Alkynyl gold(I) phosphane complexes: Evaluation of structure-activity-relationships for the phosphane ligands, effects on key signaling proteins and preliminary in-vivo studies with a nanoformulated complex

Article abstract of DOI:10.1016/j.jinorgbio.2015.12.020

Gold alkynyl complexes with phosphane ligands of the type (alkynyl)Au(I)(phosphane) represent a group of bioorganometallics, which has only recently been evaluated biologically in more detail. Structure-activity-relationship studies regarding the residues

Full text of DOI:10.1016/j.jinorgbio.2015.12.020

JIB-09880; No of Pages 9
Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Alkynyl gold(I) phosphane complexes: Evaluation of structure–activity-relationships
for the phosphane ligands, effects on key signaling proteins and preliminary in-vivo
studies with a nanoformulated complex
Vincent Andermark a,b, Katrin Göke b,c, Malte Kokoschka d,e, Mohamed A. Abu el Maaty f, Ching Tung Lum g,h
,
Taotao Zou g,h, Raymond Wai-Yin Sun g,h, Elisabet Aguiló i, Luciano Oehninger a,b, Laura Rodríguez i,
Heike Bunjes b,c, Stefan Wölfl f, Chi-Ming Che g,h, Ingo Ott a,b,
⁎
a
Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstrasse 55, 38106 Braunschweig, Germany
b
c
PVZ — Center of Pharmaceutical Engineering, Franz-Liszt-Straße 35A, 38106 Braunschweig, Germany
Institute of Pharmaceutical Technology, Technische Universität Braunschweig, Mendelssohnstrasse 1, 38106 Braunschweig, Germany
Gilead Sciences & IOCB Research Center, Department of Computational Chemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
d
16610 Prague 6, Czech Republic
e
Department of Physical Chemistry, Palacky University, 77146 Olomouc, Czech Republic
Department of Biology, Institut für Pharmazie und Molekulare Biotechnologie, Ruperto-Carola University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
f
g
State Key Laboratory of Synthetic Chemistry, Chemical Biology Center, The University of Hong Kong, 8/F, The Hong Kong Jockey Club Building for Interdisciplinary Research, 5, Sassoon Road,
Pokfulam, Hong Kong
h
Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong
Departament de Química Inorgànica, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain
i
a r t i c l e i n f o
a b s t r a c t
Article history:
Gold alkynyl complexes with phosphane ligands of the type (alkynyl)Au(I)(phosphane) represent a group of
bioorganometallics, which has only recently been evaluated biologically in more detail. Structure–activity-rela-
tionship studies regarding the residues of the phosphane ligand (P(Ph)3, P(2-furyl)3, P(DAPTA)3, P(PTA)3,
P(Et)3, P(Me)3) of complexes with an 4-ethynylanisole alkyne ligand revealed no strong differences concerning
cytotoxicity. However, a relevant preference for the heteroatom free alkyl/aryl residues concerning inhibition of
the target enzyme thioredoxin reductase was evident. Complex 1 with the triphenylphosphane ligand was se-
lected for further studies, in which clear effects on cell morphology were monitored by time-lapse microscopy.
Effects on cellular signaling were determined by ELISA microarrays and showed a significant induction of the
phosphorylation of ERK1 (extracellular signal related kinase 1), ERK2 and HSP27 (heat shock protein 27) in
HT-29 cells. Application of 1 in-vivo in a mouse xenograft model was found to be challenging due to the low sol-
ubility of the complex and required a formulation strategy based on a peanut oil nanoemulsion.
© 2015 Elsevier Inc. All rights reserved.
Received 1 October 2015
Received in revised form 8 December 2015
Accepted 28 December 2015
Available online xxxx
Keywords:
Alkyne
Cancer
Formulation
Gold
Microarray
Xenograft
1. Introduction
other gold(I) species are registered drugs for the treatment of rheuma-
toid arthritis and strong evidence for their efficacy against different dis-
Gold based therapeutics have a long tradition in medicine lasting
from ancient times over alchemy into modern ages [1–6]. While gold
and its salts have been used for hundreds to thousands of years, modern
research has witnessed the development of more sophisticated bioac-
tive complexes that contain several types of coordinated ligands (e.g.
thiolates [7,8], phosphanes [9–11], porphyrines [12], dithiocarbamates
[13,14], N-heterocyclic carbenes [15–18] or alkynes [19–25]) or
heterobimetallic species [26,27]. Currently, Auranofin (see Fig. 1) and
eases such as cancer or bacterial infections exists [2,28,29]. The renewed
interest in gold based metallodrugs has led to increasing efforts in under-
standing their biochemical mechanisms of drug action and in the rational
development of improved pharmacologically active compounds [5,6].
A single mode of action for all gold complexes unlikely exists, how-
ever, strong and selective inhibition of the enzyme thioredoxin reduc-
tase (TrxR) has been demonstrated for many gold species and might
be in general of high relevance for the pharmacology of a large number
of gold metallodrugs. Further important biochemical characteristics ob-
served frequently with gold compounds include the inhibition of tumor
cell proliferation, the induction of apoptosis, antimitochondrial effects
or the increased formation of reactive oxygen species. However, stabil-
ity of the ligands coordinated to gold is a critical issue and triggers a high
demand for gold complexes with stably coordinated ligands. Enhanced
Abbreviations: DFT, density functional theory; ERK, extracellular signal related kinase;
FAK, focal adhesion kinase; GSK, glycogen synthase kinase; HSP, heat shock protein; MAPK,
mitogen activated protein kinase; TOR, target of rapamycin; TrxR, thioredoxin reductase.
⁎
Corresponding author at: Institute of Medicinal and Pharmaceutical Chemistry, Technische
Universität Braunschweig, Beethovenstrasse 55, 38106 Braunschweig, Germany.
E-mail address: ingo.ott@tu-bs.de (I. Ott).
http://dx.doi.org/10.1016/j.jinorgbio.2015.12.020
0162-0134/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: V. Andermark, et al., Alkynyl gold(I) phosphane complexes: Evaluation of structure–activity-relationships for the
phosphane ligands, effects on key s..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2015.12.020

2
V. Andermark et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Fig. 1. The Au(I)(phosphane)thiolate complex Auranofin and the (alkynyl)Au(I)(phosphane) complex 1.
stability might be reached by the use of carbene ligands with the forma-
tion of organometallic gold species.
method for the synthesis of 1 is described in more detail in the
Experimental section.
In this context, we have recently reported on gold(I) complexes of
the type (alkynyl)Au(I)(triphenylphosphane) that contain an anionic
alkynyl group as well as a neutral phosphane ligand [19,30]. Such organ-
ometallic gold compounds promise an improved stability compared to
the traditional gold drugs based on the relatively high bond dissociation
energies around the gold center. Some of the studied complexes turned
out to be very strong and selective inhibitors of TrxR, showed high anti-
proliferative activity in tumor cells, influenced key parameters of tumor
cell metabolism, and triggered anti-angiogenic effects at non-toxic con-
centrations in zebrafish embryos [19].
Motivated by these encouraging biological properties, we selected a
highly active complex of our previous report as a lead compound for fur-
ther studies [19]. In the present study, the phosphane ligands were var-
ied with the aim to establish possible structure–activity-relationships,
and further biological properties were evaluated including effects on
cellular signaling and in-vivo studies using a xenograft animal model.
Density Functional Theory (DFT) at the RI-PBE-D3/def2-TZVPP
COSMO level was used to calculate geometries of all complexes in
vacuo and in water (Table 1). As example the calculated solution struc-
ture of 1 is shown in Fig. 2.
Table 1
Estimated bond dissociation energies (bond elongation of 5 Å) and ratios between C-Au
and P-Au bonds.
Compound
C-Au
P-Au
C/P
Kcal/mol
Kcal/mol
1 (−Ph)
72.05
74.02
72.71
71.03
67.72
68.63
53.21
48.00
50.38
51.97
58.73
57.68
1.35
1.54
1.44
1.37
1.15
1.19
2 (−2-furyl)
3 (DAPTA)
4 (PTA)
5 (ethyl)
6 (methyl)
1.1. Chemistry
Complexes 1–6 were prepared by reacting 4-ethynylanisole with the
respective chloridogold(I)phosphane under basic conditions. The com-
plexes were isolated and purified by filtration and washed as appropri-
ate (Scheme 1).
Complex formation and identity was clearly confirmed by the absence
of the terminal hydrogen signal of the alkyne, the presence of the M+ sig-
nal in mass spectrometry, and singulet resonances in 31P-NMR spectra.
13C-NMR spectra were taken (with the exception of complex 4), however,
the very low signal intensities of the alkyne carbons did not allow a com-
plete spectroscopic evaluation of these spectra. The high purities neces-
sary for biological evaluation were confirmed by elemental analyses
(deviations below 0.3% from the theoretical values).
Based on the biological screening described below, complex 1 was
selected for further studies and in this context the synthesis procedure
of 1 was stepwise improved resulting in a yield of 58%. The improved
Fig. 2. Solution structure of complex 1 calculated by DFT.
Scheme 1. Synthesis of complexes 1–6.
Please cite this article as: V. Andermark, et al., Alkynyl gold(I) phosphane complexes: Evaluation of structure–activity-relationships for the
phosphane ligands, effects on key s..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2015.12.020

V. Andermark et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
3
Table 2
2 kcal/mol for the C-Au bonds and 0.5 kcal/mol for the P-Au bonds.
The larger differences of 7.6 kcal/mol for the C-Au bonds and
10.7 kcal/mol for the P-Au bonds are likely to originate from the larger
differences in the electron donating ability of the used phosphane li-
gands. A strengthening of the P-Au bond is observed with a weakening
of the C-Au bond, as it was found for the C-Au bond of carbene
gold(I) complexes upon variation of the opposing ligand [9,33].
Concerning the weaker P-Au bonds this indicates the following order
of stability of the compounds: 2 b 3 b 4 b 1 b 6 b 5. Noteworthy, the sig-
nificantly highest values were determined for compounds 5 and 6 with
ethyl and methyl residues, respectively.
Antiproliferative effects in HT-29 and MDA-MB-231 cells and inhibition of TrxR expressed
as IC50 concentrations in micromolar (μM) units.
HT-29
MDA-MB-231
TrxR
1 (R = −Ph)
5.0 [19]
2.4 0.3
3.8 0.4
2.1 0.2
2.5 0.3
1.1 0.1
1.6 0.1
0.05 [19]
2 (R = −2-furyl)
3 (R = −DAPTA)
4 (R = PTA)
5 (R = −CH2-CH3)
6 (R = −CH3)
4.5 0.3
3.3 0.2
4.3 0.3
2.6 0.1
4.2 0.4
0.92 0.13
0.12 0.04
0.14 0.04
0.06 0.01
0.05 0.01
Subsequently high level post-SCF calculations were used to deter-
mine bond dissociation energies of the ligand-gold bonds. This allows
estimating the influence of ligand variations on the stability of their co-
ordination bonds. We chose the LPNO–CEPA [31] method and a mixed
def2-QZVP/def2-TZVP [32] basis set to perform bond dissociation
scans. The LPNO–CEPA method was recently introduced by Neese
et al. and combines a high speed with an accuracy, intermediate to
CCSD and the current gold standard CCSD(T) [31]. Our calculations
showed for both the C-Au and the P-Au bonds high bond dissociation
energies in the range of 67.72–74.02 Kcal/mol for the C-Au bond and
48.00–58.73 Kcal/mol for the P-Au bond. These differences are more
pronounced than those seen in an earlier study on alkynyl
gold(I) complexes [19], where the differences were in the range of
1.2. Effects on cell proliferation, TrxR and cell morphology
The antiproliferative effects of complexes 1–6 were evaluated in
HT-29 colon carcinoma and MDA-MB-231 breast adenocarcinoma
cells (see Table 2). The obtained IC50 values were in a rather narrow
range of 1–5 μM (2.6–5.0 μM in HT-29 cells, and 1.1–3.8 μM in MDA-
MB-231 cells), and thus did not allow clear conclusions concerning
structure–activity-relationships. The most active compound, however,
was 5, which afforded the lowest IC50 values in both cell lines indicating
some preference for the triethylphosphane ligand concerning cytotoxicity.
The disulfide reductase enzyme TrxR is an established target for gold
metallodrugs [34–36]. As expected, complexes 1–6 were efficient inhib-
itors of TrxR with IC50 values in the low nanomolar range. Compounds 1,
Fig. 3. Morphological changes in RC-124 (top) and HT-29 (bottom) cells. Cells were exposed to 1.5 μM (RC-124) or 10 μM (HT-29) of 1 and images were taken every hour over a period of
96 h. Time-lapse videos are provided as supporting information.
Please cite this article as: V. Andermark, et al., Alkynyl gold(I) phosphane complexes: Evaluation of structure–activity-relationships for the
phosphane ligands, effects on key s..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2015.12.020

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V. Andermark et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Fig. 4. ELISA microarray measurements of absolute phosphor-protein concentrations in HT-29 cells treated with 1. mock-treatment: solvent control (DMF).
5 and 6 with phenyl-, ethyl-, and methyl-substituted phosphane ligands
were highly active (IC50 values of 0.05 and 0.06 μM, respectively) and
complexes 2–4 with furyl-, DAPTA and PTA containing phosphanes
also afforded appreciable IC50 values. However, 2–4 were significantly
less active against TrxR than 1, 5 and 6 (see Table 2).
Since the differences in cytotoxicity between these most active TrxR
inhibitors (1, 5 and 6) were small and previous studies had indicated in-
teresting additional biological properties of complex 1 (e.g. anti-
angiogenic properties, effects on mitochondria), this compound was se-
lected as a well investigated example for further studies [19].
Please cite this article as: V. Andermark, et al., Alkynyl gold(I) phosphane complexes: Evaluation of structure–activity-relationships for the
phosphane ligands, effects on key s..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2015.12.020

V. Andermark et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
5
Fig. 5. Tumor volume (left) and body weight (right) in the NCI-H460 xenograft mice model.
In order to check for possible tumor selectivity, the cytotoxicity
against non-tumor L-929 mouse connective tissue fibroblasts and RC-
124 human kidney cells was evaluated. IC50 values of 3.3 1.0 μM in
L-929 cells and 1.5 0.2 μM in RC-124 cells, respectively, were obtain-
ed. This high antiproliferative activity against non-tumor cell lines indi-
cates that compound 1 does not show selectivity for tumor tissue.
Microscopic live cell imaging allows monitoring of morphological
changes in drug exposed cells under cell culture conditions. In these ex-
periments RC-124 or HT-29 cells were grown until at least 20%
confluency before 1 was added and pictures were taken every hour
for 96 h (see Fig. 3 and video files of the supporting information).
Whereas untreated RC-124 control cells showed a continuous exten-
sion of the cell layer leading to confluency, cells treated with 1.5 μM of 1
experienced major morphological alterations within the first 12 h of ex-
posure. This was most obvious after 6–10 h of incubation when the cells
were strongly deformed compared to the untreated control and round-
ed up. With longer exposure cell growth still was maintained, however,
cell morphology was substantially affected as evident by an elongated
shape of the cells.
their phosphorylated states in HT-29 colon cancer cells (see Fig. 4).
For this purpose an ELISA microarray assay was used, which had previ-
ously been developed and applied [37]. Complex 1 was administered at
concentrations of 5.0 μM and 10 μM, and measurements were done over
a period of 5 h.
A highly significant and persistent activation of the important mito-
gen activated protein kinases (MAPK) phospho-ERK1 (phosphorylated
extracellular signal related kinase 1) and phospho-ERK2 was clearly ob-
served. Both kinases play a key role in the MAPK cascade and regulate
diverse biological functions such as cell growth, differentiation and sur-
vival. Moreover, the chaperone HSP27 (heat shock protein 27) was
strongly induced by 10 μM of 1, and this can be interpreted as a response
to cytotoxic stress caused by the compound. Low effects or absence of
effects were noted for the phosphorylations of the focal adhesion kinase
(FAK), the proto-oncogene Src, target of rapamycin (TOR), p70S6K, gly-
cogen synthase kinase 3 β (GSK-3β), Akt1, p38, and Chk2.
1.4. Formulation of 1 and preliminary animal studies
In contrast analogous experiments using HT-29 cells exposed to
10 μM of 1 remained morphologically little affected for more than
30 h, after which obvious cell death occurred as evidenced by a detach-
ment and rounding up of the cells. This effect was not reversible and
cells completely detached over extended exposure.
Initial experiments to prepare solutions or suspensions of 1, which
are suitable for administration purposes in mice, were done using oil
or phosphate buffered saline. For this purpose the compound was dis-
solved in concentrations up to 0.2 mg/μL in DMF, DMSO, PET (60% poly-
ethylene glycol 400, 30% ethanol, 10% Tween 80) or Kolliphor EL and the
resulting stock solutions were diluted up to 50-fold using phosphate
buffered saline or oil. However, these attempts did not afford suitable
solutions/suspensions since visually non-homogenous precipitates
were obtained upon dilution.
1.3. ELISA microarray studies
In order to gain more insights into the mechanisms of 1 on the cellu-
lar level several important key signaling proteins were measured in
Fig. 6. NCI-H460 xenograft mice treated with 6 dosages (2.5 mg/kg) of nanoformulated 1 after 14 days.
Please cite this article as: V. Andermark, et al., Alkynyl gold(I) phosphane complexes: Evaluation of structure–activity-relationships for the
phosphane ligands, effects on key s..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2015.12.020

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V. Andermark et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Accordingly, several pharmaceutical formulations were screened to
a similar pattern like previously studied phosphane containing gold
complexes [40], but differed from a biscarbene gold(I) complex, which
had not triggered ERK activation [41]. Strong induction of HSP27 phos-
phorylation had also been observed with the two other gold
metallodrugs recently [40,41]. Small effects of 1 were noted on the
phosphorylation of FAK in the investigated period of 5 h, which is in
agreement with the observation that cell detachment of this cell line oc-
curred only after extended exposure of more than 30 h.
Taken together with previous results [19,30] the organometallic
alkynyl-gold(I)-phosphane center can be regarded as a useful organo-
metallic pharmacophore, which can be incorporated into biologically
active structures (e.g. coumarines [24] or naphthalimides [25]).
However, the in-vivo application of the model compound 1 experi-
enced major difficulties related to the insufficient solubility of the com-
plex in media used for injection. Nanoformulation of 1 in a peanut oil
nanoemulsion resulted in a formulation suitable for injection purposes
in mice. The low drug loading allowed a maximum dosage of
2.5 mg/kg, which was applied in a mouse xenograft model. The dosage
was ineffective concerning tumor growth inhibition but was well toler-
ated. The inactivity might be the consequence of the low dosage applied
or caused by an inefficient drug release from the formulation. Accord-
ingly, further studies will be required to translate the promising in-
vitro effects of alkynyl gold species into animal models. Such strategies
to enhance the in-vivo efficacy need to address the solubility problems
encountered with complex 1. Improved pharmaceutical formulations of
1 appear very promising in this aspect as well as further structural opti-
mizations leading to compounds with lower lipophilicity.
increase the solubility of complex 1. The nine used formulations includ-
ed nanoemulsions of various oils, mixed micelles, smectic nanoparticles
and liposomes (see Experimental section) [38]. After incubation with an
excess of 1 and removal of undissolved material, the amount dissolved
in each carrier was preliminarily estimated (for details see supporting
information) via atomic absorption spectroscopy (AAS) and for the
three carriers with the best loading results, the gold content was exactly
determined using AAS. These measurements yielded the following total
concentrations of 1: 0.017 mg/ml in Dynasan 112 nanoemulsion,
0.009 mg/ml in mixed micelles and 0.028 mg/ml in peanut oil
nanoemulsion.
Accordingly, peanut oil nanoemulsions, which had dissolved the
highest levels of 1, were selected for the preliminary in-vivo studies.
For these animal experiments a dedicated nanoemulsion formulation
was prepared which was handled and bottled under aseptic conditions.
Crushing complex 1 prior to incubation led to a higher drug load in this
emulsion compared to the screening results (0.098 mg/ml).
To study effects of this formulation of 1 in-vivo an established NCI-
H460 xenograft model was used. A number of six doses (2.5 mg/kg) of
formulated 1 were injected intratumorally at days 0, 2, 5, 7, 9 and 12.
The tumor volumes were measured and mice were sacrificed after
14 days (see Figs. 5 and 6). However, the treatment was not effective
as the tumor volumes did not reduce. Body weight changes in the treat-
ed group were not observed indicating that the application was well
tolerated.
2. Conclusions
3. Experimental section
Gold alkynyl phosphane complexes can be prepared in a convenient
one step procedure in high purities as required for biological and phar-
macological studies. Quantum chemical calculations indicated a higher
stability for derivatives with short alkyl residues (methyl, ethyl) at the
phosphane. The complexes trigger strong antiproliferative effects in
tumor cells. However, as exemplified for complex 1, their cytotoxicity
was not selective for tumor cells. While the cytotoxic effects were large-
ly independent of the residues at the phosphane (with a slight prefer-
ence for the ethyl group), some structure–activity-relationships could
be noted concerning TrxR inhibition indicating that alkyl/phenyl resi-
dues are preferred over those containing N/O heteroatoms.
3.1. General
All synthesis reagents were obtained from Sigma-Aldrich (Switzerland)
or Fluka Analytical. The purities of the synthesized compounds were con-
firmed by elemental analysis (Flash EA 1112, Thermo Quest) and differed
less than 0.5% from the predicted values. 1H NMR spectra, 13C NMR spectra
and 19F NMR spectra were recorded using a Bruker AV II-400 or Bruker
DRX-400 AS NMR spectrometer. Mass spectra were recorded on a
Finnigan-MAT 95 spectrometer (ionization energy for EI-MS: 70 eV). For
the absorption measurements in biological assays a Perkin Elmer 2030
Multilabel Reader VICTOR™ X4 was used.
Time-lapse video imaging showed that RC-124 kidney cells were af-
fected strongly with the first hours of exposure to 1 and showed an elon-
gated shape after longer incubation. Such phenomena might indicate
interactions of 1 with components of the cell surface or extracellular ma-
trix and interference with cell division. In contrast, HT-29 cells were af-
fected morphologically after longer exposure resulting in an irreversible
rounding-up and detachment of the cells. Altogether these observations
show that the cytotoxic effects of 1 are dependent on the type of cell
line and/or cell culture conditions. Of note, RC-124 cells were maintained
in culture using gelatin-coated cell culture materials (see Experimental
section) in order to improve adhesion to the surfaces and were treated
with a lower dosage of 1 (1.5 μM) compared to HT-29 cells (10 μM)
based on the results of the cytotoxicity assay. The effects of 1 on cell mor-
phology and adherence are of interest considering our previous report on
strong anti-angiogenic effects in zebrafish embryos at non-toxic concen-
trations [19]. Rounding up and cell detachment had also been reported
for the gallium complex KP46, which caused a loss of integrin mediated
cell adhesion [39]. Sensitivity against KP46 was found to be enhanced
when cells were grown on collagen I, which is a major ligand for integrins
[39]. Taken together, it can be speculated that some of the cytotoxic ef-
fects of 1 — especially against the highly sensitive RC-124 cells grown
on gelatine — might in a similar manner be related to interference with
cell surface proteins.
3.2. Improved synthesis method for complex 1
100.0 mg (0.757 mmol) 1-ethynyl-4-methoxybenzene and
127.3 mg (2.270 mmol) KOH were dissolved in 20 ml methanol
and stirred for 10 min. 374.7 mg (0.757 mmol) chlorido(tri-
phenylphosphane)gold(I) were added to the solution forming a sus-
pension. Dichloromethane was added dropwise to the solution until
the solid was completely solubilized. The solution was stirred for 2 h
under light protection and afterwards stored for 72 h at −20 °C.
White crystals were formed during storage and were filtered off.
The resulting solid was dissolved in CH2Cl2 and washed 2 times
with water. The organic solvent was dried with NaSO4 and then re-
moved under reduced pressure. Yield: 58% (259.0 mg); elemental
analysis (found/theor.): C(55.02/54.93), H(3.72/3.76) spectral data
(NMR and MS spectra) were as reported before [19].
3.3. General procedure for complexes 2–6
1-ethynyl-4-methoxybenzene and KOH are dissolved in methanol
or methanol/dichloromethane 2/1. After 10 min of stirring and com-
plete solution of 1 equivalent of the respective chloridogold(I)phos-
phane is added and stirring at room temperature is continued under
light protection (reaction time). After formation of an initial precipitate
the mixture is stored at −20 °C, the precipitate is isolated by filtration
Further studies on 1 confirmed clear effects on cellular signaling in
HT-29 cells with strong inductions of ERK1/2 and HSP27. In particular
concerning the effects on ERK1 and ERK2 phosphorylation, 1 showed
Please cite this article as: V. Andermark, et al., Alkynyl gold(I) phosphane complexes: Evaluation of structure–activity-relationships for the
phosphane ligands, effects on key s..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2015.12.020

V. Andermark et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
7
and, if necessary, purified by washing with water or methanol, and
dried.
1.84 (s); MS(EI): 404.1 [M + H+]+; elemental analysis [found/theor.]:
C(35.62/35.66), H(3.92/3.99).
Complex 2, [tri(2-furyl)phosphane][2-(4-methoxylphenyl)ethy-
nyl]gold(I).General method: 18.9 mg (0.143 mmol) 1-ethynyl-4-
methoxybenzene, 8.0 mg (0.143 mmol) KOH, 10 ml methanol,
66.4 mg (0.143 mmol) chloridogold(I)[tri(2-furyl)phosphane]; 2 h re-
action time, no storage at −20 °C, yield 40 mg (50%) gray powder
(m.p. 135–136 °C); 1H-NMR (CDCl3): 3.73 (s, 3 H, OCH3), 6.46 (m, 3 H,
ArH), 6.75 (m, 2 H, ArH), 7.12 (m, 3 H, ArH), 7.39 (m, 2 H, ArH), 7.70
(m, 3 H, ArH); 13C-NMR (CDCl3): 55.02 (OCH3), 111.94 (d, J = 8.9 Hz,
ArC), 113.92 (ArC), 125.02 (ArC) 132.65 (ArC), 151.02 (ArC), C ≡ C sig-
nals not observed, 31P-NMR (CDCl3): −29.03 (s); MS(EI): 561.06
[M + H]+; elemental analysis [found/theor.]: C (44.83/45.02), H(2.79/
2.88).
Complex 3, [tri[3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]
nonane]phosphane][2-(4-methoxylphenyl)ethynyl]gold(I).General
method: 15.7 mg (0.118 mmol) 1-ethynyl-4-methoxybenzene,
6.7 mg (0.118 mmol) KOH, 10 ml methanol +5.0 ml dichlorometh-
ane, 54.8 mg (0.118 mmol) chloridogold(I)[tri[3,7-diacetyl-1,3,7-
triaza-5-phosphabicyclo[3.3.1]nonane]phosphane]; 2 h reaction
time (then concentrated in vacuum), 12 h at −20 °C, yield 20 mg
(30%) white powder; 1H-NMR (CDCl3): 1.99 (s, 3 H, COCH3), 2.01
(s, 3 H, COCH3), 3.68 (m, 1 H), 3.72 (s, 3 H, OCH3), 3.93 (m, 3 H),
4.18 (m, 1 H), 4.49 (m, 1 H), 4.63 (m, 1 H), 4.80 (m, 1 H), 5.53 (m,
1 H), 5.63 (m, 1 H), 6.73 (m, 2 H, ArH), 7.30 (m, 2 H, ArH); 13C-
NMR (CDCl3): 21.25 (s, COCH3), 21.52 (s, COCH3), 39.59 (d, PCH2N,
J = 26.9 Hz), 44.79 (d, PCH2N, J = 26.1 Hz), 49.26 (d, PCH2N, J =
27.3 Hz), 55.23 (OCH3), 61.93 (s, NCH2-N), 67.18 (s, NCH2N),
113.99 (ArC), 133.45 (ArC), 169.68 (s, C = O), 170.11 (s, C = O),
C ≡ C signals not observed; 31P-NMR (CDCl3): 1.64 (s); MS(EI):
591.12 [M + H]+; elemental analysis [found/theor.]: C(38.59/38.79),
H(4.11/4.16), N(7.80/7.54).
3.4. Computational chemistry
Geometries of all complexes were calculated using the DFT func-
tional PBE [43–46] in conjunction with the Resolution of Identity (RI)
[47,48] technique and the def2-TZVPP [32] basis set in vacuo and in
water, simulated by the COSMO [49] solvent model. Dispersive inter-
actions were included via Grimme's atom-pair wise dispersion cor-
rection [50] (D3) with Becke–Johnson damping (BJ). The stationary
point was confirmed as minimum, by a frequency analysis. Bond dis-
sociation scans (10 points) for a bond elongation of 5 Å were per-
formed using LPNO–CEPA/1 [31,51,52] with the def2-QZVP [32]
basis set on gold and def2-TZVP [32] on all other atoms. The solvent
(water) was again simulated by the COSMO solvent model. After an
elongation of 5 Å the energies did not change significantly any longer
and the differences were used as bond dissociation energy. All quan-
tum mechanical calculations were performed using ORCA (version
3.03) [53].
3.5. Cell culture
HT-29 colon carcinoma cells and L-929 mouse fibroblasts were
maintained in Dulbecco's Modified Eagle Medium (4.5 g/L D-
Glucose, L-Glutamine, Pyruvate), which was supplemented with
gentamycin (12.5 mg/L) and fetal bovine serum (Biochrom
GmbH, Berlin) (10% V/V), and were passaged once a week. RC-
124 healthy human kidney cells were maintained in McCoy's 5A
(modified, with L-Glutamine) medium, which was supplemented
with gentamycin (12.5 mg/L) and fetal bovine serum (Biochrom
GmbH, Berlin) (10% V/V), and were also passaged once a week.
For experiments with RC-124 cells, microtiter plates had been
pretreated in the following way: 30 μL of a sterilized gelatine solu-
tion (1.5% (m/V)) were added to each well of flat bottom 96-well
plates, the plates were covered with their lids, incubated for 1 h
at 37 °C, the excess solution was removed, the wells were washed
with PBS 7.4 pH, and the new cell-culture medium was added.
175 cm2 cell culture flasks used for cultivation of RC-124 cells
were pretreated analogously.
Complex 4, [tri[1,3,5-triaza-7-phosphaadamantane)]phosphane][2-
(4-methoxylphenyl)ethyn-yl]gold(I).General method: 18.6 mg
(0.140 mmol) 1-ethynyl-4-methoxybenzene, 7.9 mg (0.140 mmol)
KOH, 10 ml methanol +5.0 ml dichloromethane, 54.8 mg
(0.140 mmol) chloridogold(I)[tri[1,3,5-triaza-7-phosphaadamantane)]
phosphane]; 2 h reaction time (then concentrated in vacuum), 12 h at
−20 °C, yield 10 mg (14%) white powder; 1H-NMR (CDCl3): 3.71
(3 H, s, OCH3), 4.18 (6 H, s, NCH2N), 4.46 (6 H, m, NCH2P), 6.72 (2 H,
m, ArH), 7.30 (2 H, m, ArH); 31P-NMR (CDCl3): −12.61(s), MS(ESI)
(m/z): 486.10 [M + H]+ elemental analysis [found/theor.]: C(37.01/
37.13), H(4.13/3.95), N(8.90/8.66).
Complex 5, (triethylphosphane)[2-(4-methoxylphenyl)ethynyl]
gold(I).
3.6. Cell proliferation inhibition (crystal violet assay)
General method: 32.6 mg (0.246 mmol) 1-ethynyl-4-
methoxybenzene, 41.5 mg mg (0.768 mmol) KOH, 4.0 ml methanol,
86.5 mg (0.2.46 mmol) chloridogold(I)(triethylphosphane); 18 h reac-
tion time, 72 h at −20 °C, yield 12 mg (11%) gray powder (m.p. 79–
80 °C); 1H NMR (CDCl3): 1.20 (m, 9 H, −CH3), 1.80 (m, 6 H, PCH2);
3.78 (s, 3 H, OCH3); 6.78 (m, 2 H, ArH), 7.43 (m, 2 H, ArH); 13C NMR
(CDCl3): 8.92 (CH3), 17.86 (d, CH2, J = 33.0 Hz), 55.16 (OCH3), 113.57
(ArC), 117 (ArC), 133.67 (ArC), 158.41 (ArC), C ≡ C signals not observed;
31P-NMR (CDCl3): 38.62 (s); MS(EI): 446.1 [M + H+]+; elemental anal-
ysis [found/theor.]: C(40.65/40.37), H(4.78/4.97).
A volume of 100 μL of HT-29 cells (2565 cells/ml), L-929 cells
(8100 cells/mL) or RC-124 cells (1460 cells/ml) was transferred into
the wells of 96-well plates (note: for RC-124 pretreated plates were
used, see above) and incubated at 37 °C/5% CO2 for 48 h (HT-29, L-
929) or 72 h (RC-124). Stock solutions of the compounds in
dimethylformamide (DMF) were freshly prepared and diluted with
the respective cell culture medium to graded concentrations (final con-
centration of DMF: 0.1% V/V). After 72 h (HT-29, L-929) or 96 h (RC-
124) of exposure, the cell biomass was determined by crystal violet
staining and the IC50 value was determined as the concentration that
caused 50% inhibition of cell proliferation compared to an untreated
control. Results were calculated as the mean of three independent
experiments.
Complex 6, [2-(4-methoxylphenyl)ethynyl](trimethylphosphane)
gold(I) [42].
General method: 20.62 mg (0.156 mmol) 1-ethynyl-4-
methoxybenzene, 26.26 mg (0.468 mmol) KOH, 7.2 ml methanol,
48.13 mg (0.156 mmol) chloridogold(I)trimethylphosphane; 2 h reac-
tion time, overnight at −20 °C; yield: 49.6 mg (79%) light yellow crys-
3.7. TrxR inhibition
tals (m.p. 129–132 °C); 1H NMR (CDCL3, 400 MHz): 1.52 (d, J2
=
10.1 Hz, 9 H, P-CH3); 3.78 (s, 3 H, −OCH3), 6.78 (m, 2 H, ArH), 7.41
(m, 2 H, ArH); 13C NMR (CDCl3, 400 MHz): 15.72 (d, J = 36,1 Hz,
−CH3), 55.54 (OCH3), 113.49 (ArC), 117.20 (ArC), 133.92 (ArC),
158.35 (ArC), C ≡ C signals not observed; 31P-NMR (CDCl3, 400 MHz):
The inhibition of isolated rat TrxR was determined as described in
previous reports [9,15,33]. IC50 values were calculated from 2–3 inde-
pendent experiments and are indicated as mean values with standard
errors.
Please cite this article as: V. Andermark, et al., Alkynyl gold(I) phosphane complexes: Evaluation of structure–activity-relationships for the
phosphane ligands, effects on key s..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2015.12.020

8
V. Andermark et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
3.8. Microscopic live cell imaging
Two batches of peanut oil nanoemulsion for the animal studies were
prepared separately as described above (median particle size around
130 nm). For loading, approximately 1 mg of crushed 1 was incubated
with 1 ml of this sterile filtered nanoemulsion under nitrogen atmo-
sphere in sterile glass vials. Following one week of incubation, undis-
solved material was removed by filtration through a 0.22 μm sterile
filter. Finally, the drug-loaded as well as unloaded nanoemulsion (sol-
vent control) were filtered (0.22 μm) into heat sterilized glass vials
and flushed with sterile filtered nitrogen. All bottling steps were carried
out under a clean bench.
RC-124 cells or HT-29 cells were grown in 175 cm2 cell culture flasks
(gelatine pretreated flasks were used in case of RC-124 cells, see above)
as described above until at least 30% confluency. The compounds were
prepared freshly as stock solutions in DMF and diluted 1:1000 with
cell culture medium. The cell culture medium of the flasks was replaced
with fresh medium containing the test compounds at the indicated con-
centration. Imaging was performed using a JuLIBr live cell movie analyz-
er (NanoEnTek) equipped with two microscope units. In each
experiment one microscope unit was used to monitor an untreated con-
trol and one microscope unit was used to monitor the respective drug
treated cells. The microscope units were placed in a CO2-incubator,
loaded with the respective tissue culture flasks, and images were
taken in 1 h intervals for a period of 96 h. Each experiment was per-
formed twice on separate days and afforded comparable results.
3.11. Atomic absorption spectroscopy (AAS)
Sample preparation was done as follows. Samples: to 180 μL of the
respective drug-loaded nanodispersion each 20 μL twice distilled
water, Triton X-100 (1%) and ascorbic acid (1%) were added; standard
solutions containing 1 were prepared analogously using drug-free
nanodispersion and aqueous suspensions of 1 instead of distilled
water (matrix matched calibration). The gold levels were measured
using a high-resolution continuum source atomic absorption spectrom-
eter (ContrAA 700, AnalytikJena AG). For this purpose a volume of 25 μL
of the respective sample or standard solution was injected into a stan-
dard graphite tube, thermally processed according to an established fur-
nace program [19,33], and absorbances were read at 242.7950 nm for
5 s. All samples were measured in triplicate and the mean values were
used for further calculations.
3.9. ELISA microarrays
Proteins were quantified using sandwich ELISA microarrays. The mi-
croarrays are based on the ArrayStrip™ platform (Alere Technologies
GmbH, Jena, Germany). A detailed description of the assay protocol
has been previously reported [54]. Briefly, HT-29 colon carcinoma cells
(ATCC) were cultivated at standard cell growth conditions and treated
with the indicated concentration of the compound freshly dissolved in
dimethylformamide (DMF). For mock-treatment, cells were incubated
with the solvent control containing the same amount of DMF as the
samples (0.1%). Cells were collected at indicated time points and total
protein concentration was determined in cell lysates using the BCA Pro-
tein Assay (Pierce Biotechnology, Rockford, USA). Cellular samples were
incubated with the microarrays for 60 min. A detection cocktail of 15
biotin-labeled phospho-specific detection antibodies (R&D Systems)
was used, with the concentration of each antibody at 18 ng/ml. Colori-
metric signals were detected by transmission measurements with the
Arraymate™ reader (Alere Technologies GmbH). Total protein concen-
trations were used for normalization.
3.12. NCI-H460 xenograft experiments
Three and a half million NCI-H460 cells suspended in 100 μl of PBS
were injected into the right back flanks of female 4–5 week old BALB/
cAnN-nu (Nude) mice by subcutaneous injection. When the tumor vol-
umes reached about 50–100 mm3 (2 days after tumor inoculation), the
mice were randomly divided into 2 groups. Solvent control (refined
peanut oil nanoemulsion) or 2.5 mg/kg formulated 1 were injected
into the mice by intratumoral injection at days 0, 2, 5, 7, 9, 12 (6 doses
in total) after treatment. Tumor volumes were measured. None of the
mice died during the experiment. They were sacrificed on day 14 after
treatment.
3.10. Formulation of 1
For the screening experiments, nine colloidal dispersions were pre-
pared: The nanoemulsions contained 10% of the respective oil (soybean
oil, refined peanut oil, refined castor oil, Miglyol 812® (all Ph. Eur.),
Dynasan® 110 (Hüls AG), Dynasan® 112 (Condea)) and the aqueous
phase, which consisted of 5% poloxamer 188 (Kolliphor® P188; BASF)
as emulsifier and 2.25% glycerol as isotonizing agent dissolved in
bidistilled water. Mixing of the lipid and aqueous phase by Ultra–
Turrax-vortexing and processing in a high pressure homogenizer
(Microfluidizer M110-PS, Microfluidics) resulted in nanoemulsions
(the solid triglycerides Dynasan® 110 and Dynasan® 112 were proc-
essed at 45 °C and 55 °C respectively, i.e. above their melting tempera-
ture). The median particle size was measured using laser diffraction
with PIDS technology (Beckman Coulter LS13320) and was below
160 nm in all emulsions. Smectic cholesteryl myristate particles were
analogously prepared (2.5% cholesteryl myristate (TCI), 2% poloxamer
188; median particle size 134 nm) at 95 °C. Mixed micelles were pre-
pared by vigorous shaking of 13.4% of the phospholipid Lipoid S100®
(Lipoid GmbH) and 7.4% sodium glycocholate hydrate (Sigma) in
phosphate buffer pH 7.4 until translucent. For the liposomes, 15% of
Lipoid S100® dispersed in phosphate buffer pH 7.4 was extruded 21
times through a 100 nm PC (polycarbonate membrane, Whatman®
Nucleopore) membrane. The liposomes had a median size of 118 nm.
Each preformed carrier was incubated with excess of 1 on a vertical
shaker at 20 °C for 10 days. Then, undissolved 1 was filtered off and
the dispersions were subjected to AAS measurements. All carriers
retained their initial particle size during the screening experiments.
Acknowledgments
The support by COST action CM1105, Deutsche Forschungs-
gemeinschaft (DFG), Deutscher Akademischer Austauschdienst (DAAD),
and the state of Lower Saxony (graduate program SynFoBiA) as well as
technical assistance by Petra Lippmann are gratefully acknowledged. Fi-
nancial support of M. K. by a POSTUP2 grant (European Social Fund in
the Czech Republic, European Union, Czech ministry of education and
sports, operational program for the advancement of the competitive ca-
pacity) is gratefully acknowledged (CZ.1.07/2.3.00/30.0041).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2015.12.020.
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Please cite this article as: V. Andermark, et al., Alkynyl gold(I) phosphane complexes: Evaluation of structure–activity-relationships for the
phosphane ligands, effects on key s..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2015.12.020