Mono and dinuclear phosphinegold(I) sulfanylcarboxylates: Influence of nuclearity and substitution of PPh3 for PEt3 on cytotoxicity

Article abstract of DOI:10.1016/j.jinorgbio.2014.05.010

Gold complexes of the type [Au(PEt₃)(Hxspa)] were prepared by reacting triethylphosphinegold(I) chloride in ethanol/water (8:1) with the 3-(aryl)-2-sulfanylpropenoic acids H₂xspa [x = p = 3-phenyl-; f = 3-(2-furyl)-; t = 3-(2-thienyl)-; py = 3-(2-pyridyl); Clp = 3-(2-Chlorophenyl)-; -o-mp = 3-(2-methoxyphenyl)-; -p-mp = 3-(4-methoxyphenyl)-; -o-hp = 3-(2-hydroxyphenyl)-; -p-hp = 3-(4-hydroxyphenyl)-; -diBr-o-hp = 3-(3,5-dibromo-2-hidroxyphenyl-); spa = 2-sulfanylpropenoato] or 2-cyclopentylidene-2-sulfanylacetic acid (H₂cpa) and KOH in a 1:1:1 mole ratio. The compounds were characterized by IR spectroscopy and FAB mass spectrometry and by ¹H, ¹³C and ³¹P NMR spectroscopy. The in vitro antitumor activity of these and of the previously described dinuclear [(AuPEt₃)₂(xspa)] complexes against the HeLa-229, A2780 and A2780cis cell lines was determined and compared with those of the analogous PPh₃ complexes. The results show that the substitution of the PPh₃ ligand by PEt₃ is particularly effective in increasing the cytotoxicity of the dinuclear [(AuPR ₃)₂(xspa)] complexes, giving rise to compounds that are significantly more active than cisplatin against the aforementioned cell lines. In addition, and as a preliminary test for nephrotoxicity, the cytotoxicity of the most active compounds against the normal renal LCC-PK1 cell line was evaluated and compared with that of cisplatin.

Full text of DOI:10.1016/j.jinorgbio.2014.05.010

Mono and dinuclear phosphinegold(I) sulfanylcarboxylates: Influence of
nuclearity and substitution of PPh3 for PEt3 on cytotoxicity
Elena Barreiro, Jose´ S. Casas, Mar´ıa D. Couce, Agust´ın Sa´nchez, An-
geles Sa´nchez-Gonzalez, Jose´ Sordo, Ezequiel M. Va´zquez-Lo´pez
PII:
DOI:
Reference:
S0162-0134(14)00147-0
doi: 10.1016/j.jinorgbio.2014.05.010
JIB 9530
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
17 January 2014
6 May 2014
6 May 2014
Please cite this article as: Elena Barreiro, Jos´e S. Casas, Mar´ıa D. Couce, Agust´ın
S´anchez, Angeles Sa´nchez-Gonzalez, Jos´e Sordo, Ezequiel M. V´azquez-L´opez, Mono and
dinuclear phosphinegold(I) sulfanylcarboxylates: Influence of nuclearity and substitu-
tion of PPh3 for PEt3 on cytotoxicity, Journal of Inorganic Biochemistry (2014), doi:
10.1016/j.jinorgbio.2014.05.010
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Mono and dinuclear phosphinegold(I) sulfanylcarboxylates: influence of
nuclearity and substitution of PPh3 for PEt3 on cytotoxicity
Elena Barreiroa, José S. Casasa, María D. Couceb,*, Agustín Sáncheza, Angeles Sánchez-
Gonzaleza, José Sordoa,*, Ezequiel M. Vázquez-Lópezb
aDepartamento de Química Inorgánica, Facultade de Farmacia, Universidade de Santiago de
Compostela, 15782 Santiago de Compostela, Galicia, Spain. bDepartamento de Química
Inorgánica, Facultade de Química, Universidade de Vigo, 36310 Vigo, Galicia, Spain
ABSTRACT
Gold complexes of the type [Au(PEt3)(Hxspa)] were prepared by reacting
triethylphosphinegold(I) chloride in ethanol/water (8:1) with the 3-(aryl)-2-sulfanylpropenoic
acids H2xspa [x = p = 3-phenyl-; f = 3-(2-furyl)-; t = 3-(2-thienyl)-; py = 3-(2-pyridyl); Clp =
3-(2-Chlorophenyl)-; -o-mp = 3-(2-methoxyphenyl)-; -p-mp = 3-(4-methoxyphenyl)-; -o-hp =
3-(2-hydroxyphenyl)-; -p-hp = 3-(4-hydroxyphenyl)-; -diBr-o-hp = 3-(3,5-dibromo-2-
hidroxyphenyl-); spa = 2-sulfanylpropenoato] or 2-cyclopentylidene-2-sulfanylacetic acid
(H2cpa) and KOH in a 1:1:1 mole ratio. The compounds were characterized by IR
31
spectroscopy and FAB mass spectrometry and by 1H, 13C and P NMR spectroscopy. The in
vitro antitumor activity of these and of the previously described dinuclear [(AuPEt3)2(xspa)]
complexes against the HeLa-229, A2780 and A2780cis cell lines was determined and
compared with those of the analogous PPh3 complexes. The results show that the substitution
of the PPh3 ligand by PEt3 is particularly effective in increasing the cytotoxicity of the
dinuclear [(AuPR3)2(xspa)] complexes, giving rise to compounds that are significantly more
active than cisplatin against the aforementioned cell lines. In addition, and as a preliminary
test for nephrotoxicity, the cytotoxicity of the most active compounds against the normal renal
LCC-PK1 cell line was evaluated and compared with that of cisplatin.
Keywords: Gold(I) complexes; sulfanylpropenoic acids; 2-cyclopentylidene-2-sulfanylacetic
acid; antitumor activities
Corresponding author: J. Sordo: E-mail: jose.sordo@usc.es; Tel: +34981528074; Fax: +34981547102; M. D.
Couce: E-mail: delfina@uvigo.es; Tel: +34986812323; Fax +34986812556.

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1. Introduction
A considerable number of new metal-based therapeutic agents, including antitumoral
ones, are being prepared and biologically tested [1]; the search for effective alternatives to the
widely used platinum-based anticancer drugs [2-4] is a particularly interesting and active field
[5]. Recent examples of homo- [6,7] and heteronuclear [8,9] compounds are indicative of this
activity.
Another metal with a great deal of potential included in this field is gold, which has
been present in medicine since ancient history [10]. Interest has been considerably focused on
Au(III) compounds [11-13] due to its structural and electronic similarity to the widely used
cisplatin and cisplatin-related antitumor drugs. On the other hand, Au(I) complexes also
exhibit cytotoxic activity against cells from several tumor cell lines, some of which are
resistant to cisplatin, [12, 13]. This ability can be due to the different electronic/structural
properties of these complexes, which lead them to interact with other biological targets such
as proteins [14-16], and the thioredoxin reductase is now considered to be the most relevant
[17-23].
Auranofin, triethylphosphine(2,3,4,6-tetra-o-acetyl-β,1-D-thiopyranosato-S)gold(I) is
a well known antiarthritic drug that also shows significant cytotoxic activity [13, 24]; it is an
interesting example of a group of Au(I) thiolate compounds, which contain the S-Au-P
fragment and have also attracted interest as potential antitumor agents [12, 13]. The
replacement of the thiolate ligand by other biological ligands is presumed to modulate the
biological activity of these compounds, [10, 25] and consequently it is attractive to explore
the activity of members of this family in which the S-Au bond has been stabilized. This
stabilization can be achieved by endowing the thiolate ligand with other groups that are also
capable of binding to the metal.
On this basis we have drawn up a concise compilation in which structural
modifications could be related to changes in the in vitro antitumoral activity; thus, we have
selected a number of sulfanylcarboxylic acids, R-CH-C(SH)-COOH, H2xspa, and 2-
cyclopentylidene-2-sulfanylacetic acid (H2cpa) in order to prepare gold(I) complexes which
could also contain a phosphine ligand. The sulfanylcarboxylic ligands (Scheme 1) present a
wide spectrum of R groups which can modulate the hydrophilicity/lipophilicity of the
complexes, a property of great importance for drug action [10, 25, 26]. The possibility of
intra/intermolecular interactions and their associated structural effects is yet another point of
interest for the selection of these groups. We initially prepared compounds of the type

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[Au(PPh3)(Hxspa)] [27], which included an S-Au-P fragment, and maintained the COOH
group protonated. These compounds were subsequently structurally modified by
deprotonating this group; this was achieved by reacting them either with diisopropylamine or
triethylamine, which affords compounds of the type [HQ][Au(PPh3)(xspa)] and
[HP][Au(PPh3)(xspa)] (HQ = diisopropylammonium; HP = triethylammonium) [28] or with
Au(PPh3)Cl, thus including a new AuPPh3 group to give dinuclear [(AuPPh3)2(xspa)]
compounds [29]. In addition, we have incorporated the AgPPh3 fragment, thus preparing
heteronuclear complexes containing Ag(I) and Au(I) centers [30, 31].
We now initiate the study of the effect that the substitution of the PPh3 phosphine can
have on the cytotoxicity of the complexes. This paper describes the synthesis, characterization
and the study of the cytotoxic activity of complexes of the type [Au(PEt3)(Hxspa)] against the
human cervical carcinoma cell line HeLa-229 and the ovarian carcinoma cell line A2780 and
its cisplatin-resistant mutant A2780cis. The activity of the previously described
[(AuPEt3)2(xspa)] complexes [32] was also investigated and the results obtained for both
types of complexes were comparatively analyzed and contrasted with those of the equivalent
PPh3 complexes.
Scheme 1
In an attempt to get information about possible adverse effects limiting the potential
therapeutic utility of these compounds, a preliminary study of toxicity on normal cells was
also carried out. Thus, some of the compounds showing the best activity against the A2780cis
cell line were selected to be tested on the normal epithelial renal LLC-PK1 cell line.
2. Experimental Section
2.1. Material and methods
2-Chlorobenzaldehyde,
hydroxybenzaldehyde, 4-hydroxybenzaldehyde, 3,5-dibromo-2-hydroxybenzaldehyde, 2-
furancarboxaldehyde, 2-thiophenecarboxaldehyde, 2-pyridinecarboxaldehyde,
2-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2-
cyclopentanone (all from Aldrich), benzaldehyde (Probus), rhodanine (Aldrich) and
triethylphosphinegold(I) chloride (Aldrich) were used as supplied. The 3-(aryl)-2-
sulfanylpropenoic acids H2xspa [x = f = 3-(2-furyl)-; t = 3-(2-thienyl)-; p = 3-phenyl-; Clp =

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3-(2-Chlorophenyl)-; -o-mp = 3-(2-methoxyphenyl)-; -p-mp = 3-(4-methoxyphenyl)-; o-hp =
3-(2-hydroxyphenyl)-; p-hp = 3-(4-hydroxyphenyl)-;-diBr-o-hp 3-(3,5-dibromo-2-
=
hydroxyphenyl-); py = 3-(2-pyridyl); spa = 2-sulfanylpropenoate] were prepared by
condensation of the appropriate aldehyde with rhodanine [33], followed by hydrolysis in an
alkaline medium and acidification with aqueous HCl [34-36]. For the preparation of 2-
cyclopentyliden-2-sulfanylacetic acid (H2cpa), cyclopentanone was used instead of an
aldehyde.
Elemental analyses were performed with a Fisons 1108 microanalyser. Melting points
were determined with a Büchi apparatus. Mass spectra were recorded using FAB (fast atom
bombardment) (Micro mass Autospec spectrometer connected to a DS90 system, m-
-15
nitrobenzyl alcohol, Xe, 8 eV; ca. 1.28x10
J) and ESI-TOF (electrospray ionization time-
of-flight) (positive-ion mode, Microtof from Bruker Daltonics, methanol, capillary voltage
4500 V) ionization methods.
IR spectra (KBr pellets) were recorded on a Bruker IFS66V FT-IR spectrometer and
are reported in the synthesis section using the following abbreviations: vs = very strong, s =
1
strong, m = medium, w = weak, sh = shoulder, br = broad. H and 13C NMR spectra in
solution were recorded in dmso-d6 or CDCl3 at room temperature on a Bruker AMX 300
operating at 300.14 and 75.40 MHz, respectively, using 5 mm o.d. tubes; chemical shifts are
reported relative to TMS (tetramethylsilane) using the solvent signal ( 1H = 2.50 ppm;  13C
1
1
= 39.50 ppm or  H = 7.26 ppm;  13C = 77.00 ppm) as reference. The H-1H COSY
1
(correlated spectroscopy) NMR spectra and H-13C HMBC (heteronuclear multiple bond
correlation) and HMQC (heteronuclear multiple quantun coherence) experiments were
31
measured using a Varian Inova 400 spectrometer. P NMR spectra were recorded at 202.46
MHz on a Bruker AMX 500 spectrometer using 5 mm o.d. tubes and are reported relative to
external H3PO4 (85%). The following abbreviations were used as s = singlet, d = doublet, t =
1
triplet, pst = pseudotriplet, m = multiplet, qn = quintet. H, and 13C NMR data were obtained
from freshly prepared concentrated solutions.
2.2. X- ray crystallography
Data collection was carried out with a Bruker CCD Smart 1000 automatic
diffractometer, 293 K,  scan technique, Mok radiation ( = 0.71073 Å) and corrected for
Lorentz and polarization effects [37]. MultiScan (SADABS) [38] correction was also

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performed. The structure was solved by direct methods [39] and subsequent Fourier maps,
and refined on F2 by a full-matrix least-squares procedure using anisotropic displacement
parameters [40]. Atomic scattering factors were taken from International Tables for X-ray
Crystallography [41]. Molecular graphics were generated with MERCURY [42]. Although
the X-ray experiment afforded the molecular connectivity in compound 9, the lack of quality
of the crystals and the disorder associated with the PEt3 groups prevented a complete
refinement of the model. Crystal data: Monoclinic, P2(1)/n, Unit cell dimensions: a =
20.416(4) Å; b = 13.055(3) Å; c = 20.490(4) Å;  = 102.393(4)º; V = 5333.9(19) Å3; Z = 8; D
(calculated) =1.603 Mg/m3.
2.3. In vitro antitumor activity
2.3.1. Cell line and growth conditions
The human cervix carcinoma cell line HeLa-229 used in this study was kindly
provided by Dra. Guadalupe Mengod (CSIC-IDIBAPS of Barcelona, Spain), the human
ovarian cancer cell line A2780 and its cisplatin-resistant mutant A2780cis were both obtained
from the European Collection of Cell Cultures through Sigma-Aldrich. The cells were grown
in Dulbecco’s Modified Eagle’s medium (DMEM, HeLa-229, pH = 7.4) or RPMI 1640
medium (A2780, A2780cis) supplemented with 10% foetal calf serum (FCS) and 2 mM L-
glutamine (pH = 7.4). Cells were maintained in continuous logarithmic culture in a
humidified atmosphere of 5% CO2 at 37 ºC and were harvested using trypsin-
ethylenediaminetetraacetic acid. Pig proximal tubule LLC-PK1 cells were purchased from the
American Tissue Culture Collection (ATCC) and were grown in Medium 199 supplemented
with 3% foetal bovine serum (FBS) and 1.5 g/L NaHCO3 (pH = 7.2) and maintained at 37 ºC
in a 5% CO2 atmosphere, with the medium replaced twice a week.
All media and supplements were purchased from Sigma-RBI, Spain.
2.3.2. In vitro chemosensitivity assay
The tumor cells were seeded into 96-well plates (Beckton-Dickinson, Spain) in a
volume of 100 μL at a density of 4000 cells/well and were incubated for 4–6 h (HeLa-229) or
24 h (A2780, A2780cis) prior to dosage.
Solutions of the complexes in ethanol were added to the cells using the same
concentration of ethanol per well (1%). After the appropriate incubation time, i.e. 48 h for
HeLa-229 and 96 h for A2780 and A2780cis, the cells were fixed by adding 10 μL of 11%

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glutaraldehyde per well for 15 min. The fixative was then removed and the wells were washed
four times with distilled water. Cell biomass was determined by a crystal violet staining
technique [43] and the optical density was measured at 595 nm with a Tecan Ultra Evolution
microplate reader.
The renal LCC-PK1 cells were seeded in a 96-well microplate (1·104 cells/well) in 100
µL of growth medium and the samples were maintained at 37 ºC in a 5% CO2 atmosphere
during 24 h. The growth medium was then replaced by fresh medium containing different
concentrations of the compounds to be assayed and the samples were maintained at 37 ºC in a
5% CO2 atmosphere for 48 h. After this time, cells were fixed to the plate with 10 µL of 11%
glutaraldehyde for 15 min at room temperature and the medium was then removed and cells
were washed 4 times with deionized water.
Cells were stained with 100 µL of a 0.1% crystal violet solution (0.1 g of crystal violet
in 100 mL of 200 mM phosphoric acid, 200 mM formic acid and 200 mM MES (2-(N-
morpholino)ethanesulfonic acid); pH = 6) for 15 min at room temperature. This solution was
removed and the plate was washed four times with deionized water and dried. In order to
enable homogenous coloration in all wells, 100 µL of 10% acetic acid were added to the wells
and the plate was gently shaken at room temperature for 15 min. The crystal violet staining
technique was also used and absorbance was read at 595 nm in a microplate reader (BioRad
Microplate Reader model 680).
Each gold complex was tested using six or seven consecutive dilutions ranging from
50 μM to 0.025 μM. The effect of 1% ethanol on the cell viability (máx. 8%) was evaluated
and corrected in all cases. The pH value remains essentially constant throughout the
experiments.The compound concentration able to inhibit cell growth by 50% with respect to
controls, IC50, was then determined from semi-logarithmic dose-response sigmoid curves
using GraphPad Prism Ver. 2.01 software (GraphPad Software Inc.).
The cytotoxicity of cisplatin (dissolved in water) was evaluated for comparison
purposes under the same experimental conditions. All compounds were tested in three
independent experiments with triplicate points. Results were expressed as meanS.E. In order
to test for differences between the appropriate complexes a Student´s T test was used (p <
0.05). The in vitro studies were performed at the Unit for the Evaluation of Pharmacological
Activities of Chemical Compounds of the University of Santiago de Compostela.

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2.4. Synthesis
The mononuclear complexes of the type [Au(PEt3)(Hxspa)] (1-11) were prepared by
adding Au(PEt3)Cl in a 1:1 mole ratio to a solution of the appropriate sulfanylcarboxylic acid
and KOH in ethanol/water (8:1). After stirring at room temperature for one hour, the solvent
was evaporated under vacuum and the oil formed was dissolved in chloroform, filtered and
the solvent removed under vacuum.
The dinuclear complexes of the type [(AuPEt3)2(xspa)] were prepared as previously
described [32] by adding Au(PEt3)Cl in a 2:1 mole ratio to a solution of the appropriate
sulfanylcarboxylic acid and NaOH in methanol/water (8:1 v/v). After stirring and refluxing for
1 h, the methanol was evaporated and the crude product was dissolved in chloroform, filtered
and the solvent removed.
[Au(PEt3)(Hpspa)] (1). H2pspa (0.077 g, 4.3 mmol), Au(PEt3)Cl (0.150 g 4.3 mmol),
ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), yellow oil. Yield: 82%. Anal.
Found: C 36.5, H 4.6, S 6.2 %. Calc. for C15H22O2SPAu: C 36.4, H 4.5, S 6.5 %. MS (FAB):
the main metalated signals are at m/z (%) 977 (100), [(AuPEt3)3S]+; 808 (10),
[(AuPEt3)2pspa]+; and 315 (56), [(PEt3)Au]+.IR (cm-1): 1720vs, br, (C=O); 1454vs, 1413s,
1381s, (PEt3). NMR (dmso-d6): 1H,  7.44 (s, 1H, C(3)H), 7.99 (d, 2H, C(5)H, C(9)H), 7.35
(pst, 2H, C(6)H, C(8)H), 7.22 (m, 1H, C(7)H), 1.80 (m, 6H, H(PCH2)), 1.07 (m, 9H,
H(PCH3)); 13C,  170.5 C(1), 127.2 C(2), 136.5 C(3), 134.4 C(4), 130.3 C(5) and C(9), 127.6
C(6) and C(8), 133.6 C(7), 17.3 (d, J(C-P) = 33.9 (PCH2)), 9.0 (PCH3); 31P {1H},  41.00(s).
1
NMR (CDCl3): H,  7.94 (s, 1H, C(3)H), 8.08 (d, 2H, C(5)H, C(9)H), 7.35 (pst, 2H, C(6)H,
13
C(8)H), 7.25 (m, 1H, C(7)H), 1.72 (m, 6H, H(PCH2)), 1.07 (m, 9H, H(PCH3)); C,  169.5
C(1), 128.6 C(2), 139.1 C(3), 136.0 C(4), 130.9 C(5) and C(9), 127.6 C(6) and C(8), 128.3
C(7), 18.0 (d, J(C-P) = 33.9 (PCH2)), 9.0 (PCH3); 31P {1H},  34.9(s).
[Au(PEt3)(Hfspa)] (2). H2fspa (0.073 g, 4.3 mmol), Au(PEt3)Cl (0.150 g 4.3 mmol),
ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), brown oil. Yield: 80%. Anal.
Found: C 31.9, H 4.2, S 6.6 %. Calc. for C13H20O3SPAu: C 32.2, H 4.1, S 6.6 %. MS (FAB):
the main metalated signals are at m/z(%) 977 (50), [(AuPEt3)3S]+; 799 (83), [(AuPEt3)2fspa]+;
484 (48), [M]+; and 315 (100), [(PEt3)Au]+. IR (cm-1): 1720vs, 1695vs, br, (C=O); 1456s,
1412s, 1381s (PEt3). NMR (dmso-d6): 1H,  7.65 (s, 1H, C(3)H), 7.41 (d, 1H, C(5)H) 6.58 (t,
1H, C(6)H) 7.44 (d, 1H, C(7)H), 1.86 (qn, 6H, H(PCH2)) 1.10 (t,12H, H(PCH3)); 13C,  170.5
C(1), 123.6 C(2), 132.7 C(3), 153.2 C(4), 114.1 C(5), 112.8 C(6), 143.2 C(7), 18.1 (d, J(C-P)
31
= 34.5 (PCH2)), 9.8 (PCH3); P {1H},  41.1 (s), 55.1 (s). NMR (CDCl3): 1H,  7.89 (s, 1H,

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C(3)H), 7.68 (d, 1H, C(5)H) 6.52 (t, 1H, C(6)H) 7.45 (d, 1H, C(7)H), 1.78 (qn, 6H, H(PCH2))
13
1.12 (t,12H, H(PCH3)); C,  169.3 C(1), 125.6 C(2), 128.5 C(3), 152.9 C(4), 115.2 C(5),
111.9 C(6), 143.0 C(7), 18.0 (d, J(C-P) = 33.8 (PCH2)), 8.9 (PCH3); 31P {1H},  36.6 (s), 55.1
(s).
[Au(PEt3)(Htspa)] (3). H2tspa (0.08 g, 4.3 mmol), Au(PEt3)Cl (0.150 g 4.3 mmol),
ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), yellow oil. Yield: 85%. Anal.
Found: C 31.0, H 4.3, S 12.5 %. Calc. for C13H20O2S2PAu: C 31.2, H 4.0, S 12.8 %. MS
(FAB): the main metalated signals are at m/z (%) 977 (13), [(AuPEt3)3S]+; 815 (48),
[(AuPEt3)2tspa]+; 499 (11), [M]+; and 315 (74), [(PEt3)Au]+. ESI-MS (+) peaks at m/z (%):
977 (6), [(AuPEt3)3S]+; 815 (56), [(AuPEt3)2(tspa)+H]+; 663 (14), [(AuPEt3)2S+H]+; 523
(0.4), [Au(PEt3)(Htspa)+Na]+; 501 (0.1), [Au(PEt3)(Htspa)+H]+; 455 (3), [Au(PEt3)2+Na-
H]+ ; 433 (100), [Au(PEt3)2]+ ; [Au(PEt3)]+ and 135 (4), [OPEt3+H]+. IR (cm-1): 1721sh,
1707vs, br, (C=O); 1455s, 1413s, 1381m, (PEt3). NMR (dmso-d6): 7.88 (s, 1H, C(3)H),
7.40 (d, 1H, C(5)H) 7.10 (t, 1H, C(6)H), 7.57 (d, 1H, C(7)H), 1.83 (m, 6H, H(PCH2)), 1.11
13
(m, 9H, H(PCH3)); C,  169.9 C(1), 129.9 C(2), 131.3 C(3), 140.1 C(4), 130.3 C(5), 126.1
C(6), 128.6 C(7), 17.3 (d, J(C-P) = 34.1 (PCH2)), 8.9 (PCH3); 31P {1H},  38.3 (s), 52.2 (s).
1
NMR (CDCl3): H,  8.27 (s, 1H, C(3)H), 7.45 (d, 1H, C(5)H) 7.10 (t, 1H, C(6)H), 7.49 (d,
13
1H, C(7)H), 1.76 (q, 6H, H(PCH2)) 1.01 (t,12H, H(PCH3)); C,  169.0 C(1), 124.6 C(2),
134.6 C(3), 140.8 C(4), 132.8 C(5), 126.1 C(6), 129.2 C(7), 17.9 (d, J(C-P) = 34.2 (PCH2)),
8.9 (PCH3); 31P {1H},  36.9 (s), 58.1 (s).
[Au(PEt3)(H-o-pyspa)] (4). H2-o-pyspa (0.078 g, 4.3 mmol), Au(PEt3)Cl (0.150 g, 4.3
mmol), ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), orange oil. Yield: 87%.
Anal. Found: C 33.6, H 4.0, N 2.6, S 6.2 %. Calc. for C14H21O2SNPAu: C 33.9, H 4.3, N 2.8,
S 6.5 %. MS (FAB): the main metalated signals are at m/z (%) 977 (100), [(AuPEt3)3S]+; 810
(8) [(AuPEt3)2-o-pyspa]+; 662 (3), [(AuPEt3)2S]+; 495 (12), [M]+; 433 (28), [(PEt3)2Au]+ and
315 (34), [(PEt3)Au]+. IR (cm-1): 1669s, ν (C═O); 1455vs, 1414m, 1361m, br, ν(PEt3). NMR
(dmso-d6): 1H, δ 7.45 (s, 1H, C(3)H), 8.52 (d, 1H, C(5)H), 7.74 (st, 1H, C(6)H), 7.12 (st, 1H,
13
C(7)H), 8.60 (d, 1H, C(8)H), 1.84 (m, 6H, H(PCH2)), 1.08 (m, 9H, H(PCH3)); C,  171.1
C(1), 142.2 C(2), 135.3 C(3), 156.4 C(4), 148.9 C(5), 132.1 C(6), 121.0 C(7), 124.2 C(8),
17.3 (d, J(C-P) = 34.1 (PCH2)), 8.9 (PCH3); 31P {1H},  37.3 (s).
[Au(PEt3)(HClpspa)] (5). H2Clpspa (0.092 g, 4.3 mmol), Au(PEt3)Cl (0.150 g, 4.3
mmol), ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), yellow oil. Yield: 89%.
Anal. Found: C 33.9, H 4.0, S 5.8 %. Calc. for C15H21O2SClPAu: C 34.1, H 4.0, S 6.0 %. MS

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(FAB): the main metalated signals are at m/z (%) 977 (100), [(AuPEt3)3S]+; 859 (15),
[(AuPEt3)2AuS]+; 433 (35), [(PEt3)2Au]+ and 315 (30), [(PEt3)Au]+. IR (cm-1): 1766vs, 1722
1
vs, br, ν(C═O); 1456vs, 1414vs, 1381vs, ν(PEt3). NMR (dmso-d6): H, δ 12.50 (s, br, 1H,
C(1)OH), 7.66 (s, 1H, C(3)H), 7.40 (d, 1H, C(6)H) 7.35 (pst, 1H, C(7)H), 7.19 (m, 1H,
C(8)H), 8.10 (d, 1H, C(9)H), 1.85 (m, 6H, H(PCH2)), 1.07 (m, 9H, H(PCH3)). 13C,  169.0
C(1), 126.5 C(2), 132.3 C(3), 135.2 C(4), 135.0 C(5), 128.7 C(6), 130.1 C(7), 128.4 C(8),
132.0 C(9), 17.5 (d, J(C-P) = 33.8 (PCH2)), 9.1 (PCH3); 31P {1H},  34.7 (s); NMR (CDCl3):
1H, δ 8.08 (s, 1H, C(3)H), 7.36 (d, 1H, C(6)H), 7.45 (st, 1H, C(7)H), 7.14 (m, 1H, C(8)H),
8.45 (d, 1H, C(9)H), 1.76 (m, 6H, H(PCH2)), 1.10 (m, 9H, H(PCH3)); 13C,  168.9 C(1), 125.7
C(2), 134.9 C(3), 135.6 C(4), 135.1 C(5), 129.2 C(6), 129.0 C(7), 128.5 C(8), 131.8 C(9),
18.0 (d, J(C-P) = 34.1 (PCH2)), 9.0 (PCH3); 31P {1H},  36.4 (s).
[Au(PEt3)(H-o-mpspa)] (6). H2-o-mpspa (0.090 g, 4.32 mmol), Au(PEt3)Cl (0.150 g,
4.3 mmol), ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), orange oil. Yield: 82%.
Anal. Found: C 36.2, H 4.5, S 5.9 %. Calc. for C16H24O3SPAu: C 36.6, H 4.6, S 6.1 %. MS
(FAB): the main metalated signals are at m/z (%) 1154 (3), [(AuPEt3)3-o-mpspa]+; 977 (68),
[(AuPEt3)3S]+; 839 (100), [(AuPEt3)2-o-mpspa]+; 524 (3), [M]+; 433 (20), [(PEt3)2Au]+ and
315 (44), [(PEt3)Au]+. IR (cm-1): 1724vs, 1674vs, br, ν(C═O); 1461vs, 1413vs, 1381s ν(PEt3).
NMR (dmso-d6): 1H, δ 12.08 (s, vbr, 1H, C(1)OH), 8.26 (s, 1H, C(3)H), 8.49 (d, 1H, C(6)H),
6.93 (st, 2H, C(7)H, C(9)H), 7.21 (st, 1H, C(8)H), 3.76 (s, 3H, OCH3), 1.83 (q, 6H, H(PCH2))
13
1.01 (t,12H, H(PCH3)); C,  170.1 C(1), 119.2 C(2), 133.6 C(3), 125.3 C(4), 156.9 C(5),
110.3 C(6), 130.1 C(7), 128.2 C(8), 128.9 C(9), 55.3 C(OCH3), 17.2 (d, J(C-P) = 34.1
(PCH2)), 8.9 (PCH3); 31P {1H},  38.5 (s); NMR (CDCl3): 1H, δ 8.25 (s, 1H, C(3)H), 8.70 (d,
1H, C(6)H), 6.86 (st, 1H, C(7)H, C(9)H), 7.00 (st, 1H, C(8)H), 3.82 (s, 3H, OCH3), 1.75 (q,
6H, H(PCH2)) 1.16 (t,12H, H(PCH3)).
[Au(PEt3)(H-p-mpspa)] (7). H2-p-mpspa (0.090 g, 4.32 mmol), Au(PEt3)Cl (0.150 g, 4.3
mmol), ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), yellow oil. Yield: 85%.
Anal. Found: C 36.8, H 4.8, S 6.3 %. Calc. for C16H24O3SPAu: C 36.6, H 4.6, S 6.1 %. MS
(FAB): the main metalated signals are at m/z (%) 977 (100), [(AuPEt3)3S]+; 859 (5)
[(AuPEt3)2AuS]+; 839 (2), [(AuPEt3)2-p-mpspa]+; 662 (4), [(AuPEt3)2S]+; 433 (11),
[(PEt3)2Au]+ y 315 (18), [PEt3Au]+. IR (cm-1): 1720vs, br, ν (C═O); 1456m, 14117m, 1380w,
1
ν(PEt3). NMR (dmso-d6): H, δ 12.00 (s, br, 1H, C(1)OH), 7.42 (s, 1H, C(3)H), 8.04 (d, 2H,
C(5)H, C(9)H), 6.90 (d, 2H, C(6)H, C(8)H), 3.75 (s, 3H, OCH3), 1.83 (q, 6H, H(PCH2)) 1.07
(t,12H, H(PCH3)); 13C,  170.8 C(1), 129.3 C(2), 134.1 C(3), 130.9 C(4), 131.9 C(5) and

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C(9), 113.1 C(6) and C(8), 158.5 C(7), 55.1 C(OCH3), 17.3 (d, J(C-P) = 33.4 (PCH2)), 9.0
(PCH3); 31P {1H},  41.5 (s), 54.8 (s).
[Au(PEt3)(H-o-hpspa)] (8). H2-o-hpspa (0.084 g, 4.3 mmol), Au(PEt3)Cl (0.150 g, 4.3
mmol), ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), orange solid. Yield: 80%.
Anal. Found: C 34.9, H 4.1, S 5.9 %. Calc. for C15H22O3SPAu: C 35.3, H 4.3, S 6.3 %. MS
(FAB): the main metalated signals are at m/z (%) 977 (100), [(AuPEt3)3S]+; 825 (33),
[(AuPEt3)2-o-hpspa]+; 510 (12), [M]+; 433 (22), [(PEt3)2Au]+ and 315 (68), [(PEt3)Au]+. IR
(cm-1): 1725vs, 1716vs, 1689m, ν(C═O); 1451s, 1413m, 1383m, ν(PEt3). NMR (dmso-d6):
1H, δ 12.20 (s, br, 1H, C(1)OH), 8.06 (s, 1H, C(3)H), 8.22 (s, 1H, C(5)OH), 7.07 (d, 1H,
C(6)H), 7.27 (st, 1H, C(7)H), 6.72 (st, 1H, C(8)H), 7.50 (d, 1H, C(9)H), 1.89 (m, 6H,
13
H(PCH2)), 1.11 (m, 9H, H(PCH3)); C,  170.1 C(1), 123.3 C(2), 126.6 C(3), 125.1 C(4),
155.9 C(5), 116.3 C(6), 129.3 C(7), 118.0 C(8), 129.7 C(9), 18.2 (d, J(C-P) = 34.1 (PCH2)),
10.0 (PCH3); 31P {1H},  35.1 (s).
[Au(PEt3)(H-p-hpspa)] (9). H2-p-hpspa (0.084 g, 4.3 mmol), Au(PEt3)Cl (0.150 g, 4.3
mmol), ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), orange solid. Yield: 92%.
Anal. Found: C 34.9, H 4.0, S 5.9 %. Calc. for C15H22O3SPAu: C 35.3, H 4.3, S 6.3 %. MS
(FAB): the main metalated signals are at m/z (%) 977 (40), [(AuPEt3)3S]+; 825 (50),
[(AuPEt3)2-p-hpspa]+; 510 (50), [M]+; 433 (29), [(PEt3)2Au]+ and 315 (100), [(PEt3)Au]+.
ESI-MS (+) peaks at m/z (%): 977 (100), [(AuPEt3)3S]+; 859 (20), [(AuPEt3)2AuS]+; 825 (12),
[Au(PEt3)2(H-p-hpspa+H]+; 663 (7), [(AuPEt3)2S+H]+; 433 (67), [Au(PEt3)2]+; 315 (35),
[Au(PEt3)]+ and 135 (1), [OPEt3+H]+. IR (cm-1): 1700sh, 1687vs, ν(C═O); 1452s, 1413w,
1383s, ν(PEt3). NMR (dmso-d6): 1H, δ 12.81 (s, vbr, 1H, C(1)OH), 7.41 (s, 1H, C(3)H), 7.94
(d, 2H, C(5)H, C(9)H), 6.73 (d, 2H, C(6)H, C(8)H), 9.74 (s, 1H, OH), 1.84 (m, 6H,
13
H(PCH2)), 1.04 (m, 9H, H(PCH3)); C,  171.0 C(1), 127.8 C(2), 129.6 C(3), 134.8 C(4),
132.1 C(5) and C(9), 114.6 C(6) and C(8), 157.2 C(7), 17.2 (d, J(C-P) = 34.1 (PCH2)), 8.9
(PCH3); 31P {1H},  38.3 (s), 52.0 (s). Slow concentration of a chloroform solution afforded
crystals suitable for X-ray diffractometry of formulae [Au(PEt3)(H-p-hpspa)])·OPEt3,
(9)·OPEt3.
[Au(PEt3)(HdiBr-o-hpspa)] (10). H2diBr-o-hpspa (0.151 g, 4.3 mmol), Au(PEt3)Cl
(0.150 g, 4.3 mmol), ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), yellow solid.
Yield: 95%. Anal. Found: C 26.8, H 3.0, S 4.6%. Calc. for C15H20O3SBr2PAu: C 27.0, H 3.0,
S 4.8 %. MS (FAB): the main metalated signals are at m/z (%) 982 (5) [(AuPEt3)2-diBr-o-
hpspa]+; 977 (100), [(AuPEt3)3S]+; 662 (20), [(AuPEt3)2S]+; 666 (3), [M]+; 433 (46),

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[(PEt3)2Au]+ and 315 (83), [(PEt3)Au]+. IR (cm-1): 1734vs, 1708m, ν(C═O); 1449m, 1414w,
1
1384w, ν(PEt3). NMR (CDCl3): H, δ 13.00 (s, 1H, C(1)OH), 7.77 (s, 1H, C(3)H), 9.80 (s,
1H, C(5)OH), 7.87 (s, 1H, C(7)H), 7.66 (s, 1H, C(9)H), 1.82 (m, 6H, H(PCH2)), 1.15 (m, 9H,
H(PCH3)); 13C,  169.8 C(1), 123.1 C(2), 132.9 C(3), 127.2 C(4), 151.0 C(5), 110.5 C(6),
136.8 C(7), 113.6 C(8), 134.2 C(9), 18.7 (d, J(C-P) = 33.4 (PCH2)), 9.7 (PCH3); 31P {1H}, 
35.1 (s).
[Au(PEt3)(Hcpa)] (11). H2cpa (0.067 g, 4.3 mmol), Au(PEt3)Cl (0.150 g, 4.3 mmol),
ethanol (8 cm3), KOH (0.024 g, 4.3 mmol), H2O (1 cm3), brown oil. Yield: 85%. Anal.
Found: C 33.2, H 5.0, S 6.5 %. Calc. for C13H24O2SPAu: C 33.0, H 5.1, S 6.8 %. MS (FAB):
the main metalated signals are at m/z (%) 977 (100), [(AuPEt3)3S]+; 859 (11), [(AuPEt3)2S]+;
786 (14), [(AuPEt3)2cpa]+; 662 (6), [(AuPEt3)2S]+; 472 (2), [M]+; 433 (16), [Au(PEt3)2]+ and
315 (28), [AuPEt3]+. IR (cm-1): 1699vs, br, ν(C═O); 1452m, 1409m, 1383m, ν(PEt3). NMR
(dmso-d6): 1H, δ 3.50 (m, 2H, C(4)H2), 1.60 (m, 2H, C(5)H2), 1.60 (m, 2H, C(6)H2), 3.50 (m,
2H, C(7)H2), 1.87 (m, 6H, H(PCH2)), 1.10 (m, 9H, H(PCH3)); 13C,  171.8 C(1), 122.6 C(2),
151.6 C(3), 36.4 C(4), 28.2 C(5), 26.3 C(6), 34.6 C(7), 18.1 (d, J(C-P) = 33.4 (PCH2)), 8.9
(PCH3); 31P {1H},  38.5 (s).
3. Results and discussion
3.1. Synthesis and characterization
The complexes of the type [Au(PEt3)(Hxspa)] (1-11) were prepared by adding
Au(PEt3)Cl to a solution of the appropriate sulfanyl carboxylic acid and KOH (1:1:1 molar
ratio) in ethanol/water. This solution was stirred for one hour, after this time the solvent was
evaporated and the resulting oil was dissolved in chloroform. Complexes 8, 9 and 10 were
obtained as solids but in the other cases the evaporation of the chloroform again afforded oily
products in high yields. These oils were also obtained in several attempts using different
solvents.
The FAB(+) spectra of these complexes show peaks due to the M+, except for 1, 5 and 7,
in which this peak was absent. Besides, the spectra show metalated peaks due to the cleavage
of the Au-S and Au-P bonds; dimetalated and trimetallated species were also identified, as
previously described for the equivalent triphenylphosphine derivatives [27].
3.2. Crystal and molecular structure of [Au(PEt3)(H-p-hpspa)])·OPEt3, (9)·OPEt3

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Attempts to obtain suitable crystals for an X-ray study by using different solvents were, in
general, unsuccessful. However, in the case of [Au(PEt3)(H-p-hpspa)] (9), a chloroform
solution left standing after several weeks liberated an oily solid from which a crystal was
selected by hand. Although suitable for X-ray diffraction, the crystal was of poor quality and
had a weak diffraction power. It contained two Au(PEt3)(H-p-hpspa) and two OPEt3
molecules in the asymmetric unit. The poor quality of the crystal and the considerable
disorder of the ethyl groups made a rigorous refinement of the structure difficult but the
connectivity of the non-hydrogen atoms has been proved beyond any doubt and is shown in
Figure 1 for the Au(PEt3)(H-p-hpspa) unit. As seen in the figure, the Au atom is S-bonded
(Au-S: ca. 2.30 Å) to the S atom of the sulfanylcarboxylate ligand and P-bonded (Au-P: ca.
2.25Å) to the P atom of the triethylphosphine ligand in a practically linear environment
resembling the situation in the equivalent triphenylphosphine complexes [27]. The
aforementioned limitations preclude a more detailed discussion of the bond distances and
angles.
Figure 1
3.3. IR spectra
The IR spectra of these complexes, when compared with the corresponding spectra of
their free ligands, show the absence of the (SH) band, which is present in the spectrum of the
free ligands between 2590-2560 cm-1, consistent with the deprotonation of this group and the
S-coordination to the Au atom. The (C=O) band of the COOH group for the free ligands is
located at 1670 (H2pspa) , 1673 (H2fspa), 1665 (H2tspa) , 1627 (H2-o-pyspa), 1683
(H2Clpspa) , 1664 (H2-o-mpspa), 1662 (H2-p-mpspa) , 1682 (H2-o-hpspa), 1686 (H2-p-hpspa),
1682 (H2-diBr-hpspa) and 1659 cm-1 (H2cpa). These positions are compatible with the
involvement of the C=O group in hydrogen bonds with the –OH fragment of a neighbour
COOH group to form dimers, a usual symptom in carboxylic acids [44, 45]. The S-metalation
of these acids with AuPPh3 fragments does not significantly change the position of the
(C=O) band in R-CS(AuPPh3)-COOH complexes, where the presence of the aforementioned
interaction was identified by X-ray diffraction [27]; nevertheless, small changes in the
position with respect to that in the free acid were detected and attributed to changes in the
hydrogen bond pattern. The position of this band, however, significantly varies in the IR
spectra of the complexes described here. For [Au(PEt3)(H-o-pyspa)] (4) and [Au(PEt3)(Hcpa)]

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(11), the position of the (C=O) band is indicative of the presence of dimeric species, formed
by the interaction of COOH groups, as occurs in the PPh3 derivatives. On the contrary, in the
IR of the complexes [Au(PEt3)(Hpspa)] (1), [Au(PEt3)(Htspa)] (3), [Au(PEt3)(HClpspa)] (5),
[Au(PEt3)(H-p-mpspa)] (7) and [Au(PEt3)(HdiBr-o-hspa)] (10) this band is located at wave
numbers higher than 1700 cm-1, shifting to positions found in complexes in which the –OH
fragment of the COOH group is involved in an intramolecular hydrogen bond with the S
atom, thus leading to monomeric units [46]. Similar monomeric R-CS(AuPEt3)-COOH units
can be present here. Furthermore, in the IR spectra of [Au(PEt3)(Hfspa)] (2), [Au(PEt3)(H-o-
mpspa)] (6), [Au(PEt3)(H-o-hpspa)] (8) and [Au(PEt3)(H-p-hpspa)] (9) we observe the
presence of bands in these two positions, suggesting the coexistence of dimers and monomers.
The fact that some of these bands are very broad suggests that the COOH groups can be
involved in interactions of a different type and intensity.
3.4. Solution studies
1
13
31
In solution the compounds were studied by H, C and P NMR. We tried to study the
13
1H and C spectra of all ligands and complexes in solvents of different donor character, like
dmso-d6 and CDCl3, but the low solubility of some of them prevented the study. The signals
were assigned on the basis of previous data for similar compounds [27, 32, 35] and, when
necessary, 1H-1H COSY, 1H-13C gHSQC and gHMBC NMR experiments were carried out.
1
In the H NMR spectra of the complexes, the broad signal that in the spectra of the free
ligands is located between 4 and 5 ppm, assigned to the thiol group, was not present,
according to the deprotonation of this group and the subsequent coordination of the S atom to
the metal. This coordination also produces changes in the chemical shifts of the ligand
protons, in particular, a shielding of the C(3)H signal, although in the complexes
[Au(PEt3)(H-o-hpspa)] (8), [Au(PEt3)(Hfspa)] (2) and [Au(PEt3)(H-o-pyspa)] (4) this signal is
only slightly affected. In addition, the very broad signal at low field in the spectra of the
ligands persists in that of the complexes, in accordance with the non-deprotonation of the
COOH group. All the spectra show signals located around 1.80 and 1.05 ppm, attributed to
the CH2 and CH3 groups of the PEt3 ligand, respectively.
In the 13C NMR spectra, the C signals of the ligands are also affected by the metal
coordination. These changes are very significant in [Au(PEt3)(H-o-pyspa)] (4), where C(2)
and C(3) are shielded and deshielded more than 20 ppm respectively, as a consequence of the
thione-thiol evolution when the ligand coordinates to the metal, and they are also indicative of
the S-coordination. In the other complexes, coordination also produces changes, although

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smaller, in the C(2), C(3), and C(4) positions, whereas the C(1) signals - with similar
positions in dmso-d6 and CDCl3 - are located close to those previously found in the equivalent
complexes [27], where the COOH group remains protonated. A singlet at 9 ppm and a doublet
at 18 ppm, that are shifted slightly upfield with respect to AuPEt3Cl, denote the presence of
the phosphine ligand. [32, 47]
31
The P{1H} spectra consist of one singlet located between 34 and 42 ppm in dmso-d6 -
more shielded in CDCl3 - that is attributed to the coordinated PEt3. The chemical shift is
similar to that previously found in complexes where S-Au-PEt3 fragments are present [31, 47-
52], where the 13C chemical shifts and 1J(13C-31P) values of the ethyl group are consistent with
this connectivity [47, 48, 52]. When the spectra of the complexes were not immediately
recorded, in some of them a very weak singlet appears between 50 and 60 ppm, which can be
attributed [48, 53] to the presence of OPEt3, formed after the decomposition of the complexes
and further oxidation. No peak around -19 ppm that would be attributable to the free PEt3 [47,
54] was observed, which implies that the phosphine oxidation process occurs very fast.
Keeping in mind that OPEt3 was identified in the X-ray study of [Au(PEt3)(H-p-
hpspa)]·OPEt3 (9)·OPEt3, and that this compound was proposed as one of the metabolites
formed in the biological mechanism of action of auranofin [47, 48, 50, 53, 55], the evolution
with time of this compound towards the oxide formation was investigated by recording its 31P
NMR spectrum in dmso-d6. At the same time, and due to the interest to compare the evolution
of a compound of the type [Au(PEt3)(HL)] with that of the equivalent [(AuPEt3)2(L)], we also
selected [Au(PEt3)(Htspa)] (3) because the equivalent dinuclear complex was previously
studied [32].
The 31P spectra of a freshly prepared solution of both compounds show a very intense
peak at 38 ppm attributable to the coordinated PEt3 [32]. After a few minutes, a low intensity
peak rises at 51.5 ppm; this signal is assigned to OPEt3 [50, 53, 56, 57]. The amount of oxide
present after 20 min, estimated from the integrated 31P peaks, represents about 3% for
compound 9, and 5% for compound 3, related to the coordinated PEt3. Neither after 5 h nor
24 h, was a significant increase in the oxide formation detected and after 55 days the integral
values were 8% for compound 9 and 5% for compound 3. During the experiment, no signal at
-19 ppm, attributable to the free PEt3, was observed, meaning that the oxidation of the
phosphine is very fast. Neither was any signal localized that could be assigned to the
[Au(PEt3)2]+ ion.
When the data for 3 are compared with those of the equivalent dinuclear complex, we
found that the formation of the oxide is initially more significant for 3 (5% against 1.5% after

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20 min) but the overall evolution with time seems to be less significant (5% against 10.8%
after 55 days). In addition, a signal for [Au(PEt3)2]+ was not identified for 3 but it was present
in the spectra of the dinuclear complex [32]. The formation of this ion was, however, detected
in the ESI/MS(+) electrospray spectrum (experimental part), where the remanence of the
mononuclear complex is also evident together with the formation of dinuclear or even
trinuclear species. This technique also visualizes the formation of OPEt3, which contrasts with
recent electrospray results obtained for Auranofin [58], a compound which also contains an S-
Au-P fragment with PEt3 as the P-donor ligand. It should be noted that the oxide was detected
when Auranofin was incubated with glutathione or human serum albumin, both thiol ligands
favouring the breaking of the Au-P bond and the subsequent formation of S-Au-S bonds. This
additional support seems to be unnecessary in the present case.
3.5. Cytotoxic activity against tumor and normal cells
The human HeLa-229 cervix carcinoma cells and A2780 cells, together with its cisplatin-
resistant mutant A2780cis, were used to analyze the effect that the replacement of the PPh3
group with PEt3 produces on the cytotoxicity of the previously studied mononuclear
[Au(PPh3)(Hxspa)] complexes [27]. In addition, we have selected the equivalent dinuclear
[(AuPEt3)2(xspa)] complexes. These complexes were previously [32] investigated in vivo,
dissolved in water/polyethylene glycol (3:2) and intraperitoneally administered at well-
tolerated doses of 2.5 and 5 mg/kg to Sprague-Dawley rats, showing significant anti-
inflammatory activity against plantar edema induced by carrageenan. The election of these
compounds enables us to compare their cytotoxic activity with those of the equivalent
mononuclear [Au(PEt3)(Hxspa)] (1-11) and the dinuclear [(AuPPh3)2(xspa)] complexes [29],
these latter containing the triphenylphosphine ligand.
The sulfanylcarboxylate ligands, as previously described [27], are inactive against these
cell lines, except H2tspa, which is slightly active against the HeLa culture cell. We have
previously shown [29] the low activity of Au(PPh3)Cl against these three cell cultures
[IC50(µM) values of 25(1), 8.02(0.1) and 14(1) µM against HeLa, A2780 and A2780cis,
respectively]; Au(PEt3)Cl was shown to be more active than the equivalent PPh3 derivative, as
the following IC50(µM) values against the same lines show: Au(PEt3)Cl: 1.7(0.06);
0.33(0.02); 1.8(0.06) µM.
Table S2 (Supplementary Information) shows the values of the IC50 parameter for the
[(AuPEt3)2(xspa)] complexes against the HeLa cell line. The complexes of the type
[Au(PEt3)(Hxspa)] (1-11) are scarcely active against these cells, showing very low values of

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cellular growth inhibition and therefore the IC50 parameter was not determined. The 1:2
complexes are significantly active against this line, except in the cases of [(AuPEt3)2(diBr-o-
hpspa)] and [(AuPEt3)2(o-pyspa], for which the low inhibition of the cellular growth
precludes the determination of the IC50 value (note that the triphenylphosphine complexes of
these two acids were the least active against this cell line when they were studied [29]). As
seen in Figure 2, the incorporation of PEt3 instead of PPh3 increases the activity of the
equivalent compounds, the effect being particularly relevant for the H2fspa, H2Clpspa and H2-
p-mpspa derivatives.
The [Au(PEt3)(Hxspa)] (1-11) complexes are more effective against the A2780 cell line
than against the HeLa cell line - [Au(PEt3)(H-p-mpspa)] (7) showed a low inhibition of the
cellular growth and the IC50 parameter was not calculated. However, as graphically shown in
Figure 3, only in the case of the H2pspa, H2fspa and H2tspa derivatives were the PEt3
derivatives more active than the PPh3 ones. In addition, all of them are less active than other
mononuclear gold(I) derivatives of a similar type, like [Au(PEt3)(L)] {HL = menadione
hydrogen bisulfite thiosemicarbazone [51]; 5-(hydroxymethyl)-8-methyl-3-thiol-7-
azacoumarin [52]} and also than cisplatin. The situation is very different for the 1:2
complexes. As Figure 4 shows, except for the H2-o-pyspa derivative, the introduction of a
second AuPEt3 fragment significantly improves the activity of the compounds, with IC50
values for these slightly better than those of cisplatin. All of these compounds are also more
active than the equivalent PPh3 derivatives, which are already significantly active.
Against the A2780cis cell line only four compounds of the type [Au(PEt3)(Hxspa)] (1-
11), namely the H2pspa, H2fspa, H2Clpspa and H2-o-mpspa derivatives, have IC50 values
revealing a better behaviour than cisplatin (although not better than the previously cited
gold(I) complexes [51, 52] tested under the same conditions); furthermore, except for the
H2Clpspa derivative, they also have better values than the equivalent PPh3 derivatives (Figure
5). No relevant increase in activity was obtained by substituting PPh3 by PEt3 in the other
cases. With the exception of [(AuPEt3)2(o-pyspa)], that showed a low inhibition of the cellular
growth, the situation is different for the compounds of the type [(AuPEt3)2(xspa)], all of
which have IC50 values that are significantly better than the equivalent 1:1 complexes and the
equivalent 1:2 PPh3 complexes and they also display a behaviour that is better than that of
cisplatin in all cases (Figure 6). The H2diBr-o-hpspa derivative is the exception to this rule.
Whereas these promising activities are interesting, potential candidates to be used in
clinic require an optimal ratio between the cancer killing dose and systemic toxicity [59]. A

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preliminary study of this systemic toxicity can be made in vitro by comparing cytotoxicity
against cancer cells and normal cells, the objective being a drug less toxic to normal cells than
to tumor cells.
As nephrotoxicity is an adverse effect of cisplatin and a new drug should reduce this
effect, the cell line selected was the normal epithelial renal LLC-PK1, the use of which
showed to be a very effective method for the in vitro elucidation of the nephrotoxic
mechanism caused by cisplatin [60]. Compounds showing some of the best values of activity
against the A2780cis cell line were tested against this normal renal cell line and for
comparison, cisplatin was evaluated under the same experimental conditions.
Table 2 shows the values of the IC50 parameter against this cell line. Here the pspa
derivative shows a value which is not statistically significantly different to that of cisplatin
whereas the other compounds are slightly more active. However, the higher activity of the
gold complexes against the A2780 and A2780cis cell lines compared to cisplatin led to better
values of the SI parameter (SI = selectivity index = IC50 for normal cells/IC50 for cancer
cells). The values are different for both cell lines and it should be underlined that in the case
of the A2780cis cell line, all the complexes show values (38.4, 7.0, 6.6 and 10.5 for the
H2pspa, H2tspa, H2-o-hpspa and H2-p-hpspa, respectively) significantly better than the value
found for cisplatin (1.4). These values are encouraging and qualify these compounds for
further studies.
4. Conclusions
In conclusion, the screening of the cytotoxic activity of the triethylphosphine
complexes against the HeLa-229, A2780 and A2780cis cell lines reveals that the
[(AuPEt3)2(xspa)] compounds are highly effective, particularly against the A2780cis line,
showing a high ability to circumvent cellular resistance to cisplatin. These compounds are the
most active of a series of gold(I) complexes prepared to correlate structure and activity. The
series was initiated with compounds of the type R-CH-C(SAuPPh3)-COOH, labelled as
[Au(PPh3)(Hxspa)]; the reaction of these with diisopropylamine or triethylamine lead to
compounds of the type [HQ][Au(PPh3)(xspa)] and [HP][Au(PPh3)(xspa)] (HQ =
diisopropylammonium; HP = triethylammonium) which showed to be more active than the
precursors with the COOH group protonated. The deprotonation of this group, incorporating a
new AuPPh3 group to give dinuclear [(AuPPh3)2(xspa)] compounds, produces a new increase
in activity, which is again increased when these dinuclear complexes include PEt3 instead of

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PPh3 as described in this paper. The screening of the cytotoxic activity against the normal
renal LLC-PK1 cell line led to promising values of the selectivity index in the case of the
A2780cis cell line, resistant to cisplatin.
Structural changes like deprotonation, metalation or changing the phosphine ligand,
are useful to increase the activity of the compounds, reaching the maximum value for the
[(AuPEt3)2xspa] species. From this accumulated experience, these dinuclear complexes seem
to be the most promising compounds. The closeness of the IC50 values makes it difficult to
identify one specific R-substituent on the sulfanylcarboxylate fragment responsible for the
activity in this type of compounds. Additional studies including other cell lines can perhaps
reveal new discriminatory aspects by which some specific compounds can be selected for
further studies and thus increase the knowledge of the basis of its biological effects.
Acknowledgements
We wish to thank the Dirección Xeral de I+D, Xunta de Galicia, Spain, for financial
support (IN845B-2010/121) and the Consellería de Cultura, Educación e Ordenación
Universitaria, Xunta de Galicia, Spain, for a postdoctoral fellowship (I2C plan) to E.B.

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Table 1
In vitro cytotoxicity against the A2780 and A2780cis cell line expressed as IC50 (μM) for the
complexes [Au(PEt3)(Hxspa)] (1-11) and [(AuPEt3)2(xspa)]. The resistance factor, RF, is
expressed as IC50(A2780cis)/IC50(A2780).
[Au(PEt3)(Hxspa)](1-11)
[(AuPEt3)2(xspa)]
Ligand (H2xspa)
A2780
2.3 (0.4)
A2780cis
1.3 (0.1)
0.82 (0.04)
3.06 (0.11)
6 (1)
RF
0.56
0.91
2.35 0.064 (0.003)*
A2780
0.19 (0.02)*
0.17 (0.02)*
A2780cis
RF
H2pspa
0.15 (0.03)*
0.36 (0.02)*
0.31 (0.02)*
-
0.79
2.12
4.84
-
H2fspa
0.90 (0.07)
1.3 (0.1)
13(3)
H2tspa
H2-o-pyspa
H2Clpspa
H2-o-mpspa
H2-p-mpspa
H2-o-hpspa
H2-p-hpspa
H2-diBr-o-hpspa
H2cpa
0.46
0.65
0.26
-
11 (2)
3.2 (0.5)
4.6 (0.2)
-
2.1 (0.2)
1.2 (0.1)
-
0.23 (0.02)*
0.20 (0.03)*
0.12 (0.02)
0.34 (0.02)*
0.30 (0.02)*
0.69 (0.05)*
0.23 (0.02)*
0.44 (0.04)*
0.22 (0.02)*
0.11 (0.02)
0.47 (0.05)*
0.38 (0.04)*
3.58 (0.04)*
0.75 (0.04)*
1.91
1.10
0.92
1.38
1.27
5.19
3.26
6.5 (0.4)
12 (4)
9.7 (0.3)
8.1 (0.3)
8.4 (0.5)
12 (2)
1.49
0.67
0.64
0.70
13(2)
17(4)
Cisplatin
0.44 (0.06)
3.6 (0.5)
8.18
* Significant with respect to the 1:1 complexes (P<0.05)

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Table 2
In vitro cytotoxicity against the LLC-PK1 cell line expressed as IC50 (µM) for [(AuPEt3)2
(xspa)] complexes.
Ligand (H2xpspa) [(AuPEt3)2(xspa)]
H2pspa
H2tspa
5.76 (0.08)
2.17 (0.03)
H2-o-hpspa
H2-p-hpspa
Cisplatin
3.11 (0.03)
3.98 (0.08)
5.13(0.39)

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CAPTION TO THE FIGURES
Fig. 1. A view of the structure of the Au(PEt3)(H-p-hpspa) (9) unit present in the crystal of
[Au(PEt3)(H-p-hpspa)]·OPEt3, (9)·OPEt3.
Fig. 2. The in vitro cytotoxicity of complexes [(AuPPh3)2(xspa)] and [(AuPEt3)2(xspa)]
against the HeLa-229 cell line.
Fig. 3. The in vitro cytotoxicity of complexes [Au(PPh3)(Hxspa)] and [Au(PEt3)(Hxspa)] (1-
11) against the A2780 cell line.
Fig. 4. The in vitro cytotoxicity of complexes [(AuPPh3)2(xspa)] and [(AuPEt3)2(xspa)]
against the A2780 cell line.
Fig. 5. The in vitro cytotoxicity of complexes [Au(PPh3)(Hxspa)] and [Au(PEt3)(Hxspa)] (1-
11) against the A2780cis cell line.
Fig. 6. The in vitro cytotoxicity of complexes [(AuPPh3)2(xspa)] and [(AuPEt3)2(xspa)]
against the A2780cis cell line.

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Scheme 1

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Fig. 1

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Fig. 2

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Fig. 3