Gold(I) thiotetrazolates as thioredoxin reductase inhibitors and antiproliferative agents

Article abstract of DOI:10.1039/c4dt03105a

Gold(I) complexes with phosphane and thiotetrazolate ligands were prepared and investigated as a new type of bioactive gold metallodrugs. The complexes triggered very efficient inhibition of the enzyme thioredoxin reductase (TrxR), which is an important molecular target for gold species. Strong cytotoxic effects were observed in MDA-MB-231 breast adenocarcinoma and HT-29 colon carcinoma cells, and the complexes also caused strong effects in vincristine resistant Nalm-6 leukemia cells. Cellular uptake studies showed elevated cellular gold levels for complexes containing a triphenylphosphane ligand, whereas trifurylphosphane analogues accumulated at significantly lower cellular concentrations.

Full text of DOI:10.1039/c4dt03105a

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Gold(I) thiotetrazolates as thioredoxin reductase
inhibitors and antiproliferative agents†
Cite this: Dalton Trans., 2015, 44,
1161
Tatiyana V. Serebryanskaya,a,b,c Alexander S. Lyakhov,b Ludmila S. Ivashkevich,b
Julia Schur,a Corazon Frias,d Aram Prokopd and Ingo Ott*a
Gold(I) complexes with phosphane and thiotetrazolate ligands were prepared and investigated as a new
type of bioactive gold metallodrugs. The complexes triggered very efficient inhibition of the enzyme
thioredoxin reductase (TrxR), which is an important molecular target for gold species. Strong cytotoxic
effects were observed in MDA-MB-231 breast adenocarcinoma and HT-29 colon carcinoma cells, and
the complexes also caused strong effects in vincristine resistant Nalm-6 leukemia cells. Cellular uptake
studies showed elevated cellular gold levels for complexes containing a triphenylphosphane ligand,
whereas trifurylphosphane analogues accumulated at significantly lower cellular concentrations.
Received 8th October 2014,
Accepted 4th November 2014
DOI: 10.1039/c4dt03105a
www.rsc.org/dalton
formations via interaction with numerous sulfur containing
proteins and peptides in vivo.2 Therefore, stability is a crucial
Introduction
The therapeutic use of gold and its complexes dates back to point in the design of gold(I) species, and usually, typical “soft
thousands of years ago and was also a hot topic in alchemy bases” such as phosphorus- and sulphur-containing nucleo-
during medieval ages and the Renaissance.1 However, the ela- philes are utilized as ligands to enhance the stability of the
boration of gold based antiproliferative agents is a relatively resulting gold(I) complexes under physiological conditions.
young and intensively developing area of medicinal and Gold(I) phosphane complexes are the most frequently investi-
pharmaceutical chemistry.2–5 The precise mechanisms of anti- gated group of gold based compounds.3,9–15 Many of them,
tumor activity of gold compounds have not been completely including auranofin (Fig. 1) and chlorido gold(I) phosphane
understood. As a result of a series of mechanistic studies it (1, Fig. 1) complexes, were shown to be effective antiprolifera-
was suggested that binding of gold to the selenoenzyme thio- tive agents and potent TrxR inhibitors.6,7,16
redoxin reductase (TrxR) leads to its inhibition, which results in
Here we report on the synthesis and biological evaluation of
the alteration of mitochondrial functions, increased formation a new series of tetrazole-containing gold(I) phosphane com-
of reactive oxygen species and finally causes cell death via plexes (2, Fig. 1). Being rather new ligands in bioinorganic
apoptosis.6,7 Both gold(I) and gold(III) derivatives were found to chemistry, tetrazole derivatives have been gaining increasing
be efficient inhibitors of thioredoxin reductase,6 yet gold(III) attention in recent years due to the development of more
based complexes are nowadays considered as prodrugs that are efficient synthesis methods.17 Moreover, the tetrazole ring is a
reduced before binding to give gold(I) species.8
well known bioisoster of the carboxylic group and several tetra-
The choice of ligands appears to be one of the most chal- zole-containing platinum complexes have earlier been reported
lenging tasks in the design of novel gold based antitumor as possessing a promising antiproliferative profile and low
agents. Owing to the “soft acidic” nature of gold(I), its coordi- toxicity in vivo.18–20
nation compounds are highly prone to rapid metabolic trans-
Gold(I) complexes with polynitrogen-containing azoles have
been relatively little studied so far. Among them, mixed-ligand
phosphane gold(I) 5-thiotetrazolates 2 are among the most
investigated groups.21 The obvious structural analogy between
this group of gold(I) complexes and auranofin makes them
interesting subjects for antitumor drug design. Despite this
fact, there have been to date no data reported in the literature
on their antiproliferative or other biological effects. In order to
elucidate a possible structure–activity relationships within this
group of gold(I) complexes, a panel of (phosphane)gold(I) 1-R-
5-thiotetrazolates has been synthesized and characterized by
means of spectroscopic and structural methods. Their antipro-
aInstitute of Medicinal and Pharmaceutical Chemistry, Technische Universität
Braunschweig, Beethovenstrasse 55, 38106 Braunschweig, Germany.
E-mail: ingo.ott@tu-bs.de
bResearch Institute for Physical Chemical Problems of the Belarusian State
University, 14 Leningradskaya st., 220030 Minsk, Belarus
cInstitute of Chemistry, Saint Petersburg State University, 198504 Stary Petergof,
Russian Federation
dDepartment of Paedriatric Oncology, Children’s Hospital Cologne, Amsterdamer
Strasse 59, 50735 Cologne, Germany
†CCDC 1027451. For crystallographic data in CIF or other electronic format see
DOI: 10.1039/c4dt03105a
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Fig. 1 Examples of gold(I) phosphane complexes.
liferative activity against two human cancer cell lines as well as of NH protons that are observed in the spectra of uncoordi-
the inhibitory effects toward TrxR and glutathione reductase nated tetrazolylthiones 3a–c at approx. 14 ppm. Coordination
(GR) have been evaluated, whereupon cellular uptake studies also leads to significant upfield shifts (approx. 5–6 ppm) of
were performed using atomic absorption spectroscopy (AAS). the C(5) signals, which are observed at approx. 158 ppm in the
Additionally, the antiproliferative activity of the best candi- 13C NMR spectra of 2a–e (163–164 ppm in the spectra of 3a–c).
dates was investigated against resistant leukemia cell lines.
Other resonances remain mainly unchanged when compared
to the spectra of the starting materials. Due to 13C–31P coup-
ling, phenyl and furyl carbons of the phosphane ligands
appear as doublet resonances.
Results and discussion
When compared with the spectra of the free ligands 3a–c,
IR spectra of 2a–e show the same pattern of changes including
disappearance of the intensive bands at 1510, 1350–1360,
1040–1050, and 790–800 cm−1 characteristic of the stretching
and deformation vibrations of CvS and N–CvS frag-
ments.22,23 New partially split bands at 690–710 cm−1 assigned
to the stretching vibrations of the C–S bond23 were observed.
Altogether, these changes support the formation of a covalent
Au–S bond between the triarylphosphane gold(I) moiety and
the thiolate function of the tetrazole-containing ligands.
According to the results of DSC/TG, all the complexes were
found to be thermally stable up to 180–200 °C. Exothermal
decomposition of 2a–c and 2e at higher temperatures is
preceded by their melting at 180–195 °C, while 2d melts at a
noticeably lower temperature (approx. 120 °C).
Synthesis and characterization
Tetrazole-containing gold(I) complexes 2a–e bearing triphenyl-
phosphane or trifurylphosphane ligands were synthesized,
starting from the corresponding 1-substituted 5-thiotetrazoles
3a–c and chloridogold(I) phosphanes ClAuPR3 [R = Ph (1a),
2-furyl (1b)] in the presence of triethylamine according to
Scheme 1. The synthesis of compounds 2a and 2b under
similar conditions was reported elsewhere earlier.22,23 Com-
plexes 2c–e were obtained for the first time.
The composition and structure of the products were
confirmed by elemental analysis, ESI mass spectrometry, and
NMR spectroscopy (1H and 13C). Complexes were additionally
characterized by means of IR spectroscopy and thermal ana-
lysis (TG/DSC). The molecular and crystal structure of 2e was
additionally confirmed by single crystal X-ray diffraction. In
general the spectral and structural characteristics of complexes
2a and 2b were found to be consistent with previously
published results.23,24
Crystal and molecular structures of complex 2e
Complex 2e crystallizes in the monoclinic space group C2/c,
with eight formula units in the unit cell. Crystal data and
structure refinement details for this compound are summar-
ized in Table 1. Similarly to the previously reported complexes
2a and 2b,24 complex 2e presents a molecular complex
showing a bidentate linear coordination environment of the
gold atoms (Fig. 2a).
ESI(+) mass spectra of 2a–e showed pseudomolecular ions
[M + H]+ along with the respective [Au(PR3)2]+ species. Assign-
ments of signals observed in 1H and 13C NMR spectra of all
complexes were made based on previously published data23,25
and are in agreement with the suggested structures (see
1
Scheme 1). In particular, H NMR spectra of 2a–e lack signals
In complex 2e, the gold atom is in a standard linear coordi-
nation, being attached to the phosphorus atom with an
Au1–P1 distance of 2.2475(6) Å and to the sulfur atom with
an Au1–S1 distance of 2.3051(7) Å. The coordination angle
P1–Au1–S1 takes on a value of 170.93(3)°. The geometries
of the coordination units are very close in three compounds,
2a, 2b and 2e (see Table 2).
In complex 2e, the oxygen atoms of the furane rings are
oriented away from the gold atom. The angles Au1–P1–C12
[116.21(8)°] and Au1–P1–C22 [114.05(9)°] are larger than
the tetrahedral standard, whereas the angle Au1–P1–C32
[107.76(9)°] is close to that but is somewhat smaller. The
Scheme 1 Synthesis of thiotetrazole-containing gold(I) phosphane
complexes 2a–e.
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Table 1 Main crystal data and structure refinement details for complex
2e
Formula
Formula weight
T/K
C19H14AuN4O3PS
606.34
296(2)
Crystal system
Space group
Crystal size/mm
Monoclinic
C2/c
0.35 × 0.34 × 0.12
a/Å
b/Å
c/Å
13.79810(10)
13.08130(10)
22.1996(3)
91.0696(5)
4006.27(7)
8
2.011
7.557
5371
0
β/°
V/Å3
Z
Dc/g cm−3
μ/mm−1
Reflections
Restraints
Parameters
R1/wR2[I > 2σ(I)]
R1/wR2 [all data]
Goodness-of-fit
262
0.0210/0.0484
0.0283/0.0505
1.004
phosphane C–P–C valence angles, lying in
a range of
103.83(12)–107.55(12)°, are smaller than the tetrahedral one.
These geometric features are very similar to those in complexes
2a and 2b. The fragment AuPR3 (R = 2-furyl) displays a dis-
torted non-propeller-like structure due to the large variation in
dihedral angles Au–P–C–C [–5.8(3), 8.1(3) and 16.6(4)°].
As for the tetrazole ring geometry, it is typical of 1H-tetra-
zoles in the three complexes 2a, 2b and 2e (see Table 2), with
the shortest bond N2–N3 and remaining bond distances
lying in rather narrow ranges. In the crystal structure of
complex 2e, there are non-classic intermolecular hydrogen
bonds C35–H35⋯O11b between two neighboring complex
molecules [symmetry transformation: (b) −x, y, −z + 1/2; hydro-
gen bond geometry: H35⋯O11b 2.55 Å, C35⋯O11b 3.424(3) Å,
C35–H35⋯O11b 157°]. These bonds form dimeric entities
comprising R22(14) hydrogen-bonded rings (Fig. 2b).
Fig. 2 (a) Complex 2e, with the atom numbering scheme and displace-
ment ellipsoids drawn at the 30% probability level; (b) hydrogen-bonded
dimer in the crystal structure of 2e; (c) dimeric unit formed by aurophilic
bonding in the crystal structure of 2e [symmetry transformation:
(c) 1 − x, y, 1/2 − z].
The complex molecules are bonded into dimeric units
through very weak aurophilic bonding,26 with a separation of
the gold atoms Au1⋯Au1c of 3.3892(5) Å (symmetry transform-
ation as in Fig. 2c). These units are connected via the above
mentioned hydrogen bonds to give polymeric chains running
along the a axis.
TrxR and GR inhibition activity
Table 2 Some geometric features (Å, °) of complexes 2a, 2b and 2e
As mentioned above, inhibition of the selenoenzyme thio-
redoxin reductase (TrxR) is considered to be an important
mechanism of bioactivity of gold(I) species.3,6,7 Therefore, the
potential of tetrazole-based gold(I) complexes to inhibit TrxR
was studied on isolated rat liver TrxR using the DTNB (dithio-
bisnitrobenzoic acid) reduction assay. As far as selenocysteine
is considered to be the relevant binding site for gold com-
plexes, another structurally and functionally similar disulfide
reductase, i.e. glutathione reductase (GR, from yeast), was
included in the test as a reference. Instead of selenocysteine,
GR contains a cysteine residue in its active site and thus can
Compound
2aa
2ba
2e
Au1–P1
Au1–S1
P1–Au1–S1
C5–S1–Au1
S1–C5
N1–C5
N1–N2
N2–N3
N3–N4
N4–C5
2.261(1)
2.325(1)
170.11(3)
97.5(1)
1.740(4)
1.342(5)
1.354(5)
1.289(5)
1.354(5)
1.331(5)
2.265(2)
2.304(2)
172.68(5)
105.3(2)
1.733(7)
1.346(9)
1.378(8)
1.295(9)
1.366(8)
1.346(8)
2.2475(6)
2.3051(7)
170.93(3)
99.28(9)
1.726(3)
1.350(3)
1.361(3)
1.285(4)
1.359(4)
1.326(3)
a From ref. 24.
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Table 3 Antiproliferative activity and inhibition activity against TrxR and GR of gold(I) phosphane complexes 1a–b and 2a–e, n.d.: not determined
IC50 (μM)
IC50 (μM)
Selectivity GR/TrxR
Compound
TrxR
GR
4.2 0.727
(x-fold)
MDA-MB-231
HT-29
1a
1b
2a
2b
2c
2d
2e
3a
3b
3c
0.256 0.00227
0.207 0.021
0.037 0.010
0.046 0.012
0.165 0.065
0.033 0.005
0.053 0.001
n.d.
16
9.5 0.7
9.9 0.7
7.9 1.9
7.8 2.0
8.9 1.1
8.5 1.5
9.3 0.2
>100
4.2 0.910
10.0 1.8
12.0 2.7
10.2 1.5
8.6 0.7
13.3 2.9
11.5 1.7
>100
n.d.
0.97 0.25
0.55 0.04
0.66 0.19
0.43 0.05
0.51 0.07
n.d.
26
12
4
13
10
—
—
—
n.d.
n.d.
n.d.
n.d.
>100
>100
>100
>100
be used to check the specificity of TrxR inhibition by the
tested compounds.28,29
According to the results given in Table 3, tetrazole-contain-
ing gold(I) complexes turned out to be very effective inhibitors
of TrxR with IC50 values in the low nanomolar range. With the
exception of 2c, all complexes showed IC50 values in the
range 30–55 nM and at least 10-fold selectivity for inhibition of
TrxR over GR. In all cases 2a–e were more effective enzyme
inhibitors than the respective chlorido complexes 1a and 1b.
Antiproliferative activity
Antiproliferative activity of thiotetrazole-containing gold(I)
complexes 2a–e against two human cancer cell lines
(MDA-MB-231 and HT-29) was assessed using the crystal violet
assay. Chlorido(triphenylphosphane)gold(I) (1a) and chlorido-
[tri(2-furyl)phosphane]gold(I) (1b) were included in the test as
positive controls. According to the results (Table 3), the tetra-
zole-containing complexes 2a–e demonstrate considerable
antiproliferative activity against both human cancer cell lines
with IC50 values found in the micromolar or low micromolar
range (approx. 8.0–13.0 μM). These values are comparable to
those of the positive controls and the platinum anticancer
drug cisplatin (IC50 values of 7.0 μM in HT-29 cells30 and
4.0 μM in MDA-MB-231 cells31). The metal-free thiotetrazole
derivatives 3a–c were investigated as negative controls and
showed no inhibition of tumor cell proliferation.
Fig. 3 Cellular gold content of HT-29 cells exposed to 10 µM of gold(I)
complexes 2a–2e.
complexes 2a–c. The content of gold in HT-29 cells reached by
these complexes after 1 h of exposure correlated to intracellu-
lar concentrations of 150 (2a), 265 (2b) and 210 (2c) μM,
respectively. Taking into account the exposure concentration
of 10.0 μM, one can conclude that for these complexes at least
15-fold accumulation can be achieved already within the first
hour of the experiment. This is markedly higher than the
8-fold accumulation reported for the chlorido derivative 1a
after 6 h of exposure.10 Drastically lower levels of cellular
uptake were found for [tri(2-furyl)phosphane]gold(I) complexes
2d and 2e, which can be explained by their lower lipophilicity.
Intracellular concentrations of 10 and 40 μM observed for 2d
and 2e, respectively, after 1 h of exposure are 7–15 times lower
in comparison with their triphenylphosphane-based analogs
2a and 2b.
Cellular uptake
In order to evaluate the extent of cellular uptake, we measured
the gold content in HT-29 cells exposed to 10.0 μM (approx.
equal to the IC50 calculated for this cell line) solutions of the
tested compounds for a period of 1, 4 and 8 h by a method
based on high resolution continuum source atomic absorption
spectroscopy (HR-CS AAS).32,33 Results were corrected for the
respective protein contents of the samples and are presented
in Fig. 3 as nmol gold per milligram cellular protein. Besides,
based on certain biophysical parameters of HT-29 cells,32 the
Antiproliferative activity in a resistant cell line
nmol Au per mg protein values obtained were used to estimate Resistance to therapeutic agents is a major problem in current
the respective intracellular molar concentrations. tumor chemotherapy. Hence, the development of drugs that
As shown in Fig. 3, the highest values of intracellular can overcome drug resistance in tumor tissues is of major
accumulation were observed for triphenylphosphine gold(I) interest. Here we used wild type and vincristine resistant
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analogues 1a and 1b indicated that the thiotetrazolato ligand
was important for this effect. The lowest activity in the series
2a–e was observed for 2c, which contains an aliphatic side
chain with a tertiary nitrogen at the thiotetrazolato ligand.
Notably, strong TrxR inhibition with thiophenolate containing
gold(I) carbene complexes was also observed in a previous
study.34 However, the enhanced activity against TrxR did not
translate into elevated cytotoxicity since complexes 2a–e were
of comparable activity with 1a and 1b in cell proliferation
assays.
Cellular uptake studies showed a very high accumulation of
the triphenylphosphane containing complexes 2a–c, whereas
the triphenylfurane analogues 2d and 2e were found at sig-
nificantly lower levels in the cells. It can be speculated that
possible dimer formation by hydrogen bonding of the trifuryl-
phosphane moiety might contribute to this effect besides
differences in the lipophilicity. It should be mentioned that we
observed higher cellular accumulation of triphenylphosphane
gold complexes compared to trialkylphosphane derivatives in
a number of recent studies10,11,35 and that lipophilicity is
generally considered to be an important parameter for cellular
uptake of gold phosphane species.36
Importantly, selected compounds 2a and 2d showed
decent activity in p-glycoprotein overexpressing vincristine-
resistant Nalm-6 cells, which indicates that this type of gold
compounds might be useful for the design of resistance over-
coming drugs.
In summary, gold complexes with thiotetrazolate represent
a new promising class of gold species, which trigger important
biological effects such as TrxR inhibition and cytotoxicity.
Fig. 4 Effects of 2a (top) and 2d (bottom) on DNA fragmentation in
wild type and vincristine resistant Nalm-6 cells after 72 h of exposure;
Co: control; DMSO: cells treated with the same amount of DMSO; VCR:
vincristine (20 nM).
NALM-6 cells, which overexpress the drug efflux pump p-glyco-
protein,33 to evaluate the possible overcoming of drug resist-
ance by the gold complexes under study. Complexes 2a and 2d
were selected for these experiments as examples with high (2a)
and low (2d) cellular uptake but comparable toxicity and TrxR
inhibitory potential (see above). Apoptosis induction was
assessed as DNA fragmentation in the cells.
In fact, both 2a and 2d triggered strong effects in wild type
and vincristine resistant NALM-6 cells already in low micro-
molar concentrations (>50% DNA fragmentation at 1.0 µM
with 2a and 2.5 µM with 2d), which clearly demonstrated
that both complexes could overcome p-glycoprotein related
multidrug resistance (see Fig. 4).
Experimental
Materials and methods
All reagents and solvents were used as received from Sigma,
Aldrich, or Acros Organics. 1H and 13C NMR spectra were
recorded on a Bruker DRX-400 AS NMR system. ESI(+) mass
spectra were recorded on a Bruker micrOTOF instrument using
dichloromethane as a solvent. The purity of the target com-
pounds (>95%) was confirmed by elemental analysis. For all
compounds undergoing biological evaluation, the experi-
mental values differed less than 0.5% from the calculated
ones.
IR spectra of powder samples were registered on a Nicolet
Thermo Avatar 330 spectrometer using a Smart Diffuse Reflec-
tion accessory in the range of 4000–400 cm−1. The TG and DSC
curves were obtained with a NETZSCH STA 429 thermoanalyzer
under a dynamic nitrogen atmosphere (heating rate of 10 K
min−1, aluminum oxide, mass 5–6 mg and temperature range
from room temperature to 600 °C).
Conclusions
A
series of different gold(I) phosphane complexes with
thiotetrazolato ligands was prepared and biologically evalu-
ated. The crystal structure of complex 2e showed the expected
linear coordination geometry around the gold center. Interest-
ingly, dimers were formed by intermolecular hydrogen
bonding between the furane moieties.
Synthesis of gold(I) complexes 2a–e
Compounds 2a–c were obtained using the following general
Complexes 2a–e turned out to be very effective inhibitors of procedure. 0.5 mmol (248 mg) of chlorido(triphenylphos-
the enzyme TrxR. Comparison with the respective chloride phane)gold(I) and 0.5 mmol of the corresponding 1-R′-5-thio-
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2
4
tetrazole 3a–c were dissolved in 20 ml of CH2Cl2. Triethyl- 134.3 (d, J(C,P) = 13.8, ortho-C from PPh3); 131.9 (d, J(C,P) =
amine (0.1 mL) was added dropwise to the prepared solution, 2.5, para-C from PPh3); 129.3 (d, 3J(C,P) = 11.7, meta-C from
and the mixture was stirred overnight at room temperature. PPh3); 128.9 (d, 1J(C,P) = 59.0, ipso-C from PPh3); 57.5 (s,
Then the mixture was evaporated to dryness, and the residue α-CH2); 45.5 (s, N(CH3)2); 45.4 (s, β-CH2). FT-IR (ν, cm−1):
was dissolved in hot THF. After cooling to room temperature, 3067 (m), 3049 (m), ν(CHaryl); 2965 (s), 2940 (s), 2856 (m),
needle-like crystals of triethylammonium chloride formed and 2825 (s), 2791 (m), ν(CHalkyl); 1585 (m), 1480 (s), ν(CCaryl);
were separated by filtration. The filtrate was evaporated again, 1437 (vs), 1380 (s), δ(CHaryl) + δ(CHalkyl); 1301 (s), 1260 (m),
and the solid residue was recrystallized from CH2Cl2– 1250 (m), 1179 (s), 1160 (m), ν(NvNtz) + ν(N–Ntz); 1100 (s),
hexane (2a and 2b) or acetonitrile (2c) to obtain the colorless 1066 (m), 1023 (m), 997 (m), 982 (m), 920 (m), δ(CN4)
product.
+ δ(C6H5); 777 (m), ν(C–N); 750 (vs), ν(C–P); 710 (s), 690 (vs)
(1-Methyltetrazol-5-ylthiolato)(triphenylphosphane)gold(I) ν(C–S).
(2a). Yield (from CH2Cl2–hexane): 235 mg (82%). Anal. Calc.
Complexes 2d and 2e were obtained using the same pro-
for C20H18N4PSAu: C, 41.82; H, 3.16; N, 9.75; S, 5.58. Found: C, cedure starting from chlorido[tri(2-furyl)phosphane]gold(I)
42.31; H, 3.30; N, 9.97; S, 5.32%. MS (ESI+, 100 V, CH2Cl2, m/z): and the corresponding 1-R-5-thiotetrazole (R = Me (3a),
575.07 [M + H]+; 721.15 [Au(PPh3)2]+. DSC/TG: 189–190 °C Ph (3b)).
(mp); 282 °C (Tmax, exothermal dec.). 1H NMR [400 MHz,
(1-Methyltetrazol-5-ylthiolato)[tri(2-furyl)phosphane]gold(I)
CDCl3, δ (ppm)]: 7.62–7.47 (m, 15H, C6H5); 3.96 (s, 3H, CH3). (2d). Yield (from THF–hexane): 220 mg (81%). Anal. Calc. for
13C NMR [100 MHz, CDCl3, δ (ppm), J (Hz)]: 158.0 (s, C(5)tz); C14H12N4O3PSAu: C, 30.89; H, 2.22; N, 10.29. Found: C, 30.97;
2
4
134.3 (d, J(C,P) = 13.8, ortho-C from PPh3); 131.9 (d, J(C,P) = H, 2.38; N, 10.71%. MS (ESI+, 100 V, CH2Cl2, m/z): 545.01
2.5, para-C from PPh3); 129.3 (d, 3J(C,P) = 11.7, meta-C from [M + H]+; 661.03 [Au(2-furyl)2]+. DSC/TG: 120–122 °C (mp);
PPh3); 128.9 (d, 1J(C,P) = 59.0, ipso-C from PPh3); 34.0 (s, 232 °C (Tmax, exothermal dec.). 1H NMR [400 MHz, CDCl3,
CH3tz). FT-IR (ν, cm−1): 3054 (s), ν(CHaryl); 2942 (m), ν(CHalkyl); δ (ppm)]: 7.80 (m, 3H, CHfuryl); 7.34 (m, 3H, CHfuryl); 6.57 (m,
1478 (s), ν(CCaryl); 1436 (vs), 1377 (s), δ(CHaryl) + δ(CHalkyl); 3H, CHfuryl); 3.97 (s, 3H, CH3). 13C NMR [100 MHz, CDCl3,
1332 (m), 1313 (m), 1264 (s), 1222 (m), 1162 (s), ν(NvNtz) + δ (ppm), J (Hz)]: 157.8 (s, C(5)tz); 150.0 (d, 3J(C,P) = 6.4, C(5)
1
ν(N–Ntz); 1101 (s), 1077 (m), 1026 (m), 996 (m), 969 (m), δ(CN4) from furyl); 141.3 (d, J(C,P) = 93.8, C(2) from furyl); 125.8 (d,
+ δ(C6H5); 749 (vs), ν(C–P); 693 (vs), ν(C–S).
2J(C,P) = 27.1, C(3) from furyl); 111.7 (d, 3J(C,P) = 9.8, C(4)
(1-Phenyltetrazol-5-ylthiolato)(triphenylphosphane)gold(I) from furyl); 34.1 (s, CH3). FT-IR (ν, cm−1): 3115 (m), 2981 (w),
(2b). Yield (from CH2Cl2–hexane): 290 mg (91%). Anal. Calc. ν(CHaryl); 2951 (w), ν(CHalkyl); 1546 (m), 1465 (m), 1450 (m),
for C25H20N4PSAu: C, 47.18; H, 3.17; N, 8.80; S, 5.04. Found: C, ν(CCaryl); 1381 (m), 1366 (m), δ(CHaryl) + δ(CHalkyl); 1271 (m),
47.32; H, 3.22; N, 9.08; S, 4.88%. MS (ESI+, 100 V, CH2Cl2, m/z): 1215 (m), ν(NvNtz) + ν(N–Ntz); 1170 (m), ν(C–O); 1130 (m),
637.09 [M + H]+; 721.15 [Au(PPh3)2]+. DSC/TG: 195–196 °C 1110 (s), 910 (m), 881 (m), δ(CN4) + δ(furyl); 760 (s), ν(C–P);
(mp); 213 °C (Tmax, exothermal dec.). 1H NMR [400 MHz, 705 (m) ν(C–S).
CDCl3, δ (ppm)]: 7.76–7.74 (m, 2H, CH from C6H5tz); 7.59–7.40
(1-Phenyltetrazol-5-ylthiolato)[tri(2-furyl)phosphane]gold(I)
tz
(m, 18H, CH from C6H5 and PPh3); 3.96 (s, 3H, CH3). 13C (2e). Yield (from CH2Cl2–hexane): 270 mg (89%). Anal. Calc.
NMR [100 MHz, CDCl3, δ (ppm), J (Hz)]: 158.1 (s, C(5)tz); 135.2 for C19H14N4O3PSAu: C, 37.64; H, 2.33; N, 9.24. Found: C,
(s, ipso-C from C6H5tz); 134.3 (d, 2J(C,P) = 13.8, ortho-C from 37.43; H, 2.27; N, 9.12%. MS (ESI+, 100 V, CH2Cl2, m/z): 607.03
PPh3); 131.9 (s, meta-C from C6H5tz); 131.9 (d, 4J(C,P) = 2.3, [M + H]+; 661.03 [Au(2-furyl)2]+. DSC/TG: 179–180 °C (mp);
3
para-C from PPh3); 129.3 (d, J(C,P) = 11.7, meta-C from PPh3); 214 °C (Tmax, exothermal dec.). 1H NMR [400 MHz, CDCl3,
129.1 (s, para-C from C6H5tz); 128.9 (d, 1J(C,P) = 59.0, ipso-C δ (ppm)]: 7.80–7.79 (m, 3H, CHfuryl); 7.78–7.76 (m, 2H, C6H5tz);
from PPh3); 124.6 (s, ortho-C from C6H5tz). FT-IR (ν, cm−1): 7.54–7.51 (m, 2H, C6H5tz); 7.48–7.45 (m, 1H, C6H5tz); 7.34–7.32
3074 (w), 3051 (w), 3032 (w), ν(CHaryl); 1595 (m), 1500 (s), 1481 (m, 3H, CHfuryl); 6.57–6.56 (m, 3H, CHfuryl). 13C NMR
(m), 1435 (vs), 1418 (m), 1402 (m), 1377 (s), ν(CCaryl) + [100 MHz, CDCl3, δ (ppm), J (Hz)]: 157.8 (C(5)tz); 150.0 (d,
δ(CHaryl); 1330 (w), 1311 (w), 1271 (s), 1229 (s), 1182 (m), 3J(C,P) = 6.3, C(5) from furyl); 141.4 (d, 1J(C,P) = 92.5, C(2)
1159 (m), ν(NvNtz) + ν(N–Ntz); 1103 (vs), 1079 (m), 1028 (m), from furyl); 135.2 (s, ipso-C from C6H5tz); 129.3 (s, meta-C from
998 (m), 980 (w), δ(CN4) + δ(C6H5); 758 (vs), 748 (vs), ν(C–P); C6H5tz); 129.2 (s, para-C from C6H5tz); 125.7 (d, J(C,P) = 27.1,
2
713 (s), 696 (vs) ν(C–S).
C(3) from furyl); 124.5 (s, ortho-C from C6H5tz); 111.7 (d,
[1-(2-(N-Dimethylamino)ethyl)tetrazol-5-ylthiolato](triphenyl- 3J(C,P) = 9.8, C(4) from furyl). FT-IR (ν, cm−1): 3158 (m),
phosphane)gold(I) (2c). Yield (from acetonitrile): 265 mg 3122 (m), ν(CHaryl); 1597 (m), 1550 (m), 1496 (s), ν(CCaryl);
(84%). Anal. Calc. for C23H25N5PSAu: C, 43.75; H, 3.99; N, 1459 (m), 1397 (m), 1369 (s), δ(CHaryl); 1313 (m), 1266 (m),
11.09; S, 5.08. Found: C, 43.72; H, 3.44; N, 11.07; S, 5.01%. MS 1217 (m), ν(NNtz) + ν(N–Ntz); 1170 (s), (C–O); 1130 (s),
(ESI+, 100 V, CH2Cl2, m/z): 632.13 [M
+
H]+; 721.15 1100 (m), 1080 (m), 1040 (m), 1010 (vs), 909 (m), 881 (m),
[Au(PPh3)2]+. DSC/TG: 195–196 °C (mp); 256 °C (Tmax, exother- 830 (m), δ(CN4) + δ(furyl); 752 (vs), ν(C–P); 692 (s) ν(C–S).
mal dec.). 1H NMR [400 MHz, CDCl3, δ (ppm), J (Hz)]:
Crystallographic studies
7.62–7.47 (m, 15H, C6H5 from PPh3); 4.41 (t, 3J(H,H) = 7.2, 2H,
α-CH2); 2.85 (t, 3J(H,H) = 7.2, 2H, β-CH2); 2.32 (s, 6H, N(CH3)2). Single crystal X-ray data of complex 2e were collected at room
13C NMR [100 MHz, CDCl3, δ (ppm), J (Hz)]: 157.6 (s, C(5)tz); temperature on a SMART APEX II diffractometer using graphite
1166 | Dalton Trans., 2015, 44, 1161–1169
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monochromated Mo-Kα radiation (λ = 0.71073 Å). The reflec- tions in DMF and diluted with the cell culture medium to the
tion data were corrected on absorption. The structures were final assay concentrations (0.1% v/v DMF). The assay pro-
solved by direct methods with the program SIR200437 and cedure is described in more detail in our recent papers.10,40
refined on F2 by the full-matrix least squares technique with The IC50 value was described as that concentration reducing
SHELXL-2013.38 All non-hydrogen atoms were refined anisotro- the proliferation of untreated control cells by 50%.
pically. The hydrogen atoms were placed at calculated posi-
Cellular uptake studies
tions and refined using a “riding” model, with Uiso(H) =
1.2Ueq(C). Molecular graphics was performed with the
Sample preparation for cellular uptake studies. The cellular
program PLATON.39 Crystallographic data of complex 2e uptake was measured according to a previously described pro-
(excluding structure factors) have been deposited with the cedure.32 In short, cells were grown until at least 70% con-
Cambridge Crystallographic Data Centre.
fluency in 75 cm2 cell culture flasks. Stock solutions of the
gold complexes in DMF were freshly prepared and diluted with
cell culture medium to the desired concentration (final DMF
TrxR/GR inhibition assay
To determine the inhibition of TrxR and GR an established concentration: 0.1% v/v; final gold complex concentration:
microplate reader based assay was performed with minor 10 µM). The cell culture medium of the cell culture flasks was
modifications.12 For this purpose commercially available rat replaced with 4.0 mL of the cell culture medium solutions con-
liver TrxR and baker yeast GR (both from Sigma-Aldrich) were taining the compounds and the flasks were incubated at
used and diluted with distilled water to achieve concentrations 37 °C/5% CO2 for 1, 4 or 8 h. The cell pellets were isolated by
of 0.4 U mL−1 (TrxR) and 20.0 U mL−1 (GR). The compounds trypsinisation and centrifugation (room temperature, 3500g,
were freshly dissolved as stock solutions in DMF. To each 5 min), resuspended in double distilled water, lysed by soni-
25 μL aliquots of the enzyme solution, each 25 μL of potassium cation and appropriately diluted using double distilled water.
phosphate buffer pH 7.0 containing the compounds in graded An aliquot was removed for the purpose of protein quantifi-
concentrations or the vehicle (DMF) without compounds cation by the Bradford method. The determination of the gold
(control probe) were added and the resulting solutions (final content of the samples was performed by high resolution con-
concentration of DMF: max. 0.5% v/v) were incubated with tinuum source atomic absorption spectroscopy (see below).
moderate shaking for 75 min at 37 °C in a 96 well plate. To Results were calculated from the data of 2–3 independent
each well, 225 µL of the reaction mixture (1000 µL of the reac- experiments and are given as nmol gold per mg cellular
tion mixture consisted of 500 µL potassium phosphate buffer protein.
pH 7.0, 80 µL of 100 mM EDTA solution pH 7.5, 20 µL bovine
Atomic absorption spectroscopy (AAS). Gold contents were
serum albumin solution 0.05%, 100 µL of 20 mM NADPH solu- measured with a graphite furnace high resolution continuum
tion and 300 µL of distilled water) were added and the reaction source atomic absorption spectrometer (contrAA®700, Analytik
was started by addition of 25 µL of an 20 mM ethanolic dithio- Jena AG) at 242.795 nm according to a recently described
bis-2-nitrobenzoic acid (DTNB) solution. After proper mixing, method with minor modifications.32,33 In short, to 200 µL
the formation of 5-thio-2-nitrobenzoic acid (5-TNB) was moni- aliquots of the diluted lysates 20 μL Triton X-100 (1%) and
tored with a microplate reader (Perkin Elmer Victor X4) at 20 μL ascorbic acid (1%) were added. The gold content of the
405 nm in 35 s intervals for 350 s. For each tested compound samples was accessed using freshly prepared matrix-matched
the non-interference with the assay components was con- solutions of test compounds for calibration. Probes were
firmed by a negative control experiment using an enzyme free injected at a volume of 25 μL into standard graphite wall
solution. The IC50 values were calculated as the concentration tubes. The mean absorbance of triplicate injections was used
of the compound decreasing the enzymatic activity of the throughout the study. Drying, atomization and tube cleaning
untreated control by 50% and are given as the means and steps were performed as outlined in more detail in the
error of 2–3 independent experiments.
literature.10
Concentration dependent effects on drug resistant human
leukemia cells. The following human cell line and its chemo-
resistant subline were used in this study: leukemic B-cell
Cell culture and cytotoxicity assay in MDA-MB-231 and HT-29
cells
The antiproliferative effects in MDA-MB-231 and HT-29 cells precursor (Nalm-6) and its vincristine (Nalm/VCR) resistant
after 72 h (HT-29) or 96 h (MDA-MB-231) exposure to the gold subline. All cell lines were cultured in RPMI 1640 sup-
complexes were evaluated using the crystal violet assay.
plemented with 10% fetal calf serum. Apoptotic cell death was
In short, HT-29 human colon carcinoma cells and determined by a modified cell cycle analysis, which detects
MDA-MB-231 breast cancer cells were maintained in cell DNA fragmentation at the single cell level. For the measure-
culture medium (minimum essential eagle medium sup- ment of DNA fragmentation cells were seeded at a density of
plemented with 2.2 g NaHCO3, 110 mg L−1 sodium pyruvate 1 × 105 cells mL−1 and treated with different concentrations of
and 50 mg L−1 gentamicin sulfate adjusted to pH 7.4) contain- compound 2a or 2d (stock solutions were prepared in DMSO
ing 10% (v/v) fetal calf serum at 37 °C/5% CO2 and passaged in these experiments). After 72 h of incubation, cells were col-
once a week according to standard procedures. For the experi- lected by centrifugation at 300g for 5 min, washed with PBS at
ments, the compounds were prepared freshly as stock solu- 4 °C, and fixed in PBS/2% (v/v) formaldehyde on ice for
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30 min. After fixation, cells were incubated with ethanol–PBS 16 W. Fiskus, N. Saba, M. Shen, M. Ghias, J. Liu, S. D. Gupta,
(2 : 1, v/v) for 15 min, pelleted, and resuspended in PBS con-
taining 40 μg per mL of RNase A. After incubation for 30 min
at 37 °C, cells were pelleted again and finally resuspended in
PBS containing 50 μg per mL of propidium iodide. Nuclear
L. Chauhan, R. Rao, S. Gunewardena, K. Schorno,
C. P. Austin, K. Maddocks, J. Byrd, A. Melnick, P. Huang,
A. Wiestner and K. N. Bhalla, Cancer Res., 2014, 74, 2520–
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DNA fragmentation was then quantified by flow cytometric 17 V. A. Ostrovskii, G. I. Koldobskii and R. E. Trifonov,
determination of hypodiploid DNA. Data were collected and
analyzed using an FACScan (Becton Dickinson, Heidelberg,
Comprehensive Heterocyclic Chemistry III, Elsevier Ltd.,
2008, vol. 15, pp. 257–423.
Germany) equipped with the CELLQuest software. Data are 18 M. Uemura, T. Suzuki, K. Nishio, M. Chikuma and
given in % hypoploidy (subG1), which reflects the number of
apoptotic cells.
S. Komeda, Metallomics, 2012, 4, 686–692.
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Acknowledgements
The postdoctoral fellowships for T.V.S. by German Academic
Exchange Service (DAAD) and Saint Petersburg State University
(12.50.1560.2013) as well as the financial support by Dr Kleist-
Foundation (Berlin) are gratefully acknowledged. Mass-
spectrometry was performed at the Center for Chemical Analy-
sis and Material Research of St Petersburg State University.
23 A. Ilie, C. I. Rat, S. Scheutzow, C. Kiske, K. Lux,
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