Comparative study of the antitumoral activity of phosphine-thiosemicarbazone gold(I) complexes obtained by different methodologies

Article abstract of DOI:10.1016/j.jinorgbio.2019.110931

A series of phosphino-thiosemicarbazone gold(I) dinuclear complexes obtained by two different synthetic procedures have been prepared. All the compounds have been spectroscopically characterized including single crystal X ray diffraction analysis in some of cases. [Au₂(HL¹)Cl₂] (1), [Au₂(HL²)₂]Cl₂ (2) and [Au₂(HL³)₂]Cl₂ (3) have been prepared by chemical synthesis using a gold(III) salt as precursor; while [Au₂(L¹)₂] (4), [Au₂(L²)₂]?2CH₃CN (5) and [Au₂(L³)₂] (6) have been isolated from an electrochemical synthesis (HLⁿ = 2-[2-(diphenylphosphanyl)-benzylidene]-N-R-thiosemicarbazone; HL¹: R = methyl, HL²: R = methoxyphenyl, HL³: R = nitrophenyl). The in vitro cytotoxic activity of these gold(I) complexes was tested against some human tumor cell lines: HeLa 229 (cervical epithelial carcinoma), MCF-7 (ovarian adenocarcinoma), NCI-H460 (non-small-cell lung cancer) and MRC5 (normal human lung fibroblast), and the IC₅₀ values compared with those of cisplatin. The neutral methyl-substituted complexes 1 and 4 and methoxyphenyl 5 displayed significant cytotoxic activities in all investigated cancer cell lines, being 1 and 4 the most effective. The ability of complexes 1 and 4 to induce cell death by apoptosis in Hela 229 was also investigated by fluorescence microscopy using the apoptotic DNA fragmentation as marker. These results indicated that the inhibition of cell proliferation is mainly due to an apoptotic process. In order to obtain more information about the mechanism of action of these metallocompounds, the interactions of complexes 1 and 4 with the thioredoxin reductase (TrxR) enzyme were analyzed. Both complexes exhibited a strong inhibition of the thioredoxin reductase activity.

Full text of DOI:10.1016/j.jinorgbio.2019.110931

Journal Pre-proof
Comparative study of the antitumoral activity of phosphine-
thiosemicarbazone gold(I) complexes obtained by different
methodologies
Luis M. González-Barcia, Sandra Fernández-Fariña, Laura
Rodríguez-Silva, Manuel R. Bermejo, Ana M. González-Noya,
Rosa Pedrido
PII:
S0162-0134(19)30514-8
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110931
JIB 110931
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
30 July 2019
14 November 2019
15 November 2019
Please cite this article as: L.M. González-Barcia, S. Fernández-Fariña, L. Rodríguez-Silva,
et al., Comparative study of the antitumoral activity of phosphine-thiosemicarbazone
gold(I) complexes obtained by different methodologies, Journal of Inorganic
Biochemistry (2018), https://doi.org/10.1016/j.jinorgbio.2019.110931
This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
not yet the definitive version of record. This version will undergo additional copyediting,
typesetting and review before it is published in its final form, but we are providing this
version to give early visibility of the article. Please note that, during the production
process, errors may be discovered which could affect the content, and all legal disclaimers
that apply to the journal pertain.
© 2018 Published by Elsevier.

Journal Pre-proof
Comparative study of the antitumoral activity
of phosphine-thiosemicarbazone gold(I)
complexes obtained by different
methodologies
Luis M. González-Barciaa, Sandra Fernández-Fariñaa, Laura Rodríguez-Silvab,
Manuel R. Bermejoa, Ana M. González-Noyaa,*, Rosa Pedridoa,*
aDepartamento de Química Inorgánica, Facultade de Química, Universidade de Santiago de
Compostela, Campus Vida, E-15782 Santiago de Compostela, Spain
bDepartamento de Química Inorgánica, Facultade de Ciencias, Universidade de Santiago de
Compostela, Campus Terra, E-27002 Lugo, Spain
Dedication: In memoriam of Professor Juan Manuel Salas Peregrín and his
contribution to the research on bioinorganic chemistry in Spain
* Corresponding authors.
Tel.: +34 881814258; fax: +34 981597525
E-mail addresses: ana.gonzalez.noya@usc.es (A.M. González-Noya),
rosa.pedrido@usc.es (R. Pedrido)
1

Journal Pre-proof
2

Journal Pre-proof
Keywords
Gold(I)
Phosphine-thiosemicarbazone ligands
Antitumor properties
Apoptosis
Thioredoxin reductase
Graphical Abstract
Chemical
synthesis
Electrochemical
synthesis
Cytotoxicity
Apoptosis
TrxR activity
3

Journal Pre-proof
Abstract
A series of phosphino-thiosemicarbazone gold(I) dinuclear complexes obtained by
two different synthetic procedures have been prepared. All the compounds have been
spectroscopically characterized including single crystal X ray diffraction analysis in
some of cases. [Au2(HL1)Cl2] (1), [Au2(HL2)2]Cl2 (2) and [Au2(HL3)2]Cl2 (3) have
been prepared by chemical synthesis using a gold(III) salt as precursor; while
[Au2(L1)2] (4), [Au2(L2)2]∙2CH3CN (5) and [Au2(L3)2] (6) have been isolated from an
electrochemical
synthesis
(HLn=2-[2-(diphenylphosphanyl)-benzylidene]-N-R-
thiosemicarbazone; HL1: R=methyl, HL2: R=methoxyphenyl, HL3: R=nitrophenyl).
The in vitro cytotoxic activity of these gold(I) complexes was tested against some
human tumor cell lines: HeLa 229 (cervical epithelial carcinoma), MCF-7 (ovarian
adenocarcinoma), NCI-H460 (non-small-cell lung cancer) and MRC5 (normal
human lung fibroblast), and the IC50 values compared with those of cisplatin. The
neutral methyl-substituted complexes 1 and 4 and methoxyphenyl 5 displayed
significant cytotoxic activities in all investigated cancer cell lines, being 1 and 4 the
most effective. The ability of complexes 1 and 4 to induce cell death by apoptosis in
Hela 229 was also investigated by fluorescence microscopy using the apoptotic DNA
fragmentation as marker. These results indicated that the inhibition of cell
proliferation is mainly due to an apoptotic process. In order to obtain more
information about the mechanism of action of these metallocompounds, the
interactions of complexes 1 and 4 with the thioredoxin reductase (TrxR) enzyme
were analyzed. Both complexes exhibited a strong inhibition of the thioredoxin
reductase activity.
4

Journal Pre-proof
1. Introduction
The use of gold complexes in medicine is documented since ancient times for the
treatment of rheumatoid arthritis and inflammatory skin disorders such as urticaria
and psoriasis [1,2]. Nowadays the Auranofin [3], 2,3,4,6-tetra-o-acetyl-L-thio-β-D-
glyco-pyranosato-S-(triethyl-phosphine)gold(I), used for the clinical treatment of
rheumatoid arthritis, can be considered one of the leading gold metallodrugs.
Recently this compound has showed promising induced apoptosis in ovarium cancer
cells [4,5], and this finding has inspired the scientific community to investigate the
anticancer properties of many other gold compounds [6,7].
In addition, the available information about the mechanism of action of gold
complexes is still scarce. The results obtained pointed to a weak interaction of the
gold complexes with DNA or RNA targets [8], which may preclude a cis-platinum
covalent mode of action. Other investigations demonstrated that gold complexes
have a great preference for interacting with thiol-containing proteins like thioredoxin
reductases [9], or cysteine proteases, ribonuclease A (RNase A), deoxyribonuclease I
(DNase I) [10] or react with zinc finger domains [11] leading to relevant cellular
alterations.
In particular, thioredoxin reductase (TrxR) is a relevant enzyme with antioxidant
properties necessary for cell growth and development [12,13,14]. Nowadays, it is
well known that in tumor cells TrxR is overexpress avoiding apoptosis or necrosis
process, causing tumor growth and drug resistance [15,16,17]. Therefore, TrxR
enzyme has recently emerged as a main target in the development of anticancer
therapeutics [12,18,19,20]. In this sense, diphosphine [21,22,23,24,25,26], N-
heterocyclic carbine [27], benzimidazole [28], thiolate [29,30], alkynyl [31],
thiosemicarbazone [32,33] and dithiocarbamate [34] gold complexes have been
probed to inhibit the TrxR enzyme at low micromolar levels. The available X-ray
5

Journal Pre-proof
data of gold complexes with TrxR derivatives show that the gold complexes strongly
bind to the sulphur or selenium atoms of cysteinyl thiols and selenocysteines of
active site of the enzyme, and these bonds may be responsible for strong inhibition of
this enzyme.
All these evidences lead us to think that it is worthy to explore the arsenal of gold
complexes as potential efficient anti-cancer gold metallodrugs because they may
display distinct modes of action from those of platinum anticancer compounds
[35,36,37].
On the other hand the coordination chemistry of gold has expanded continuously
due to the development of new synthetic approaches that have enabled great
diversification in the field of gold research [38]. In this context we have recently
reported that an electrochemical procedure can be successfully applied to the
efficient isolation of neutral gold(I) compounds for the first time [39]. The absence
of counterions, bases or co-ligands makes possible the assembly of gold complexes
whose nature and architectures are different from those obtained by traditional
chemical synthesis. This achievement is relevant because it makes available a wider
variety of gold compounds that are in high demand for biomedical or catalytic
applications.
Our electrochemical approach for gold(I) complexes employs phosphine-
thiosemicarbazone ligands, a type of organic strands that combines two skeletons
that have been successfully applied in the design of anticancer drugs,
thiosemicarbazone [40] and phosphine [6]. The functionalization of
a
thiosemicarbazone chain with a triphenylphosphine group not only facilitate the
stabilization of gold(I) complexes [41,42,] but also overcome one of the major
inconveniences associated with thiosemicarbazone complexes, their partial
6

Journal Pre-proof
insolubility. Moreover, the introduction of a phosphine group is expected to facilitate
the solubility in the cell membrane of the resulting gold(I) complexes.
With both exposed arguments in mind we decided to apply an electrochemical
methodology for the synthesis of gold(I) complexes using a series of three
phosphino-thiosemicarbazone ligands. For our purposes we chose phosphino-
thiosemicarbazone ligands substituted by aliphatic (methyl) and aromatic
(methoxyphenyl, nitrophenyl) groups. The same ligands were employed to prepare
the corresponding gold(I) compounds by traditional chemical synthesis. Our aim is
comparing the cytotoxicity activity of a series of phosphine-thiosemicarbazone
gold(I) compounds obtained by two different methodologies, electrochemical and
chemical synthesis. With thus we hope to demonstrate that electrochemical synthesis
can provide a new arsenal of neutral gold(I) compounds with potential biological
activity.
2. Experimental section
2.1. Materials and methods
All
solvents,
2-diphenylphosphinobenzaldehyde,
4-methoxyphenyl-
isothiocyanate, gold plates, cisplatin and staurosporine are commercially available
and were used without further purification.
Elemental analysis of C, H, N and S were performed on a FISONS EA 1108
analyzer. Melting points were determined using a BUCHI 560 instrument and a
1
13
31
capillary apparatus. H, C and P NMR were registered in a VARIAN MERCURY
300 and BRUKER AVIII-500 spectrometers. Infrared spectra were measured from
KBr pellets on a BRUKER FT-MIR VERTEX 70V spectrophotometer in the ranges
4000-500 or 650-100 cm-1. Electrospray ionization mass spectra (ESI) were recorded
7

Journal Pre-proof
on an API4000 Applied Biosystems mass spectrometer equipped with a triple-
quadrupole analyzer. The MALDI-TOF mass spectra were measured using a
BRUKER MICROTOF mass spectrometer. Conductivity was measured at 25 ºC
from 103 M solutions in DMF on a Crison micro CM 2200 conductivimeter. UV-
Vis absorption spectra were recorded from solutions ca. 10−5 M in methanol at room
temperature using a UV-vis Jasco spectrophotometer. Fluorescence emission spectra
were recorded with a Fluoromax-3 spectrofluorimeter using ca. 10−5 M solutions in
methanol.
2.2. General synthesis of ligands HL1-HL3
The three ligands were previously reported by us [39]. They were synthesized by
condensation of 2-diphenylphosphinobenzaldehyde with the stoichiometric amount
of methyl-thiosemicarbazide (HL1), methoxyphenyl-thiosemicarbazide (HL2) or
nitrophenyl-thiosemicarbazide (HL3) in absolute ethanol. The solution was heated
under reflux for 4 h and concentrated with a Dean–Stark trap. The yellow
precipitates were collected by filtration. The resulting solids were finally washed
with diethyl ether (3 × 10 mL) and dried in vacuo.
8

Journal Pre-proof
Scheme 1. Chemical synthesis of the gold(I) complexes with HL1-HL3.
Scheme 2. Electrochemical synthesis of the gold(I) complexes with HL1-HL3.
2.3. General synthesis of Au(I) complexes 1-3
Complexes 1-3 were prepared by reaction of the corresponding ligand with
reduced [HAuCl4]. The synthesis and the experimental data for [Au2(HL1)Cl2] (1)
were previously reported by us [43].
9

Journal Pre-proof
The complexes [Au2(HL2)2]Cl2 (2) and [Au2(HL3)2]Cl2 (3) were synthesized
following the process summarized below:
2,2-Thiodiethanol (3 drops) was added to [HAuCl4]·3H2O (83 mg, 0.210 mmol),
previously dissolved in water (ca. 1 mL). The transparent solution formed was mixed
with the ligand HL2 (100 mg, 0.210 mmol) or HL3 (100 mg, 0.210 mmol) dissolved
in methanol (30 mL). The resulting pale-yellow solution was refluxed for 4 h.
Afterwards, the solution was concentrated to a small volume (5 mL) and cooled
overnight at 5ºC. The solid suspended was filtered off, washed with diethyl ether,
and finally dried under vacuo.
2.3.1. [Au2(HL2)2]Cl2 (2)
Yellow solid. Yield: 55 mg, 35%. M.P.: 196-198 °C. Anal Calcd. (%) for
C58H54Cl2N8O2P2S2Au2 (1486.02 g·mol-1): C, 46.9; N, 7.5; H, 3.7; S, 4.3. Found:
C, 46.8; N, 7.4; H, 3.6; S, 4.3. IR (KBr, cm-1): ν(N-H) 3323 (w), ν(C=N + C-N) 1514
(vs), 1464 (m), 1437 (s), ν(C=S) 1099 (m), 748 (m). Mass spectrometry (ESI+): m/z,
1
666.1 [M(HL)]+, 1331.2 [M2(HL)2-H]+. H NMR (300 MHz, DMSO-d6) δ: 12.11 (s,
2H, NH), 9.89 (s, 2H, NH), 8.81 (s, 2H, Himine), 8.58 (m, 2H, Ar-H), 7.69-6.79 (m,
34H, Ar-H), 3.76 (s, 6H, CH3). 31P NMR (202 MHz, DMSO-d6) δ: 29.92. M= 130.4
μS·cm-1. UV-vis, max nm: 332.
Recrystallization of 2 from acetonitrile yielded colourless crystals suitable for X-
ray crystallography ([Au2(L2)2]Cl2·CH3CN·H2O 2*).
2.3.2. [Au2(HL3)2]Cl2 (3)
Yellow solid. Yield: 71 mg, 50%. M.P.: >250 °C. Anal. Calcd. (%) for
C52H42Cl2N8O4P2S2Au2 (1433.86 g·mol-1): C, 43.6; N, 7.8; H, 3.0; S, 4.5. Found: C;
43.6; N, 7.7; H, 3.0; S, 4.4. IR (KBr, cm-1): ν(N-H) 3300 (m), ν(C=N + C-N) 1539
10

Journal Pre-proof
(vs), 1504 (s), 1437 (w), ν(N=O) 1333 (vs), ν(C=S) 1099 (m), 750 (w). Mass
1
Spectrometry (MALDI-TOF): m/z, 680.0 [M(HL)-H], 1360.0 [M2(HL)2-2H]. H
NMR (300 MHz, DMSO-d6) δ: 12.49 (s, 2H, NH), 10.36 (s, 2H, NH), 8.88 (s, 2H,
Himine), 8.58 (dd, J= 8.8 Hz, 4.7 Hz, 2H, Ar-H), 8.30-7.35 (m, 32H, Ar-H), 6.80 (dd,
J= 13.1 Hz, 7.9 Hz, 2H, Ar-H). 31P NMR (202 MHz, DMSO-d6) δ: 29.21. M= 144.8
μS·cm-1. UV-vis, max nm: 388.
Slow evaporation of the mother liquors afforded two type of crystals from which
the molecular structures of two compounds were determined by X-ray
crystallography: yellow crystals of [Au(HL3)2]Cl·CH3OH 3* and orange crystals of
[Au2(L3)2]·CH3OH·H2O 3**.
11

Journal Pre-proof
2.4. General synthesis of Au(I) complexes 4-6
The complexes [Au2(L1)2] (4), [Au2(L2)2]∙2CH3CN (5) and [Au2(L3)2] (6) were
previously reported by us [39] as they are the first series of gold(I) complexes
obtained by means of an electrochemical methodology.
All the neutral gold(I) complexes (4-6) were synthesized by an electrochemical
synthetic procedure. The ligand was dissolved in acetonitrile and a small amount of
tetraethylammonium perchlorate was added as supporting electrolyte. Then, the
solution was electrolyzed using a platinum wire as the cathode and a gold plate as the
anode. The cell can be represented as Pt(-) | ligand + CH3CN | Au (+). As an
example, the synthesis of complex 6 is described below:
A solution (0.1 g, 0.206 mmol) of the ligand H2L3 in acetonitrile (ca. 80 mL) with
a small amount of tetraethylammonium perchlorate was electrolyzed at 10 mA and
10.4 V for 66 min. The resulting yellow solid was filtered off, washed with diethyl
ether and dried under vacuum. Electronic efficiency (Ef= 1.0 mol/F-1)
Recrystallization of 6 from DMSO yielded yellow crystals suitable for X-ray
crystallography ([Au2(L3)2]2·2DMSO 6*).
The same procedure was followed for the synthesis of the gold(I) complexes 4 (10
mA and 9.5 V for 85 min, Ef= 0.9 mol/F-1) and 5 (10 mA and 13.5 V for 68 min, Ef=
0.9 mol/F-1).
2.5. Crystal structure determinations
Crystals 2* and 6* were obtained by recrystallization of solids 2 and 6 in
acetonitrile or dimethylsulfoxide, respectively. Crystals 3* and 3** were isolated
from the mother liquors resulting from the synthesis of solid 3.
Data for 2*, 3*, 3** and 6* were collected on a BRUKER APPEX-II CCD
Diffractometer, all using graphite-monochromated Mo-K radiation (k = 0.71073
A˚) from a fine-focus sealed tube source. The computing data and reduction was
12

Journal Pre-proof
made by APPEX2 BRUKER AXS software in all cases and finally all of them
refined by full-matrix, least-squares based on F2 by SHELXL-97 [44]. In all cases an
empirical absorption correction was applied using SADABS [45]. All hydrogen
atoms were included in the model at geometrically calculated positions and refined
using a riding model. Structures were depicted in ORTEP [46].
2.6. Cytotoxicity by the MTT assay
The in vitro cytotoxic activity of gold(I) complexes 1-6 was evaluated against HeLa
229 (cervical carcinoma), MCF-7 (ovarian adenocarcinoma), NCI-H460 (non-small-
cell lung cancer) and MRC-5(normal lung human fibroblast), by the
MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric
assay. In these experiments, cells were maintained in DMEM (Dulbecco’s Modified
Eagle’s Medium), supplemented with 10% FBS (Fetal Bovine Serum) and L-
Glutamine 2mM, at 37ºC in 5% CO2 atmosphere. All cell lines were provided by the
USEF platform, University of Santiago de Compostela).
Cells were seeded in a 96-well plate at a density of 4 x 103 cells per well with 100
µL of medium. The plate was incubated for 4-6 h before treated with a particular
gold(I) complex solution at different concentrations. Since the complexes are
insoluble in aqueous media, they were predissolved in DMSO (final concentration
DMSO ≤ 5%). After been incubated for 48 h at 37ºC and 5% CO2 atmosphere, MTT
(10 µL, 5 mg mL-1 in PBS (Phosphate Buffered Saline)) was added to each well, and
the plate was incubated for 4 hours. Later, SDS (Sodium Dodecyl Sulphate) was
added (100 µL, 10% HCl 0.01 M) and the plate was incubated for 12-14 h under the
same experimental conditions. The ability of living cells to reduce MTT to formazan
was used to detect cell viability. The absorbance was measured at 595 nm using a
13

Journal Pre-proof
microplate reader (Tecan infinite M1000 PRO), considering three replicates per
sample. IC50 values were calculated from dose-response curves using GraphPad
Prism V2.01, 1996 (GraphPad Software Inc.).
2.7. Induction of apoptosis in Hela 229 cell line
The ability of gold(I) complexes 1 and 4 to induce cell death by apoptosis in
Hela 229 was investigated by detecting apoptotic DNA fragmentation using the
TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay.
104 Hela 229 cells were seeded on a 96 well culture plate (Perkin Elmer 6005558)
the day before the assay. The medium was replaced by fresh medium (Sigma D5671)
containing test compounds (Thermo Fisher BP2541) in a final volume of 100
µM/well and incubated for 4 h at 37ºC and 5% CO2 atmosphere.
Then, compounds were removed and cells were washed once with DPBS
(Dulbecco’s Phosphate Buffered Saline) (Sigma D8537). Next, cells were stained by
following the instructions of the Click-iT® TUNEL Alexa Fluor® Imaging Assay
(Thermo Fisher C10247). Finally, fluorescence microscopy images were obtained by
employing an Operetta high-content imaging system (Perkin Elmer). To validate the
assay, staurosporine percentage of apoptosis formation value was determined and
compared with those described in the literature [47].
2.8. Inhibition of thioredoxin reductase activity
The ability of complexes 1 and 4 to inhibit the thioredoxin reductase (TrxR)
activity was studied by DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) reduction assay
using isolated rat liver TrxR enzyme. The experiment was carried out following the
information sheet of Sigma product T9698.
14

Journal Pre-proof
Previously, the TrxR enzyme solution was diluted with KH2PO4 (68.8 mM) and
NaH2PO4 (272.8 mM) buffers pH 7.0. Then, solutions of different concentrations of
a particular gold(I) complex predissolved in DMSO (final concentration DMSO ≤
5%) with the enzyme (1 unit/mL) were incubated for 75 min at 37 ºC with shaking.
The solutions were transferred to a 96-well plate (Thermo-Fisher 9502227) and
the reaction mixture (34.7 mM KH2PO4, pH 7.0, 137.8 mM NaH2PO4, 20 mM
β-Nicotinamide adenine dinucleotide 2′-phosphate (NADPH), 0.05 % bovine serum
albumin (BSA), 100 mM EDTA pH 7.5) was added together with the colorimetric
reagent dithionitrobenzoic acid (DTNB). After mixing, the ability of TrxR to reduce
DTNB to TNB (2-nitro-5-thiobenzoic acid) was monitored at 405 nm in 10 s
intervals for 6 min. IC50 values were calculated as the concentrations of a drug
required to decrease the TrxR activity by 50%.
3. Results and Discussion
3.1. Synthesis and spectroscopic characterization
The ligands employed in this study, HLn [2-[2-(diphenylphosphino)benzylidene]-
N-R-thiosemicarbazone] (Scheme 1), are potentially monoanionic and tridentate
through the nitrogen, sulfur and phosphorous donor atoms, [NSP]. These three
ligands have been synthesized following a method previously described by us [39].
The gold(I) complexes 1-6 have been synthesized using different methodologies
as shown in Schemes 1 and 2. Complexes 1-3 have been prepared by an
equimolecular reaction of L1–L3 with an aqueous solution of H[AuCl4]3H2O,
previously reduced with 2,2’-thiodiethanol (Scheme 1). Complex 1 has been
previously reported by us [43], but its biological activity has never been explored.
15

Journal Pre-proof
Compounds 4-6 were used as models to report for the first time the synthetic
electrochemical methodology for the preparation of Au(I) compounds from a noble
metal like gold [39]. However, they have also not been explored in terms of their
biological properties.
All complexes have been characterized by means of elemental analysis, IR, NMR
spectroscopy, mass spectrometry and conductivity measurements. Complete
spectroscopic information for 2 and 3 has been collected in the Experimental section.
The three complexes 1-3 obtained by chemical synthesis are dinuclear entities,
with the HLn ligands acting in their protonate mode (see Scheme 1). However,
compound 1 differs notably from 2 and 3. In fact, 2 and 3 are ionic compounds that
incorporate two ligand threads and two chloride ions acting as counterions, while 1 is
a neutral compound that contains two gold(I) ions, one ligand unit and two chloride
atoms bound to the metal centers.
The complexes 4-6 prepared by an electrochemical method are dinuclear neutral
compounds that contain the ligands in their monoanionic mode Ln- (see Scheme 2).
These differences in the structure of the gold(I) complexes described herein may
be relevant in order to analyze the biological activity of this series of compounds.
The IR spectra of complexes 2 and 3 show the presence of the υ(NH) bands in the
range 3400-3200 cm-1, which suggest that the ligands are coordinated to the metal
centers in their neutral form. The bands assigned to the C=S group undergo a slight
displacement respect to the free ligands, being indicative of the C=S bond weakening
after coordination of the sulphur atom to the metal center. Furthermore, the
υ(C=N + CN) bands exhibit some displacements although the coordination through
the nitrogen atoms is unlikely for Au(I) atoms.
1
The H NMR spectra show the characteristic hydrazidic NH signal around 12
ppm, shifted downfield with respect to the free ligands. The presence of this signal
16

Journal Pre-proof
confirms the neutral character of the ligands in these complexes. The signal due to
the imine proton at 8.8 ppm does not modify its position, which could be indicative
that the imine nitrogen is not coordinated to the gold centers.
The 31P NMR spectra for these Au(I) complexes exhibit a single signal around 30
ppm, shifted downfield respect to the free ligands. This fact indicates that the two
phosphorous atoms coordinated to the metal centers are magnetically equivalent.
The formation of the complexes 2 and 3 is also supported by the appearance of
fragments [M(HL)]+, [M2(HL)2-H]+ (ESI+, complex 2) and [M(HL)-H], [M2(HL)2-
2H] (MALDI, complex 3) in their mass spectra.
The molar conductivity values of 130.4 and 144.8 S cm-1 for 2 and 3,
respectively, are in agreement with 1:2 electrolyte compounds.
3.2. Description of the crystal structures
The structures of 2*, 3*, 3** and 6* have been determined by single crystal X-ray
crystallography.
The molecular structure of 2*, [Au2(HL2)2]Cl2·CH3CN·H2O (Figure 1), shows
that this compound is ionic, formed by a catioinic dinuclear gold(I) complex and two
chloride counterions. The crystal also exhibits acetonitrile and water molecules.
Every Au(I) atom exhibit a distorted linear geometry, coordinated to the phosphorous
atom of a ligand molecule and the thiocarbonyl sulphur atom of a second ligand unit
[P1-Au1-S1 162.84(5)º]. Both ligands do not cross and the resulting structure can be
named as mesocate or meso-helicate [48]. However, the box formed (Figure 2) is a
18 membered auromacrocycle that shows a certain torsion. The distance between the
Au(I) centres is large [Au1-Au1i ≈ 5.8 Å], which rule out the existence of aurophilic
interactions.
17

Journal Pre-proof
C19
N1
P1
N2
C20
Au1
N3
Au1i
S1
Figure 1. Ortep representation of compound 2*. Thermal ellipsoids are shown at the
40% probability level. Chloride counterions, solvent molecules and hydrogen atoms
are omitted for clarity.
Figure 2. Representation of the auromacrocicle in complex 2*.
The recrystallization of compound 3 in methanol afforded two different crystals
3* and 3**, which do not correspond with the stoichiometry of the solid isolated
from the reaction. The structure of complex 3*, [Au(HL3)2]Cl·CH3OH (Figure 3),
reveals the formation of an ionic mononuclear complex in which two neutral ligands
are coordinated to the Au(I) center and chloride atom acts as counterion. The gold(I)
18

Journal Pre-proof
atom is coordinated to the phosphorous atoms of both ligand units, showing a
distorted linear geometry [P2-Au1-P1 166.76(8)°]. Likewise, the presence of a weak
interaction between the sulfur atom of a thiosemicarbazone ligand and the gold(I)
center (2.988 Å) justifies the distortion of the P1-Au1-P1 pseudo linear arrangement.
Cl1
N2
S1
C7
N4
N6
C8
N3
C34
P2
N7
C33
N8
S2
P1
Au1
Figure 3. Ortep representation of compound 3*. Thermal ellipsoids are shown at the
40% probability level. Solvent molecules and hydrogen atoms are omitted for clarity.
19

Journal Pre-proof
P1
C34
Au1
S1
C8
P2
N4
N8
Au2
N7
C7
N2
N3
S2
C33
N6
Figure 4. Ortep representation of compound 3**. Thermal ellipsoids are shown at
the 40% probability level. Solvent molecules and hydrogen atoms are omitted for
clarity.
Complex 3** [Au2(L3)2]·CH3OH·H2O (Figure 4) consists of a neutral dinuclear
compound in which each gold(I) atom is coordinated to the thioamide sulfur atom
from one ligand unit and the phosphorous atom from the second ligand unit. This
coordination generates a distorted linear geometry around Au(I) centers [P1-Au1-S1
164.95(5)° and P2-Au2-S2 167.56(5)°]. This structure is a mesocate, similar to that
exhibit by the cation in complex 2. The distance between Au(I) atoms (≈5.7 Å) is
longer than the usual distances for aurophilic interactions (2.76-3.40 Å) [49,50]
Nevertheless, weak interactions between the nitrogen imine atoms (N4 and N8) and
the gold(I) ions cannot be ruled out [Au1-N4 2.689(5), Au2-N8 2.722(5) Å].
20

Journal Pre-proof
P2
Au1
C45
C19
S2
N5
N6
C20
N3
N1
N2
C46
P1
N7
Au2
S1
Figure 5. Ortep representation of compound 6*. Thermal ellipsoids are shown at the
50% probability level. Solvent molecules and hydrogen atoms are omitted for clarity.
The X-ray structure of complex 6*, [Au2(L3)2] 2DMSO, is shown in Figure 5.
Each ligand strand uses the thioamide sulfur atom to bind one gold(I) center, while
the remaining phosphorus atom is coordinated to the second gold(I) ion, thus
generating a box-type structure. The gold(I) ions have distorted linear [PS] kernels.
A weak interaction with the imine nitrogen atoms cannot be discarded [N1-Au1
2.620 Å, N5-Au2 2.840 Å]. The intramolecular distance between the gold(I) centers
is 5.8 Å, which precludes any aurophilic interaction. The two ligand threads remain
antiparallel in order to avoid unfavorable steric interactions and this arrangement
leads to a box-type structure.
In summary, structures of 2* and 6*·confirm the dinuclear stoichiometries
proposed for the complexes. In the case of 3 the crystallization process in the
methanol mother liquors after one month gave rise to a two different crystalline
complexes, 3* and 3**. This type of process is not rare in gold(I) chemistry, as the
21

Journal Pre-proof
evolution of the solid complex to other compounds along the crystallization process
has been observed before [41,42,43].
2.3. Absorption and emission spectra
UV–vis spectra for the three ligands and the six Au(I) complexes were recorded at
ca. 10−5 M concentration in methanol. The absorption spectra of the ligands display a
strong absorption in the range 321-336 nm and a shoulder at ca. 240-260 nm. We
attribute these bands to intraligand π→π* or n→π* transition characteristic of
aromatic systems. The similarities between the spectra of these ligands indicate that
the 4-N-terminal substituents exercise a minor influence on their spectroscopic
properties.
The absorption spectra for all the complexes exhibit a single band in the range
325-388 nm, with a slight variation in the energy respect to the free ligands. These
emissions can be assigned to intraligand transitions modified by coordination to the
gold(I) center.
Table 1
Excitation and emission values for the ligands and the gold(I) complexes in 10−5 M
methanol solutions.
Complex
Excitation (nm)
Emission (nm)
426, 406
427, 402
428, 405
378, 340
419
427, 404
427, 405
428, 403
428, 404
HL1
331
331
336
325
332
388
325
336
348
HL2
HL3
1
2
3
4
5
6
The emission spectra of the ligands HL1-HL3 and their corresponding Au(I)
complexes 1-6 were recorded at room temperature in ca. 10−5 M methanol solution.
22

Journal Pre-proof
These spectra exhibit intense bands, with maxima at ca. 419-428 nm except in the
case of compound 1 where both bands are displaced to a lower wavelength. The
emissions in the complexes can be assigned to intraligand or S-to-Au LMCT
transitions.
3.4. Cytotoxic studies
The in vitro cytotoxic activity of the gold(I) complexes 1-6 was tested against the
human tumor cell lines HeLa 229 (cervix epithelial carcinoma), MCF-7 (ovarian
adenocarcinoma), NCI-H460 (non-small-cell lung cancer) and MRC-5 (normal lung
human fibroblast), and the IC50 values compared with those of cisplatin [51,52]. As
these compounds are not soluble in water, a mixture DMSO/water (DMSO ≤5%)
was used in the cytotoxicity tests with PBS (1 mM) as buffer.
The IC50 values (concentrations of a drug required to inhibit tumor cell
proliferation by 50%, compared to the control viability) of these compounds at 48 h
are all summarized in Table 2 and represented in Figure 6. As can be seen, complex
1 is the most active in the three cancer lines tested. Complexes 4 and 5 show IC50
values, between 5 to 8 times lower activity than the most potent complex 1.
23

Journal Pre-proof
Table 2. IC50 values determined for HeLa 229, MCF-7 and NCI-H460 cancer cell
lines after 48 h incubation at 37ºC and 5% CO2 atmosphere, with gold(I) complexes,
in comparison to cisplatin.
Compound
1
Hela 229
1.79 ± 0.04
MCF-7
21.3 ± 0.7
NCI-H460
10.4 ± 0.9
65 ± 1
76 ± 1
45 ± 1
2
69 ± 1
81 ± 1
N.D.[a]
3
5.58 ± 0.17
8.54 ± 0.09
46 ± 0.11
0.96 ± 0.02
29.2 ± 0.4
36.3 ± 0.2
72 ± 1
14.1 ± 0.3
22.3 ± 0.4
N.D.[a]
4
5
6
Cisplatin
13 ± 1
7.71 ± 0.67
[a] N.D.= not determined
The cytotoxicity results of the complexes 1-6 against three different cancer cell
lines let to analyze some correlations structure-activity. Firstly, the 4-N-methyl
substituted complexes exhibit the best values of IC50 in the three cancer cell lines,
indicating that a small alkyl group favors the cytotoxic activity whereas big aromatic
substituents clearly gives rise to worse results. On the other hand, the influence of the
ionic/neutral nature of the complexes is clear, as the neutral 1, 4, 5 and 6 compounds
are more effective than the ionic complexes 2 and 3. In fact, the neutral
methoxyphenyl-substituted complex 4 obtained by electrochemical synthesis, shows
IC50 values of 5.58 (HeLa 229), 29.2 (MCF-7) and 14.1 (NCI-H460) µM whereas its
analogous ionic complex 2 exhibits a lower cytotoxicity with a IC50 values of 65
(HeLa 229), 76 (MCF-7) and 45 (NCI-H460) µM.
24

Journal Pre-proof
90
80
70
60
50
40
30
20
10
0
HeLa
MCF-7
NCl-H4640
1
2
3
4
5
6
Cisplatin
Compound
Figure 6. Representation of the IC50 values for complexes 1-6 and cisplatin against
HeLa 229, MCF-7 and NCI-H460 cancer cell lines.
Because metal complexes may induce a high cytotoxicity in normal cells, the effect
of complexes 1-6 in normal lung human fibroblasts MRC-5 was assessed. The
concentrations found for the series 1-6 were >100 M, indicating that the complexes
produce a minimal reduction of the normal cell viability, which is suggestive of
certain selectivity against cancer cells over the normal ones.
3.5. Detection of cell death
The neutral methyl substituted compounds 1 and 4 exhibit the better IC50 values.
For that reason they have been selected in order to study more in depth the possible
mechanism of action for these complexes.
Apoptosis is a cell death process in which a large number of morphological and
chemical changes occur in the cellular structure, such as cell contraction,
cytoplasmic and organelles condensation, or the fragmentation of DNA. Among the
different types of programmed cell death, the design of drugs that induce this process
is interesting due to the contribution of apoptosis to cancer [53].
25

Journal Pre-proof
In order to check that gold(I) complexes 1 and 4 induced apoptosis in HeLa 229
cell line, DNA fragmentation was used as a marker of cell death by using the
TUNEL assay.
Cells were stained incorporating modified nucleotides with fluorophores by the
enzyme terminal deoxynucleotidyl transferase (TdT) at the 3’-OH ends of
fragmented DNA. EC50 values (concentrations of a drug where 50% of its maximal
effect is observed) were calculated from concentration-response curves by using an
Operetta high-content imaging system (Perkin Elmer). To study the induction of
apoptosis, Hela 229 cells were exposed for 4 h to both gold(I) complexes. Complex 1
induced cell death by apoptosis in HeLa 229 with an EC50 value of 12.76 µM.
Figure 7. HeLa 229 cells were prepared and examined by fluorescence microscopy
to identify TUNEL stained apoptotic cells (red) and Hoechst stained live cells (blue).
HeLa 229 cells treated with gold(I) complexes 1 and 4 (100 µM) for 4 h are
TUNEL-positive and show apoptotic bodies.
Complex 4 exhibited a low efficiency at the maximum concentration tested (100
µM), being not possible to calculate an EC50 value due to an incomplete
concentration-response curve. However, results showed that complex 4 has a
percentage of apoptosis formation of 57 ± 10%. Therefore, these results show that
both gold(I) complexes induce apoptosis being compound 1 more effective. This
26

Journal Pre-proof
evidence can also be seen in fluorescence microscopy images where compound 1
presents more apoptotic cells (red) than compound 4 (Figure 7).
2.6. Inhibition of the thioredoxin reductase activity
It is known the ability of gold complexes to inhibit the activity of the thioredoxin
reductase (TrxR) enzyme as mentioned in the introduction. In order to evaluate the
relation between the potent cytotoxicity of complexes 1 and 4 and the activity of the
TrxR, the inhibitory potency of these compounds on the isolated rat liver thioredoxin
reductase enzyme was explored by DTNB reduction assay. IC50 values of both
gold(I) complexes exhibited a strong inhibitory character of the TrxR activity at
micromolar concentrations. Particularly, complex 1 is the most active showing an
IC50 value of 0.083 µM, 10 times higher activity than the IC50 value of complex 4,
0.857 µM. This fact could be related with the structure of both complexes. Thus,
complex 1 contains hemilabile ligands (Cl-) that can be easily displaced allowing a
better union to the active center of the enzyme, and this fact could explain its better
activity.
However, although the structures of complexes 1 and 4 are different, both
compounds are effective in promoting the cell death. For that reason, a more
complex action mechanism, including not only the inhibition of the thioredoxin
reductase enzyme, is suggested. It is likely that both compounds not only produce
inhibition of this enzyme but also others that should be investigated in the future.
3. Conclusions
Two series of phosphino-thiosemicarbazone gold(I) complexes obtained by
different synthetic procedures have been studied in order to compare their biological
properties. The chemical synthesis afforded ionic compounds [Au2(HL2)2]Cl2 (2) and
27

Journal Pre-proof
[Au2(HL3)2]Cl2 (3), except for the methyl-substituted ligand. In this case the neutral
complex [Au2(HL1)Cl2] (1) was obtained. The electrochemical methodology yielded
neutral complexes [Au2(L1)2] (4), [Au2(L2)2]∙2CH3CN (5) and [Au2(L3)2] (6).
The in vitro cytotoxic activity of these gold(I) complexes was tested against the
HeLa 229, MCF-7 and NCI-H460 tumor cell lines by the MTT colorimetric assay,
and these results let to analyze some correlations structure-activity. The 4-N-methyl
substituted complexes 1 and 4 exhibited the best IC50 values in three different tumor
lines, indicating that a small alkyl group like methyl favors the cytotoxic activity
whereas big aromatic substituents clearly gets worse results. On the other hand,
neutral compounds are more effective than the corresponding cationic species. Thus,
the neutral dinuclear compound 5 (containing a methoxyphenyl group), obtained by
an electrochemical route, was much more effective than the analogue cationic
dinuclear complex 2. This fact corroborates that the electrochemical synthesis makes
available a new arsenal of neutral gold(I) compounds with potential biological
activity.
Fluorescence microscopy studies of HeLa cells exposed to complexes 1 and 4
demonstrated that the mechanism of inhibition of cell proliferation that induce cell
death is mainly apoptotic.
Complexes 1 and 4 have been tested as inhibitors of the thioredoxin reductase
(TrxR) enzyme, showing that both complexes exhibit a strong inhibition of the
thioredoxin reductase activity, being compound 1 notably more effective.
28

Journal Pre-proof
Abbreviations
DMEM
DMSO-d6
DNA
DPBS
DTNB
EC50
EDTA
Ef
Dulbecco’s Modified Eagle’s Medium
deuterated dimetylsulphoxide
deoxyribonucleic acid
Dulbecco’s Phosphate Buffered Saline
5,5'-dithiobis-(2-nitrobenzoic acid)
half maximal effective concentration
ethylenediaminetetraacetic acid
Electronic efficiency
FBS
Fetal Bovine Serum
IC50
half maximal inhibitory concentration
infrared
IR
m
medium infrared signal
MALDI-TOF matrix-assisted laser desorption ionization time of flight
MTT
NMR
PBS
RNA
r.t.
3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
nuclear magnetic resonance
Phosphate Buffered Saline
ribonucleic acid
room temperature
s
strong infrared signal
SDS
Sodium Dodecyl Sulphate
29