y. Sun, C.-M. Che, Coordination Chemistry Reviews 253 (2009) 1682e
1691.
[26] I. Kostova, Anti-Cancer Agents in Medicinal Chemistry 6 (2006) 19e32.
[27] P.J. Barnard, S.J. Berners-Price, Coordination Chemistry Reviews 251 (2007)
1889e1902.
[28] A. Casini, Journal of Inorganic Biochemistry 109 (2012) 97e106.
[29] G.D. Hoke, G.F. Rush, G.E. Bossard, J.V. McArdle, B.D. Jensen, C.K. Mirabelli,
Journal of Biological Chemistry 263 (1988) 11203e11210.
[30] G.D. Hoke, R.A. Macia, P.C. Meunier, P.J. Bugelski, C.K. Mirabelli, G.F. Rush,
W.D. Matthews, Toxicology and Applied Pharmacology 100 (1989) 293e306.
[31] P.F. Smith, G.D. Hoke, D.W. Alberts, P.J. Bugelski, S. Lupo, C.K. Mirabelli,
G.F. Rush, Journal of Pharmacology and Experimental Therapeutics 249 (1989)
944e950.
[32] J.J. Liu, P. Galettis, A. Farr, L. Maharaj, H. Samarasinha, A.C. McGechan,
B.C. Baguley, R.J. Bowen, S.J. Berners-Price, M.J. McKeage, Journal of Inorganic
Biochemistry 102 (2008) 303e310.
[33] A.S. Humphreys, A. Filipovska, S.J. Berners-Price, G.A. Koutsantonis,
B.W. Skelton, A.H. White, Dalton Transactions (2007) 4943e4950.
[34] S.J. Berners-Price, R.J. Bowen, P. Galettis, P.C. Healy, M.J. McKeage, Coordina-
tion Chemistry Reviews 185-6 (1999) 823e836.
4.4.3. Propidium iodide staining of DNA content and cell cycle
analyses
Human Caco-2 cell line PD7 and TC7 clones were exposed for
72 h with 20 mM of the metallic compounds. Cells were fixed in 70%
ice-cold ethanol and stored at 4 ꢂC for 24 h. After centrifugation,
cells were rehydrated in PBS (phosphated buffered saline) and
stained in propidium iodide (PI, 50
mg/mL) solution containing
RNase A (100 g/mL). PI stained cells were analyzed for DNA con-
m
tent in a FACSARRAY BD equipped with an argon ion laser. The red
fluorescence emitted by PI was collected by 620 nm longer pass
filter, as a measure of the amount of DNA-bound PI and displayed
on a linear scale. Cell cycle distribution was determined on a linear
scale. The percentage of cells in cycle phases was determined using
MODIFIT 3.0 verity software.
[35] M.J. McKeage, S. Berners-Price, P. Galettis, R.J. Bowen, W. Brouwer, L. Ding,
L. Zhuang, B.C. Baguley, Cancer Chemotherapy and Pharmacology 46 (2000)
343e350.
[36] D. Screnci, M.J. McKeage, P. Galettis, T.W. Hambley, B.D. Palmer, B.C. Baguley,
British Journal of Cancer 82 (2000) 966e972.
4.4.4. Solution chemistry
The stability of [Au(Spyrim)(PTAeCH2Ph)]Br (4a) in buffer so-
lution was analyzed by absorption UVevisible spectroscopy. The
complex was dissolved in DMSO (10 mM) and diluted in the
reference PBS buffer, at pH 7.4, to a final concentration of
[37] E. Vergara, E. Cerrada, C. Clavel, A. Casini, M. Laguna, Dalton Transactions 40
(2011) 10927e10935.

172
E. García-Moreno et al. / European Journal of Medicinal Chemistry 79 (2014) 164e172
[38] S. Miranda, E. Vergara, F. Mohr, D. de Vos, E. Cerrada, A. Mendia, M. Laguna,
Inorganic Chemistry 47 (2008) 5641e5648.
[50] E. Cerrada, A. Moreno, M. Laguna, Dalton Transactions (2009) 6825e6835.
[51] M.N. Xanthopoulou, S.K. Hadjikakou, N. Hadjiliadis, M. Kubicki, S. Skoulika,
T. Bakas, M. Baril, I.S. Butler, Inorganic Chemistry 46 (2007) 1187e1195.
[52] C.K. Mirabelli, C. Sung, M.H. Whitman, D.T. Hill, S. Mong, S.T. Crooke,
Biochemical Pharmacology 35 (1986) 1427.
[39] E. Vergara, A. Casini, F. Sorrentino, O. Zava, E. Cerrada, M.P. Rigobello,
A. Bindoli, M. Laguna, P.J. Dyson, ChemMedChem 5 (2010) 96e102.
[40] E. Vergara, S. Miranda, F. Mohr, E. Cerrada, E.R.T. Tiekink, P. Romero,
A. Mendia, M. Laguna, European Journal of Inorganic Chemistry (2007) 2926e
2933.
[53] A. de Almeida, B.L. Oliveira, J.D.G. Correia, G. Soveral, A. Casini, Coordination
Chemistry Reviews 257 (2009) 2689e2704.
[41] E. Vergara, E. Cerrada, A. Casini, O. Zava, M. Laguna, P.J. Dyson, Organome-
tallics 29 (2010) 2596e2603.
[42] E. Garcia-Moreno, S. Gascon, M.J. Rodriguez-Yoldi, E. Cerrada, M. Laguna,
Organometallics 32 (2013) 3710e3720.
[43] E. García-Moreno, E. Cerrada, M.J. Bolsa, A. Luquin, M. Laguna, European
Journal of Inorganic Chemistry (2013) 2020e2030.
[44] S. Schäfer, W. Frey, A.S.K. Hashmi, V. Cmrecki, A. Luquin, M. Laguna, Poly-
hedron 29 (2010) 1925e1932.
[54] A. Casini, Journal of Inorganic Biochemistry 109 (2013) 97e106.
[55] K.P. Bhabak, B.J. Bhuyan, G. Mugesh, Dalton Transactions 40 (2011) 2099e
2111.
[56] A. Bindoli, M.P. Rigobello, G. Scutarib, C. Gabbiani, A. Casini, L. Messori, Co-
ordination Chemistry Reviews 253 (2009) 1692e1707.
[57] L. Oehninger, R. Rubbiani, I. Ott, Dalton Transactions 42 (2013) 3269e3284.
[58] A. Citta, E. Schuh, F. Mohr, A. Folda, M.L. Massimino, A. Bindoli, A. Casini,
M.P. Rigobello, Metallomics 5 (2013) 1006e1015.
[45] A. Mena-Cruz, P. Lorenzo-Luis, A. Romerosa, M. Saoud, M. Serrano-Ruiz,
Inorganic Chemistry 46 (2007) 6120e6128.
[46] A. Mena-Cruz, P. Lorenzo-Luis, A. Romerosa, M. Serrano-Ruiz, Inorganic
Chemistry 47 (2008) 2246e2248.
[47] E. Cerrada, E.J. Fernandez, M.C. Gimeno, A. Laguna, M. Laguna, R. Terroba,
M.D. Villacampa, Journal of Organometallic Chemistry 492 (1995) 105e110.
[48] D.W. Allen, R. Berridge, N. Bricklebank, E. Cerrada, M.E. Light, M.B. Hursthouse,
M. Laguna, A. Moreno, P.J. Skabara, Journal of the Chemical Society Dalton
Transactions (2002) 2654e2659.
[59] E. Schuh, C. Pfluger, A. Citta, A. Folda, M.P. Rigobello, A. Bindoli, A. Casini,
F. Mohr, Journal of Medicinal Chemistry 55 (2012) 5518e5528.
[60] I. Romero-Canelon, P.J. Sadler, Inorganic Chemistry 52 (2013) 12276e12291.
[61] E. Martin, C. Spendley, A.J. Mountford, S.J. Coles, P.N. Horton, D.L. Hughes,
M.B. Hursthouse, S.J. Lancaster, Organometallics 27 (2008) 1436e1446.
[62] C. Wetzel, P.C. Kunz, M.U. Kassak, A. Hamacher, P. Böhler, W. Watjen, I. Ott,
R. Rubbiani, B. Spingler, Dalton Transactions 40 (2011) 9212e9220.
[63] J. Carmichael, W.G. Degraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Cancer
Research 47 (1987) 936e942.
[49] M. Cera, E. Cerrada, M. Laguna, J.A. Mata, H. Teruel, Organometallics 21 (2002)
121e126.