S -Propargylthiopyridine phosphane derivatives as anticancer agents: Characterization and antitumor activity

Article abstract of DOI:10.1021/om400340a

S-Propargylthiopyridine phosphane gold(I) derivatives with the water-soluble phosphanes PTA (1,3,5-triaza-7-phosphaadamantane), DAPTA (3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane), and TPPTS (sodium triphenylphosphane trisulfonate) are described and used as metalloligands by coordination to copper(I). New heteronuclear gold(I) and copper(I) complexes of the type [Au₂Cu(C≡CCH₂SC₅H ₄N)₂L₂] (L = PTA, DAPTA, PPh₃, TPPTS) and [AuCu(C≡CCH₂SC₅H₄N)(L) (PPh₃)₂]NO₃ (L = PTA, DAPTA) are reported. The X-ray crystal structure of [Au(C≡CCH₂SC₅H ₄N)(PTA)] (1), which confirms the coordination of the metallic center to the alkyne unit, displays a zigzag polymeric chain with short gold-gold contacts of 3.2680(16) A. Strong antiproliferative effects are found for most of the new complexes in human colon cancer cell lines (Caco-2, PD7, and TC7 clones), with these effects being more pronounced than for the reference drugs cisplatin and auranofin, especially for the heterodimetallic derivatives, which are markedly more active than the corresponding mononuclear precursors. Apoptosis-induced cell death is found for all compounds, as shown by an annexin-V/propidium iodide double-staining assay.

Full text of DOI:10.1021/om400340a

Article
pubs.acs.org/Organometallics
S‑Propargylthiopyridine Phosphane Derivatives As Anticancer
Agents: Characterization and Antitumor Activity
Elena García-Moreno,† Sonia Gascon,‡ Ma Jesus Rodriguez-Yoldi,‡ Elena Cerrada,†
́
and Mariano Laguna*,†
†Departamento de Química Inorgan
E-50009 Zaragoza, Spain
́ ́ ́
ica, Instituto de Síntesis Química y Catalisis Homogenea, Universidad de Zaragoza-CSIC,
‡Departamento de Farmacología y Fisiología, Unidad de Fisiología, Facultad de Veterinaria, Universidad de Zaragoza, 50013,
Zaragoza, CIBERobn, Spain
S
* Supporting Information
ABSTRACT: S-Propargylthiopyridine phosphane gold(I) de-
rivatives with the water-soluble phosphanes PTA (1,3,5-triaza-
7-phosphaadamantane), DAPTA (3,7-diacetyl-1,3,7-triaza-5-
phosphabicyclo[3.3.1]nonane), and TPPTS (sodium triphe-
nylphosphane trisulfonate) are described and used as metal-
loligands by coordination to copper(I). New heteronuclear
gold(I) and copper(I) complexes of the type [Au2Cu(C
CCH2SC5H4N)2L2] (L = PTA, DAPTA, PPh3, TPPTS) and
[AuCu(CCCH2SC5H4N)(L)(PPh3)2]NO3 (L = PTA,
DAPTA) are reported. The X-ray crystal structure of
[Au(CCCH2SC5H4N)(PTA)] (1), which confirms the
coordination of the metallic center to the alkyne unit, displays
a zigzag polymeric chain with short gold−gold contacts of
3.2680(16) Å. Strong antiproliferative effects are found for
most of the new complexes in human colon cancer cell lines
(Caco-2, PD7, and TC7 clones), with these effects being more pronounced than for the reference drugs cisplatin and auranofin,
especially for the heterodimetallic derivatives, which are markedly more active than the corresponding mononuclear precursors.
Apoptosis-induced cell death is found for all compounds, as shown by an annexin-V/propidium iodide double-staining assay.
(I), also known as auranofin, which was found to possess
antiarthritic activity24 and subsequently similar, or even better,
anticancer properties than cisplatin.25 However, although this
derivative has been successfully tested in vitro in different cancer
cell lines,26,27 it has shown good in vivo antitumor activity only
in murine models against P388 leukemia.28 The mechanism of
action of auranofin appears to differ from that of cisplatin since
the former does not damage DNA.24,26,27 Instead, auranofin
and related derivatives induce cell death as a result of their
effects on mitochondrial integrity.27
In this context, we have recently reported a series of
thiolate29−31 or alkyne32 gold(I) compounds that exhibit potent
cytotoxicity against different cancer cell lines, with this activity
being higher than cisplatin in several cases. Additionally, alkynyl
gold(I) derivatives with biological activity have been recently
published.33−35 The presence of the water-soluble phosphanes
PTA (1,3,5-triaza-7-phosphaadamantane), DAPTA (3,7-diace-
tyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane), TPPMS (so-
dium triphenylphosphane monosulfonate), or TPPTS (sodium
triphenylphosphane trisulfonate) confers a moderate to high
INTRODUCTION
■
Cisplatin is one of the most widely used metallic complexes for
cancer treatment, especially for cancers of the ovary, head and
neck, bladder, and cervix, melanoma, and lymphomas.1
However, the effectiveness of cisplatin is hindered by toxic
side-effects and the occurrence of tumor resistance.2−5 In an
attempt to overcome these drawbacks, new platinum-based
anticancer agents similar to cisplatin have been developed and
entered human clinical trials, although only a few of these have
reached routine clinical use.6 As a consequence, there is
considerable interest in the search for new metallic complexes
for use in cancer treatment. Among the new non-platinum
compounds with potential as anticancer drugs, gold derivatives
have gained increasing attention due to their generally strong
tumor cell growth inhibiting effects and the observation that
many such compounds inhibit the enzyme thioredoxin
reductase (TrxR).7−10 This enzyme is relevant for the
proliferation of tumor tissues and has emerged as a new target
for drug development.8,11 Several reviews12−23 concerning
gold(I) and gold(III) derivatives with anticarcinogenic activity
have been published in the past few years. One of the most
widely studied such gold(I) derivatives is (1-thio-β-D-
glucopyranose-2,3,4,6-tetraacetato-S)(triethylphosphane) gold-
Received: April 19, 2013
Published: June 21, 2013
© 2013 American Chemical Society
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Scheme 1. (i) [AuCl(PR3)]/KOEt, (ii) 1/2[Cu(MeCN)4]PF6, and (iii) [Cu(NO3)(PPh3)2]
Table 1. Selected Bond Lengths and Angles for [Au(CCCH2SC5H4N)(PTA)] (1)
bond
bond length (Å)
bond angle (deg)
Au(1)−C(1)
Au(1)−P(1)
Au(1)−Au(1)
Au(1)−Au(1)
S(2)−C(4)
1.970(17)
2.272(4)
3.2680(16)
3.2680(16)
1.782(18)
1.845(17)
1.24(3)
C(1)−Au(1)−P(1)
C(1)−Au(1)−Au(1)
P(1)−Au(1)−Au(1)
C(1)−Au(1)−Au(1)
P(1)−Au(1)−Au(1)
176.9(5)
92.6(5)
a
a
b
b
b
a
b
89.59(11)
89.9(5)
89.74(11)
142.67(4)
S(2)−C(3)
Au(1)#1−Au(1)−Au(1)
C(2)−C(1)
P(1)−C(9)
P(1)−C(11)
P(1)−C(10)
1.80(2)
1.87(2)
1.87(2)
a
b
−x+y+1, −x+1, z+1/3. −y+1, x−y, z−1/3.
water solubility on these complexes that could facilitate drug
administration and transport in the body.
improve their antitumor activity in comparison with their
mononuclear precursor probably as a result of interaction of
both metals with different biological targets.
The occurrence of anticancer activity in both thiolato
phosphane and alkyne phosphane gold(I) derivatives encour-
aged us to synthesize new phosphane complexes with the ligand
2-propargylthiopyridine, which was previously described by
some of us,36 by combining the thiol and alkyne units in the
same molecule. Moreover, we also evaluated their behavior as
metalloligands for group 11 metal ions, such as copper, via the
N and S atoms of the thiol unit.
Working with this idea, here we report on the synthesis and
characterization of new propargylthiopyridine gold(I) deriva-
tives with the water-soluble phosphanes PTA and DAPTA and
the sulfonated triphenylphosphane derivative TPPTS, as well as
their reaction with copper(I) derivatives to give heteropolynu-
clear compounds. An evaluation of the cytotoxic activity of the
resulting compounds toward two human colon cancer cell lines
has also been undertaken. In addition, as apoptosis is the
predominant mechanism by which cancer cells die, we have also
evaluated the apoptic activity of these new complexes and their
implication in cell-cycle progression. The results of this study
suggested that S-propargylthiopyridine phosphane derivatives
could find a use as therapeutic drugs for colon cancer.
Our interest in the synthesis of copper derivatives is not
casual, as copper is a crucial trace element in redox chemistry
that is present in all living organisms. Many copper complexes
have been investigated as therapeutic agents.37 As examples,
copper thiosemicarbazones have demonstrated therapeutic
potential in Alzheimer’s disease,38,39 copper aspirinate is used
in cardiovascular dysfunction,40 and derivatives with non-
steroidal anti-inflammatory drugs have exhibited increased anti-
inflammatory properties and reduced toxicity.41 In addition,
metabolic changes occurring in cancer cells have been
associated, among other factors, with copper, which plays an
important role in angiognesis42 and in proteasome inhibition.43
Consequently, many examples of Cu(I and II) complexes have
RESULTS AND DISCUSSION
■
2-Propargylthiopyridine reacts with the chlorophosphane
gold(I) derivatives [AuCl(PR3)] (PR3 = PTA, DAPTA, PPh3,
TPPTS) in the presence of KOH in MeOH as deprotonating
agent to give air-stable alkynylgold(I) derivatives with the
formula [Au(CCCH2SC5H4N)(PR3)] (PR3 = PTA, 1;
DAPTA, 2; PPh3, 3; TPPTS, 4; Scheme 1, i). The absence
recently been investigated for their antitumor activity.44−56
A
1
combination of two different cytotoxic metals in the same
molecule is scarcely represented in the literature. Only
examples of bimetallic Ti−Ru,57 Ti−Au,58 Ti−Pd,59 and Ti−
Pt59 have recently been described. Most of these derivatives
of the CC−H signal in the H NMR spectra of the four
derivatives confirmed the presence of the anionic form of the
ligand and subsequent coordination to the metallic center. The
1H NMR spectra exhibit multiplets in the aromatic region
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corresponding to the pyridine fragment, together with the
characteristic pattern of the phosphanes PTA and DAPTA in 1
and 2 and additional multiplets due to the triphenyl
phosphanes PPh3 and TPPTS. The 31P{1H} NMR spectra of
the complexes show only one resonance, which is shifted
downfield by about 50 ppm with respect to that for the free
phosphanes.
Slow diffusion of a dichloromethane solution of [Au(C
CCH2SC5H4N)(PTA)] (1) into diethyl ether gave crystals
suitable for X-ray diffraction. Selected bond lengths and angles
are listed in Table 1. The crystal structure (Figure 1), which
confirms the coordination of gold to the alkyne unit, consists of
a molecule associated with two additional symmetry-related
units in the unit cell. The molecules are connected in a head-to-
tail arrangement, with a torsion angle of 116.24°, via short
intermolecular gold−gold contacts of 3.2680(16) Å to give a
zigzag polymeric chain (Figure 2). These contacts are longer
than those observed in gold(I) derivatives with PTA (3.047−
3.1164 Å)32,60−62 but shorter than in recently reported
thiolatogold(I) complexes with PTA (ca. 3.35 Å).29,31 A similar
polymeric chain has been found in [Au(PTA)4][Au(CN2)],63
although with a longer separation between the metallic units
(3.45 Å).
The independent molecule displays a typical linear geometry
around the metal, with a C1−Au1−P1 angle of 176.9(5)°. Only
two examples of alkyne gold(I) with PTA, namely, rac-
[Au{CCC(Et)(Me)OH}(PTA)]60 and [Au(CC-3-
SC4H3)(PTA)],32 have been characterized by X-ray analysis.
In the former, three molecules are connected by intermolecular
gold−gold contacts to form a trinuclear chain, with an
additional single molecule, which is laterally connected,
affording an infinite polymeric chain with gold side-chains.
Only two units are connected to each other in the case of
[Au(CC-3-SC4H3)(PTA)]. Additional differences are ob-
served for the Au−C and C−C distances in the alkyne unit,
with the former being slightly shorter in complex 1 (Au1−C1
1.970(17) Å) than in previously reported alkyne derivatives
(1.997−2.079 Å)32,60 and the latter (C1−C2 1.24 Å) longer
than in the same complexes (1.20 and 1.21 Å on average).
The presence of uncoordinated nitrogen and sulfur atoms in
these new derivatives means that they could act as metal-
loligands and form heterodimetallic complexes. Thus, the
reaction of two equivalents of 1−4 with [Cu(MeCN)4]PF6
affords the cationic gold(I)/Cu(I) compounds [Cu{Au(C
CCH2SC5H4N)(L)}2]PF6 (L = PTA, 5; DAPTA, 6; PPh3, 7;
TPPTS, 8) (Scheme 1, ii). The presence of the new metallic
center in these heterodimetallic derivatives was confirmed by
1
the high-field shift in the H NMR spectra of the PTA and
DAPTA derivatives and the downfield shift found for their
PPh3 and TPPTS counterparts, with respect to complexes 1−4.
Furthermore, downfield shifts in the signals due to the
Figure 1. ORTEP plot of the X-ray structure of complex 1. View of the
three symmetry-related units. H atoms have been omitted for clarity.
Thermal ellipsoids are drawn at 50% probability.
1
thiopyridine moiety are observed in the H NMR spectra of
all complexes. In addition, a septuplet at about −144 ppm is
also observed in the 31P{1H} NMR spectra due to the presence
Figure 2. Polymeric structure of [Au(CCCH2SC5H4N)(PTA)].
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Table 2. IC50 Values of Metallic Complexes against PD7 and TC7 Colon Cancer Cell Lines Compared with Auranofin and
Cisplatin
a
IC50 (μM)
compound
log D7.4
PD7
TC7
[Au(CCCH2SC5H4N)(PTA)] (1)
[Au(CCCH2SC5H4N)(DAPTA)] (2)
[Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5)
[Cu{Au(CCCH2SC5H4N)(DAPTA)}2]PF6 (6)
[AuCu(CCCH2SC5H4N)(PTA)(PPh3)2]NO3 (9)
[AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10)
[Cu(NO3)(PPh3)]
−0.07
−0.03
−0.01
−0.40
+0.10
+0.07
3.65 0.34
4.53 0.18
13.12 1.65
2 × 10−4 10−5
0.02 9.9 × 10−3
0.47 0.06
14.91 0.74
0.03 5.6 × 10−3
0.02 2.9 × 10−3
0.6 2 × 10−2
3.4 × 10−3 10−4
2.91 0.06
1.17 0.12
1.15 0.34
cisplatin
37.24 5.15
1.8 0.1
45.6 8.08
auranofin
2.1 0.4
a
Mean SE of at least three determinations.
of the PF6 anion, which is confirmed by the appearance of two
intense bands at ca. 840 and 555 cm−1 in the IR spectra,
assignable to the stretching vibration of this anion. Since
attempts to grow suitable crystals for X-ray analysis failed, we
can only propose the structure of complexes 5−8. Considering
the presence of both uncoordinated N and S atoms, and the
tendency of copper(I) to prefer N-ligand coordination and a
tetrahedral geometry,64 we suggest the structure depicted in
Scheme 1, in which both [Au(CCCH2SC5H4N)(L)] units
are coordinated to the Cu(I) center. The presence of low-field
displacement in the NMR protons of the thiopyridine unit in
complexes 5−8 in comparison with the starting gold derivatives
1−4 and similar ν(CC) vibration values in the IR spectra for
all the complexes should be in accordance with our hypothesis.
The reaction of 1 and 2 with [Cu(NO3)(PPh3)2] in a 1:1
molar ratio affords air-stable solids with the stoichiometry
[AuCu(CCCH2SC5H4N)L(PPh3)2]NO3 (L = PTA, 9;
same as the remaining two doublets, thus indicating that the
former signal is due to two of the four possible isomers. In the
absence of an X-ray structure, we can only propose a
hypothetical structure for these dinuclear derivatives, which is
depicted in Scheme 1.
Cytotoxicity of the S-Propargylthiopyridine Com-
plexes. The cellular effects of some of these new derivatives
have been tested against human colon cancer cell lines. Two
clones were selected for further analysis: Caco-2/PD7 (from
early passage) and Caco-2/TC7 (from late passage). These
clones exhibited a well-organized brush border at late
confluence and no differences in the levels of dipeptidylpepti-
dase IV activity. Expression of the brush border-associated
hydrolate sucrase-isomaltase was shown to increase from early
to late passages of Caco-2 cells, concomitant with a decrease in
the rates of glucose consumption.72 The results are expressed in
terms of IC50 values (Table 2), which were obtained after
exposure to the drug for 72 h, using the well-established MTT
assay (see Experimental Section for details). We also evaluated
the cell proliferation inhibition induced by auranofin, cisplatin,
and the copper starting material [Cu(NO3)(PPh3)2] used to
prepare complexes 9 and 10 under the same experimental
conditions. Exposure of PD7 cell lines to increasing
concentrations (0−20 μM) of complexes 1−10 led to a
remarkable inhibition of cell growth. Thus, complex [Au(C
CCH2SC5H4N)(PTA)] (1) displayed a similar IC50 value to
that found for auranofin, and its counterpart complex 2, which
contains DAPTA, exhibited an almost 4-fold higher value than
1. Both complexes show markedly lower values than that found
for cisplatin. Notably, the heteronuclear gold and copper
derivatives 5−10 exhibited particularly potent cytotoxicity, with
IC50 values ranging from 10−4 to 1.17 μM, much lower than the
values observed for the reference drugs and for the
mononuclear Au and Cu precursors. [AuCu(C
CCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10) exhibited a sim-
ilar cytotoxicity to the precursor [Cu(NO3)(PPh3)2], whereas
the related complex 9, which contains PTA as ligand, exhibited
higher cytotoxicity. Complexes [Cu{Au(CCCH2SC5H4N)-
L}2]PF6 (L = PTA, 5; DAPTA, 6) showed the highest
antitumor efficacy, with IC50 values of around 10−2 and 10−4 for
the PD7 cell line and 10−2 for TC7 clones. Generally speaking,
similar or slightly higher IC50 values were observed for the TC7
cell lines than for the PD7 lines, except in the case of complex
5, which had a value 2 orders of magnitude higher for the
former than for the latter, and complex 10, where the lowest
value (by 3 orders of magnitude) was found for TC7 cell lines.
1
DAPTA, 10). Their H NMR spectra are in accordance with
the presence of the phosphane PPh3 and the propargylthiopyr-
idine unit, together with the water-soluble phosphane PTA or
DAPTA, in a relative 2:1 proportion. The 31P{1H} NMR
spectra show three broad signals at room temperature. At −60
°C these signals split into a singlet at about 40 ppm, which has
also been observed in gold(I) acetylides with triphenylphos-
phine,65−68 two doublets at ca. 0 ppm, one of which has a lower
intensity (in 9), in a similar region to that observed for the
CuPPh3 starting material, and three doublets in the region of
−90 ppm in 9 and −69 ppm in 10, which are characteristic of
Cu-PTA69−71 or Cu-DAPTA units, respectively. The two sets
of doublets centered at 0 and −90 ppm, respectively, which
have the same coupling constants, are coupled, as confirmed by
P,P-COSY NMR experiments. Integration of the three sets of
resonances gives a 1:1:1 ratio, thus pointing to the presence of
one Au-PPh3 unit and a copper(I) center simultaneously
coordinated to one PPh3 and one PTA or DAPTA molecule as
the result of PPh3 migration from the Cu(I) to the Au(I)
center. This fact was corroborated by the detection of a peak at
m/z 608 in the mass spectrum, which corresponds to a [M −
(CuLPPh3)]+, in this case [Au(CCCH2SC5H4N)(PPh3)]+,
unit and indicates the presence of AuPPh3 coordinated to the
propargylthiopyridine anion. The two doublets in the region of
0 ppm in 9 agree with the presence of two epimers, since the
copper center has an asymmetric environment. The occurrence
of further doublets in the Cu−L (L = PTA or DAPTA) region
could be due to the presence of other isomers arising from the
relative position of the Au-PPh3 unit, in addition to their
corresponding epimers. One of these doublets integrates the
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Figure 3. Quantitative flow cytometry analyses using propidium iodide (PI) uptake and annexin V staining in PD7 and TC7 colon cancer cells
treated with DMSO, 20 μM of complexes 1−10, cisplatin, auranofin, and [Cu(NO3)(PPh3)] after 72 h. [Au(CCCH2SC5H4N)(PTA)] (1);
[Au(CCCH2SC5H4N)(DAPTA)] (2); [Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5); [Cu{Au(CCCH2SC5H4N)(DAPTA)}2]PF6 (6);
[AuCu(CCCH2SC5H4N)L(PPh3)}]NO3 L = PTA (9) and L = DAPTA (10).
Cisplatin and its derivatives are among the most effective
anticancer agents and, as such, are used to treat different types
of tumors, including colon cancer.73−75 Indeed, the combina-
tion of oxaliplatin, a third-generation platinum derivative, with
5-fluorouracil and leuvocorin, as standard adjuvants, is among
the most effective chemotherapies for advanced76 or metastatic
colorectal cancer.77,78 It should be noted that our new
derivatives exhibit higher cytotoxicities than cisplatin in both
cell lines, with a difference of up to 5 orders of magnitude in the
best case, [Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5) (IC50
of 2 × 10−4 μM).
Drug delivery, including absorption, distribution, and
elimination, can be favored by a balanced relationship between
lipophilicity and hydrophilicity since the drug must be
transported through multiple mixed water−lipid layers. As
poor water solubility may lead to poor absorption and
bioavailability following oral dosing, solubility is an important
physicochemical property in drug discovery.79 Furthermore, a
direct relationship between hepatocyte uptake and lipophilicity
has been reported recently in vitro.80 As well, it has also been
shown that gold derivatives with a highly lipophilic character
provoke severe adverse effects as a result of drug accumulation
in the mitochondria.81,82 Thus, intravenous administration of
the highly liphophilic compound [Au(dppe)2]Cl (dppe = 1,2-
bis(diphenylphosphane)ethane) caused myocardial lesions83
and severe hepatotoxicity84 attributed to alteration in
mitochondrial function.85 The lipophilicity/hydrophilicity of
our derivatives covers a narrow range, with n-octanol/water
(log D7.4) distribution coefficients of between −0.4 and +0.1
(Table 2), thus indicating a balanced relationship between their
lipophilic and hydrophilic character.
Apoptosis Studies. As cancer is characterized by
uncontrolled cellular proliferation, there is considerable interest
in chemotherapy-induced apoptosis.86,87 In light of this, we
tested the ability of our metal derivatives to inhibit human
tumor cell proliferation by inducing apoptosis using the
annexin-V/propidium iodide double-staining assay, which are
well-established biomarkers of cell death,88 over 72 h (Figure
3). We also measured the effects of cisplatin and auranofin for
comparison purposes. During the early stages of apoptosis, the
cell membrane loses asymmetry and the membrane phospho-
lipid phosphatidylserine (PS) is translocated from the
cytoplasmic to the extracellular side. Annexin-V (FITC
conjugated) specifically binds to PS exposed on the cell surface
and can be detected by flow cytometry, thereby allowing the
identification of early stage apoptic cells. Viable cells with intact
membranes and cells undergoing early apoptosis but with
relatively intact membranes exclude propidium iodide (PI),
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Table 3. Summary of the Effects of Treating PD7 and PC7 Cell Lines with the Metallic Compounds 1−10, Cisplatin, and
Auranofin
cells
complex
live (%)
early apoptic (%)
late apoptic (%)
necrotic (%)
PD7
TC7
PD7
TC7
PD7
TC7
PD7
TC7
PD7
TC7
PD7
TC7
PD7
TC7
PD7
TC7
PD7
TC7
PD7
TC7
DMSO
DMSO
89.9
88.1
0.8
3.2
2.7
5.8
5.7
1.1
3.5
1.9
4.2
3.2
2.5
3.6
5.1
12.5
5.1
3.0
3.3
2.6
0.7
3.7
4.4
14.6
6.0
22.7
0.2
[Au(CCCH2SC5H4N)(PTA)] (1)
[Au(CCCH2SC5H4N)(PTA)] (1)
[Au(CCCH2SC5H4N)(DAPTA)] (2)
[Au(CCCH2SC5H4N)(DAPTA)] (2)
[Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5)
[Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5)
[Cu{Au(CCCH2SC5H4N)(DAPTA)}2]PF6 (6)
[Cu{Au(CCCH2SC5H4N)(DAPTA)}2]PF6 (6)
[AuCu(CCCH2SC5H4N)(PTA)(PPh3)2]NO3 (9)
[AuCu(CCCH2SC5H4N)(PTA)(PPh3)2]NO3 (9)
[AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10)
[AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10)
[Cu(NO3)(PPh3)2]
60.7
61.5
18.8
4.9
36.6
32.7
9.1
1.6
68.9
87.6
0.6
4.9
0.7
94.8
92.9
85.7
92.3
94.2
91.4
61.0
67.3
88.0
86.5
22.4
8.8
0.7
1.2
1.3
0.5
1.7
0.6
0.9
1.9
1.6
3.7
1.6
34.8
31.6
6.2
0.4
2.0
[Cu(NO3)(PPh3)2]
3.0
6.1
cisplatin
55.4
82.0
37.4
0.0
7.6
cisplatin
3.3
auranofin
7.7
32.2
81.5
auranofin
18.3
Figure 4. Cell-cycle analysis after treatment with the metal complexes for 72 h. Cell cycle and DNA fragmentation were determined by propidium
iodide staining. Percentages of G1-, S-, and G2-phase cells are shown when possible.
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whereas the membranes of dead and damaged cells are
permeable to PI and are also stained with annexin-V.
cisplatin, auranofin, and [Cu(NO3)(PPh3)2]. Cell-cycle pro-
gression could be determined only for complex [Au(C
CCH2SC5H4N)(DAPTA)] (2) and cisplatin. In contrast,
treatment of both cell lines with complexes 1, 3, 5−10,
[Cu(NO3)(PPh3)2], and auranofin led to a markedly perturbed
flow cytometry profile, most likely due to extensive cell death
(see Table 3), with values of less than 2% for live cells. These
disturbances in the cell cycle could inhibit the cell proliferation;
therefore these compounds have an anticancer effect by
stimulating apoptosis, as is shown in Table 3 and Figure 3.
Nonsignificant alterations in cell-cycle progression are observed
after complex treatment with complex 2 and cisplatin. Although
classical Pt(II) complexes are known to induce alterations in
the cell cycle, particularly an important increase in the G2/M
cell fraction,90 both complex 2 and cisplatin barely induce cell-
cycle changes for TC7 cells and only insignificant changes for
PD7 cells.
The data reported in Table 3 and Figure 3 reveal that
treatment with complexes 1−10 for 72 h produces much higher
amounts of early and late apoptosis than in cells treated with
DMSO for both cancer cell lines (PD7 and TC7), thus
indicating their ability to induce cell death by an apoptotic
pathway. In particular, compound [Au(CCCH2SC5H4N)-
(PTA)] (1) is the only example that induces a significantly
higher degree of early apoptosis (approximately 10-fold in PD7
and 22-fold in TC7 cell lines) compared with DMSO
treatment, whereas [AuCu(CCCH2SC5H4N)(DAPTA)-
(PPh3)2]NO3 (10) causes a 11-fold increase in both early
and late apoptosis in PD7 and a 12-fold increase in early and
late apoptosis in TC7 cell lines. Complex [Au(C
CCH2SC5H4N)(DAPTA)] (2) increases the degree of early
apoptosis 6-fold, with a similar population of live cells to that
found in DMSO medium in both cell lines, which is in
accordance with the high IC50 value measured. Complexes 5, 6,
and 9 exhibit similar behavior, with most of the cell population
(values ranging from 85% to 94%) in late-stage apoptosis.
Similar values were found for the [Cu(NO3)(PPh3)2] starting
material. Treatment of the cells with auranofin produces
different behavior in the two clones. Thus, the percentage of
live cells with PD7 is much higher than with TC7, where no
living cells remain after 72 h and only apoptotic cells are
detected. For cisplatin, similarly to complex 2, most of the
population remains as live cells and only a small degree of
apoptosis is observed. In addition, the lack of PI uptake for all
complexes supports the conclusion that they do not cause
necrosis. The only exception to this is complex [Cu{Au(C
CCH2SC5H4N)(DAPTA)}2]PF6 (6), with 12.5% of necrosis
for the PD7 cell line. In contrast, both cisplatin and auranofin
produce similar percentages or even twice that of DMSO,
except in the case of auranofin with TC7 cells, where no
necrosis is detected and live cells remain after 72 h.
The above findings highlight the fact that these new
derivatives are able to induce cell death by activating apoptic
pathways, thereby reducing their ability to nonselectively react
with biological targets to cause necrosis and its related side-
effects.89 A comparison of all the complexes studied shows that
[Au(CCCH2SC5H4N)(PTA)] (1) displays a markedly
different behavior since most of the population is found at an
early apoptotic stage. Similarly high population values are found
for complex [AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]-
NO3 (10), although in this case for late apoptosis.
We subsequently analyzed the effects of the metal complexes
on cell-cycle progression in both cancer cell lines after 72 h of
treatment. Cell-cycle analysis was performed by using flow
cytometry to assess the DNA content of cells stained with
propidium iodide. The fluorescence intensity of the stained
cells at certain wavelengths correlates with the amount of DNA
contained therein. As the DNA content of cells doubles during
the S phase of the cell cycle, the relative amount of cells in the
G0 phase and G1 phase (before the S phase), in the S phase,
and in the G2 phase and M phase (after the S phase) can be
determined, as the fluorescence of cells in the G2/M phase will
be twice as high as that of cells in the G0/G1 phase. Cell-cycle
anomalies can indicate various kinds of cell damage, for
example DNA damage, which cause the cell to interrupt the cell
cycle at certain checkpoints to prevent transformation into a
cancer cell (carcinogenesis). Figure 4 shows a histogram of
PD7 and TC7 cells treated with DMSO, complexes 1−10,
CONCLUSION
■
The reaction of chlorophosphane gold(I) derivatives bearing
water-soluble phosphanes with 2-propargylthiopyridine leads to
the formation of mononuclear complexes that can act as
metalloligands upon coordination to copper(I). Trinuclear Au−
Cu compounds are obtained by using starting copper
derivatives containing labile ligands. However, in addition to
PPh3 migration, a mixture of different possible isomers is
detected when triphenylphosphane copper(I) is employed as
starting material. Most of the compounds synthesized were
tested for their antiproliferative activity against the human
colon cancer cell line Caco-2 (PD7 and TC7 clones). Although
all derivatives showed a better cytotoxicity than cisplatin, the
corresponding heteronuclear compounds displayed the lowest
IC50 value, with a value of 2 × 10−4 μM being detected in the
best case, thus indicating remarkably more activity than the
mononuclear precursors. A balanced relationship between
lipophilicity and hydrophilicity, which is one of the
prerequisites for the synthesis of an anticancer drug in order
to facilitate its administration and transport in the body and
avoid host toxicity, is found for these derivatives. Apoptosis-
related cell death is activated in Caco-2 cells after treatment
with all complexes.
The present study provides a better understanding of the
mechanisms underlying the effect of propargylthiopyridine
phosphane derivatives on cancer cells. These advances may lead
to therapeutic strategies to protect and improve cancer
treatment. Further studies in this respect are currently under
way in our group.
EXPERIMENTAL SECTION
■
1H, 31P{1H}, and 13C{1H} NMR spectra were recorded on 400 or 300
MHz Bruker Avance spectrometers. Chemical shifts are quoted relative
to external TMS (1H, 13C) or 85% H3PO4 (31P); coupling constants
are reported in Hz. MALDI mass spectra were measured on a
Micromass Autospec spectrometer in positive ion mode using DCTB
as matrix. IR spectra were recorded on a Perkin-Elmer Spectrum 100
FTIR (far-IR) spectrometer. Elemental analyses were obtained in-
house using a LECO CHNS-932 microanalyzer. The starting materials
phosphanes PTA91 and DAPTA,92,93 C5H4NSCH2CCH,36 [AuCl-
(PR′3)],93,94 and auranofin95 were prepared according to published
methods. A sample of TPPTS was kindly provided by European Oxo
GmbH. Cisplatin was purchased from Sigma-Aldrich.
Synthesis of the [Au(CCCH2SC5H4N)(L)] Complexes. To a
solution of KOH (0.014 g, 0.25 mmol) in MeOH (ca. 10 mL)
containing 2-propargylthiopiridine (0.2 mmol) was added [AuCl(L)]
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(L = PTA, DAPTA, PPh3, TPPTS) (0.2 mmol). Light brown solids
precipitated in the ethanolic solution, which were isolated by filtration
after 20 h of stirring at room temperature, washed with ethanol and
diethyl ether, and dried in vacuo. Using this method the following
complexes were prepared:
CH2−SC5H4N)PTA)]+. Anal. Calcd (%) for C28H36Au2CuF6N8P3S2
(1212.02): C 27.72, H 2.99, N 9.24. Found: C 27.41, H 3.03, N 9.59.
[Cu{Au(CCCH2SC5H4N)2(DAPTA)}2]PF6 (6): 63% yield, brown
1
solid. H NMR (400 MHz, CD2Cl2, 25 °C): δ (ppm) 1.98 (s, 12H,
−CH3CO), 3.56−3.62 (m, 2H, NCH2P), 3.76−3.93 (m, 8H, NCH2P
+ −SCH2), 4.08−4.01 (m, 2H, NCH2P), 4.15−4.22 (m, 2H NCH2N)
4.55−4.71 (m, 4H, NCH2N + NCH2P), 4.83−4.87 (m, 2H, NCH2N),
5.38−5.53 (m, 2H, NCH2P), 5.64 (d, 2H, J = 14.4 Hz, NCH2N),
7.25−7.33 (m, 2H, H5), 7.46−7.52 (m, 2H, H3), 7.68−7.72 (m, 2H,
H4), 8.47−8.54 (m, 2H, H6). 31P{1H} NMR (161.97 MHz, dmso-d6):
1
[Au(CCCH2SC5H4N)(PTA)] (1): 75% yield, pale brown solid. H
NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 3.98 (s, 2H, −SCH2), 4.21
(s, 6H, PCH2N), 4.48 and 5.54 (AB system, 6H, JAB = 13.2 Hz,
NCH2N), 6.98 (dd, 1H, J = 6.9, 5.4 Hz, H5), 7.16 (d, 1H; J = 8 Hz,
H3), 7.42 (ddd, 1H, J = 8, 7.1, 1.8 Hz, H4), 8.34(d, 1H, J = 4.4 Hz, H6).
31P{1H} NMR (161.97 MHz, CDCl3): δ −51.2 (s) ppm. 13C{1H}
NMR (100 MHz, MeOD): δ 149.4 (s, C6); 136.1 (s, C4); 121.6 (s,
C3); 119.4 (s, C5); 77.1 (s, CC); 73.1 (s, NCH2N); 52.1 (s,
PCH2N); 20.2 (s, SCH2). IR (KBr): 2158 cm−1 ν(CC). MALDI
MS: m/z (%) 503 (40.45) [M]+. Anal. Calcd (%) for C14H18AuN4PS
(502.07): C 33.47, H 3.61, N 11.15. Found: C 33.08, H 3.42, N 10.85.
−
δ −26.6 (s), −144.2 (sept, PF6 ) ppm. IR (KBr): 2163 cm−1 ν(C
−
C), 833, 556 cm−1 ν(PF6 ). MALDI MS: m/z (%) 637 (47) [M-(C
C−CH2−SC5H4N)DAPTA]+; 575 (100) [M − (Cu(CC−CH2−
SC5H4N)DAPTA)]+. Anal. Calcd (%) for C34H44Au2CuF6N8O4P3S2
(1356.07): C 30.09, H 3.27, N 8.26. Found: C 30.30, H 3.23, N 8.32.
[Cu{Au(CCCH2SC5H4N)2(PPh3)}2]PF6 (7): 75% yield, brown solid.
1H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 3.75 (s, 4H, −SCH2),
7.20−7.59 (m, 34H, PPh3+ H5 + H3), 7.74 (td, 2H, J = 7.9, 1.8 Hz,
H4), 8.71 (dd, 2H, J = 6, 1.8 Hz, H6). 31P{1H} NMR (161.97 MHz,
S25°,H2O: 83 g/L.
[Au(CCCH2SC5H4N)(DAPTA)] (2): 79% yield, pale brown solid. 1H
NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 2.01 (s, 6H, −CH3CO),
3.48 (d, 1H, J = 13.2 Hz, NCH2P), 3.82 (s, 2H, NCH2P), 3.96−4.08
(m, 3H, NCH2P + −SCH2), 4.12 (d, 1H, J = 11.3 Hz, NCH2N), 4.51−
4.59 (m, 2H NCH2N + NCH2P), 4.83 (d, 1H, J = 13.23 Hz, NCH2N),
5.67 (d, 1H, J = 13.2 Hz, NCH2P), 5.45 (dd, 1H, J = 11.1 Hz, J = 1.9
Hz, NCH2N), 6.91 (dd, 1H, J = 6.8, 5.3 Hz, H5), 7.14 (d, 1H, J = 8 Hz,
H3), 7.42 (ddd, 1H; J = 7.5, 7.2, 1.6 Hz, H4), 8.34 (d, 1H, J = 4.1 Hz,
H6). 31P{1H} NMR (161.97 MHz, CDCl3): δ −25.03 (s) ppm.
13C{1H} NMR (100 MHz, MeOD): δ 170.1 (s, CO); 149.5 (s, C6);
136.2 (s, C4); 121.7 (s, C3); 119.7 (s, C5), 77.1 (s, CC), 67.1 (s,
NCH2N), 61.8 (s, NCH2N), 49.4 (s, PCH2N), 44.9 (s, PCH2N), 39.3
(s, PCH2N), 21.4 (s, CH3), 20.2 (s, SCH2). IR (KBr): 1646 ν(CO),
2160 cm−1 ν(CC). Anal. Calcd (%) for C17H22AuN4O2PS (574.09):
C 35.55, H 3.88, N 9.75. Found: C 35.80, H 3.58, N 10.02. S25°,H2O <
0.1 g/L.
−
dmso-d6): δ 45 (s), −144.2 (sept, PF6 ) ppm. 13C{1H} NMR (75.4
MHz, CDCl3): δ 139.4 (s, C6), 134.2, 134, 132.2 (s, Ph), 130.1 (s, C4),
129.7 (s, C3), 129.3 (s, C5), 77.0 (s, CC), 22.8 (s, CH2) ppm. IR
(KBr): 2158 cm−1 ν(CC). IR (KBr): 2151 cm−1 ν(CC), 832, 557
−
cm−1 ν(PF6 ). MALDI MS: m/z (%) 670 (50) [M − (CC−CH2−
SC5H4N)PPh3]+; 607 (100) [M − (Cu(CC−CH2−SC5H4N)-
PPh3)]+. Anal. Calcd (%) for C52H42Au2CuF6N2P3S2 (1423.42): C
43.88, H 2.97, N 1.97. Found: C 43.59, H 3.21, N 2.00.
[Cu{Au(CCCH2SC5H4N)2(TPPTS)}2]PF6 (8): 73% yield, brown
1
solid. H NMR (400 MHz, CD2Cl2, 25 °C): δ (ppm) 3.75 (s, 4H,
−SCH2), 7.15−7.29 (m, 2H, H5), 7.27−7.32 (m, 2H, H3), 7.52−7.77
(m, 24H, TPPTS), 7.89 (ddd, 2H, J = 8.4, 7.8, 1.6 Hz, H4), 8.54−8.65
(m, 2H, H6). 31P{1H} NMR (161.97 MHz, dmso-d6): δ 45 (s), −144.2
−
(sept, PF6 ) ppm. 13C{1H} NMR (100 MHz, MeOD): δ (ppm) 134.2
(TPPTS); 134.0 (s, C6), 132.0 (s, C4), 129.5 (s, C3), 116.7 (s, C5), 77.0
1
[Au(CCCH2SC5H4N)(PPh3)] (3): 68% yield, pale brown solid. H
(m, CC), 20 (s, SCH2). IR (KBr): 2157 cm−1 ν(CC), 832, 556
NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 4.14 (s, 2H, −SCH2), 6.97
(ddd, 1H, J = 8, 5.2, 0.8 Hz, H5), 7.28 (t, 1H; J = 1.8 Hz, H3), 7.45−
7.66 (m, 16H, H4 + PPh3), 8.45 (d, 1H, J = 1.7 Hz, H6). 31P{1H}
NMR (161.97 MHz, CDCl3): δ 42.1 (s) ppm. 13C{1H} NMR (75.4
MHz, CDCl3): δ 149.4 (s, C6), 135.9, 134.3, 131.5 (s, Ph), 129.1 (s,
C4), 121.7 (s, C3), 119.3 (s, C5), 99.2 (s, CC), 20.2 (s, CH2) ppm.
IR (KBr): 2158 cm−1 ν(CC). MALDI MS: m/z (%): 608 (90)
[M]+. Anal. Calcd (%) for C26H21AuNPS (607.08): C 51.41, H 3.48,
N 2.31. Found: C 50.97, H 3.32, N 2.55. S25°,H2O < 0.1 g/L.
−
cm−1 ν(PF6 ). MALDI MS: m/z (%) 975 (63) [M − (CC−CH2−
SC5H4N) TPPTS]+; 912 (100) [M − (Cu(CC−CH2−SC5H4N)-
TPPTS]+. Anal. Calcd (%) for C52H36Au2CuF6N2Na6P3S8 (2035.69):
C 30.68, H 1.78, N 1.38. Found: C 30.35, H 1.91, N 1.30.
[AuCu(CCCH2SC5H4N)(PTA)(PPh3)2]NO3 (9): 70% yield, brown
1
solid. H NMR (400 MHz; CDCl3; 25 °C): δ (ppm) 3.77 (s, 6H,
NCH2N), 3.87 (s, 2H, −SCH2), 4.34 and 4.12 (AB system, 6H, JAB
=
14.2 Hz, NCH2P), 7.01 (t, 1H, J = 3 Hz, H3), 7.19−7.34 (m, 31H,
PPh3 + H5), 7.53 (td, 1H, J = 5.4, 1.2 Hz, H4), 8.51 (d, 1H, J = 3.3 Hz,
H6). 31P{1H} NMR (121.45 MHz, CDCl3, −60 °C): δ 40.3 (s, Au-
PPh3), 0.5 (d, minor isomer, Cu-PPh3), 0.16 (d, mayor isomer, Cu-
PPh3), −89.9 (d, Cu-PTA), −85.4 (d, Cu-PTA), −96.2 (d, Cu-PTA)
(JPP = 85 Hz) ppm. 13C{1H} NMR (100 MHz, MeOD): δ 142.4 (s,
C6), 138.7 (s, PPh3 + C4), 134.1 (s, PPh3 + C3), 126.1 (s, C5), 77.0 (s,
CC), 70.1 (s, CC), 44.7 (m, NCH2N + PCH2N), 25 (s, SCH2).
1
[Au(CCCH2SC5H4N)(TPPTS)] (4): 89% yield, pale brown solid. H
NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 3.96 (s, 2H, −CH2), 7.12
(dd, 1H, J = 7.2, 54.9 Hz, H5), 7.33 (d, 1H; J = 8.1 Hz, H3), 7.43−7.61
(m, 12H, TPPTS), 7.69−7.67 (m, H4), 8.44 (d, 1H, J = 4.7 Hz, H6).
31P{1H} NMR (161.97 MHz, CDCl3): δ 41.7 (s) ppm. IR (KBr): 2161
cm−1 ν(CC). Anal. Calcd (%) for C26H18AuNNa3O9PS4 (912.9): C
34.18, H 1.99, N 1.53. Found: C 34.30, H 2.11, N 1.75. S25°,H2O < 0.1
g/L.
−
IR ν: 2160 (CC), 1098, 1014, 691 (NO3 ) cm−1. MALDI MS (m/
z): [M − CuPTAPPh3]+ 608 (100%). Anal. Calcd (%) for
C50H48AuCuN5O3P3S (1152.45): C 52.11, H 4.88, N 6.08. Found:
C 52.30, H 4.57, N 6.30.
Synthesis of the [Cu{Au(CCCH2SC5H4N)2(L)}2]PF6 Com-
plexes. To a solution of [Au(CCCH2SC5H4N)(L)] (L = PTA,
DAPTA, PPh3, TPPTS) (0.2 mmol) in dichloromethane/acetonitrile
(5/5 mL) under argon atmosphere was added [Cu(MeCN)4]PF6 (0.1
mmol). The solution was concentrated after 3 h of stirring at room
temperature. Addition of diethyl ether gave pale brown solids, which
were isolated by filtration.
[AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10): 70% yield,
1
brown solid. H NMR (400 MHz; CDCl3; 25 °C): δ (ppm) 1.55
(s, 3H, CH3CO), 1.94 (s, 3H, CH3CO), 3.15 (m, 1H, PCH2N), 3.40
(m, 1H, PCH2N), 3.50 (m, 1H, NCH2N), 3.71 (m, 2H, PCH2N), 3.84
(s, 2H, SCH2), 3.88 (m, 1H, NCH2N), 4.33 (m, 1H, NCH2N), 4.73
(d, 1H, J = 14 Hz, PCH2N), 5.10 (d, 1H, J = 15.6 Hz, PCH2N), 5.57
(d, 1H, J = 14.4 Hz, NCH2N), 7.07 (t, 1H, J = 5.6 Hz H5), 7.20−7.41
(m, 31H, PPh3 + H3), 7.52 (td, 1H, J = 1.8 Hz, J = 7.8 Hz, H4), 8.58
(d, 1H, J = 4.3 Hz, H6). 31P{1H} NMR (121.45 MHz, CDCl3, −60
°C): δ 39.7 (s, Au-PPh3), 0.17 (d, Cu-PPh3), −64.5 (d, Cu-DAPTA),
−68.9 (d, Cu-DAPTA), −69.6 (d, Cu-DAPTA) ppm (JPP = 83 Hz).
13C{1H} NMR (100 MHz, MeOD): δ 170.0 (s, CO), 133.7 (s, C6);
129.2 (s, C4), 125.7 (s, C3), 119.7 (s, C5), 77.1 (s, CC); 70.0 (s,
NCH2N); 45.2 (s, PCH2N), 21.0 (s, CH3), 20.1 (s, SCH2). MALDI
MS (m/z): [M − Cu(DAPTA)PPh3] 608 (66%), [M − (DAPTA)-
Using this method the following complexes were prepared.
[Cu{Au(CCCH2SC5H4N)2(PTA)}2]PF6 (5): 78% yield, pale brown
1
solid. H NMR (400 MHz, dmso-d6, 25 °C): δ (ppm) 3.94 (s, 4H,
−SCH2), 4.27 (s, 12H, PCH2N), 4.51 and 4.36 (AB system, 12H, JAB
=
4 Hz, NCH2N), 7.36−7.37 (m, 2H, H5), 7.57−7.58 (d, 2H, H3), 7.83−
7.87 (m, 2H, H4), 8.51 (s,br, 2H, H6). 31P{1H} NMR (161.97 MHz,
−
CD2Cl2): δ −37 (s) ppm, −144.2 (sept, PF6 ). 13C{1H} NMR (100
MHz, MeOD): δ 142.4 (s, C6), 138.7 (s, C4), 134.1 (s, C3), 126.1 (s,
C5), 77.0 (s, CC), 70.1 (s, CC), 44.7 (m, NCH2N + PCH2N),
25.1 (s, SCH2). IR (KBr): 2163 cm−1 ν(CC), 830, 555 cm−1
−
ν(PF6 ). MALDI MS: m/z (%): 1067 (10) [M]+; 565 (63%) [M −
−
(CC−CH2−SC5H4N)PTA]+; 503 (100%) [M − (Cu(CC−
PPh3] 671 (12%). IR ν: 1098, 1027, 692 (NO3 ) cm−1. Anal. Calcd
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(%) for C53H52AuCuN5O5P3S (1224.51): C 51.99, H 4.28, N 5.72.
Found: C 51.60, H 4.31, N 5.40.
Measurements of Apoptosis. Human Caco-2 cell line PD7 and
TC7 clones were exposed for 72 h with 20 μM of the metallic
compounds, collected, and stained with Annexin V-FTIC according to
the manufacturer’s recommendation. A negative control was prepared
by unreacted cells, which was used to define the basal level of apoptic
and necrotic or dead cells. After incubation, cells were transferred to
flow-cytometry tubes, washed twice with temperate phosphate-
buffered saline (PBS), and resuspended in 100 μL of annexin V
binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5
mM CaCl2), 5 μL of the annexin V-FITC, and 5 μL of PI to each 100
μL of cell suspension. After incubation for 15 min at room
temperature in the dark, 400 μL of 1× annexin binding buffer was
added and analyzed by flow cytometry within one hour. The signal
intensity was measured using a FACSARIA BD and analyzed using
FASCDIVA BD.
Cell-Cycle Analyses. Human Caco-2 cell line PD7 and TC7 clones
were exposed for 72 h with 20 μM of the metallic compounds. Cells
were fixed in 70% ice-cold ethanol and stored at 4 °C for 24 h. After
centrifugation, cells were rehydrated in PBS and stained in PI (50 μg/
mL) solution containing RNase A (100 μg/mL). PI-stained cells were
analyzed for DNA content 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.
X-ray Structure Determination of 1. Pale brown crystals of 1,
suitable for X-ray diffraction, were grown by slow diffusion of diethyl
ether over dichloromethane solution. A crystal was mounted on a glass
fiber with inert oil and centered in a Bruker-Smart CCD area-detector
diffractometer with graphite-monochromated Mo Ka radiation (λ =
0.7107 Å) at 100 K. The diffraction frames were corrected for
absorption with SADABS.96 The structure was solved by sir97.97 Full-
matrix least-squares refinement was carried out using SHELXL98
2
minimizing w(Fo * − Fc2)2. Hydrogen atoms were included by using a
riding model. Weighted R factors (Rw) and all goodness of fit S values
are based on F2; conventional R factors are based on F. The complete
crystallographic data have been deposited with the Cambridge
Crystallographic Data Centre [CCDC 934115]. A copy of this
material can be obtained free of charge from CCDC via www.ccdc.
cam.ac.uk/data-request/cif or e-mail: deposit@ccdc.cam.ac.uk. Princi-
ple crystal and refinement data: empirical formula, C14H18AuN4PS;
formula weight, 502.32; crystal system and space group, hexagonal,
P3(2); a = 12.357(5) Å, b = 12.357(5) Å, c = 9.110(5) Å; volume,
1204.7(10) Å3; Z, 3; density (calculated), 2.077 Mg/m3; absorption
coefficient, 9.386 mm−1; theta range for data collection, 1.90° to
24.97°; reflections collected, 6533; independent reflections, 2764
[R(int) = 0.1282]; R1(F2 > 2σ(F2)), 0.0720; wR2(all data), 0.1873;
S(all data), 1.086.
Distribution Coefficients (log D7.4). The n-octanol−water coef-
ficients of the complexes were determined as previously reported99
using a shake-flask method. PBS-buffered distilled water (100 mL,
phosphate buffer [PO43−] = 10 μM, [NaCl] = 0.15 M, pH 7.4) and n-
octanol (100 mL) were shaken together for 72 h to allow saturation of
both phases. A 1 mg sample of the complexes was mixed in 1 mL of
the aqueous and organic phase, respectively, for 10 min. The resultant
emulsion was centrifuged to separate the phases. The concentrations
of the compound in each phase were determined using UV absorbance
spectroscopy. Log D7.4 was defined as [compound(organic)]/[com-
pound(aqueous)].
Antiproliferative Assays. Human Caco-2 cell line PD7 and TC7
clones were kindly provided by Dr. Edith Brot-Laroche (Universite
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 °C. Cells (passages 50−80) were grown in Dulbecco’s modified
Eagle’s medium (DMEM) (Gibco Invitrogen, Paisley, UK)
supplemented with 20% fetal bovine serum (FBS), 1% nonessential
amino acids, 1% penicillin (1000 U/mL), 1% streptomycin (1000 μg/
mL), and 1% amphoterycin (250 U/mL). Experiments were
performed 24 h postseeding.
ASSOCIATED CONTENT
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*(M. Laguna) E-mail: mlaguna@unizar.es. Fax: (+34) 976-
761187.
■
Notes
́
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This article is dedicated to Prof. Dr. Ma Pilar Garcia, in
memoriam. We thank Centro de Investigacion
■
́
́
Biomedica de
Aragon
́
(CIBA), Espana (http://www.ics.aragon.es), for the
̃
technical assistance and Torrecid S.A. for a generous donation
of H[AuCl4].
Stock solutions of the complexes (saline solution or DMSO) were
diluted in complete medium to the required concentration. DMSO at
similar concentrations did not shown any effects on cytotoxicity. For
cytotoxicity screening cells were seeded in 96-well plates at a density of
4 × 103 cells/well. The culture medium was replaced with fresh
medium (without FBS) containing the complexes at concentrations
varying from 0 to 20 μM, with an exposure time of 72 h. Thereafter,
the cell survivals were measured using the MTT test as previously
described.100 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
complexes, 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 at 37 °C for 4 h.
Thereafter the medium and MTT were removed, and DMSO was
added in each well. At the end, the results are obtained by measuring
absorbance with a scanning multiwell spectrophotometer (BIOTEX
SINERGY HT SIAFRTD) at a 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.
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