Synthesis and characterization of azolate gold(i) phosphane complexes as thioredoxin reductase inhibiting antitumor agents

Article abstract of DOI:10.1039/c2dt11781a

Following an increasing interest in the gold drug therapy field, nine new neutral azolate gold(i) phosphane compounds have been synthesized and tested as anticancer agents. The azolate ligands used in this study are pyrazolates and imidazolates substituted with deactivating groups such as trifluoromethyl, nitro or chloride moieties, whereas the phosphane co-ligand is the triphenylphosphane or the more hydrophilic TPA (TPA = 1,3,5-triazaphosphaadamantane). The studied gold(i) complexes are: (3,5-bis-trifluoromethyl-1H-pyrazolate-1-yl)- triphenylphosphane-gold(i) (1), (3,5-dinitro-1H-pyrazolate-1-yl)- triphenylphosphane-gold(i) (2), (4-nitro-1H-pyrazolate-1-yl)-triphenylphosphane- gold(i) (5), (4,5-dichloro-1H-imidazolate-1-yl)-triphenylphosphane-gold(i) (7), with the related TPA complexes (3), (4), (6) and (8) and (1-benzyl-4,5-di- chloro-2H-imidazolate-2-yl)-triphenylphosphane-gold(i) (9). The presence of deactivating groups on the azole rings improves the solubility of these complexes in polar media. Compounds 1-8 contain the N-Au-P environment, whilst compound 9 is the only one to contain a C-Au-P environment. Crystal structures for compounds 1 and 2 have been obtained and discussed. Interestingly, the newly synthesized gold(i) compounds were found to possess a pronounced cytotoxic activity on several human cancer cells, some of which were endowed with cis-platin or multidrug resistance. In particular, among azolate gold(i) complexes, 1 and 2 proved to be the most promising derivatives eliciting an antiproliferative effect up to 70 times higher than cis-platin. Mechanistic experiments indicated that the inhibition of thioredoxin reductase (TrxR) might be involved in the pharmacodynamic behavior of these gold species.

Full text of DOI:10.1039/c2dt11781a

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PAPER
Synthesis and characterization of azolate gold(I) phosphane complexes as
thioredoxin reductase inhibiting antitumor agents†
Rossana Galassi,*a Alfredo Burini,a Simone Ricci,a Maura Pellei,a Maria Pia Rigobello,b Anna Citta,b
Alessandro Dolmella,c Valentina Gandinc and Cristina Marzano*c
Received 20th September 2011, Accepted 8th February 2012
DOI: 10.1039/c2dt11781a
Following an increasing interest in the gold drug therapy field, nine new neutral azolate gold(I) phosphane
compounds have been synthesized and tested as anticancer agents. The azolate ligands used in this study
are pyrazolates and imidazolates substituted with deactivating groups such as trifluoromethyl, nitro or
chloride moieties, whereas the phosphane co-ligand is the triphenylphosphane or the more hydrophilic
TPA (TPA = 1,3,5-triazaphosphaadamantane). The studied gold(I) complexes are: (3,5-bis-
trifluoromethyl-1H-pyrazolate-1-yl)-triphenylphosphane–gold(I) (1), (3,5-dinitro-1H-pyrazolate-1-yl)-
triphenylphosphane–gold(I) (2), (4-nitro-1H-pyrazolate-1-yl)-triphenylphosphane–gold(I) (5), (4,5-
dichloro-1H-imidazolate-1-yl)-triphenylphosphane–gold(I) (7), with the related TPA complexes (3), (4),
(6) and (8) and (1-benzyl-4,5-di-chloro-2H-imidazolate-2-yl)-triphenylphosphane–gold(I) (9). The
presence of deactivating groups on the azole rings improves the solubility of these complexes in polar
media. Compounds 1–8 contain the N–Au–P environment, whilst compound 9 is the only one to contain
a C–Au–P environment. Crystal structures for compounds 1 and 2 have been obtained and discussed.
Interestingly, the newly synthesized gold(I) compounds were found to possess a pronounced cytotoxic
activity on several human cancer cells, some of which were endowed with cis-platin or multidrug
resistance. In particular, among azolate gold(I) complexes, 1 and 2 proved to be the most promising
derivatives eliciting an antiproliferative effect up to 70 times higher than cis-platin. Mechanistic
experiments indicated that the inhibition of thioredoxin reductase (TrxR) might be involved in the
pharmacodynamic behavior of these gold species.
against inflammatory diseases such as rheumatoid arthritis, and
then for their ascertained antineoplastic activity. Simultaneously
Introduction
The consolidated and exceptional antitumor activity of platinum
(II) compounds on the clinical treatment of different types of
cancers is unfortunately accompanied by serious side effects and
by the occurrence of cis-platin resistance.1 The continuous
struggle to overcome these latter drawbacks led to two other
platinum based drugs oxaliplatin and carboplatin.2 However,
chemists are still seeking new and more potent metal based
drugs to reach a good compromise between an optimal cytotoxic
activity and minor liver and kidney toxicities, typical for these
kinds of drugs.3 Coinage metals have been taken in consideration
as alternative metals in the design of metal based drugs; among
them, some gold(I)complexes had success first for their efficacy
with the cytotoxicity studies, a great effort has been made to
identify the molecular targets for gold(I) based drugs that are
comparable to platinum based drugs, but this topic is still a chal-
lenge for researchers.4,5 Classes of gold(I) compounds containing
phosphanes as ligands6–8 or ancillary ligands9–13 have been
studied, which are similar to the well-studied auranofin, consist-
ing of a sugar frame bound to a gold(I) triethylphosphane ligand.
Some examples of phosphane complexes, being the most active
in vivo, reached the pre-clinical pharmacological evaluation.14
Unfortunately, severe hepatotoxicity was verified and stopped
their development. The conclusion of this promising study was
that the hydro/lipophilicity balance of the gold complex is the
likely key to be focused on in order to achieve the goal.15–17
Hence, the design of gold complexes for biological testing
should consider the involvement of more hydrophilic phosphane
and/or other polar or more bio-compatible co-ligands. Based on
these previous studies, a new class of gold(I) complexes with
phosphane ligands and biomolecules such as azoles has been
designed in this study. This choice was particularly important
given that pyrazoles and imidazoles play a pivotal role in the
coordination chemistry of gold in the +1 oxidation state. More-
over, in pyrazoles, substitution at the 3 and/or 5 position adjacent
aSchool of Science and Technology, Chemistry Division, University of
Camerino, via S. Agostino, 1, 62032 Camerino, Italy. E-mail: rossana.
galassi@unicam.it; Fax: +39 737 637345; Tel: +39 737 402243
bDipartimento di Chimica Biologica, Università di Padova, viale
G. Colombo 3, 35131 Padova, Italy
cDipartimento di Scienze Farmaceutiche, Università di Padova, via
Marzolo 5, 35131 Padova, Italy
†CCDC 847347 and 847346 for compounds 1 and 2. For crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/
c2dt11781a.
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 5307–5318 | 5307

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Table 1 List of the compounds
to the nitrogen atoms is particularly easy to achieve, and can
strongly affect the steric environment around the N-donors and
any metal ions coordinated to them.18 Pyrazolates can coordinate
as monodentate, exo-(k2) and endo-bidentate (k1 : k1, μ). In the
bridging mode, pyrazolate ligands can give rise to nine-mem-
bered cyclic trinuclear Au(I) derivatives in a rather high yield by
treating the pyrazolate ligands with LAuCl precursors, where L
is a labile ligand. In the literature several structures of these
[μ-pz–Au3] compounds are reported19–21 and the success of their
syntheses does not seem to be affected by the steric hindrance of
the substituents in the 3, 4 and 5 positions of the pyrazole ring,
as highlighted by the synthesis of cyclic [μ-pz–Au3] compounds
with a very hindered 3,5-disubstituted-pyrazolate.22,23 However,
if L in the starting gold complex is dimethylsulfide, tetrahy-
drothiophene or triphenylarsine ligands, dinuclear,24,25 trinuc-
lear19,20 or polynuclear derivatives26 can be obtained. On the
other hand, when the gold(I) chloride precursor contains a phos-
phane as ligand, the exchange reaction is more difficult and
mononuclear derivatives can be readily obtained.19,27–29
Yield,
%
Solvents used in the
synthesis
Compound
Formula
1
2
3
4
5
6
7
8
9
3,5-pzCF3AuPPh3
3,5-pzNO2AuPPh3
3,5-pzCF3AuTPA
3,5-pzNO2AuTPA
4-pzNO2AuPPh3
4-pzNO2AuTPA
75
90
75
76
55
59
84
78
40
CH3OH–THF
THF
CH3CN–CH3OH
CH3OH–H2O
THF–CH3OH
THF–CH3OH–CH3CN
CH3OH
4,5-imClAuPPh3
4,5-imClAuTPA
4,5-im1-Bz,ClAuPPh3
CH3OH
THF
the pyrazole or imidazole rings, reactions between substituted-
azolates and Ph3PAuCl or TPAAuCl were carried out to obtain
mononuclear gold(I) derivatives. In this study pyrazole and imi-
dazole phosphane gold(I) compounds have been synthesized,
characterized and biologically evaluated for their cytotoxic
effects and for some specific enzyme inhibition actions. The
azoles chosen for these complexes were pyrazoles and imida-
zoles containing deactivating groups (nitro-, trifluoromethyl- or
halide groups) and phosphane as the co-ligands such as triphe-
nylphosphane (PPh3) and 1,3,5-triaza-phosphaadamantane (TPA).
The newly synthesized azolate gold(I) compounds have been
tested for their antitumor properties on a wide panel of human
cancer cell lines including cis-platin resistant and multidrug
resistant (MDR) phenotypes. Based on the cytotoxicity screen-
ing, the most promising drug candidates, gold(I)-triphenylpho-
sphane derivatives, have been also evaluated for their capacity to
inhibit cytosolic and mitochondrial isoforms of TrxR, both in
vitro (in a cell-free system) and in human ovarian 2008 cancer
cells. In 2008 cells, the inactivation of the closely related
flavoenzymes glutathione reductase (GR) and glutathione peroxi-
dase (GPx) was also examined with the aim of proving their
selectivity towards TrxR.
AzNa þ LAuCl ) AzAuL þ NaCl
ð1Þ
(Az = N-pyrazolate or N-imidazolate; L = PPh3 or TPA = 1,3,5-
triazaphosphaadamantane)
Imidazolates are heterocycles similar to pyrazolates and their
coordination modes on gold(I) can lead to mononuclear30–32 or
trinuclear cyclic gold(I) complexes.33,34 However, contrary to
pyrazolates, different synthesis strategies can be adopted. Mono-
nuclear imidazole or pyrazole phosphane Au(I) compounds have
been studied for their antimicrobial activity27 while the cytotoxic
activity of phosphane Au(I) compounds and several pyridylpho-
sphane Au(I) complexes has been ascertained and evaluated on
many cancer cell panels. Moreover, phosphane complexes such
as [Au(d2pypp)2]Cl where d2pypp = 1,3-bis(di-2-pyridylpho-
sphino)propane, at sub-micromolar concentrations, selectively
induce apoptosis in breast cancer cells but not in normal breast
cells. Apoptosis was induced via the mitochondrial pathway,
which involved mitochondrial membrane potential depolaris-
ation, as well as the depletion of the glutathione pool and the
activation of capsase-3 and capsase-9.7,16,35 The mechanism of
action for gold(I) phosphane complexes is still a challenge and
several likely molecular targets have been identified. In general,
these complexes are powerful inhibitors of thioredoxin reductase
(TrxR).5 This enzyme plays a crucial role in the intracellular
redox balance and takes part in a wide variety of cell activities
including cell proliferation, cell signalling, apoptosis, angiogen-
esis, and embryogenesis.36 TrxR is involved in many aspects of
tumor pathophysiology and many cancer cell lines and tumors
exhibit increased production of TrxR. Furthermore, upregulation
of TrxR has been associated with resistance to chemotherapy,37
in addition to auranofin having been recently shown to open
mitochondrial pathways to apoptosis, through the selective inhi-
bition of mitochondrial thioredoxin reductase, even in human
ovarian cancer cells resistant to cis-platin.38
Results and discussion
Syntheses and characterizations
The list of synthesized compounds, yields and solvent conditions
are reported in Table 1 and their schematic structures are shown
in the Chart 1. The syntheses of complexes 1–8 have been per-
formed by following the eqn (1) reaction scheme. Complex 9 is
the only one possessing the C–Au–P environment and it was
obtained by the imidazole C2-lithiation at −40 °C in THF fol-
lowed by the addition of Ph3PAuCl. The starting materials used
to obtain complexes 1–8 show different solubility in most
common solvents. In order to overcome this disadvantage, mix-
tures of solvents were sometime used. When the starting azole
was neutral, the first step of the synthesis was the N–H deproto-
nation with methanolic NaOH or KOH (compounds 1, 3, 7 and
8) or with NaH/THF (compounds 5 and 6). The synthesis yields
range from 55 to 90% with the lowest yields for complexes with
4-nitropyrazole as the ligand. Several methods and different sol-
vents were tested to obtain a soluble product from the reaction of
3,5-dinitropyrazolate and TPAAuCl. At any rate, the scarce
soluble compound 4 was obtained with a good yield.
Gold(I) derivatives with 3,5-dialkyl or diaryl pyrazolate as
ligands show peculiar features in the field of emission proper-
ties25,39 and material science,28 while their low solubility in
polar solvents makes it difficult to consider these compounds for
biological testing. With the introduction of more polar groups to
5308 | Dalton Trans., 2012, 41, 5307–5318
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Chart 1 Schematic view of the compounds synthesized in this study.
Compounds 1–9 were characterized by elemental analysis, IR,
1H and 31P NMR spectroscopies and by ESI mass spectrometry.
Compounds 1 and 2 were isolated as single crystals and the
crystal structure determinations were performed by single-crystal
X-ray Diffraction. The NMR characterization of compounds 1–9
C–Cl groups upon coordination to the gold–phosphane moieties.
The band due to Pquat–Cph stretching is visible at 1100 cm−1 in
all the compounds.40
Crystal structures descriptions
1
shows the expected signals in the H NMR spectra, and singlets
in the 31P NMR spectra. Substitution of the chloride with the
azolate ligand does not introduce significant shifts in the 31P
NMR signals of the phosphane moieties. Only in the case of
compound 5, a signal broadening was observed at room tempera-
ture, which was likely due to linkage isomerism. Phosphane co-
ligand fast exchange likely occurs in acetone solution. In fact, by
adding a molar amount of free PPh3 to compound 2 in the NMR
tube, only a slight shift was observed (from 31.06 to
31.58 ppm). This behavior had already been observed in CD2Cl2
by Oda in [Au(L)PPh3] where HL was either imidazole or pyra-
zole.27 The ESI-MS characterization reveals good ionization in
both positive and negative fields in acetonitrile solutions. In the
former, bis-phosphanegold(I) cations were recorded for most of
the complexes, while in the negative field monoanionic (Az)2Au
moieties were observed. Compound 8 was not investigated by
ESI due to its low solubility in CH3CN or other ESI compatible
solvents. In the IR spectra the characteristic bands for azoles
were found in the range 3050–3170 cm−1 (C–H azole stretch-
ing), 1550–1650 cm−1 and 1460–1510 cm−1 attributable to
CvC and CvN stretchings. Small shifts were also recorded for
the bands attributed to the vibrational modes of NO2, CF3 and
The crystal structure of complexes 1 (molecules I and II) and 2
are shown in Fig. 1. Crystal data are reported Table 2 while
selected bond distances and bond angles are in Table 3. The
overall shapes of the three molecules are similar and this is
revealed by an inspection made with the Molecule Overlay
routine in Mercury.41 The fit of the skeleton of 2 with those of
molecules I and II of 1 returns RMSDs of 0.11 and 0.06 Å,
respectively.
The geometries are dictated by the linear coordination of the
gold atoms, and the P–Au–N angles are 174.5(2)° in 2 and
178.0(1)° and 175.1(1)° in molecules I and II of 1, respectively.
This kind of deviation from the ideal 180° for linear coordination
is not unusual for a two-coordinated gold complex. In fact, a
search in the CCDC42 database reveals that the mean P–Au–N
angle is 175.4° in about 200 reported complexes. The unit cell is
filled in such a way that no Au–Au interaction can be detected in
either complex.
By looking at the P–Au–pyrazolate moieties, all the atoms of
this fragment are coplanar within 0.10 Å in 2 and within 0.05 Å
in the two independent molecules of 1. The pyrazolate ligands
themselves are almost perfectly planar in both complexes. They
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Fig. 1 ORTEP view of the complexes (1) (left) and (2) (right), together with the numbering scheme. Ellipsoids are at the 40% probability level; the
hydrogen atoms have been omitted. In the view of 1, the C-F bonds of the trifluoromethyl residue of molecule II with refined site occupancy of
0.48 have been drawn with dashed lines and the labeling has been limited for clarity.
compares quite well with the mean (1.816 Å) for nearly
1600 monodentate –PPh3 ligands bound to a gold centre in the
CCDC database.
Table 2 Crystallographic data for the two complexes (1) and (2)
1
2
The CCDC repository was also inspected for mononuclear 2-
coordinate (N, P) gold complexes in which the nitrogen and the
phosphorous atoms belong, respectively, to a penta-atomic ring
and to a triphenylphosphane ligand. This search returned only a
tenth of the entries from few research groups.20,27,29,43–48 In
these structures, the ranges for the Au–P, A u –N distances are
2.229–2.243 and 1.971–2.077 Å, respectively. Bond lengths in
the present work fit within these ranges except for the Au–P
length in 2 (see Table 3), which is just a little bit shorter (2.224
(2) Å). Among known structures, those showing more similarity
to 1 and 2 (that is, showing exactly a pyrazolate (1-) and a triphe-
nylphosphane ligand) are those with the CCDC codes
LAHMAJ,20 namely 3,5-bis-[(4-octyloxy)phenyl]-1H-pyrazo-
late)(triphenylphosphine)gold and QESRUB,27 [(triphenylpho-
sphine)-(pyrazolyl)–gold(I)]; however, the structures found in
this work with the codes WULFIT43 are the ones that better
resemble the Au–P, A u –N bonds and they were [(1H-benzotria-
zol-1-yl)-triphenylphosphine–gold(I)], and MALRIA,47 [tetrazo-
lyl-triphenylphosphine–gold(I)] (with 1-I), which also shows a
negatively charged nitrogen ligand, and OBIVUR48 [(3,5-bis(4-
n-butoxyphenyl)pyrazole-N)-(triphenylphosphine)–gold(I)p-tolu-
enesulfonate] (with 1-II).
Empirical formula
Formula weight
Wavelength (Å)/
temperature (K)
Crystal system
Crystal size
C23H16N2F6PAu
662.31
0.71073/293(2)
C21H16N4O4PAu
616.31
0.71073/293(2)
Monoclinic
0.35 × 0.15 ×
0.15
P 21/c (No. 14)
14.633(4)
9.092(2)
35.389(5)
97. 79(3)
4664.8(17)
8
Monoclinic
0.20 × 0.15 ×
0.12
P 21/c (No. 14)
8.1300(16)
11.393(2)
23.339(5)
97.60(3)
2142.8(7)
4
Space group
a (Å)
b (Å)
c (Å)
β (°)
Volume (Å3)
Z (molecules/unit cell)
Calculated density (Mg m−3
Absorption coefficient, μ
)
1.886
6.437
1.910
6.980
(cm−1
)
F (000)
Independent (unique)
reflections
Observed reflections
[I > 2σ(I)]
Data/parameters/restraints
Goodness-of-fita on F2
Final R indices [I > 2σ(I)]
2528
9147
1184
4165
6221
3856
9147/593/0
0.932
4165/280/0
1.364
R1b = 0.0368;
wR2c = 0.0891
R1b = 0.0343;
wR2c = 0.0887
Largest difference peak and
0.991 and −0.588 0.847 and −1.085
hole (eÅ–3
)
The examination of the packing diagram did not reveal any
classic hydrogen bonds for either of the complexes. In 2, an
inspection of the shortest non-bonding contacts showed that the
O(2) and O(3) atoms of one molecule loosely (O–H > 2.50 Å)
interact, respectively, with the hydrogen atoms bonded to the C
(5) and C(14) of a nearby molecule. The motif develops along
the crystallographic b axis and appears to be a consequence of
π–π stacking effects. The molecules of 2 are piled along the
same axis in a head-to-tail arrangement so that the pyrazolate
ligand of one molecule faces a second pyrazolate on one side.
On the other side, it faces the phenyl ring of the PPh3 moiety,
which is almost coplanar with it. The distance between the cen-
troids of the facing pyrazolate ligands is 3.50 Å, and 3.86 Å
between the pyrazolate and the phenyl rings (compared with
3.40 Å in graphite).
a Goodness-of-fit = [∑(w(Fo − Fc ) )/(Nobsevns − Nparams)]1/2, based on
2
2 2
all data; b R1 = ∑(|Fo| − |Fc|)/∑|Fo|; c wR2 = [∑[w(Fo − Fc ) ]/
2
2 2
∑[w(Fo ) ]]1/2
2 2
.
are arranged in such a way that the mean plane of the ligand is
close to the mean plane of one of the three phenyl rings of the
PPh3 ligands and roughly orthogonal to the remaining two. In 2,
the dihedral angles made by these planes are 9.8, 63.9, 83.6°; in
1, the corresponding values for molecules I and II are 31.9, 69.7,
84.9° and 22.0, 69.4, 71.7°, respectively. As for the trigonal
pyramidal PPh3 moiety, it shows the expected propeller-like
arrangement of the three phenyl rings in both 1 and 2. The
average planes encompassing the three phenyl rings in the two
complexes are roughly orthogonal, showing a mean value
of 77°. The average P–Cipso bond length is 1.813 Å, and this
In 1, the shortest non-bonding contacts are those coupling
molecules I and II via the N(3) and N(4) atoms if the disordered
5310 | Dalton Trans., 2012, 41, 5307–5318
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Table 3 Selected bond lengths (Å) and angles (°) for complexes (2) and (1). The column for 1 reports the parameters for the two independent
molecules (I and II) within the asymmetric unit
2
1
Molecule I
Molecule II
Au–P
2.224 (2)
2.065 (5)
1.339 (7)
1.339 (8)
Au(1)–P(1)
Au(1)–N(1)
N(1)–N(3)
N(1)–C(1)
2.239 (1)
2.048 (5)
1.340 (7)
1.356 (7)
Au(2)–P(2)
Au(2)–N(2)
N(2)–N(4)
N(2)–C(24)
2.243 (2)
2.058 (5)
1.356 (6)
1.340 (8)
Au–N(1)
N(1)–N(2)
N(1)–C(3)
a
a
a
P–CPh
1.810
P–CPh
1.818
P–CPh
1.811
P–Au–N(1)
Au–P–C(4)
Au–P–C(10)
Au–P–C(16)
Au–N(1)–N(2)
Au–N(1)–C(3)
174.5 (2)
110.9 (2)
111.0 (2)
115.9 (2)
122.1 (4)
128.8 (4)
P(1)–Au(1)–N(1)
Au(1)–P(1)–C(6)
Au(1)–P(1)–C(12)
Au(1)–P(1)–C(18)
Au(1)–N(1)–N(3)
Au(1)–N(1)–C(1)
178.0 (1)
113.1 (2)
111.6 (2)
113.2 (2)
118.8 (4)
131.7 (4)
P(2)–Au(2)–N(2)
Au(2)–P(2)–C(29)
Au(2)–P(2)–C(35)
Au(2)–P(2)–C(41)
Au(2)–N(2)–N(4)
Au(2)–N(2)–C(24)
175.1 (1)
111.7 (2)
109.4 (2)
114.8 (2)
121.3 (4)
127.5 (4)
a Mean of three values.
CF3 residue is not considered. The latter are, again, loosely
connected (N–H > 2.48 Å) with the hydrogen atoms bonded
to the C(9) and C(46) of a proximal molecule. Contrary to 2
above, however, these contacts do not originate π–π stacking
motifs.
than those of cis-platin (average IC50 of 0.89 and 0.68 μM for 1
and 2, respectively). Interestingly, these complexes reached anti-
tumor activities in the low micromolar range, which were even
lower than those obtained with the phosphine gold(I) drug
auranofin.38,48
With all the azolate gold(I) complexes, very interesting results
have been obtained against the ovarian carcinoma cis-platin-
resistant (C13*) cells. In C13* cells, cis-platin resistance has
been correlated with reduced cell drug uptake, high cellular
TrxR and glutathione levels, and enhanced repair of DNA
damage.49 Cytotoxicity assays testing all the gold(I) derivatives
1–9 against the 2008/C13* cell line pair showed a similar
pattern of response across the parental and resistant sub-lines and
allowed the calculation of RF values (RF = Resistant Factor,
which is defined as the ratio between IC50 values calculated for
the resistant cells and those arising from the sensitive ones)
roughly 7.5-fold lower than that obtained with cis-platin (RF =
9.0) (see Table 4). These data clearly reveal no cross-resistance
phenomena.
Additionally, gold(I) complexes were tested against a multi-
drug resistant (MDR) colon carcinoma subline, LoVo MDR
cells. It is well known that acquired MDR, whereby cells
become refractory to multiple drugs, represents a major barrier
to the success of chemotherapy. Although most metal complexes
are not P-glycoprotein substrates, several multidrug resistance
proteins (MRP1, MRP2, MRP4) have been found to be involved
in Pt uptake and are responsible for its efflux/afflux from
cells.49–51 The resistance of LoVo MDR cells to doxorubicin, a
drug belonging to the MDR spectrum, is associated with an
overexpression of the multi-specific drug transporters, such as,
for example, the 170 kDa P-glycoprotein (P-gp).52 Tested on a
LoVo/LoVo MDR cell line pair, pyrazolategold(I)-triphenylpho-
sphane and -TPA derivatives allow the calculation of an RF
value up to 10 times lower than that obtained with doxorubicin,
clearly suggesting that pyrazolategold(I) complexes are not
potential MDR substrates. Conversely, 4,5-dichloro-1-imidazo-
late–gold(I) derivatives 7 and 8 showed a significantly lower
cytotoxicity potency against resistant sub-line LoVo MDR,
attesting they do not overcome MDR phenomena.
Biological studies
The antiproliferative effects of gold(I) azolate compounds were
investigated in a panel of various human cancer cells containing
samples of breast (MCF-7), lung (A549), cervical (A431), colon
(LoVo and LoVo MDR), and ovarian (2008 and C13*) cancers.
Cytotoxicity was evaluated by means of an MTT test after 72 h
of treatment with increasing concentrations of the tested com-
pounds. For comparison purposes, the cytotoxicity of cis-platin,
the most widely used metallodrug, was evaluated in the same
experimental conditions. The IC50 values, calculated from dose-
survival curves, have been reported in Table 4. Uncoordinated
azole ligands proved to be scarcely effective in decreasing
cancer cell viability over 7 cell lines. Compound 4 was not
tested for its cytotoxicity owing to the poor solubility in DMSO
and in other solvents suitable for biological testing. Among the
gold(I) coordination compounds, triphenylphosphane derivatives
proved to be more effective with respect to the TPA analogues.
The TPA derivatives 3, 6 and 8 showed mean IC50 (μM) values
(excluding those calculated for LoVo MDR and C13*cells) of
32.81 (27.11–43.44), 17.92 (10.31–24.35) and 12.15
(6.48–17.45), respectively, which were from 2 to 4 times higher
than that of cis-platin (7.63 μM). The triphenylphosphane
derivatives 5 and 7 showed a relevant antiproliferative activity
eliciting IC50 values (μM) within the same order of magnitude
(average IC50 of 1.03 and 1.21 μM for 5 and 7, respectively) and
about 6 times lower than cis-platin. Compound 9, possessing the
C–Au–P environment, despite retaining an antiproliferative
activity slightly lower than that of the relative compound 7 with
an N–Au–P environment, displayed a cytotoxic potency about 2-
fold higher than that of the reference metallodrug cis-platin.
Notably, compounds 1 and 2 emerged as the most effective in
killing cancer cells, with IC50 averages roughly 10 times lower
This journal is © The Royal Society of Chemistry 2012
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Table 4 In vitro antitumor activity
IC50 (μM) S.D.
MCF-7 A549
Compound
A431
LoVo
LoVo MDR
R.F.
2008
C13*
R.F.
PPh3Au(3,5-pzCF3) 1
PPh3Au(3,5-pzNO2) 2
TPAAu(3,5-pzCF3) 3
TPAAu(3,5-pzNO2) 4
PPh3Au(4-pzNO2) 5
TPAAu(4-pzNO2) 6
PPh3Au(4,5-imCl) 7
TPAAu(4,5-imCl) 8
1.03 0.59 0.96 0.34 0.63 0.31 0.90 0.52
0.53 0.35 0.49 0.21 0.82 0.40 0.73 0.30
43.44 2.15 32.93 2.38 29.47 1.22 31.12 3.42 54.81 2.12
1.49 2.54
1.23 0.95
1.7
1.7
1.7
—
1.1
1.7
9.1
4.1
2.0
1.3
1.8
—
0.91 1.01 0.72 0.19 0.8
0.81 0.32 0.52 0.22 0.8
27.11 2.31 33.36 1.18 1.2
ND
ND
ND
ND
ND
0.98 1.21
ND
ND
—
1.62 0.97 1.03 0.25 0.51 0.21 0.86 0.67
24.35 2.35 23.24 2.31 15.32 1.54 10.13 0.91 17.12 3.35
1.12 1.33 2.01 1.18 1.9
16.54 3.96 27.75 3.01 1.7
2.17 0.87 1.21 0.94 0.6
15.76 1.73 9.45 1.93 0.6
3.78 1.21 4.04 1.12 1.7
39.97 1.11 51.55 2.24 1.3
33.38 1.13 58.63 3.14 1.7
1.13 0.74 3.05 1.52 1.13 0.49 1.05 0.34
13.63 2.08 17.45 2.11 7.45 1.05 6.48 1.27
9.58 1.34
26.96 3.51
6.66 1.02
PPh3Au(4,5-im1-Bz,Cl) 9 4.16 0.97 3.99 1.25 1.98 0.52 3.19 0.74
3,5-pzCF3
3,5-pzNO2
4-pzNO2
61.63 2.62 38.61 2.20 49.92 2.27 41.13 3.42 53.21 3.47
45.63 0.93 44.49 0.91 38.24 2.55 30.34 1.77 54.87 2.96
>100
67.12 1.64 56.72 1.54 52.51 1.83 51.12 1.32 >100
8.51 1.37
(1.11 0.86) (19.21 2.37)
>100
>100
>100
>100
>100
>100
—
4,5-ImCl
—
60.34 3.04 44.52 1.24 —
cis-platin (doxorubicin) 10.31 1.36 13.42 1.96 2.08 1.06 8.03 1.23
1.1 (17.3) 4.35 1.59 39.34 2.92 9.0
S.D. = standard deviation. IC50 values were calculated by four parameter logistic model (P < 0.05). Cells (3–8 × 104 mL−1) were treated for 72 h with
increasing concentrations of tested compounds. Cytotoxicity was assessed by MTT test. R.F. (resistance factor) = IC50 resistant cells/IC50 sensitive
cells.
many other gold(I) complexes, including auranofin and other
phosphine gold(I) complexes,55 and may be consistent with the
different sequence of the two TrxR isoforms and the greater
acidity of TrxR1.56
Table 5 In vitro inhibition of purified TrxR and GR
TrxR1
TrxR2
IC50 (nM)
GR
IC50 (μM)
Compound
IC50 (nM)
Tested against GR, compounds 5 and 7 revealed a significant
ability in inhibiting GR (IC50 of 0.8 and 0.13 μM for 5 and 7,
respectively). Differently, 1 and 2 proved to have a lower effect
with IC50 values in the micromolar range, one order of magni-
tude higher than those detected for TrxR isoforms. This result
clearly attests a significant selectivity for TrxR inhibition over
GR inhibition, similarly to data previously obtained with other
phosphane gold derivatives.55,56
1
2
5
7
3.50
38.12
48.12
258.30
33.57
1.96
1.78
0.08
0.13
15.80
86.54
7.14
S.D. = standard deviation. TrxR1 and TrxR2 activity was assessed by
measuring NADPH-dependent reduction of DTNB at 412 nm; GR
activity was followed at 340 nm. IC50 values were calculated by four
parameter logistic model (P < 0.05).
Inhibition of redox enzymes in human ovarian cancer cells
In vitro inhibition of purified TrxR and GR
TrxR and GR inhibition were also evaluated in 2008 cells treated
for 12 h with IC50 concentrations of gold(I)-triphenylphosphane
derivatives. TrxR activity was assayed by measuring at 412 nm
NADPH-dependent reduction of DTNB (Fig. 2, panel A) as well
as TrxR-mediated insulin reduction (Fig. 2 panel A, insert a).
Compounds 5 and 7 decreased TrxR activity by roughly 60%
whereas 1 and 2 were found to be able to decrease cellular TrxR
activity by approximately 85% with respect to untreated cells.
Conversely, disulfide isomerase GR was less intensely inhibited
by these gold(I) complexes (Fig. 2, panel B and C). The tested
compounds were also evaluated as potential inhibitors of GPx, a
different Sec-containing redox enzyme. Interestingly, none of the
tested complexes caused a significant decrease in GPx activity,
attesting the gold(I)-triphenylphosphane derivative selectivity
towards the TrxR enzyme. This difference shown by gold(I)
derivatives in the inhibitory potencies of TrxR and GPx may be
correlated with Sec accessibility in the selenoenzyme active
sites. Indeed, unlike the flexible C-terminus active site of TrxR,
the active site of GPx is located at the N-terminal end of
α-helices, surrounded by aromatic side chains.57 That context
may protect Sec from a gold(I) electrophilic attack.
Based on the cytotoxicity test results, gold(I)-triphenylphosphane
derivatives (1, 2, 5 and 7) were selected for further molecular
pharmacological studies. In reality, as the selenoenzyme TrxR
has been generally recognized as the most relevant molecular
target for gold complexes,53 the ability of the azole gold(I)-tri-
phenylphosphane complexes to inhibit TrxR activity has been
evaluated. Moreover, their inhibitory activity towards disulfide
reductase GR, which is structurally closely related to TrxR, has
been assessed.
The inhibitory effects of gold(I) complexes on rat isolated
cytosolic (TrxR1) or mitochondrial (TrxR2) isoforms of TrxR
were measured according to standard procedures.54 Gold(I) com-
plexes were tested at increasing concentrations and IC50 values
were calculated from the dose-effect curves (Table 5). The cyto-
solic isoform appeared markedly inhibited by 1, 2 and 7 deriva-
tives at nanomolar concentrations (IC50 of 15.8 nM, 3.5 nM and
7.14 for 1, 2 and 7, respectively). On the other hand, higher con-
centrations were required for a reduction of 50% of TrxR2
activity for all the derivatives compared with TrxR1 (Table 5).
This difference in sensitivity had been previously underlined for
5312 | Dalton Trans., 2012, 41, 5307–5318
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the 4- and 5-position in addition to the use of phosphane ligands
with different cone angle (PPh3 = 145° and TPA = 102°), gave
mononuclear derivatives as the main product. The introduction
of deactivating groups in the azolate rings had a double function.
The first aim was to avoid the typical cyclization resulting in
nine-membered cycles or polynuclear derivatives and the second
was to get a better solubility in polar solvents. Moreover, for the
latter scope, a more polar phosphane co-ligand as the TPA has
been employed too. However, in the biological tests, triphenyl-
phosphane derivatives possess better in vitro antitumor proper-
ties compared with the TPA analogues. By increasing the
deactivation potency of the substituents on the pyrazole ring an
increase of the antiproliferative activity has been achieved (2 > 1
> 5) among the pyrazolate gold(I)-triphenylphosphane series.
Complexes 1 and 2 were tested for their ability to inhibit cytoso-
lic and mitochondrial TrxR in vitro and they were found to
efficiently hamper enzyme activity at concentrations that did not
affect the related oxidoreductase GR. Moreover, in 2008 human
ovarian cancer cells, the scarce effect in inactivating the flavoen-
zymes GR and the Sec-containing enzyme GPx revealed for 1
and 2 a certain selectivity towards TrxR.
Experimental section
Synthesis
Elemental analyses (C, H, N, and S) were performed in-house
with a Fisons Instruments 1108 CHNS-O Elemental Analyser.
Melting points were taken on an SMP3 Stuart Scientific Instru-
ment. IR spectra were recorded from 4000 to 600 cm−1 with a
Perkin-Elmer SPECTRUM ONE System FT-IR instrument. IR
annotations used: br = broad, m = medium, mbr = medium
broad, s = strong, sh = shoulder, vs = very strong, w = weak and
1
vw = very weak. H and 31P NMR spectra were recorded on an
Oxford-400 Varian spectrometer (400.4 MHz for 1H and
162.1 MHz for 31P). Chemical shifts, in ppm, for 1H NMR
spectra are relative to internal Me4Si. 31P NMR chemical shifts
were referenced to a 85% H3PO4 standard. The 31P NMR spec-
troscopic data were accumulated with 1H decoupling. NMR
annotations used: br = broad, d = doublet, m = multiplet, s =
singlet. Electrospray mass spectra (ESI-MS) were obtained in
positive- or negative-ion mode on a Series 1100 MSD detector
HP spectrometer (1100 Series MSD detector), using an aceto-
nitrile or methanol mobile phase. The compounds were added to
the reagent grade acetonitrile to give solutions of approximate
concentration 0.1 mM. These solutions were injected (1 μl) into
the spectrometer via a HPLC HP 1090 Series II fitted with an
autosampler. The pump delivered the solutions to the mass spec-
trometer source at a flow rate of 300 μl min−1, and nitrogen was
employed both as a drying and nebulizing gas. Capillary vol-
tages were typically 4000 V and 3500 V for the positive- and
negative-ion mode, respectively. Confirmation of all major
species in this ESI-MS study was aided by comparison of the
observed and predicted isotope distribution patterns, the latter
calculated using the IsoPro 3.0 computer program. The used sol-
vents were HPLC grade and they were used as purchased, unless
water and oxygen sensitive reactions were led. In this last case
anhydrous and radicals free THF was obtained by treating the
solvent with Na/acetophenone under a N2 atmosphere.
Fig. 2 Effects of gold(I)–triphenylphosphane compounds on redox
enzymes in human ovarian cancer cells. 2008 cells were incubated for
12 hours with IC50 of tested compounds. Subsequently, cells were
washed twice with PBS and lysed. TrxR activity was tested by measur-
ing NADPH-dependent reduction of DTNB (A) and by insulin reduction
assay (insert a) at 412 nm; GR activity (B) and GPx (C) activities were
followed at 340 nm. All the values are the means SD of not less than
three measurements. Multiple comparisons were made by the one-way
analysis of variance followed by the Tukey–Kramer multiple comparison
test or ANOVA test (**p < 0.01; *p < 0.05).
Conclusions
The synthesis of nine new neutral azolate gold(I) phosphane
compounds has been performed with medium to good yields.
The use of pyrazoles substituted with deactivating groups in the
3- and 5- or 4-position and that of imidazole with chlorides in
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1075.3 (30) [Au(PPh3)2 + 3,5(NO2)2pzAu]+; ESI (−) (CH3CN)
m/z %: 156.9 (100) [3,5-(NO2)2pz]−, 376.6 (30). Elemental
analysis for C21H16AuN4O4P · 0.5THF, calcd %: C, 42.35; H,
3.09, N, 8.59. Found %: C, 42.50; H, 3.09; N, 8.40.
Materials
3,5-bis(trifluoromethyl)pyrazole and other chemicals were pur-
chased and used without further purification. The complex
Ph3PAuCl was synthesized from tetrachloridegold(I) acid and a
double molar amount of PPh3 in ethyl alcohol as previously
reported.58 The synthesis of the 3,5-dinitropyrazolate ligand
was performed as previously reported in literature,59 only the
purification was led with a slight modification to obtain the
pure sodium salt. In fact, after the second pyrolysis, the mixture
of 3,5-dinitropyrazole and 3-nitropyrazole was treated with
NaOH 1M. The resulting salts were then acidified by glacial
CH3COOH until pH 5 to protonate only the 3-nitropyrazole.
The pure sodium 3,5-dinitropyrazolate was extracted with
hot benzene and obtained as a solid with a yield of more
than 70%.
Synthesis of [(3,5-bis-trifluoromethyl-1H-pyrazolate-1-yl)-(1,3-
5-triaza-phosphaadamantane)–gold(I)] (3). To a CH3CN sol-
ution (6 mL) of 3,5-bis(trifluoromethyl)pyrazole (0.031 g,
0.15 mmol), 0.85 mL of 1% methanolic solution of KOH
(0.15 mmol) was added. To this solution, 1,3-5-triaza-phosphaa-
damantanegold(I)chloride (0.060 mg, 0.157 mmol) dissolved in
30 mL of CH3CN was added. The mixture reaction was stirred
for two hours at room temperature and for an hour at 40 °C.
After filtration, the solution was concentrated to dryness under
vacuum to obtain a pale yellow solid. The crude product was
then dissolved in CH2Cl2 (10 mL) and washed with water (3 ×
5 mL). The dichloromethane solution was then dried over a bed
of anhydrous Na2SO4 and evaporated to dryness to obtain a
white solid (65 mg) in 75% of yield. M. p. 200.1–202.3 °C.
1H-NMR (acetone-d6, 293 K): δ 6.99 (s, 1H, pz), 4.74 (d, 6H,
2JP-H = 12.8 Hz, PCH2N), 4.58 (s, 4H, NCH2Neq), 4.54 (s, 2H,
NCH2Nax). 31P{1H} NMR (acetone-d6, 293 K): δ −57.94 (s). IR
(cm−1): 3109w (C-Hpz), 3060w, 2952w, 2916w, 2867m, 1711m,
1672m, 1546m, 1497sh, 1438m, 1417m, 1411m, 1362m,
1338m, 1257vs, 1243vs, 1217vs, 1155m, 1108vs, 1094vs,
1040m, 1009vs, 972vs, 964vs, 945vs, 900m, 854m, 807vs,
752m, 738vs, 718m. ESI(+) (CH3OH), m/z (%): 242.3 (100)
[3,5(CF3)2pz + K]+. ESI(−) (CH3OH), m/z (%): 602.8 (100)
[(3,5(CF3)2pz)2 + Au]−. Elemental analysis for C11H13Au
F6N5P, calcd %: C, 23.71; H, 2.35; N, 12.57. Found %: C,
24.30; H, 2.28, N, 12.35.
Synthesis of [(3,5-bis-trifluoromethyl-1H-pyrazolate-1-yl)-tri-
phenylphosphane–gold(I)] (1). To a solution of CH3OH (6 mL)
and 3,5-bis(trifluoromethyl)pyrazole (0.1 g, 0.49 mmol),
2.75 mL of 1% methanolic solution of KOH (0.49 mmol) was
added. After a few minutes, 5 mL of a solution of Ph3PAuBF4 in
THF (0.49 mmol) was added. After magnetic stirring of an hour,
the suspension was filtered. The colorless solution was then con-
centrated to dryness under vacuum and an oily product was
obtained. The crude product was washed with hexane and crys-
tallized
by
a
mixture of CH2Cl2–hexane.
Yield
75%. M. p. 124.2–125.8 °C. 1H NMR (CD2Cl2, 293 K): δ
7.62–7.56 (m, 15H, PPh3), 6.96 (s, 1H, pz). 31P NMR (CD2Cl2,
293 K): δ 31.79 (s). IR (cm−1): 3144vw (C-Hpz), 3059vw,
3048vw, 1621vw, 1589w, 1546m, 1530m, 1497m, 1482m,
1436s (NO2), 1352m, 1332w, 1310w, 1253vs (NO2), 1220s,
1157s sh, 1139vs, 1101vs, 1075s, 1010vs, 974s, 925m, 849m,
816m, 745s, 712s, 689vs. ESI(+) (CH3CN) m/z %: 242.3 (18)
[3,5(CF3)2pz + K]+, 500.1 (30) [Au(PPh3) + CH3CN]+, 721.3
(25) [Au(PPh3)2]+, 1121 (100) [Au(PPh3)2 + 3,5(CF3)2pzAu]+;
ESI(−) (CH3CN) m/z, %: 202.9 (70) [3,5-(CF3)2pz)]−, 468.5
(55), 602.3 (100) [3,5(CF3)2pz)2Au]−. Elemental analysis for
C23H16AuF6N2P, calcd %: C, 41.71; H, 2.43; N, 4.23. Found %:
C, 41.82; H, 2.17; N, 4.54.
Synthesis of [(3,5-bis-dinitro-1H-pyrazolate-1-yl)-(1,3-5-triaza-
phosphaadamantane)–gold(I)] (4). To an aqueous methanol sol-
ution (20 mL of CH3OH and 1 mL of H2O) of sodium 3,5-dini-
tropyrazolate (0.046 g, 0.25 mmol), solid 1,3-5-triaza-
phosphaadamantanegold(I)chloride (0.100 g, 0.25 mmol) was
added. A lemon yellow precipitate was readily obtained. The
mixture reaction was stirred overnight. After filtration over a
paper filter a yellow solid was collected. The crude product
showed scarce solubility in most common solvents and it was
purified by washing with water (2 mL × 5 times) and further
drying under vacuum (0.101 g). Yield 76%. M. p.
213.1–214.8 °C with decomposition. IR (cm−1): 3149m
(C-Hpz), 2949m, 2911m, 2880m, 1547s, 1532m (NO2), 1490s,
1449m, 1418m, 1354s, 1321s, 1286m (NO2), 1246m, 1184m,
1097m, 1078m, 1043m, 1014s, 995m, 973vs, 946vs, 907m,
900m, 832m, 815m, 801m, 737vs. Elemental analysis for
C9H13AuN7O4P · CH3OH · NaCl, calcd %: C, 19.96; H, 2.85; N,
16.30. Found %: C, 20.66; H, 2.67, N, 16.56.
Synthesis of [(3,5-dinitro-1H-pyrazolate-1-yl)-triphenylpho-
sphane–gold(I)] (2). Solid Ph3PAuCl (0.494 g, 1 mmol) was dis-
solved in 10 mL of THF. To this solution solid AgBF4 (0.194 g,
1 mmol) was added. The suspension was stirred for 10 minutes
and sodium 3,5-dinitropyrazolate was then added (0.180 g,
1 mmol). The pale yellow suspension was stirred for another
hour. After filtration the solution was concentrated to dryness
under vacuum to obtain a pale yellow solid. The crude product
was washed with water to eliminate salts and the unreacted pyra-
zolate. The crystallization of the crude solid was performed by
dissolving the solid in THF and by layering hexane. Yield
90%. M. p. 162.4–163.5 °C with decomposition. 1H NMR
(acetone-d6, 293 K): δ 7.82–7.60 (m, 16H, PPh3 + pz), 3.63 (m,
2H, THF), 1.79 (m, 2H, THF). 31P NMR (acetone-d6, 293 K): δ
31.06 (s). IR (cm−1): 3168vw (C-Hpz), 3054vw, 3030vw,
1677w, 1585w, 1543s, 1489vs, 1449s, 1435vs, 1361vs, 1325vs,
1294s, 1181s, 1098vs, 1067m, 1046m, 1025m, 995m, 923w,
831s, 812s, 740vs, 711s, 679vs. ESI (+) (CH3CN) m/z %: 500.3
(37) [AuPPh3 + CH3CN]+, 721.3 (100) [Au(PPh3)2]+, 935 (10),
Synthesis
of
[(4-nitro-1H-pyrazolate-1-yl)-triphenylpho-
sphane–gold(I)] (5). To a THF solution (8 mL) of 4-nitropyra-
zole (100 mg, 1.13 mmol) solid NaH (0.0027 g, 1.13 mmol)
was added. To this suspension solid triphenylphosphanegold(I)
chloride (0.558 g, 1.13 mmol) was added. The suspension was
filtered after two hours of stirring. After filtration, the solution
was concentrated to dryness under vacuum and a white solid was
obtained. After dissolution in CH2Cl2, extractions with water (3
× 5 mL) were performed to remove the excess of nitropyrazole
5314 | Dalton Trans., 2012, 41, 5307–5318
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and sodium chloride. The organic phase was dried by anhydrous
Na2SO4 and evaporated to dryness to obtain the product as a
white solid (217 mg) in 55% yield. M. p. 170.8–172.1 °C with
decomposition. 1H-NMR (acetone-d6, 293 K): δ 8.31 (s, 2H),
7.71–7.61 (m, 15H, PPh3). 31P{1H} NMR (acetone-d6, 293 K):
δ 32.12 (s). IR (cm−1): 3119w (C-Hpz), 3075w, 3053w,
2958m, 2924w, 2856m, 1586w, 1549w, 1495s (NO2), 1479s,
1435s, 1400s, 1331m, 1311m, 1272vs (NO2), 1157m, 1100m,
1064m, 1023m, 997s, 943m, 860m, 812s, 744vs, 710s, 689vs.
ESI(+) (CH3CN), m/z (%): 721 (100) [Au(PPh3)2]+. ESI(−)
(CH3CN), m/z (%): 112.1 (20) [4-NO2pz]−, 180 (100) [4-
NO2pzH + 4-NO2pz]−. Elemental analysis for C21H17AuN3O2P,
calcd %: C, 44.15; H, 3.00; N, 7.35. Found %: C, 44.38; H,
3.09, N, 6.29.
1188m, 1101s, 1041m, 1011s, 997m, 963m, 920m, 812m, 743s,
711s, 689vs. ESI(+) (CH3CN), m/z (%): 1053.1 (100) [Au
(PPh3)2 + 4,5-Cl2Im]+. ESI(−) (CH3CN), m/z (%): 134.9 (100)
[4,5-Cl2Im]−, 468.9 (40) [Au(4,5-Cl-Im)2]−. Elemental analysis
for C21H16AuCl2N2P · H2O, calcd %: C, 41.13, H, 2.96, N, 4.57.
Found %: C, 41.25, H, 2.64, N, 4.91.
Synthesis of [(4,5-dichloro-1H-imidazolate-1-yl)-(1,3-5-triaza-
phosphaadamantane)–gold(I)] (8). 40 mg of 4,5-dichloroimida-
zole (0.29 mmol) were dissolved in 10 mL of CH3OH. To this
solution 0.29 mL of a 1 M methanolic solution of NaOH
(0.29 mmol) was added. After stirring for 10 minutes, solid
TPAAuCl (0.113 g, 0.29 mmol) was also added. Awhite precipi-
tate was readily formed and the suspension was stirred at room
temperature for 5 h. The suspension was filtered off through a
paper filter. The white solid was washed with water (4 × 3 mL)
and dried by vacuum (112 mg). Yield: 78%. M.p. >250 °C with
Synthesis of [(4-nitro-1H-pyrazolate-1-yl)-(1,3-5-triaza-phos-
phaadamantane)–gold(I)] (6). To a THF solution (5 mL) of 4-
nitropyrazole (17.7 mg, 0.157 mmol) solid NaH (5.6 mg,
0.235 mmol) was added. After observation of gas evolution and
magnetic stirring for half an hour, the suspension was filtered
over a celite bed (1.5 cm) and dropped into a solution of 1,3-5-
triaza-phosphaadamantanegold(I)chloride (0.060 g; 0.157 mmol)
dissolved in 30 mL of CH3CN. The pale white suspension was
stirred for an hour at room temperature and another hour at
40 °C. After filtration, the solution was concentrated to dryness
under vacuum and a solid white was obtained. After dissolution
in CH2Cl2, extractions with water (3 × 5 mL) were performed to
remove the excess of nitropyrazole and sodium chloride.
The organic phase was then dried over a bed of anhydrous
Na2SO4 and evaporated to dryness to obtain the product as a
white solid (43 mg) in 59% yield. M. p. 203.2–204.8 °C with
1
decomposition starting from 200 °C. H-NMR (DMSO, 293 K):
δ 7.00 (s, 1H, Im), 4.51 (d, 6H, 2JP-H = 12 Hz, PCH2N), 4.37 (s,
2H, NCH2Nax), 4.30 (s, 4H, NCH2Neq). 31P{1H} NMR (DMSO,
293 K): δ −52.52 (s). IR (cm−1): 3124w (C-Him), 2963m,
2948m, 2927m, 2833m, 1652m, 1493m, 1466m, 1439s, 1427m,
1411m, 1284s, 1241s, 1228vs, 1190s, 1099m, 1040m, 1010s,
967vs, 945vs, 901m, 824m, 801s, 738s, 668m. Elemental analy-
sis for C9H13AuCl2N5P, calcd %: C, 22.06, H, 2.67, N, 14.29.
Found %: C, 22.25, H, 2.62, N, 13.90.
Synthesis of [(1-benzyl-4,5-dichloro-2H-imidazolate-2-yl)-tri-
phenylphosphane–gold(I)] (9)
Synthesis of 1-benzyl-4,5-dichloroimidazole. 700 mg of 4,5-
dichloroimidazole (5.1 mmol) were dissolved in 10 mL of DMF.
To this solution 0.58 mL of benzylchloride (5.1 mmol) and
540 mg of Na2CO3 (5.1 mmol) were added. The suspension was
stirred at reflux overnight. After filtering on paper the solution
was concentrated to dryness under vacuum obtaining an orange
oil. The oil was treated with 30 mL of water and stirred vigor-
ously until a solid was formed. The solid was recovered by fil-
tration and dissolved in diethyl ether. The ether solution was first
filtered and then concentrated to dryness under vacuum. A pale
orange solid was obtained. Yield 65%. M. p. 60.2–61.4 °C.
1H-NMR (acetone-d6, 293 K): δ 7.81 (s, 1H, C2-H), 7.39–7.28
(m, 5H, C6H5), 5.30 (s, 2H, CH2bz). IR (cm−1): 3124m
(C-Him), 3067w, 3033w, 1519m, 1496m, 1456m, 1483m,
1389m, 1364w, 1345m, 1253s, 1215m, 1201w, 1180m, 1114m,
1078m, 975m, 951w, 911w, 854w, 806m, 782m, 717vs, 692vs,
662s. Elemental analysis for C10H8Cl2N2, calcd %: C, 52.89, H,
3.55, N, 12.34. Found %: C, 53.21, H, 3.43, N, 12.16.
1
decomposition. H-NMR (DMSO, 293 K): δ 8.34 (s, 2H), 4.49
(d, 6H, 2JP-H = 16 Hz, PCH2N), 4.35 (s, 2H, NCH2Nax), 4.33 (s,
4H, NCH2Neq). 31P{1H} NMR (DMSO, 293 K): δ −52.52 (s).
IR (cm−1): 3138w (C-Hpz), 2922m, 2853m, 1551m, 1496s
(NO2), 1444w, 1405s, 1358m, 1333m, 1275vs (NO2), 1242vs,
1162m, 1100m, 1040m, 1011vs, 969vs, 946vs, 900m, 813m,
801 vs, 752m, 733vs. ESI(+) (CH3CN), m/z (%): 242.4 (100).
ESI(−) (CH3CN), m/z (%): 112.1 (100) [4-NO2pz]−, 420.8 (25)
[Au(4-NO2pz)2]−. Elemental analysis for C9H14AuN6O2P, calcd
%: C, 23.19; H, 3.03; N, 18.03. Found %: C, 22.89; H, 2.72;
N, 17.31.
Synthesis of [(4,5-dichloro-1H-imidazolate-1-yl)-triphenylpho-
sphane–gold(I)] (7). 50 mgs (0.36 mmol) of 4,5-dichloroimida-
zole were dissolved in 10 mL of CH3OH. To this solution
0.36 mL of methanolic NaOH 1 M were added (0.36 mmol).
After stirring for 15 min, 179 mg of solid PPh3AuCl
(0.36 mmol) were added. The solution was stirred for four
hours. After filtering on paper the solution was concentrated to
dryness under vacuum obtaining a white solid. The crude
product was washed with hexane (2 × 3 mL), pumped to dryness
and then with water (2 × 5 mL) to eliminate salts and the
unreacted imidazolate. The compound was dried by vacuum
(182 mg). Yield 84%. M.p. 155.2–156.8 °C with decomposition.
1H-NMR (acetone-d6, 293 K): δ 7.72–7.62 (m, 15H, PPh3), 7.16
(s, 1H, Im), 2.87 (s, br, 2H, H2O). 31P NMR (acetone-d6,
293 K): δ 32.34 (s). IR (cm−1): 3150w (C-Him), 3054w, 3032w,
1587m, 1499m, 1480m, 1434s, 1331m, 1309m, 1259m, 1222s,
Synthesis of [(1-benzyl-4,5-dichloro-2H-imidazolate-1-yl)-triphe-
nylphosphane–gold(I)]. 136 mg of the 1-benzyl-4,5-dichloroimi-
dazole (0.6 mmol) were dissolved in 10 mL of anhydrous THF
under a nitrogen flux. To this solution 0.24 mL of n-BuLi
(0.6 mmol) were added to this solution at −40 °C and the dark
orange solution was stirred at this temperature for an hour. The
solution was heated to 0 °C and solid PPh3AuCl was added
(0.297 g; 0.6 mmol). The color of the solution readily turned to
dark brown and it was stirred for two hours. After this time the
solvent was evaporated and the oily residue was treated with
CH2Cl2 (10 mL). The dichloromethane solution was then treated
with water (4 × 10 mL) and collected in a flask and dried over
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 5307–5318 | 5315

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Experiments with human cells
anhydrous Na2SO4. After removal of the solvent the crude
product was suspended in hexane and stirred vigorously over-
night. The pale brown solid obtained was filtered and dried. The
impurities were precipitated by dissolving the crude product in
CHCl3 and by layering hexane. After the removal of the solvents
Azolate gold(I) complexes and the corresponding uncoordinated
ligands were dissolved in DMSO just before the experiment
and a calculated amount of drug solution was added to the
growth medium to a final solvent concentration of 0.5%,
which had no effect on cell killing. cis-platin was dissolved
in a 0.9% NaCl solution just before the experiment. MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and
cis-platin were obtained from Sigma Chemical Co, St. Louis,
USA.
0.182
g
of an ivory solid was recovered. Yield
42%. M. p. 152.1–153.6 °C. 1H-NMR (CDCl3, 293 K): δ
7.50–7.38 (m, 15H, PPh3), 7.25–7.21 (m, 5H, C6H5), 5.29 (s,
2H, CH2bz). 31P NMR (CDCl3, 293 K): δ 43.13 (s). IR (cm−1):
3070w (C-Him), 3047w, 3032w, 2988w, 1586w, 1519m, 1496m,
1481m, 1455m, 1435s, 1398m, 1342m, 1331m, 1310m, 1293m,
1222s, 1193m, 1181m, 1100s, 1075m, 1027m, 998m, 958m,
851m, 749s, 727s, 710s, 691vs. ESI(+) (CH3CN), m/z (%):
685.0 (70) [(4,5-Cl2bzim)AuPPh3 + H]+, 721.2 (100) [Au
(PPh3)2]+, 1108.9 (40) [(4,5-Cl2bzim)2AuPPh3 + H]+. Elemental
analysis for C28H22AuCl2N2P, calcd %: C, 48.84, H, 3.17, N,
4.07. Found %: C, 49.07, H, 3.24, N, 4.09.
Cell cultures
Human breast (MCF-7) and lung (A549) carcinoma cell lines
were obtained from American Type Culture Collection (ATCC,
Rockville, MD). A431 human cervical carcinoma cells were
kindly provided by Prof. F. Zunino (Division of Experimental
Oncology B, Istituto Nazionale dei Tumori, Milan, Italy). 2008
cells and their cis-platin resistant variant, called C13* cells, are
human ovarian cancer cell lines, and they were kindly provided
by Prof. G. Marverti (Department of Biomedical Science of
Modena University, Italy). The LoVo human colon-carcinoma
cell line and its derivative multidrug-resistant subline (LoVo
MDR) were kindly provided by Prof. F. Majone (Department of
Biology of Padova University, Italy). Cell lines were maintained
in the logarithmic phase at 37 °C in a 5% carbon dioxide atmos-
phere using the following culture media containing 10% foetal
calf serum (Euroclone, Milan, Italy), antibiotics (50 units mL−1
penicillin and 50 μg mL−1 streptomycin) and 2mM l-glutamine:
(i) RPMI-1640 medium (Euroclone) for MCF-7, A431, 2008
and C13* cells; (ii) F-12 HAM’S (Sigma Chemical Co) for
A549, LoVo and LoVo MDR cells. LoVo MDR culture medium
also contained 0.1 μg mL−1 doxorubicin.
X-Ray analyses
Single crystals of the complexes 3,5-bis(trifluoromethyl)pyrazo-
lategold(I)triphenylphosphane (1) and 3,5-bis(dinitro)pyrazolate-
gold(I)triphenylphosphane (2) of X-ray quality were grown by
slow diffusion of hexane into a solution of CH2Cl2 for 1, and by
layering hexane on a THF solution for 2. Adequate specimens
were lodged on glass fibers and centred on the goniometer head
of two different instruments, thanks to the generosity of Col-
leagues from the Chemistry Department of the University of
Trieste, Italy (1) and of the C.N.R.–I.C.I.S. Institute of Padua,
Italy (2). The crystal chosen for 1 was mounted on an Enraf-
Nonius DIP1030 image plate diffractometer, and the selected
specimen for 2 was mounted on a Philips PW1100 diffract-
ometer. The raw data were collected, in both cases, at room
temperature, by using graphite–monochromated Mo Kα radiation
(λ = 0.71073 Å) and corrected for Lorentz/polarization effects,
as well as for absorption. In the case of 2, the absorption correc-
tion was performed by means of ψ-scans,60 while a cubic fit to
sin θ/λ values (24 parameters)61 was used for 1. Data were col-
lected with ω-2θ scans (2) or φ scans (1). In 2, the unit cell par-
ameters were determined by least-squares refinement of 30 well
centred high-angle reflections, and two standard reflections were
checked every 150 measurements to ensure for crystal stability.
In 1, unit-cell parameters were determined by least-squares
refinement during the whole data collection. In both collections
no sign of deterioration was detected. The structures were solved
by direct methods, with SIR9762 (2) or SHELXTL NT63 (1) and
Cytotoxicity assays
The growth inhibitory effect on tumor cell lines was evaluated
by means of MTT (tetrazolium salt reduction) assay.66 Briefly,
3–8 × 103 cells per well, dependent upon the growth character-
istics of the cell line, were seeded in 96-well microplates in
growth medium (100 μL) and then incubated at 37 °C in a 5%
carbon dioxide atmosphere. After 24 h, the medium was
removed and replaced with a fresh one containing the compound
to be studied at the appropriate concentration. Triplicate cultures
were established for each treatment. After 72 h, each well was
treated with 10 μL of a 5 mg mL−1 MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide) saline solution, and
following 5 h of incubation, 100 μL of a 30 mg mL−1 sodium
dodecylsulfate (SDS) solution in HCl 0.01 M were added. After
overnight incubation, the inhibition of cell growth induced by
the tested complexes was detected by measuring the absorbance
of each well at 570 nm using a Bio-Rad 680 microplate reader.
Mean absorbance for each drug dose was expressed as a percen-
tage of the control untreated well absorbance and plotted vs.
drug concentration. IC50 values represent the drug concentrations
that reduced the mean absorbance at 570 nm to 50% of those in
the untreated control wells.
2
refined by standard full-matrix least-squares based on Fo with
the SHELXL-9764 and OLEX265 programs.
In 1, two independent molecules (from now on, I and II)
make the asymmetric unit. During the refinement of molecule II,
some peaks appeared in positions compatible with a second
arrangement of one of the trifluoromethyl groups. At the end of
the refinement, the involved atoms were modelled as disordered
over two sites (F(10), F(11), F(12); F(10A), F(11A), F(12A)).
The two positions were refined isotropically, with partial occu-
pancies of 0.52 and 0.48, respectively. With this exception, all
non-H atoms of 1 and 2 were allowed to vibrate anisotropically
in the last cycles of refinement; H atoms were placed instead in
calculated positions and refined as “riding model”.
5316 | Dalton Trans., 2012, 41, 5307–5318
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Enzyme inhibition
generous contribution of Prof. E. Zangrando from the Chemistry
Department of University of Trieste, Italy, who allowed access to
the PW1100 and DIP1030 diffractometers, supervised the X-ray
data collection process and helped with the structure interpret-
ation. The authors are grateful to CIRCSMB (Consorzio Interu-
niversitario per la Ricerca in Chimica dei Metalli nei Sistemi
Biologici).
Isolation and purification of thioredoxin reductases from rat
liver cytosol and mitochondria. Highly purified cytosolic thiore-
doxin reductase (TrxR1) was prepared according to Luthman
and Holmgren55 starting from rat liver cytosol obtained after
centrifugation of the liver homogenate at 45 000 g for 1 h.
Mitochondrial thioredoxin reductase (TrxR2) was purified from
liver mitochondria following the procedure of Rigobello et al.67
After affinity chromatography (2′, 5′-ADP-sepharose), the
enzymes were further purified by chromatography through a
ω-aminohexyl-sepharose 4B column. The fractions obtained
after the affinity chromatography step were concentrated by
ultrafiltration and then applied on a ω-aminohexyl-sepharose 4B
column equilibrated with 50 mM Tris-HCl buffer (pH 7.5) and
the enzymatic fraction was eluted with a linear gradient of NaCl
(from 0.0 to 0.8 M). The enzyme showed a unique band on
SDS-PAGE.
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The authors gratefully thank Dr. F. Benetollo of the C.N.R.–I.C.
I.S. Institute, Padua, Italy. We are particularly indebted to the
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